Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps

The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in... ARTICLE The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps 1 2 1 1 Sébastien Jaillon, Giuseppe Peri, Yves Delneste, Isabelle Frémaux, 2 2 2 3 Andrea Doni, Federica Moalli, Cecilia Garlanda, Luigina Romani, 4 3 3 5 Hugues Gascan, Silvia Bellocchio, Silvia Bozza, Marco A. Cassatella, 1,6 2,7 Pascale Jeannin, and Alberto Mantovani Institut National de la Santé et de la Recherche Médicale, Equipe Avenir, Unité 564, University Hospital of Angers, University of Angers, Angers 49933, France Istituto Clinico Humanitas, Istituto di Ricovero e Cura a Carattere Scientifi co, 20089 Rozzano, Italy Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, 05122 Perugia, Italy Institut National de la Santé et de la Recherche Médicale, Unité 564, University of Angers, 49933 Angers, France Department of Pathology, University of Verona, 37134 Verona, Italy Immunology and Allergology Laboratory, University Hospital of Angers, 49933 Angers, France Institute of General Pathology, Faculty of Medicine, University of Milan, 20100 Milan, Italy The long pentraxin (PTX) 3 is produced by macrophages and myeloid dendritic cells in response to Toll-like receptor agonists and represents a nonredundant component of humoral innate immunity against selected pathogens. We report that, unexpectedly, PTX3 is stored in specifi c granules and undergoes release in response to microbial recognition and infl ammatory signals. Released PTX3 can partially localize in neutrophil extracellular traps formed by extruded DNA. Eosinophils and basophils do not contain preformed PTX3. PTX3- defi cient neutrophils have defective microbial recognition and phagocytosis, and PTX3 is nonredundant for neutrophil-mediated resistance against Aspergillus fumigatus. Thus, neutrophils serve as a reservoir, ready for rapid release, of the long PTX3, a key component of humoral innate immunity with opsonic activity. Innate immunity is the fi rst line of defense arm of the innate immunity includes soluble CORRESPONDENCE Alberto Mantovani: against pathogens and plays a key role in the PRRs, such as collectins, fi colins, complement [email protected] initiation, activation, and orientation of adaptive components, and pentraxins (PTXs) (3). OR immunity. Innate immunity receptors, also Members of the PTX superfamily are usu- Pascale Jeannin: called pattern recognition receptors (PRRs), ally characterized by a pentameric structure and [email protected] recognize a few highly conserved structures, are highly conserved during evolution (3–6). Abbreviations used: CHO, called pathogen-associated molecular patterns, This family is subdivided into two subclasses Chinese hamster ovary; CRP, expressed by microorganisms (1). PRRs are that depend on the length and structure of the C-reactive protein; MMP-9, matrix metalloproteinase 9; either cell associated (expressed intracellularly or molecules. The classical short PTXs C- reactive MPO, myeloperoxidase; NET, on the cell surface) or present in body fl uids. protein (CRP) and serum amyloid P component neutrophil extracellular trap; There are two functional classes of cell- associated (SAP) are acute-phase proteins in humans and OmpA, outer membrane protein A; PRR, pattern PRRs: endocytic PRRs (i.e., scavenger recep- mice, respectively (7, 8), that are produced in recognition receptor; PTX, tors and mannose receptors) involved in micro- the liver in response to infl ammatory mediators, pentraxin; SAP, serum amy- organism binding and uptake; and signaling most prominently IL-6. CRP and SAP bind, in loid P component; TLR, PRRs (members of the Toll-like receptor [TLR], a calcium-dependent manner, diff erent ligands Toll-like receptor. nucleotide-binding oligomeri zation domain, and are involved in innate resistance to microbes and helicase families) involved in cell activation and scavenging of cellular debris and extra- upon contact with pathogens (2). The humoral cellular matrix components (4, 7). Long PTXs are characterized by an unrelated N-terminal domain coupled to a PTX-like C-terminal do- S. Jaillon, G. Peri, P. Jeannin, and A. Mantovani contributed main (3, 6, 9). The prototypic long PTX3 is rap- equally to this work. The online version of this article contains supplemental material. idly produced and released by diverse cell types, JEM © The Rockefeller University Press $15.00 793 Vol. 204, No. 4, April 16, 2007 793–804 www.jem.org/cgi/doi/10.1084/jem.20061301 The Journal of Experimental Medicine in particular by mononuclear phagocytes, DCs, and endothe- observed similar levels of intracellular PTX3 using 20 and lial and epithelial cells in response to primary infl ammatory 200 μg/ml of human IgG for saturation, showing that the signals (e.g., TLR engagement, TNFα, and IL-1β) (10–14). detection of intracellular PTX3 in neutrophils is not related With high affi nity, PTX3 binds the complement component to nonspecifi c binding of the anti-PTX3 mAb (not depicted). To confi rm this observation, PTX3 expression was analyzed C1q, the extracellular matrix TNF-inducible protein 6, and selected microorganisms (e.g., Aspergillus fumigatus, Pseudomonas by Western blotting. Human neutrophils and DCs express aeruginosa, Salmonella typhimurium, Paracoccidioides brasiliensis, two or three immunoreactive forms of PTX3, depending and zymosan) (15–18). on the donor (with one major band at 47 kD and two −/− Recent studies in ptx3-defi cient (PTX3 ) mice have minor bands at 44 and 42 kD; Fig. 1 B). The presence of shown that this molecule performs complex nonredundant intracellular PTX3 in neutrophils was also confi rmed by con- functions in vivo, ranging from the assembly of a hyaluronic focal microscopy (Fig. 1 C). We assessed whether other cir- acid–rich extracellular matrix to female fertility to innate im- culating elements containing granules store PTX3. PTX3 munity against diverse microorganisms (15, 16, 19, 20). PTX3 could not be detected in eosinophils and basophils (Fig. 1 C), also binds apoptotic cells (21) and may contribute to editing nor in large granular lymphocytes (NK cells; not depicted). PTX3 was detected in LPS-activated DCs (Fig. 1 B) (10, recognition of apoptotic cells versus infectious nonself (22). In addition, there is evidence for a regulatory role of PTX3 35). By ELISA, resting human neutrophils contain 24.9 ± in noninfectious infl ammatory reactions (Latini, R., personal 3.8 ng PTX3 per 10 cells (n = 4) corresponding to 0.55 communication) (23). Outer membrane protein A (OmpA) is pmol (Fig. 1 D), calculated based on the protomer molecular a conserved constituent of the outer membrane of Enterobacte- mass. Over a period of 24 h, DCs release 50.2 ± 8.1 ng riaceae. A search of moieties recognized by PTX3 has discov- PTX3 per 10 cells (n = 5; Fig. 1 D) (10). These results show ered that it binds OmpA from Klebsiella pneumoniae and that it that neutrophils contain considerable amounts of preformed represents a nonredundant humoral amplifi cation loop of the PTX3. Finally, unlike PTX3, the short PTXs CRP and SAP innate immunity response to this microbial moiety (24). could not be localized in neutrophils (not depicted). Among innate cells, neutrophils play an important role In agreement with a previous study (11), PTX3 mRNA is not expressed in freshly isolated human neutrophils, as as- because of their ability to be rapidly recruited in tissues during infections and to produce mediators that kill or inhibit micro- sessed by RT-PCR analysis (Fig. 1 E). As a positive control, bial growth (25–27). As PTX3 is important in infectious PTX3 mRNA was evident in LPS-stimulated DCs (Fig. and infl ammatory responses (3, 28), we evaluated whether 1 E), as previously reported (10). We thus evaluated whether neutrophils produce PTX3. In previous studies, PTX3 was neutrophil precursors express PTX3 mRNA. Promyelocytes, found to be expressed in HL60 cells (11) and bone marrow myelocytes/metamyelocytes, and bone marrow–segmented myelocytes (29) as mRNA and, by proteomic analysis, in neutrophils were isolated by density centrifugation on a neutrophil granules (30). In this paper, we report that PTX3 Percoll gradient, followed by magnetic cell sorting (29, 36). is stored in a ready-made form in neutrophils but not in RT-PCR analysis revealed that PTX3 mRNA is expressed in eosinophils and basophils. PTX3 is localized in specifi c gran- promyelocytes and myelocytes/metamyelocytes but not or at low levels in bone marrow–segmented neutrophils (Fig. 1 F, ules and is secreted in response to recognition of microbial moieties and infl ammatory signals. PTX3 can localize in neu- left), a result in accordance with the absence of PTX3 mRNA trophil extracellular traps (NETs) (31), and PTX3-defi cient expression in mature peripheral neutrophils. As a positive neutrophils have defective phagocytic activity. In addition, control, myeloperoxidase (MPO) mRNA was mainly detected injection of wild-type neutrophils restores protective immunity in promyelocytes and, at a lower level, in myelocytes but not in −/− against A. fumigatus in PTX3 mice. Thus, neutrophils serve bone marrow–segmented neutrophils, as previously reported as a reservoir, ready for rapid release, of a key component of (29, 36), confi rming the purity of the isolated cell populations. humoral innate immunity and complement its subsequent Western blot analysis showed that PTX3 is expressed in the delayed neosynthesis by macrophages and DCs. three populations of neutrophil precursors (Fig. 1 F, right). In agreement with previous observations (11), the human pro- myelocytic cell line HL60 constitutively expresses PTX3 RESULTS Storage of preformed PTX3 in resting neutrophils mRNA (Fig. S1, available at http://www.jem.org/cgi/content/ FACS analysis revealed constitutive expression of PTX3 full/jem.20061301/DC1). HL60 cells express mRNA encoding in freshly isolated human neutrophils, as assessed by intracell- MPO, a marker of primary granules, but not mRNA encoding ular labeling (mean fl uorescence intensity = 89 ± 34 and lactoferrin and matrix metalloproteinase 9 (MMP-9; Fig. S1), 4 ± 1.5, using anti-PTX3 and control mAbs, respectively; as previously reported (37, 38). Moreover, HL60 cells sponta- mean ± SD; n = 8; Fig. 1 A). No expression of PTX3 neously produce PTX3 protein (Fig. S1). was observed on the surface of human neutrophils (not depicted). Recent studies reported that nonspecifi c binding of Localization of PTX3 in neutrophil-specifi c granules polyclonal Ig within neutrophils may give false positive Confocal microscopy and subcellular fractionation of neutro- phil-derived nitrogen cavitates were used to specifi cally results, as observed using antigranzyme antibody (32–34). Following the methodologies described in these studies, we localize PTX3 within freshly isolated neutrophils. As shown 794 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE Figure 1. PTX3 is constitutively expressed in human neutrophils. by RT-PCR. Results obtained in 2 out of 10 subjects tested are presented. (A) FACS analysis of PTX3 expression in permeabilized human neutrophils LPS-stimulated DCs are used as a positive control. RNA integrity and isolated from peripheral blood. (B) Western blot analysis of PTX3 expres- cDNA synthesis were verifi ed by amplifying GAPDH cDNA. (F) Analysis sion in neutrophils and LPS-stimulated DCs. (C) Analysis of PTX3 expres- of PTX3 mRNA and protein in neutrophil precursors. Promyelocytes sion in freshly isolated neutrophils, eosinophils and basophils by confocal (PM), myelocytes/metamyelocytes (MY), and bone marrow–segmented microscopy. Fluorescence (left) and differential interference contrasts neutrophils (bm-PMN) were analyzed for PTX3 and MPO mRNA expres- (right, Nomarski technique) are shown. Bars, 10 μm. (D) Analysis of PTX3 sion (left). Expression of PTX3 was evaluated by Western blotting using content in freshly isolated neutrophils, eosinophils, and basophils, as the anti-PTX3 mAb 16B5 in the three populations of neutrophil precur- well as release by LPS-stimulated DCs, determined by ELISA (mean ± SD). sors (right), and total protein loading was evaluated by analyzing (E) Analysis of PTX3 mRNA expression in freshly isolated human neutrophils actin expression. in Fig. 2 A, confocal studies pointed to complete colocaliza- In an eff ort to confi rm these observations, subcellular tion of PTX3 with lactoferrin, a constituent of specifi c gran- fractionation of neutrophil-derived nitrogen cavitates was ules, and, at least in part, with gelatinase (tertiary granules; performed. PTX3 was detected in fractions that contain lac- Fig. 2 A). In contrast, no colocalization with MPO, a marker toferrin and part of the gelatinase distribution but not in frac- of azurophilic granules, was observed (Fig. 2 A). Confocal tions containing α-mannosidase (azurophilic granules; Fig. microscopy was combined with quantitative analysis to mea- 2 C) or albumin (secretory vesicles/light membranes; not sure the percentage of PTX3 colocalization, as assessed by depicted) (40). Collectively, these results demonstrate the Pearson’s coeffi cient of correlation. Higher degrees of colo- selective association of PTX3 with specifi c (lactoferrin and calization were measured in diff erent experiments (n = 5) in lactoferrin/gelatinase ) granules. + + lactoferrin and gelatinase vesicles, which display coeffi cients of 72.22 ± 7.32% (r = 0.78) and 27.8 ± 8.73% (r = 0.45), PTX3 release by stimulated neutrophils respectively. Little or no colocalization of PTX3 was found We next evaluated whether neutrophils release PTX3 upon with MPO granules (5.90 ± 2.21%; r = 0.08). stimulation. FACS analysis showed that the relative intracellu- Among the MPO-negative granules, 16% contain only lar level of PTX3 decreased upon 2 h of stimulation with 10 lactoferrin (specifi c) and 24% contain only gelatinase (ter- μg/ml Staphylococcus aureus and Escherichia coli (Fig. 3 A). In con- tiary), whereas 60% contain both markers (specifi c) (39). Fig. trast, the intracellular level of PTX3 increased in DCs stimu- + + 2 B shows lactoferrin granules, gelatinase granules, or double- lated for 8 h with 80 ng/ml LPS compared with nonstimulated stained granules in a representative cell. PTX3 was found to cells (Fig. 3 B). The levels of PTX3 were also quantifi ed by colocalize with lactoferrin granules and double-stained gra- ELISA in the cell culture supernatants. E. coli, S. aureus, and, to a lesser extent, zymosan increased the release of PTX3 by nules (specifi c), but no colocalization was found with tertiary granules that were only gelatinase . neutrophils in a dose-dependent manner, with a maximal eff ect JEM VOL. 204, April 16, 2007 795 + + Figure 2. Localization of PTX3 within neutrophil granules. Specifi c granules were identifi ed as lactoferrin and lactoferrin / + + (A) Localization of PTX3 in freshly isolated neutrophils by confocal micro- gelatinase ; tertiary granules were identifi ed as gelatinase . Insets scopy. Cells were fi xed and stained for human PTX3 and MPO (left), PTX3 show enlargements of the indicated areas. Bars, 10 μm. (C) Analysis and gelatinase (middle), and PTX3 and lactoferrin (right; see Materials and of PTX3 content in neutrophil subcellular fractions. PTX3, gelatinase, methods). DNA labeling is also shown (Hoechst 33258). Insets show en- lactoferrin, and α-mannosidase content in each fraction were deter- largements of the indicated areas. Bars, 10 μm. (B) Localization of PTX3 mined by ELISA or using a functional assay for α-mannosidase. in neutrophil-specifi c granules. Cells were stained for PTX3, gelatinase, Results are expressed as a percentage of the relative amount in the and lactoferrin (see Materials and methods). A representative cell is shown. collected fractions. observed with the highest concentration used (10 μg/ml; Fig. PTX3 was not a consequence of cell death (not depicted). 3 C). A similar increase of PTX3 was observed using 1 or 10 Previous experiments reported that GM-CSF increases the ng/ml PMA, 0.01 or 1 μM ionomycin, and 20 ng/ml of the sensitivity of neutrophils to TLR agonists (46). Neutrophils proinfl ammatory cytokine TNF α (Fig. 3 C). 20 ng/ml IL-1β were thus primed with 50 ng/ml GM-CSF for 2 h before stim- was inactive (not depicted). Propidium iodide staining and lactate ulation with zymosan, S. aureus, and TLR agonists. The levels dehydrogenase release assay excluded that PTX3 secretion re- of PTX3 released upon stimulation with TLR agonists or sulted from cell death (not depicted). Latex beads failed to trigger microorganisms were weakly but signifi cantly increased in PTX3 release (not depicted). The release of PTX3 is associated GM-CSF–primed neutrophils (Fig. 3 E). In addition, microor- with a decrease in the level of intracellular PTX3 in neutro- ganisms and TLR agonists induced the release of MPO (Fig. phils, as assessed by Western blot analysis (Fig. S2, available 3 F) and MMP-9 (Fig. 3 G). Independent of the stimulus used, at http://www.jem.org/cgi/content/full/jem.20061301/DC1). PTX3 mRNA was not induced in stimulated neutrophils, as PMA and S. aureus induced a time-dependent release of PTX3 assessed by RT-PCR analysis after 8 h (Fig. 3 H) or 16 h (not by neutrophils, signifi cant at 1 h and maximal at the latest time depicted) of activation. These data show that microorganisms analyzed (16 h; Fig. 3 D). It is noteworthy that TNF and PMA and TLR agonists trigger substantial release of PTX3 by hu- induce selective release of specifi c granules (41, 42). Among man neutrophils. the diff erent activators tested, microorganisms appear to be the most potent inducers of PTX3 release by human neutrophils. Association of PTX3 with NETs We therefore evaluated the ability of TLR agonists to induce NETs are extracellular structures formed by the extrusion of PTX3 release. LPS, R848, Pam CSK4, and, to a lesser extent, DNA from viable neutrophils upon stimulation, and they act fl agellin (but not poly (I:C)) induced the release of PTX3 as focal points to focus antimicrobial eff ector molecules (31). by neutrophils (Fig. 3 E). In agreement with previous studies We analyzed whether PTX3 colocalizes with NETs upon (43–45), we observed that a stimulation with TLR agonists stimulation. As shown in Fig. 4, after 40 min of stimulation delays neutrophil apoptosis, confi rming that the release of with IL-8, LPS, or PMA (Fig. 4 A) or the conidia of A. fumigatus 796 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE Figure 3. Secretion of PTX3 by activated neutrophils. (A and B) 50 ng/ml GM-CSF. Supernatants were collected at 16 h. PTX3 was FACS analysis of intracellular PTX3 expression in neutrophils stimulated quantifi ed in the supernatants by ELISA. Results are expressed as for 2 h with 10 μg/ml S. aureus or 10 μg/ml FITC-labeled E. coli (A) or ng/ml (mean ± SD; n = 6). MPO (F) and MMP-9 (G) were quantifi ed in DCs stimulated or not for 8 h with 80 ng/ml LPS (B). Representative in the supernatants of neutrophils stimulated for 16 h with the indi- results from one to fi ve experiments are shown. (C–E) Analysis of PTX3 cated stimuli; results are expressed in ng/ml, showing the mean of two release upon neutrophil stimulation. (C) 2 × 10 cells/ml of neutrophils representative experiments. (H) PTX3 mRNA expression analyzed by were activated for 16 h with 1 or 10 μg/ml E. coli, S. aureus, or zymosan; RT-PCR in neutrophils untreated or stimulated for 8 h with 100 ng/ml 1 or 10 ng/ml PMA; 0.01 or 1 μM ionomycin; or 20 ng/ml TNFα. LPS, 10 μg/ml E. coli, 10 μg/ml S. aureus, 10 μg/ml zymosan, 20 ng/ml (D) Time-dependent release of PTX3 in neutrophils (nonstimulated or TNFα, 10 ng/ml PMA, or 1 μM iononmycin. RNA integrity and cDNA stimulated with 10 μg/ml S. aureus or 10 ng/ml PMA) is shown. Super- synthesis were verifi ed by amplifying GAPDH cDNA. *, P < 0.01 for natants were collected at the indicated time points. (E) Induction of untreated versus GM-CSF–treated cells. PTX3 release by TLR agonists in neutrophils pretreated or not with (Fig. 4 B), PTX3 was found associated with NETs. PTX3 depending on the donor tested (Fig. 1 B and Fig. 5, left). Three immunoreactive bands were also evident in the super- binds conidia from A. fumigatus and plays a nonredundant role in resistance against this fungus (15). Interestingly, both natants of activated neutrophils (Fig. 5, right), with molecular PTX3 and conidia were found associated to NETs (Fig. 4 B). masses ranging from 47 to 250 kD. A previous study reported Thus, PTX3 is rapidly released by neutrophils (Fig. 4 A and that PTX3 is glycosylated at the Asn 220 residue (18). A re- Fig. 3 D) and localizes in NETs. cent characterization of the PTX3 glycosidic moiety revealed three antennary structures and a role in the interaction with Characterization of neutrophil PTX3 C1q (47). We thus analyzed the level of PTX3 glycosylation As mentioned above, Western blot analysis revealed two or in neutrophils. Treatment of neutrophil cell lysates and cul- three immunoreactive PTX3 isoforms in human neutrophils, ture supernatants with N-glycosidase F resulted in a decrease JEM VOL. 204, April 16, 2007 797 Figure 5. Biochemical analysis of PTX3 in neutrophils. Cell lysates from nonstimulated cells (left) and supernatants from S. aureus– stimulated neutrophils (right) were either untreated (−) or treated with N-glycosidase F (+). Supernatants from CHO cells transfected with wild- type or N220S mutant PTX3 were collected after a 24-h culture (right). PTX3 was analyzed by Western blotting using rabbit polyclonal anti-PTX3 and revealed by peroxidase-labeled anti–rabbit IgG antibody and ECL. sized PTX3 in LPS-activated DCs, resulting in a decrease of PTX3 release in the supernatant (Fig. S3, available at http:// www.jem.org/cgi/content/full/jem.20061301), as previously reported (48). Moreover, an Asn®Ser substitution at posi- tion 220 was introduced in PTX3 (N220S mutant). Recom- binant wild-type and N220S PTX3 were detected in the supernatants of Chinese hamster ovary (CHO) cells in a mul- timeric form (Fig. 5, right). Collectively, these data show that neutrophils contain a mature glycosylated form of PTX3 that associates in the extracellular milieu to form multimers inde- pendently of glycosylation. In vitro and in vivo relevance of PTX3 expression in neutrophils We evaluated whether neutrophil-derived PTX3 is func- tional in recognizing ligands of self or microbial origin and may play a role in microbial recognition and destruction. First, we found that PTX3 purifi ed by immunoaffi nity from human PMN lysate bound to immobilized C1q and OmpA from K. pneumoniae; similarly, neutrophil-released PTX3, obtained by concentration of PMN supernatant, bound to A. fumigatus conidia, as did the recombinant protein (Fig. S4, A and B, available at http://www.jem.org/cgi/content/full/ jem.20061301/DC1) (15, 18, 24). Second, we studied neutrophil expression of PTX3 in the mouse. Mature mouse neutrophils constitutively express PTX3, as assessed by Western blotting using anti-PTX3 pAbs Figure 4. PTX3 is localized in NETs. (A) Neutrophils were exposed to or mAbs (Fig. 6 A). Bone marrow–derived (two experiments; 100 ng/ml IL-8, 100 ng/ml LPS, or 2.5 ng/ml PMA for 40 min. (left) PTX3 not depicted) and peritoneal (fi ve experiments performed staining. (right) PTX3 and DNA staining. Bars, 10 μm. (B) Neutrophils in two diff erent laboratories; Fig. 6 B) neutrophils from exposed to conidia from A. fumigatus; a differential interference contrast −/− PTX3 mice showed a signifi cantly lower phagocytosis of (Nomarski technique) is shown in the bottom panels. In both A and B, PTX3 immunostaining was done on nonpermeabilized neutrophils. conidia from A. fumigatus, with a 31 ± 7% reduction com- Bars, 5μm. pared with PTX3-competent cells (Fig. 6 B, left). 1.1 μM of exogenous PTX3 caused a signifi cant increase in the phago- cytosis of conidia by PTX3-competent and incompetent neu- +/+ of the apparent molecular mass of PTX3 (Fig. 5). As expected trophils (phagocytic index = 102 and 76% for PTX3 cells on the basis of PTX3 synthesis during neutrophil diff erentia- with and without PTX3, respectively; phagocytic index = 99 −/− tion but not in mature PMN, tunicamycin did not modify and 64% for PTX3 cells with and without PTX3, respec- the Western blot profi le in resting and activated neutrophils. tively). In the presence of exogenous PTX3, no diff erence In contrast, it prevented the glycosylation of newly synthe- was detected in the phagocytic activity of PTX3-competent 798 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE and incompetent neutrophils (Fig. 6 B, right). Similarly, the protective eff ect was observed using macrophages or CD4 −/− +/+ +/− conidicidal activity of PTX3 PMN was severely impaired T cells. As a control, PTX3 or PTX3 neutrophils also +/+ +/+ compared with that of PTX3 PMN (10 and 45%, respec- prevented fungal growth in PTX3 mice, but the eff ect −/− tively) and was increased 30% by the addition of recombinant was less pronounced than in highly susceptible PTX3 −/− PTX3 (not depicted). mice (Fig. 6 C). No such eff ect was observed with PTX3 In an eff ort to assess the actual in vivo relevance of neu- neutrophils or macrophages (Fig. 6 C). −/− trophil-associated PTX3, cyclophosphamide-treated PTX3 +/+ and PTX3 mice were infected with Aspergillus conidia D I S C U S S I O N −/− +/+ +/− and given PTX3 , PTX3 , or PTX3 PMN, macro- The prototypic long PTX3 has long been known to be phages, or purifi ed CD4 T cells 3 h later. Mice were moni- produced by diverse cell types on demand, i.e., in a gene tored for fungal growth 2 d later by quantifying the chitin expression–dependent fashion in response to extracellular content in the lung (49). Results showed that injection of signals (e.g., LPS, IL-1β, TNFα, and TLR agonists) (3). The fi nd- +/+ +/− −/− PTX3 and PTX3 neutrophils, but not PTX3 cells, ing that PTX3 is stored in neutrophil granules is therefore −/− + in PTX3 mice decreased fungal growth (Fig. 6 C). No unexpected. PTX3 is not stored in MPO granules (primary or azurophilic). By confocal analysis among the MPO gran- ules, PTX3 was found to localize in lactoferrin and in lacto- + + + ferrin /gelatinase (specifi c) but not in gelatinase (tertiary) granules. Storage of PTX3 in neutrophil granules is selective, inasmuch as short PTXs are absent and other granulated cir- culating elements (eosinophils, basophils, and NK cells) do not contain preformed PTX3. In addition to the diversity generated during granulopoiesis, granules are secreted in a targeted manner, with a timing hierarchy in granule exocyto- sis (50, 51). PTX3 is localized in granules that are rapidly mobilized and secreted upon stimulation, in agreement with its early detection in the supernatants of stimulated neutro- phils. Expression of PTX3 transcripts is confi ned to imma- ture myeloid elements, and mature neutrophils only act as a reservoir of preformed PTX3. Thus, neutrophils represent a reservoir of this PRR and release it in response to microbial or infl ammatory signals. The release of PTX3 by neutrophils is induced by micro- organisms and TLR agonists and, to a lower extent, by pro- infl ammatory cytokines. Latex beads do not induce secretion of PTX3, suggesting that phagocytosis is not suffi cient and that TLR recruitment is required to trigger its release by neu- trophils. This dichotomy of PTX3-inducing mediators be- tween neutrophils and other cell types is of physiological signifi cance. Indeed, neutrophils are the fi rst cells recruited into tissues in response to microorganism entry. Neutrophils are thus highly sensitive to microbes and microbe-derived components, suggesting that PTX3, rapidly released by infi l- Figure 6. Functional role of neutrophil-derived PTX3. (A) Analysis trating neutrophils, interacts with microorganisms and facili- of PTX3 expression in mouse bone marrow–segmented neutrophils from tates their internalization by neutrophils themselves, as well as C57BL/6 mice by Western blotting. Results are representative of three resident and/or recruited professional APCs (19). Microor- independent experiments. A pAb or mAb (16B5) was used. (B, left) Phago- −/− ganisms and TLR agonists, the main inducers of PTX3 re- cytosis of A. fumigatus conidia by peritoneal neutrophils from PTX3 +/+ or PTX3 mice after 15 or 45 min. One representative experiment out lease, also induced the release of MPO and MMP-9, which of fi ve performed is shown. *, P < 0.01. (B, right) Effect of 50 μg/ml of are stored mainly in azurophilic and tertiary granules, respec- exogenous PTX3 in the phagocytosis of conidia by PTX3-competent tively. These results suggest that microorganisms and TLR and incompetent neutrophils (45 min of incubation). One representative agonists induce the release of the molecules stored within the experiment out of fi ve performed is shown. (C) Role of PTX3 in neutrophil- three types of granules. mediated resistance against A. fumigatus. 10 A. fumigatus conidia Upon exposure to microbial or infl ammatory signals, per 20 μl were given intranasally to PTX3-defi cient or -competent mice viable neutrophils extrude nuclear components that form an pretreated with cyclophosphamide. 3 h later, mice were given 10 + +/+ +/− extracellular DNA fi brillary network. NETs trap microbes neutrophils, macrophages, or CD4 T cells i.v. from PTX3 , PTX3 , or −/− and retain neutrophil antimicrobial molecules (31). Therefore, PTX3 mice. Chitin content was measured as a correlation of fungal growth (mean ± SEM; n = 3). *, P < 0.05 using the Student’s t test. NETs serve as a focal point to focus the action of antimicrobial JEM VOL. 204, April 16, 2007 799 molecules. In this paper, we report that part of exocytosed Finally, we evaluated the in vivo relevance of PTX3 ex- PTX3 can localize in NETs. Therefore, in addition to anti- pression in neutrophils. PTX3-defi cient mice are highly sen- microbial molecules, NETs can concentrate and focus the sitive to A. fumigatus infection (15). Eff ector mechanisms of action of PTX3, a functional ancestor of antibodies. innate immunity are crucial to prevent aspergillosis (55), and The PTX3 protein is expressed in circulating PMN in among innate cells, neutrophils are essential in the initiation the absence of transcripts, whereas NF-κB–driven PTX3 of the acute infl ammatory response. Moreover, susceptibility production (52) is induced in a variety of cells by microbial to fungal infection can be associated, among other parame- sensing and infl ammatory cytokines (3). PTX3 mRNA was ters, to neutropenia (56), and impairment of neutrophil anti- detected in promyelocytes and myelocytes/metamyelocytes fungal responses results in an increase of fungal burden (57). but not in bone marrow–segmented neutrophils. In agree- These data underline the essential role played by neutrophils ment with this result and a previous report (11), we observed in controlling A. fumigatus infection. We report that PTX3 that the promyelocytic cell line HL60 expresses PTX3 expressed by neutrophils is essential to control fungal growth mRNA. A previous study reported that PTX3 is expressed in vitro and in vivo. Innate and adaptive immunity are both only at the myelocyte stage (29), and on this basis localization essential for the development of a protective antifungal im- of PTX3 in specifi c granules was postulated, a hypothesis mune response. Generation of a Th1-oriented A. fumigatus– confi rmed by our results. Collectively, we can hypothesize specifi c immune response is associated with protection (58, 59). −/− that, in accordance with the targeting-by-timing hypothesis Injection of PTX3 in PTX3 mice favors the generation (the protein content into distinct granules is determined by of a protective Th1 anti-Aspergillus immune response (15). the time of their biosynthesis), PTX3 mRNA is expressed at Neutrophil-derived PTX3, in addition to DC-derived a late stage of promyelocyte diff erentiation and in myelo- PTX3, may be involved in the orientation of the immune cytes/metamyelocytes and that most PTX3 is synthesized at response toward a protective Th1 cell phenotype. Neutrophils, the myelocytes/metamyelocytes stage, a result in agreement an innate cell type without professional antigen- presenting with its preferential localization in secondary granules. functions, may participate in the orientation of specifi c anti- PTXs usually form multimers with a discoid arrangement microbial immune responses via the release of this preformed of fi ve subunits (3). PTX3 assembles as a decamer and can be soluble PRR. produced as 10–20 subunit multimer proteins (18). PTX3 in PTX3 is a long PTX conserved in evolution, with func- neutrophil granules is mainly in the monomer form, and mul- tional properties (e.g., complement activation and opsoniza- timeric forms are detected in the supernatants of activated tion) that qualify it as a functional ancestor of antibodies. This neutrophils. The formation of PTX3 multimers is not depen- fl uid-phase PRR binds diverse microbial agents, activates the dent on glycosylation (47). However, glycosylation appears classic pathway of complement, and facilitates ingestion by crucial for the release of neo-synthetized PTX3, as observed innate immunity cells (3, 17, 24, 28), including neutrophils in DCs (this study) and fi broblasts (48). These results show (Fig. 6 B). Gene-modifi ed mice unequivocally indicate that that human neutrophils contain a mature glycosylated form of PTX3 represents a nonredundant humoral amplifi cation loop PTX3 that assembles as multimers in the extracellular milieu. of the innate immune response to diverse microbial agents Myeloid, but not plasmacytoid, DCs and macrophages (3, 15, 19, 24). Neutrophils store a variety of constituents in are major producers of PTX3 (10). Over a period of 24 h, their granules, including adhesion receptors (e.g., CD11b DCs release 50 ng of PTX3 per 10 cells (10). We report and CD18), proteolytic enzymes (e.g., cathepsin G), eff ectors that neutrophils contain 24.9 ± 3.8 ng of this PRR per 10 and regulators of matrix degradation (e.g., gelatinase), and cells (n = 5). Upon stimulation, they release 25% of stored antimicrobial molecules (25–27). The results reported in this PTX3, with a part of it remaining cell associated, presumably study broaden the repertoire of eff ector molecules stored and with NETs. Given the abundance of neutrophils in the cir- released by neutrophils to include a humoral PRR with culation and in the early phases of infl ammatory reactions in functional properties of a predecessor of antibodies. PTX3- tissues, these cells represent a major source of PTX3 covering defi cient neutrophils have defective recognition, phagocy- a temporal window preceding gene expression–dependent tosis, and killing of conidia, completely restored by PTX3, production. Under conditions of tissue damage (e.g., myo- and this molecule is nonredundant for protection against cardial infarction) or infection (e.g., sepsis), PTX3 levels in- A. fumigatus by neutrophils. Thus, PTX3 stored in neutro- crease rapidly. For instance, in acute myocardial infarction phil granules amplifi es microbial recognition by neutrophils with ST elevation, PTX3 reaches a peak in 6–8 h, compared themselves as well as presumably by neighboring innate im- with 36–48 h for CRP (53). Under these conditions, high munity cells. PTX3 is an independent marker associated with death (54). The results reported in this paper shed new light on PTX3 MATERIALS AND METHODS elevations in pathological conditions and on their pathophys- Leukocyte purifi cation. Monocytes were isolated and diff erentiated iological implications. It is likely that rapid release of stored into DCs by a 5-d culture with 20 ng/ml IL-4 and 20 ng/ml GM-CSF PTX3 by activated neutrophils plays a role in the early phases − − (R&D Systems) (60). CD14 CD86 immature DCs were used. After of its elevation in pathology, preceding gene expression– Ficoll-Paque centrifugation, neutrophils were separated from erythrocytes by dependent production. 3% dextran (GE Healthcare) density gradient sedimentation. Purity, determined 800 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE by FACS analysis on forward scatter/side scatter parameters, was routinely FACS analysis. PTX3 expression was analyzed on fi xed (2% paraformalde- >98%. Spontaneous activation of purifi ed neutrophils was eva luated by ana- hyde) and permeabilized (0.1% saponin; Sigma-Aldrich) cells with anti- + low lyzing CD11b and l-selectin expression by FACS; only l-selectin CD11b PTX3 mAb (MNB4; Qbiogene) in PBS containing 0.01% saponin. Bound neutrophils were used. Neutrophils were also isolated from whole blood antibodies were revealed by PE-labeled anti–rat IgG mAb (BD Biosciences). collected from healthy donors using a two-step buoyant density centrifugation Isotype control mAb was obtained from BD Biosciences. Fluorescence was on a Ficoll gradient (61). analyzed using a cytofl uorometer (FACScan; BD Biosciences), and results Bone marrow cells (obtained from healthy donors) at diff erent myeloid are expressed in mean fl uorescence intensity values. diff erentiation stages were isolated as previously described (36). In brief, bone marrow cells were separated by density sedimentation on a discontinu- ELISA and Western blotting. PTX3 (53), MPO (sensitivity = 0.4 ng/ml; ous Percoll gradient (GE Healthcare) of 1.065 g/ml and 1.08 g/ml. Three HyCult Biotechnology), and total gelatinase (MMP-9, sensitivity = 30 pg/ml; bands of cells, numbered in order of decreasing density, were harvested: R&D Systems) were quantifi ed by ELISA. For Western blotting, proteins band 1 primarily contained segmented neutrophils, band 2 primarily con- (corresponding to 0.4 × 10 cells or 5 μl of supernatants) were electro- tained metamyelocytes and myelocytes, and band 3 contained promyelocytes phoretically separated on a 10% polyacrylamide gel in reducing conditions (36). The three populations were subjected to immunomagnetic depletion and transferred to a membrane (Immobilon; Millipore). After saturation, of nongranulocytic cells, as previously described (29) using MACS (Miltenyi membranes were incubated for 16 h at 4°C with 3 μg/ml anti-PTX3 pAbs Biotec). The purity of cell populations was assessed by microscopy and cell or mAbs (16B5) and with 1 μg/ml peroxydase-labeled anti–rabbit IgG anti- surface phenotype (29). Eosinophils and basophils were enriched by deple- body or peroxydase-labeled anti–rat IgG antibody (Biosource International). tion of magnetically labeled cells (MACS). Protein loading was verifi ed with an anti-actin pAb (Sigma-Aldrich) re- Blood and bone marrow samples were obtained with written informed vealed by the peroxydase-labeled anti–rabbit IgG antibody. Bound antibod- consent in accordance with the Angers University Hospital ethical com- ies were detected using the ECL system (GE Healthcare). mittee requirements. Confocal microscopy. Cytospins were fi xed with 4% paraformaldehyde Cell act ivation. 2 × 10 neutrophil cells/ml in RPMI 1640 (2% FCS) and permeabilized for 5 min with 0.2% Triton X-100 (Sigma-Aldrich) in were either nonstimulated or stimulated with 1–10 μg/ml E. coli, S. aureus, PBS, pH 7.4, before incubation for 1 h at 4°C with 10% normal goat or zymosan (all obtained from Invitrogen); 20 ng/ml IL-1β or TNFα (R&D serum (Sigma-Aldrich) and then for 2 h at 4°C with 0.5 μg/ml biotin- Systems); 1–10 ng/ml PMA; 0.01–1 μM ionomycine; 100 ng/ml LPS (from conjugated rat anti–human PTX3 mAb (1 μg/ml; MNB4), or with IgG2a E. coli serotype O55:B5; all purchased from Sigma-Aldrich); 500 ng/ml control mAb. Slides were incubated with streptavidin–Alexa Fluor 488 Pam3CSK4 (a TLR2 agonist); 5 μg/ml R848 (a TLR7/8 agonist), 2 μg/ml conjugate, followed by 1 μg/ml Hoechst 33258 or by 5 μg/ml propidium fl agellin (a TLR5 agonist; all obtained from InvivoGen); and 10 μg/ml poly iodide and RNase (Invitrogen). The following reagents were also used: (I:C) (a TLR3 agonist; Sigma-Aldrich). 2 × 10 DCs/ml were stimulated for rabbit anti-MPO pAb (Invitrogen), mouse antilactoferrin mAb (clone 8, 16, or 24 h with the stimuli indicated in the fi gures. PTX3 levels in super- NI25; Calbiochem), a rabbit antigelatinase pAb (Chemicon), and Alexa natants were quantifi ed by ELISA. Fluor 647–conjugated goat anti–rabbit and anti–mouse IgG secondary NET formation was induced as previously described (31). In brief, neutro- antibodies. In each step, cells were washed with 0.2% BSA/0.05% Tween 6 5 phils (400 μl of cells at 2 × 10 cells/ml and 8 × 10 cells/well) were seeded 20 in PBS, pH 7.4. on rounded glass coverslips treated with 1% poly-l-lysine (Sigma-Aldrich), To stain NETs, neutrophils were fi xed, blocked, and washed as de- allowed to settle, and treated for 40 min with 100 ng/ml IL-8 (PeproTech), scribed in the previous paragraph, without permeabilization. Cells were in- 2.5 ng/ml PMA, 100 ng/ml LPS, and A. fumigatus conidia at a 1:5 ratio. cubated with 1 μg/ml of biotin-conjugated PTX3 affi nity-purifi ed rabbit IgG, followed by streptavidin–Alexa Fluor 647 conjugate (Invitrogen). For Cell lysates. 10 cells were lysed in 10 mM Tris-HCl, pH 7.6, 5 mM DNA detection, Syto 13 (Invitrogen) plus RNase (Sigma-Aldrich) was used. EDTA, 1% Triton X-100, or Nonidet P40 (Sigma-Aldrich) plus protease Sections were mounted with a reagent (FluorSave; Calbiochem) and analyzed inhibitors (Roche Diagnostics). Lysates were centrifuged at 14,000 rpm for with a laser scanning confocal microscope (FluoView FV1000; Olympus). 15 min at 4°C to remove cellular debris. For N-glycosidase F treatment, Images (1,024 × 1,024 pixels) were acquired with an oil immersion objec- SDS (0.2% fi nal, vol/vol) was added to cell lysates or cell culture superna- tive (100× 1.4 NA Plan-Apochromat; Olympus). Diff erential interference tants before heating at 100°C for 5 min. Lysates were treated with 5 U/ml contrast (Nomarski technique) was also used. Overlay images were assem- N-glycosidase F (Roche Diagnostics) for 16 h at 37°C before analysis by bled, and ImarisColoc (version 4.2; Bitplane AG) software was used for Western blotting. quantitative colocalization and statistical analysis. Site-directed mutagenesis. The complete PTX3 cDNA was cloned into Analysis of PTX3 and MPO mRNA expression by RT-PCR. Total the pCDNA3.1+ vector (Invitrogen). The N220S substitution was intro- RNA from DCs and peripheral blood neutrophils was extracted using duced using a kit (QuickChange XL; Stratagene). CHO cells were trans- TRIzol reagent (Life Technologies). Total RNA from neutrophil precur- fected using Lipofectamine (Invitrogen). sors and HL60 (American Type Culture Collection) was purifi ed using the RNeasy mini kit (QIAGEN). cDNA was synthesized from 1 μg of total RNA using an oligo-dT primer and reverse transcriptase (GE Health- Subcellular fractionation. Subcellular fractionation of neutrophils was care). PCR amplifi cation was performed with an amount of cDNA corre- performed as previously described (62). In brief, fresh neutrophils were dis- sponding to 25 ng of the starting total RNA using specifi c oligonucleotides rupted by nitrogen cavitation (Parr Instruments Co.), and the resulting cavi- (PTX3, 5′-G T G C A G G G C T G G G C T G C C C G -3′ and 5′-G C C G C A C A- tates were centrifuged to eliminate pellet nuclei and the remaining intact G G T G G G T C C A C C -3′; MPO, 5′-C C T T C A T G T T C C G C C T G G A C A- cells (63). The postnuclear supernatant containing cytosol, granules, and A T C G -3′ and 5′-C G G A T C T C A T C C A C T G C A A T T T G G -3′). RNA light membranes was immediately separated by centrifugation on a three- integrity was assessed by GAPDH cDNA amplifi cation. The PCR products layer Percoll density gradient. 3-ml gradient fractions were collected after a were analyzed on a 1% agarose gel by electrophoresis and visualized with sonication, frozen, and thawed for two times and analyzed for PTX3 content ethidium bromide. and for localization of subcellular organelles by marker assays (62). An α-mannosidase functional assay was used for azurophil granules (64); gelatinase, lactoferrin, and albumin ELISA were used to identify, respectively, gelatinase Mouse neutrophils and phagocytosis assay. Neutrophils from C57BL/6 (tertiary) granules, lactoferrin (specifi c) granules, and secretory vesicles/ mice (Charles River Laboratories) were isolated from bone marrow by light membranes (40). Percoll step gradient (52, 65, and 75% Percoll). An enriched neutrophil JEM VOL. 204, April 16, 2007 801 population was recovered at the 65–75% interface. Purity was analyzed by R E F E R E N C E S morphology, Giemsa staining, and FACS using anti-Ly6G (clone 1A8; BD 1. Janeway, C.A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Biosciences), and antineutrophil mAb (clone 7/4; Serotec) was routinely Annu. Rev. Immunol. 20:197–216. −/− >96%. Cells were also isolated from wild-type and PTX3 total bone 2. Gordon, S. 2002. Pattern recognition receptors: doubling up for the innate immune response. Cell. 111:927–930. marrow (15, 65) or from the peritoneal cavity 4 h after a 1.5-ml 3% thiogly- 3. Garlanda, C., B. Bottazzi, A. Bastone, and A. Mantovani. 2005. Pentraxins collate (Difco) injection and plated at 5 × 10 cells/ml in a fi nal volume of at the crossroads between innate immunity, infl ammation, matrix depo- 0.5 ml RPMI 1640 with human serum in a 24-well plate. A. fumigatus co- 7 sition, and female fertility. Annu. Rev. Immunol. 23:337–366. nidia were added at 2.5 × 10 cells/well. After 15 or 45 min of incubation at 4. Szalai, A.J., A. Agrawal, T.J. Greenhough, and J.E. Volanakis. 1999. 37°C, phagocytosis was blocked by using NaF (fi nal concentration = 0.2 M). C-reactive protein: structural biology and host defense function. Clin. In some of the experiments, 50 μg/ml of recombinant PTX3 was added. Chem. Lab. Med. 37:265–270. Cytospins were stained with Diff Quick (Dade, Biomap). At least 200 neu- 5. Hirschfi eld, G.M., and M.B. Pepys. 2003. C-reactive protein and cardio- trophils per sample were counted under oil immersion microscopy (100× vascular disease: new insights from an old molecule. QJM. 96:793–807. objective). Results are expressed as a phagocytic index: (percentage of neu- 6. Bottazzi, B., A. Bastone, A. Doni, C. Garlanda, S. Valentino, L. Deban, trophils containing at least one conidia) × (mean number of conidia per V. Maina, A. Cotena, F. Moalli, L. Vago, et al. 2006. The long pen- positive cell). traxin PTX3 as a link among innate immunity, infl ammation, and fe- male fertility. J. Leukoc. Biol. 79:909–912. Infection. Mice were administered 150 mg/kg cyclophosphamide i.p. 2 d 7. Pepys, M.B., and M.L. Baltz. 1983. Acute phase proteins with special before the intranasal infection with 10 A. fumigatus conidia per 20 μl (15). reference to C-reactive protein and related proteins (pentaxis) and se- Mice received 10 cells per 500 μl of peritoneal neutrophils, splenic macro- rum amyloid A protein. Adv. Immunol. 34:141–212. phages, or CD4 T cells i.v. 3 h after the infection. Quantifi cation of fungal 8. Baumann, H., and J. Gauldie. 1994. The acute phase response. Immunol. growth in the lungs was done by the chitin assay (49), and results are ex- Today. 15:74–80. pressed as micrograms of glucosamine per pair of lungs. Peritoneal neutro- 9. Goodman, A.R., T. Cardozo, R. Abagyan, A. Altmeyer, H.G. phils were obtained 18 h after the i.p. injection of 1 ml of endotoxin-free Wisniewski, and J. Vilcek. 1996. Long pentraxins: an emerging group of 10% thioglycolate solution (Difco). Endotoxin was depleted from all solu- proteins with diverse functions. Cytokine Growth Factor Rev. 7:191–202. tions with Detoxi-Gel (Pierce Chemical Co.). Purity was >98%, as deter- 10. Doni, A., G. Peri, M. Chieppa, P. Allavena, F. Pasqualini, L. Vago, + + mined by Cytospin and FACS analysis (GR-1 and CD11b ). Macrophages L. Romani, C. Garlanda, and A. Mantovani. 2003. Production of the were obtained by 2 h of plastic adherence of spleen cells at 37C°. CD4 T soluble pattern recognition receptor PTX3 by myeloid, but not plasma- cells were purifi ed from spleens of mice using anti-CD4 magnetic Micro- cytoid, dendritic cells. Eur. J. Immunol. 33:2886–2893. Beads (Miltenyi Biotec). 11. Alles, V.V., B. Bottazzi, G. Peri, J. Golay, M. Introna, and A. Mantovani. 1994. Inducible expression of PTX3, a new member of the pentraxin Procedures involving animals and their care conformed to institutional family, in human mononuclear phagocytes. Blood. 84:3483–3493. guidelines in compliance with national (4D.L. N.116, G.U., suppl. 40, 18- 12. Breviario, F., E.M. d’Aniello, J. Golay, G. Peri, B. Bottazzi, A. 2-1992) and international (EEC Council Directive 86/609, OJ L 358,1,12- Bairoch, S. Saccone, R. Marzella, V. Predazzi, M. Rocchi, et al. 1992. 12-1987; National Institutes of Health Guide for the Care and Use of Interleukin-1-inducible genes in endothelial cells. Cloning of a new Laboratory Animals) law and policies. All eff orts were made to minimize the gene related to C-reactive protein and serum amyloid P component. number of animals used and their suff ering. J. Biol. Chem. 267:22190–22197. 13. Han, B., M. Mura, C.F. Andrade, D. Okutani, M. Lodyga, C.C. dos Statistical analysis. Statistical analysis was performed using the Student’s Santos, S. Keshavjee, M. Matthay, and M. Liu. 2005. TNFalpha- t test. induced long pentraxin PTX3 expression in human lung epithelial cells via JNK. J. Immunol. 175:8303–8311. Online supplemental material. Supplemental materials and methods de- 14. Vouret-Craviari, V., C. Matteucci, G. Peri, G. Poli, M. Introna, and A. scribes the binding assay and the analysis of PTX3, MPO, lactoferrin, and Mantovani. 1997. Expression of a long pentraxin, PTX3, by monocytes MMP-9 mRNA expression in HL60 cells and human bone marrow. Fig. exposed to the mycobacterial cell wall component lipoarabinomannan. S1 shows the constitutive expression of PTX3 (mRNA and protein) in the Infect. Immun. 65:1345–1350. human promyelocytic cell line HL60. Fig. S2 shows the relative content of 15. Garlanda, C., E. Hirsch, S. Bozza, A. Salustri, M. De Acetis, R. Nota, PTX3, as assessed by Western blotting, in neutrophils stimulated for 16 h with A. Maccagno, F. Riva, B. Bottazzi, G. Peri, et al. 2002. Non-redundant the indicated stimuli. Fig. S3 shows that the release of PTX3 by neutrophils role of the long pentraxin PTX3 in anti-fungal innate immune response. is not aff ected by tunicamycin, in contrast to PTX3 produced by activated Nature. 420:182–186. DCs. Fig. S4 shows PMN-released PTX3 binding to (A) immobilized C1q 16. Salustri, A., C. Garlanda, E. Hirsch, M. De Acetis, A. Maccagno, B. and OmpA and to (B) A. fumigatus conidia. Online supplemental material is Bottazzi, A. Doni, A. Bastone, G. Mantovani, P. Beck Peccoz, et al. 2004. available at http://www.jem.org/cgi/content/full/jem.20061301/DC1. PTX3 plays a key role in the organization of the cumulus oophorus extra- cellular matrix and in in vivo fertilization. Development. 131:1577–1586. We sincerely acknowledge Dr. Odile Blanchet and Ms. Irène Dobo for giving human 17. Nauta, A.J., B. Bottazzi, A. Mantovani, G. Salvatori, U. Kishore, W.J. Schwaeble, A.R. Gingras, S. Tzima, F. Vivanco, J. Egido, et al. 2003. bone marrow cells. Biochemical and functional characterization of the interaction between pentraxin 3 and C1q. Eur. J. Immunol. 33:465–473. S. Jaillon is supported by a grant from the Conseil Général du Maine et Loire. This 18. Bottazzi, B., V. Vouret-Craviari, A. Bastone, L. De Gioia, C. Matteucci, study is supported by the Institut National de la Santé et de la Recherche Médicale G. Peri, F. Spreafi co, M. Pausa, C. Dettorre, E. Gianazza, et al. 1997. (Avenir program), the Ministère de la Recherche (ACI program), Cancéropôle Grand- Multimer formation and ligand recognition by the long pentraxin PTX3. Ouest, the Sixth Research Framework Programme of the European Union (projects Similarities and diff erences with the short pentraxins C-reactive protein MUGEN LSHB-CT-2005-005203 and MUVAPRED), the Ministero dell’Istruzione, Università e della Ricerca (project FIRB), Telethon (grant GGP05095), the Fondazione and serum amyloid P component. J. Biol. Chem. 272:32817–32823. 19. Diniz, S.N., R. Nomizo, P.S. Cisalpino, M.M. Teixeira, G.D. Brown, CARIPLO (project Nobel), and the Italian Association for Cancer Research. G. Peri, A. Doni, C. Garlanda, and A. Mantovani are inventors in patent A. Mantovani, S. Gordon, L.F. Reis, and A.A. Dias. 2004. PTX3 func- tion as an opsonin for the dectin-1-dependent internalization of zymo- applications concerning PTX3. The authors have no other confl icting fi nancial interests. san by macrophages. J. Leukoc. Biol. 75:649–656. Submitted: 19 June 2006 20. Varani, S., J.A. Elvin, C. Yan, J. DeMayo, F.J. DeMayo, H.F. Horton, Accepted: 15 February 2007 M.C. Byrne, and M.M. Matzuk. 2002. Knockout of pentraxin 3, a 802 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE downstream target of growth diff erentiation factor-9, causes female following exposure to proinfl ammatory mediators. J. Leukoc. Biol. 79: subfertility. Mol. Endocrinol. 16:1154–1167. 123–132. 21. Rovere, P., G. Peri, F. Fazzini, B. Bottazzi, A. Doni, A. Bondanza, V.S. 41. Dewald, B., U. Bretz, and M. Baggiolini. 1982. Release of gelatin- Zimmermann, C. Garlanda, U. Fascio, M.G. Sabbadini, et al. 2000. ase from a novel secretory compartment of human neutrophils. J. Clin. The long pentraxin PTX3 binds to apoptotic cells and regulates their Invest. 70:518–525. clearance by antigen-presenting dendritic cells. Blood. 96:4300–4306. 42. Mocsai, A., E. Ligeti, C.A. Lowell, and G. Berton. 1999. Adhesion- 22. Baruah, P., A. Propato, I.E. Dumitriu, P. Rovere-Querini, V. Russo, R. dependent degranulation of neutrophils requires the Src family kinases Fontana, D. Accapezzato, G. Peri, A. Mantovani, V. Barnaba, and A.A. Fgr and Hck. J. Immunol. 162:1120–1126. Manfredi. 2006. The pattern recognition receptor PTX3 is recruited 43. Colotta, F., F. Re, N. Polentarutti, S. Sozzani, and A. Mantovani. 1992. at the synapse between dying and dendritic cells, and edits the cross- Modulation of granulocyte survival and programmed cell death by cyto- presentation of self, viral, and tumor antigens. Blood. 107:151–158. kines and bacterial products. Blood. 80:2012–2020. 23. Souza, D.G., A.C. Soares, V. Pinho, H. Torloni, L.F. Reis, M.M. 44. Sabroe, I., R.C. Read, M.K. Whyte, D.H. Dockrell, S.N. Vogel, and Teixeira, and A.A. Dias. 2002. Increased mortality and infl ammation in S.K. Dower. 2003. Toll-like receptors in health and disease: complex tumor necrosis factor-stimulated gene-14 transgenic mice after ischemia questions remain. J. Immunol. 171:1630–1635. and reperfusion injury. Am. J. Pathol. 160:1755–1765. 45. Francois, S., J. El Benna, P.M. Dang, E. Pedruzzi, M.A. Gougerot- 24. Jeannin, P., B. Bottazzi, M. Sironi, A. Doni, M. Rusnati, M. Presta, V. Pocidalo, and C. Elbim. 2005. Inhibition of neutrophil apoptosis by Maina, G. Magistrelli, J.F. Haeuw, G. Hoeff el, et al. 2005. Complexity TLR agonists in whole blood: involvement of the phosphoinositide and complementarity of outer membrane protein A recognition by cel- 3-kinase/Akt and NF-kappaB signaling pathways, leading to increased lular and humoral innate immunity receptors. Immunity. 22:551–560. levels of Mcl-1, A1, and phosphorylated Bad. J. Immunol. 174:3633–3642. 25. Nathan, C. 2006. Neutrophils and immunity: challenges and opportu- 46. Hayashi, F., T.K. Means, and A.D. Luster. 2003. Toll-like receptors nities. Nat. Rev. Immunol. 6:173–182. stimulate human neutrophil function. Blood. 102:2660–2669. 26. Cassatella, M.A., S. Gasperini, and M.P. Russo. 1997. Cytokine expres- 47. Inforzato, A., G. Peri, A. Doni, C. Garlanda, A. Mantovani, A. Bastone, sion and release by neutrophils. Ann. N Y Acad. Sci. 832:233–242. A. Carpentieri, A. Amoresano, P. Pucci, A. Roos, et al. 2006. Structure 27. Segal, A.W. 2005. How neutrophils kill microbes. Annu. Rev. Immunol. and function of the long pentraxin PTX3 glycosidic moiety: fi ne-tuning 23:197–223. of the interaction with C1q and complement activation. Biochemistry. 28. Bottazzi, B., C. Garlanda, G. Salvatori, P. Jeannin, A. Manfredi, and A. 45:11540–11551. Mantovani. 2006. Pentraxins as a key component of innate immunity. 48. Lee, G.W., A.R. Goodman, T.H. Lee, and J. Vilcek. 1994. Relationship Curr. Opin. Immunol. 18:10–15. of TSG-14 protein to the pentraxin family of major acute phase proteins. 29. Theilgaard-Monch, K., L.C. Jacobsen, R. Borup, T. Rasmussen, M.D. J. Immunol. 153:3700–3707. Bjerregaard, F.C. Nielsen, J.B. Cowland, and N. Borregaard. 2005. The 49. Montagnoli, C., F. Fallarino, R. Gaziano, S. Bozza, S. Bellocchio, T. transcriptional program of terminal granulocytic diff erentiation. Blood. Zelante, W.P. Kurup, L. Pitzurra, P. Puccetti, and L. Romani. 2006. 105:1785–1796. Immunity and tolerance to Aspergillus involve functionally distinct regu- 30. Lominadze, G., D.W. Powell, G.C. Luerman, A.J. Link, R.A. Ward, and latory T cells and tryptophan catabolism. J. Immunol. 176:1712–1723. K.R. McLeish. 2005. Proteomic analysis of human neutrophil granules. 50. Sengelov, H., L. Kjeldsen, and N. Borregaard. 1993. Control of exocy- Mol. Cell. Proteomics. 4:1503–1521. tosis in early neutrophil activation. J. Immunol. 150:1535–1543. 31. Brinkmann, V., U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, 51. Sengelov, H., P. Follin, L. Kjeldsen, K. Lollike, C. Dahlgren, and N. D.S. Weiss, Y. Weinrauch, and A. Zychlinsky. 2004. Neutrophil extra- Borregaard. 1995. Mobilization of granules and secretory vesicles during cellular traps kill bacteria. Science. 303:1532–1535. in vivo exudation of human neutrophils. J. Immunol. 154:4157–4165. 32. Grossman, W.J., and T.J. Ley. 2004. Granzymes A and B are not ex- 52. Basile, A., A. Sica, E. D’Aniello, F. Breviario, G. Garrido, M. Castellano, pressed in human neutrophils. Blood. 104:906–907. A. Mantovani, and M. Introna. 1997. Characterization of the promoter 33. Metkar, S.S., and C.J. Froelich. 2004. Human neutrophils lack gran- for the human long pentraxin PTX3. J. Biol. Chem. 272:8172–8178. zyme A, granzyme B, and perforin. Blood. 104:905–906. 53. Peri, G., M. Introna, D. Corradi, G. Iacuitti, S. Signorini, F. Avanzini, 34. Wagner, C., C. Iking-Konert, B. Denefl eh, S. Stegmaier, F. Hug, and F. Pizzetti, A.P. Maggioni, T. Moccetti, M. Metra, et al. 2000. PTX3, G.M. Hansch. 2004. Granzyme B and perforin: constitutive expression a prototypic long pentraxin, is an early indicator of acute myocardial in human polymorphonuclear neutrophils. Blood. 103:1099–1104. infarction in man. Circulation. 102:636–641. 35. Doni, A., M. Michela, B. Bottazzi, G. Peri, S. Valentino, N. Polentarutti, 54. Latini, R., A.P. Maggioni, G. Peri, L. Gonzini, D. Lucci, P. Mocarelli, L. C. Garlanda, and A. Mantovani. 2006. Regulation of PTX3, a key Vago, F. Pasqualini, S. Signorini, D. Soldateschi, et al. 2004. Prognostic component of humoral innate immunity in human dendritic cells: signifi cance of the long pentraxin PTX3 in acute myocardial infarction. stimulation by IL-10 and inhibition by IFN-gamma. J. Leukoc. Biol. 79: Circulation. 110:2349–2354. 797–802. 55. Schaff ner, A., H. Douglas, and A. Braude. 1982. Selective protection 36. Cowland, J.B., and N. Borregaard. 1999. Isolation of neutrophil precur- against conidia by mononuclear and against mycelia by polymorphonu- sors from bone marrow for biochemical and transcriptional analysis. clear phagocytes in resistance to Aspergillus. Observations on these two J. Immunol. Methods. 232:191–200. lines of defense in vivo and in vitro with human and mouse phagocytes. 37. Devarajan, P., J.J. Johnston, S.S. Ginsberg, H.E. Van Wart, and N. J. Clin. Invest. 69:617–631. Berliner. 1992. Structure and expression of neutrophil gelatinase cDNA. 56. Gerson, S.L., G.H. Talbot, S. Hurwitz, B.L. Strom, E.J. Lusk, and P.A. Identity with type IV collagenase from HT1080 cells. J. Biol. Chem. Cassileth. 1984. Prolonged granulocytopenia: the major risk factor for 267:25228–25232. invasive pulmonary aspergillosis in patients with acute leukemia. Ann. 38. Johnston, J.J., P. Rintels, J. Chung, J. Sather, E.J. Benz Jr., and N. Intern. Med. 100:345–351. Berliner. 1992. Lactoferrin gene promoter: structural integrity and non- 57. Romani, L., and D.H. Howard. 1995. Mechanisms of resistance to fun- expression in HL60 cells. Blood. 79:2998–3006. gal infections. Curr. Opin. Immunol. 7:517–523. 39. Kjeldsen, L., D.F. Bainton, H. Sengelov, and N. Borregaard. 1993. 58. Cenci, E., S. Perito, K. Enssle, P. Mosci, J. Latge, L. Romani, and F. Structural and functional heterogeneity among peroxidase-negative Bistoni. 1997. Th1 and Th2 cytokines in mice with invasive aspergillosis. granules in human neutrophils: identifi cation of a distinct gelatinase- Infect. Immun. 65:564–570. containing granule subset by combined immunocytochemistry and sub- 59. Nagai, H., J. Guo, H. Choi, and V. Kurup. 1995. Interferon-gamma cellular fractionation. Blood. 82:3183–3191. and tumor necrosis factor-alpha protect mice from invasive aspergillosis. 40. Cassatella, M.A., V. Huber, F. Calzetti, D. Margotto, N. Tamassia, J. Infect. Dis. 172:1554–1560. G. Peri, A. Mantovani, L. Rivoltini, and C. Tecchio. 2006. Interferon- 60. Jeannin, P., T. Renno, L. Goetsch, I. Miconnet, J.P. Aubry, Y. activated neutrophils store a TNF-related apoptosis-inducing ligand Delneste, N. Herbault, T. Baussant, G. Magistrelli, C. Soulas, et al. (TRAIL/Apo-2 ligand) intracellular pool that is readily mobilizable 2000. OmpA targets dendritic cells, induces their maturation and JEM VOL. 204, April 16, 2007 803 delivers antigen into the MHC class I presentation pathway. Nat. gamma, and GM-CSF and the necessity to overcome the action of Immunol. 1:502–509. endogenous proteases. Biochemistry. 37:13165–13173. 61. Bourke, E., D. Bosisio, J. Golay, N. Polentarutti, and A. Mantovani. 2003. 64. De Togni, P., G. Cabrini, and F. Di Virgilio. 1984. Cyclic AMP inhi- The Toll-like receptor repertoire of human B lymphocytes: inducible bition of fMet-Leu-Phe-dependent metabolic responses in human and selective expression of TLR9 and TLR10 in normal and trans- neutrophils is not due to its eff ects on cytosolic Ca2+. Biochem. J. formed cells. Blood. 102:956–963. 224:629–635. 62. Kjeldsen, L., H. Sengelov, and N. Borregaard. 1999. Subcellular frac- 65. Pruijt, J.F., P. Verzaal, R. van Os, E.J. de Kruijf, M.L. van Schie, A. tionation of human neutrophils on Percoll density gradients. J. Immunol. Mantovani, A. Vecchi, I.J. Lindley, R. Willemze, S. Starckx, et al. 2002. Methods. 232:131–143. Neutrophils are indispensable for hematopoietic stem cell mobili- 63. McDonald, P.P., C. Bovolenta, and M.A. Cassatella. 1998. Activation zation induced by interleukin-8 in mice. Proc. Natl. Acad. Sci. USA. of distinct transcription factors in neutrophils by bacterial LPS, interferon- 99:6228–6233. 804 PTX3 IN NEUTROPHILS | Jaillon et al. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps

Download PDF
12 pages

Loading next page...
 
/lp/pubmed-central/the-humoral-pattern-recognition-receptor-ptx3-is-stored-in-neutrophil-uIZqAEg66A

References (152)

Publisher
Pubmed Central
Copyright
Copyright © 2007, The Rockefeller University Press
ISSN
0022-1007
eISSN
1540-9538
DOI
10.1084/jem.20061301
Publisher site
See Article on Publisher Site

Abstract

ARTICLE The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps 1 2 1 1 Sébastien Jaillon, Giuseppe Peri, Yves Delneste, Isabelle Frémaux, 2 2 2 3 Andrea Doni, Federica Moalli, Cecilia Garlanda, Luigina Romani, 4 3 3 5 Hugues Gascan, Silvia Bellocchio, Silvia Bozza, Marco A. Cassatella, 1,6 2,7 Pascale Jeannin, and Alberto Mantovani Institut National de la Santé et de la Recherche Médicale, Equipe Avenir, Unité 564, University Hospital of Angers, University of Angers, Angers 49933, France Istituto Clinico Humanitas, Istituto di Ricovero e Cura a Carattere Scientifi co, 20089 Rozzano, Italy Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, 05122 Perugia, Italy Institut National de la Santé et de la Recherche Médicale, Unité 564, University of Angers, 49933 Angers, France Department of Pathology, University of Verona, 37134 Verona, Italy Immunology and Allergology Laboratory, University Hospital of Angers, 49933 Angers, France Institute of General Pathology, Faculty of Medicine, University of Milan, 20100 Milan, Italy The long pentraxin (PTX) 3 is produced by macrophages and myeloid dendritic cells in response to Toll-like receptor agonists and represents a nonredundant component of humoral innate immunity against selected pathogens. We report that, unexpectedly, PTX3 is stored in specifi c granules and undergoes release in response to microbial recognition and infl ammatory signals. Released PTX3 can partially localize in neutrophil extracellular traps formed by extruded DNA. Eosinophils and basophils do not contain preformed PTX3. PTX3- defi cient neutrophils have defective microbial recognition and phagocytosis, and PTX3 is nonredundant for neutrophil-mediated resistance against Aspergillus fumigatus. Thus, neutrophils serve as a reservoir, ready for rapid release, of the long PTX3, a key component of humoral innate immunity with opsonic activity. Innate immunity is the fi rst line of defense arm of the innate immunity includes soluble CORRESPONDENCE Alberto Mantovani: against pathogens and plays a key role in the PRRs, such as collectins, fi colins, complement [email protected] initiation, activation, and orientation of adaptive components, and pentraxins (PTXs) (3). OR immunity. Innate immunity receptors, also Members of the PTX superfamily are usu- Pascale Jeannin: called pattern recognition receptors (PRRs), ally characterized by a pentameric structure and [email protected] recognize a few highly conserved structures, are highly conserved during evolution (3–6). Abbreviations used: CHO, called pathogen-associated molecular patterns, This family is subdivided into two subclasses Chinese hamster ovary; CRP, expressed by microorganisms (1). PRRs are that depend on the length and structure of the C-reactive protein; MMP-9, matrix metalloproteinase 9; either cell associated (expressed intracellularly or molecules. The classical short PTXs C- reactive MPO, myeloperoxidase; NET, on the cell surface) or present in body fl uids. protein (CRP) and serum amyloid P component neutrophil extracellular trap; There are two functional classes of cell- associated (SAP) are acute-phase proteins in humans and OmpA, outer membrane protein A; PRR, pattern PRRs: endocytic PRRs (i.e., scavenger recep- mice, respectively (7, 8), that are produced in recognition receptor; PTX, tors and mannose receptors) involved in micro- the liver in response to infl ammatory mediators, pentraxin; SAP, serum amy- organism binding and uptake; and signaling most prominently IL-6. CRP and SAP bind, in loid P component; TLR, PRRs (members of the Toll-like receptor [TLR], a calcium-dependent manner, diff erent ligands Toll-like receptor. nucleotide-binding oligomeri zation domain, and are involved in innate resistance to microbes and helicase families) involved in cell activation and scavenging of cellular debris and extra- upon contact with pathogens (2). The humoral cellular matrix components (4, 7). Long PTXs are characterized by an unrelated N-terminal domain coupled to a PTX-like C-terminal do- S. Jaillon, G. Peri, P. Jeannin, and A. Mantovani contributed main (3, 6, 9). The prototypic long PTX3 is rap- equally to this work. The online version of this article contains supplemental material. idly produced and released by diverse cell types, JEM © The Rockefeller University Press $15.00 793 Vol. 204, No. 4, April 16, 2007 793–804 www.jem.org/cgi/doi/10.1084/jem.20061301 The Journal of Experimental Medicine in particular by mononuclear phagocytes, DCs, and endothe- observed similar levels of intracellular PTX3 using 20 and lial and epithelial cells in response to primary infl ammatory 200 μg/ml of human IgG for saturation, showing that the signals (e.g., TLR engagement, TNFα, and IL-1β) (10–14). detection of intracellular PTX3 in neutrophils is not related With high affi nity, PTX3 binds the complement component to nonspecifi c binding of the anti-PTX3 mAb (not depicted). To confi rm this observation, PTX3 expression was analyzed C1q, the extracellular matrix TNF-inducible protein 6, and selected microorganisms (e.g., Aspergillus fumigatus, Pseudomonas by Western blotting. Human neutrophils and DCs express aeruginosa, Salmonella typhimurium, Paracoccidioides brasiliensis, two or three immunoreactive forms of PTX3, depending and zymosan) (15–18). on the donor (with one major band at 47 kD and two −/− Recent studies in ptx3-defi cient (PTX3 ) mice have minor bands at 44 and 42 kD; Fig. 1 B). The presence of shown that this molecule performs complex nonredundant intracellular PTX3 in neutrophils was also confi rmed by con- functions in vivo, ranging from the assembly of a hyaluronic focal microscopy (Fig. 1 C). We assessed whether other cir- acid–rich extracellular matrix to female fertility to innate im- culating elements containing granules store PTX3. PTX3 munity against diverse microorganisms (15, 16, 19, 20). PTX3 could not be detected in eosinophils and basophils (Fig. 1 C), also binds apoptotic cells (21) and may contribute to editing nor in large granular lymphocytes (NK cells; not depicted). PTX3 was detected in LPS-activated DCs (Fig. 1 B) (10, recognition of apoptotic cells versus infectious nonself (22). In addition, there is evidence for a regulatory role of PTX3 35). By ELISA, resting human neutrophils contain 24.9 ± in noninfectious infl ammatory reactions (Latini, R., personal 3.8 ng PTX3 per 10 cells (n = 4) corresponding to 0.55 communication) (23). Outer membrane protein A (OmpA) is pmol (Fig. 1 D), calculated based on the protomer molecular a conserved constituent of the outer membrane of Enterobacte- mass. Over a period of 24 h, DCs release 50.2 ± 8.1 ng riaceae. A search of moieties recognized by PTX3 has discov- PTX3 per 10 cells (n = 5; Fig. 1 D) (10). These results show ered that it binds OmpA from Klebsiella pneumoniae and that it that neutrophils contain considerable amounts of preformed represents a nonredundant humoral amplifi cation loop of the PTX3. Finally, unlike PTX3, the short PTXs CRP and SAP innate immunity response to this microbial moiety (24). could not be localized in neutrophils (not depicted). Among innate cells, neutrophils play an important role In agreement with a previous study (11), PTX3 mRNA is not expressed in freshly isolated human neutrophils, as as- because of their ability to be rapidly recruited in tissues during infections and to produce mediators that kill or inhibit micro- sessed by RT-PCR analysis (Fig. 1 E). As a positive control, bial growth (25–27). As PTX3 is important in infectious PTX3 mRNA was evident in LPS-stimulated DCs (Fig. and infl ammatory responses (3, 28), we evaluated whether 1 E), as previously reported (10). We thus evaluated whether neutrophils produce PTX3. In previous studies, PTX3 was neutrophil precursors express PTX3 mRNA. Promyelocytes, found to be expressed in HL60 cells (11) and bone marrow myelocytes/metamyelocytes, and bone marrow–segmented myelocytes (29) as mRNA and, by proteomic analysis, in neutrophils were isolated by density centrifugation on a neutrophil granules (30). In this paper, we report that PTX3 Percoll gradient, followed by magnetic cell sorting (29, 36). is stored in a ready-made form in neutrophils but not in RT-PCR analysis revealed that PTX3 mRNA is expressed in eosinophils and basophils. PTX3 is localized in specifi c gran- promyelocytes and myelocytes/metamyelocytes but not or at low levels in bone marrow–segmented neutrophils (Fig. 1 F, ules and is secreted in response to recognition of microbial moieties and infl ammatory signals. PTX3 can localize in neu- left), a result in accordance with the absence of PTX3 mRNA trophil extracellular traps (NETs) (31), and PTX3-defi cient expression in mature peripheral neutrophils. As a positive neutrophils have defective phagocytic activity. In addition, control, myeloperoxidase (MPO) mRNA was mainly detected injection of wild-type neutrophils restores protective immunity in promyelocytes and, at a lower level, in myelocytes but not in −/− against A. fumigatus in PTX3 mice. Thus, neutrophils serve bone marrow–segmented neutrophils, as previously reported as a reservoir, ready for rapid release, of a key component of (29, 36), confi rming the purity of the isolated cell populations. humoral innate immunity and complement its subsequent Western blot analysis showed that PTX3 is expressed in the delayed neosynthesis by macrophages and DCs. three populations of neutrophil precursors (Fig. 1 F, right). In agreement with previous observations (11), the human pro- myelocytic cell line HL60 constitutively expresses PTX3 RESULTS Storage of preformed PTX3 in resting neutrophils mRNA (Fig. S1, available at http://www.jem.org/cgi/content/ FACS analysis revealed constitutive expression of PTX3 full/jem.20061301/DC1). HL60 cells express mRNA encoding in freshly isolated human neutrophils, as assessed by intracell- MPO, a marker of primary granules, but not mRNA encoding ular labeling (mean fl uorescence intensity = 89 ± 34 and lactoferrin and matrix metalloproteinase 9 (MMP-9; Fig. S1), 4 ± 1.5, using anti-PTX3 and control mAbs, respectively; as previously reported (37, 38). Moreover, HL60 cells sponta- mean ± SD; n = 8; Fig. 1 A). No expression of PTX3 neously produce PTX3 protein (Fig. S1). was observed on the surface of human neutrophils (not depicted). Recent studies reported that nonspecifi c binding of Localization of PTX3 in neutrophil-specifi c granules polyclonal Ig within neutrophils may give false positive Confocal microscopy and subcellular fractionation of neutro- phil-derived nitrogen cavitates were used to specifi cally results, as observed using antigranzyme antibody (32–34). Following the methodologies described in these studies, we localize PTX3 within freshly isolated neutrophils. As shown 794 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE Figure 1. PTX3 is constitutively expressed in human neutrophils. by RT-PCR. Results obtained in 2 out of 10 subjects tested are presented. (A) FACS analysis of PTX3 expression in permeabilized human neutrophils LPS-stimulated DCs are used as a positive control. RNA integrity and isolated from peripheral blood. (B) Western blot analysis of PTX3 expres- cDNA synthesis were verifi ed by amplifying GAPDH cDNA. (F) Analysis sion in neutrophils and LPS-stimulated DCs. (C) Analysis of PTX3 expres- of PTX3 mRNA and protein in neutrophil precursors. Promyelocytes sion in freshly isolated neutrophils, eosinophils and basophils by confocal (PM), myelocytes/metamyelocytes (MY), and bone marrow–segmented microscopy. Fluorescence (left) and differential interference contrasts neutrophils (bm-PMN) were analyzed for PTX3 and MPO mRNA expres- (right, Nomarski technique) are shown. Bars, 10 μm. (D) Analysis of PTX3 sion (left). Expression of PTX3 was evaluated by Western blotting using content in freshly isolated neutrophils, eosinophils, and basophils, as the anti-PTX3 mAb 16B5 in the three populations of neutrophil precur- well as release by LPS-stimulated DCs, determined by ELISA (mean ± SD). sors (right), and total protein loading was evaluated by analyzing (E) Analysis of PTX3 mRNA expression in freshly isolated human neutrophils actin expression. in Fig. 2 A, confocal studies pointed to complete colocaliza- In an eff ort to confi rm these observations, subcellular tion of PTX3 with lactoferrin, a constituent of specifi c gran- fractionation of neutrophil-derived nitrogen cavitates was ules, and, at least in part, with gelatinase (tertiary granules; performed. PTX3 was detected in fractions that contain lac- Fig. 2 A). In contrast, no colocalization with MPO, a marker toferrin and part of the gelatinase distribution but not in frac- of azurophilic granules, was observed (Fig. 2 A). Confocal tions containing α-mannosidase (azurophilic granules; Fig. microscopy was combined with quantitative analysis to mea- 2 C) or albumin (secretory vesicles/light membranes; not sure the percentage of PTX3 colocalization, as assessed by depicted) (40). Collectively, these results demonstrate the Pearson’s coeffi cient of correlation. Higher degrees of colo- selective association of PTX3 with specifi c (lactoferrin and calization were measured in diff erent experiments (n = 5) in lactoferrin/gelatinase ) granules. + + lactoferrin and gelatinase vesicles, which display coeffi cients of 72.22 ± 7.32% (r = 0.78) and 27.8 ± 8.73% (r = 0.45), PTX3 release by stimulated neutrophils respectively. Little or no colocalization of PTX3 was found We next evaluated whether neutrophils release PTX3 upon with MPO granules (5.90 ± 2.21%; r = 0.08). stimulation. FACS analysis showed that the relative intracellu- Among the MPO-negative granules, 16% contain only lar level of PTX3 decreased upon 2 h of stimulation with 10 lactoferrin (specifi c) and 24% contain only gelatinase (ter- μg/ml Staphylococcus aureus and Escherichia coli (Fig. 3 A). In con- tiary), whereas 60% contain both markers (specifi c) (39). Fig. trast, the intracellular level of PTX3 increased in DCs stimu- + + 2 B shows lactoferrin granules, gelatinase granules, or double- lated for 8 h with 80 ng/ml LPS compared with nonstimulated stained granules in a representative cell. PTX3 was found to cells (Fig. 3 B). The levels of PTX3 were also quantifi ed by colocalize with lactoferrin granules and double-stained gra- ELISA in the cell culture supernatants. E. coli, S. aureus, and, to a lesser extent, zymosan increased the release of PTX3 by nules (specifi c), but no colocalization was found with tertiary granules that were only gelatinase . neutrophils in a dose-dependent manner, with a maximal eff ect JEM VOL. 204, April 16, 2007 795 + + Figure 2. Localization of PTX3 within neutrophil granules. Specifi c granules were identifi ed as lactoferrin and lactoferrin / + + (A) Localization of PTX3 in freshly isolated neutrophils by confocal micro- gelatinase ; tertiary granules were identifi ed as gelatinase . Insets scopy. Cells were fi xed and stained for human PTX3 and MPO (left), PTX3 show enlargements of the indicated areas. Bars, 10 μm. (C) Analysis and gelatinase (middle), and PTX3 and lactoferrin (right; see Materials and of PTX3 content in neutrophil subcellular fractions. PTX3, gelatinase, methods). DNA labeling is also shown (Hoechst 33258). Insets show en- lactoferrin, and α-mannosidase content in each fraction were deter- largements of the indicated areas. Bars, 10 μm. (B) Localization of PTX3 mined by ELISA or using a functional assay for α-mannosidase. in neutrophil-specifi c granules. Cells were stained for PTX3, gelatinase, Results are expressed as a percentage of the relative amount in the and lactoferrin (see Materials and methods). A representative cell is shown. collected fractions. observed with the highest concentration used (10 μg/ml; Fig. PTX3 was not a consequence of cell death (not depicted). 3 C). A similar increase of PTX3 was observed using 1 or 10 Previous experiments reported that GM-CSF increases the ng/ml PMA, 0.01 or 1 μM ionomycin, and 20 ng/ml of the sensitivity of neutrophils to TLR agonists (46). Neutrophils proinfl ammatory cytokine TNF α (Fig. 3 C). 20 ng/ml IL-1β were thus primed with 50 ng/ml GM-CSF for 2 h before stim- was inactive (not depicted). Propidium iodide staining and lactate ulation with zymosan, S. aureus, and TLR agonists. The levels dehydrogenase release assay excluded that PTX3 secretion re- of PTX3 released upon stimulation with TLR agonists or sulted from cell death (not depicted). Latex beads failed to trigger microorganisms were weakly but signifi cantly increased in PTX3 release (not depicted). The release of PTX3 is associated GM-CSF–primed neutrophils (Fig. 3 E). In addition, microor- with a decrease in the level of intracellular PTX3 in neutro- ganisms and TLR agonists induced the release of MPO (Fig. phils, as assessed by Western blot analysis (Fig. S2, available 3 F) and MMP-9 (Fig. 3 G). Independent of the stimulus used, at http://www.jem.org/cgi/content/full/jem.20061301/DC1). PTX3 mRNA was not induced in stimulated neutrophils, as PMA and S. aureus induced a time-dependent release of PTX3 assessed by RT-PCR analysis after 8 h (Fig. 3 H) or 16 h (not by neutrophils, signifi cant at 1 h and maximal at the latest time depicted) of activation. These data show that microorganisms analyzed (16 h; Fig. 3 D). It is noteworthy that TNF and PMA and TLR agonists trigger substantial release of PTX3 by hu- induce selective release of specifi c granules (41, 42). Among man neutrophils. the diff erent activators tested, microorganisms appear to be the most potent inducers of PTX3 release by human neutrophils. Association of PTX3 with NETs We therefore evaluated the ability of TLR agonists to induce NETs are extracellular structures formed by the extrusion of PTX3 release. LPS, R848, Pam CSK4, and, to a lesser extent, DNA from viable neutrophils upon stimulation, and they act fl agellin (but not poly (I:C)) induced the release of PTX3 as focal points to focus antimicrobial eff ector molecules (31). by neutrophils (Fig. 3 E). In agreement with previous studies We analyzed whether PTX3 colocalizes with NETs upon (43–45), we observed that a stimulation with TLR agonists stimulation. As shown in Fig. 4, after 40 min of stimulation delays neutrophil apoptosis, confi rming that the release of with IL-8, LPS, or PMA (Fig. 4 A) or the conidia of A. fumigatus 796 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE Figure 3. Secretion of PTX3 by activated neutrophils. (A and B) 50 ng/ml GM-CSF. Supernatants were collected at 16 h. PTX3 was FACS analysis of intracellular PTX3 expression in neutrophils stimulated quantifi ed in the supernatants by ELISA. Results are expressed as for 2 h with 10 μg/ml S. aureus or 10 μg/ml FITC-labeled E. coli (A) or ng/ml (mean ± SD; n = 6). MPO (F) and MMP-9 (G) were quantifi ed in DCs stimulated or not for 8 h with 80 ng/ml LPS (B). Representative in the supernatants of neutrophils stimulated for 16 h with the indi- results from one to fi ve experiments are shown. (C–E) Analysis of PTX3 cated stimuli; results are expressed in ng/ml, showing the mean of two release upon neutrophil stimulation. (C) 2 × 10 cells/ml of neutrophils representative experiments. (H) PTX3 mRNA expression analyzed by were activated for 16 h with 1 or 10 μg/ml E. coli, S. aureus, or zymosan; RT-PCR in neutrophils untreated or stimulated for 8 h with 100 ng/ml 1 or 10 ng/ml PMA; 0.01 or 1 μM ionomycin; or 20 ng/ml TNFα. LPS, 10 μg/ml E. coli, 10 μg/ml S. aureus, 10 μg/ml zymosan, 20 ng/ml (D) Time-dependent release of PTX3 in neutrophils (nonstimulated or TNFα, 10 ng/ml PMA, or 1 μM iononmycin. RNA integrity and cDNA stimulated with 10 μg/ml S. aureus or 10 ng/ml PMA) is shown. Super- synthesis were verifi ed by amplifying GAPDH cDNA. *, P < 0.01 for natants were collected at the indicated time points. (E) Induction of untreated versus GM-CSF–treated cells. PTX3 release by TLR agonists in neutrophils pretreated or not with (Fig. 4 B), PTX3 was found associated with NETs. PTX3 depending on the donor tested (Fig. 1 B and Fig. 5, left). Three immunoreactive bands were also evident in the super- binds conidia from A. fumigatus and plays a nonredundant role in resistance against this fungus (15). Interestingly, both natants of activated neutrophils (Fig. 5, right), with molecular PTX3 and conidia were found associated to NETs (Fig. 4 B). masses ranging from 47 to 250 kD. A previous study reported Thus, PTX3 is rapidly released by neutrophils (Fig. 4 A and that PTX3 is glycosylated at the Asn 220 residue (18). A re- Fig. 3 D) and localizes in NETs. cent characterization of the PTX3 glycosidic moiety revealed three antennary structures and a role in the interaction with Characterization of neutrophil PTX3 C1q (47). We thus analyzed the level of PTX3 glycosylation As mentioned above, Western blot analysis revealed two or in neutrophils. Treatment of neutrophil cell lysates and cul- three immunoreactive PTX3 isoforms in human neutrophils, ture supernatants with N-glycosidase F resulted in a decrease JEM VOL. 204, April 16, 2007 797 Figure 5. Biochemical analysis of PTX3 in neutrophils. Cell lysates from nonstimulated cells (left) and supernatants from S. aureus– stimulated neutrophils (right) were either untreated (−) or treated with N-glycosidase F (+). Supernatants from CHO cells transfected with wild- type or N220S mutant PTX3 were collected after a 24-h culture (right). PTX3 was analyzed by Western blotting using rabbit polyclonal anti-PTX3 and revealed by peroxidase-labeled anti–rabbit IgG antibody and ECL. sized PTX3 in LPS-activated DCs, resulting in a decrease of PTX3 release in the supernatant (Fig. S3, available at http:// www.jem.org/cgi/content/full/jem.20061301), as previously reported (48). Moreover, an Asn®Ser substitution at posi- tion 220 was introduced in PTX3 (N220S mutant). Recom- binant wild-type and N220S PTX3 were detected in the supernatants of Chinese hamster ovary (CHO) cells in a mul- timeric form (Fig. 5, right). Collectively, these data show that neutrophils contain a mature glycosylated form of PTX3 that associates in the extracellular milieu to form multimers inde- pendently of glycosylation. In vitro and in vivo relevance of PTX3 expression in neutrophils We evaluated whether neutrophil-derived PTX3 is func- tional in recognizing ligands of self or microbial origin and may play a role in microbial recognition and destruction. First, we found that PTX3 purifi ed by immunoaffi nity from human PMN lysate bound to immobilized C1q and OmpA from K. pneumoniae; similarly, neutrophil-released PTX3, obtained by concentration of PMN supernatant, bound to A. fumigatus conidia, as did the recombinant protein (Fig. S4, A and B, available at http://www.jem.org/cgi/content/full/ jem.20061301/DC1) (15, 18, 24). Second, we studied neutrophil expression of PTX3 in the mouse. Mature mouse neutrophils constitutively express PTX3, as assessed by Western blotting using anti-PTX3 pAbs Figure 4. PTX3 is localized in NETs. (A) Neutrophils were exposed to or mAbs (Fig. 6 A). Bone marrow–derived (two experiments; 100 ng/ml IL-8, 100 ng/ml LPS, or 2.5 ng/ml PMA for 40 min. (left) PTX3 not depicted) and peritoneal (fi ve experiments performed staining. (right) PTX3 and DNA staining. Bars, 10 μm. (B) Neutrophils in two diff erent laboratories; Fig. 6 B) neutrophils from exposed to conidia from A. fumigatus; a differential interference contrast −/− PTX3 mice showed a signifi cantly lower phagocytosis of (Nomarski technique) is shown in the bottom panels. In both A and B, PTX3 immunostaining was done on nonpermeabilized neutrophils. conidia from A. fumigatus, with a 31 ± 7% reduction com- Bars, 5μm. pared with PTX3-competent cells (Fig. 6 B, left). 1.1 μM of exogenous PTX3 caused a signifi cant increase in the phago- cytosis of conidia by PTX3-competent and incompetent neu- +/+ of the apparent molecular mass of PTX3 (Fig. 5). As expected trophils (phagocytic index = 102 and 76% for PTX3 cells on the basis of PTX3 synthesis during neutrophil diff erentia- with and without PTX3, respectively; phagocytic index = 99 −/− tion but not in mature PMN, tunicamycin did not modify and 64% for PTX3 cells with and without PTX3, respec- the Western blot profi le in resting and activated neutrophils. tively). In the presence of exogenous PTX3, no diff erence In contrast, it prevented the glycosylation of newly synthe- was detected in the phagocytic activity of PTX3-competent 798 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE and incompetent neutrophils (Fig. 6 B, right). Similarly, the protective eff ect was observed using macrophages or CD4 −/− +/+ +/− conidicidal activity of PTX3 PMN was severely impaired T cells. As a control, PTX3 or PTX3 neutrophils also +/+ +/+ compared with that of PTX3 PMN (10 and 45%, respec- prevented fungal growth in PTX3 mice, but the eff ect −/− tively) and was increased 30% by the addition of recombinant was less pronounced than in highly susceptible PTX3 −/− PTX3 (not depicted). mice (Fig. 6 C). No such eff ect was observed with PTX3 In an eff ort to assess the actual in vivo relevance of neu- neutrophils or macrophages (Fig. 6 C). −/− trophil-associated PTX3, cyclophosphamide-treated PTX3 +/+ and PTX3 mice were infected with Aspergillus conidia D I S C U S S I O N −/− +/+ +/− and given PTX3 , PTX3 , or PTX3 PMN, macro- The prototypic long PTX3 has long been known to be phages, or purifi ed CD4 T cells 3 h later. Mice were moni- produced by diverse cell types on demand, i.e., in a gene tored for fungal growth 2 d later by quantifying the chitin expression–dependent fashion in response to extracellular content in the lung (49). Results showed that injection of signals (e.g., LPS, IL-1β, TNFα, and TLR agonists) (3). The fi nd- +/+ +/− −/− PTX3 and PTX3 neutrophils, but not PTX3 cells, ing that PTX3 is stored in neutrophil granules is therefore −/− + in PTX3 mice decreased fungal growth (Fig. 6 C). No unexpected. PTX3 is not stored in MPO granules (primary or azurophilic). By confocal analysis among the MPO gran- ules, PTX3 was found to localize in lactoferrin and in lacto- + + + ferrin /gelatinase (specifi c) but not in gelatinase (tertiary) granules. Storage of PTX3 in neutrophil granules is selective, inasmuch as short PTXs are absent and other granulated cir- culating elements (eosinophils, basophils, and NK cells) do not contain preformed PTX3. In addition to the diversity generated during granulopoiesis, granules are secreted in a targeted manner, with a timing hierarchy in granule exocyto- sis (50, 51). PTX3 is localized in granules that are rapidly mobilized and secreted upon stimulation, in agreement with its early detection in the supernatants of stimulated neutro- phils. Expression of PTX3 transcripts is confi ned to imma- ture myeloid elements, and mature neutrophils only act as a reservoir of preformed PTX3. Thus, neutrophils represent a reservoir of this PRR and release it in response to microbial or infl ammatory signals. The release of PTX3 by neutrophils is induced by micro- organisms and TLR agonists and, to a lower extent, by pro- infl ammatory cytokines. Latex beads do not induce secretion of PTX3, suggesting that phagocytosis is not suffi cient and that TLR recruitment is required to trigger its release by neu- trophils. This dichotomy of PTX3-inducing mediators be- tween neutrophils and other cell types is of physiological signifi cance. Indeed, neutrophils are the fi rst cells recruited into tissues in response to microorganism entry. Neutrophils are thus highly sensitive to microbes and microbe-derived components, suggesting that PTX3, rapidly released by infi l- Figure 6. Functional role of neutrophil-derived PTX3. (A) Analysis trating neutrophils, interacts with microorganisms and facili- of PTX3 expression in mouse bone marrow–segmented neutrophils from tates their internalization by neutrophils themselves, as well as C57BL/6 mice by Western blotting. Results are representative of three resident and/or recruited professional APCs (19). Microor- independent experiments. A pAb or mAb (16B5) was used. (B, left) Phago- −/− ganisms and TLR agonists, the main inducers of PTX3 re- cytosis of A. fumigatus conidia by peritoneal neutrophils from PTX3 +/+ or PTX3 mice after 15 or 45 min. One representative experiment out lease, also induced the release of MPO and MMP-9, which of fi ve performed is shown. *, P < 0.01. (B, right) Effect of 50 μg/ml of are stored mainly in azurophilic and tertiary granules, respec- exogenous PTX3 in the phagocytosis of conidia by PTX3-competent tively. These results suggest that microorganisms and TLR and incompetent neutrophils (45 min of incubation). One representative agonists induce the release of the molecules stored within the experiment out of fi ve performed is shown. (C) Role of PTX3 in neutrophil- three types of granules. mediated resistance against A. fumigatus. 10 A. fumigatus conidia Upon exposure to microbial or infl ammatory signals, per 20 μl were given intranasally to PTX3-defi cient or -competent mice viable neutrophils extrude nuclear components that form an pretreated with cyclophosphamide. 3 h later, mice were given 10 + +/+ +/− extracellular DNA fi brillary network. NETs trap microbes neutrophils, macrophages, or CD4 T cells i.v. from PTX3 , PTX3 , or −/− and retain neutrophil antimicrobial molecules (31). Therefore, PTX3 mice. Chitin content was measured as a correlation of fungal growth (mean ± SEM; n = 3). *, P < 0.05 using the Student’s t test. NETs serve as a focal point to focus the action of antimicrobial JEM VOL. 204, April 16, 2007 799 molecules. In this paper, we report that part of exocytosed Finally, we evaluated the in vivo relevance of PTX3 ex- PTX3 can localize in NETs. Therefore, in addition to anti- pression in neutrophils. PTX3-defi cient mice are highly sen- microbial molecules, NETs can concentrate and focus the sitive to A. fumigatus infection (15). Eff ector mechanisms of action of PTX3, a functional ancestor of antibodies. innate immunity are crucial to prevent aspergillosis (55), and The PTX3 protein is expressed in circulating PMN in among innate cells, neutrophils are essential in the initiation the absence of transcripts, whereas NF-κB–driven PTX3 of the acute infl ammatory response. Moreover, susceptibility production (52) is induced in a variety of cells by microbial to fungal infection can be associated, among other parame- sensing and infl ammatory cytokines (3). PTX3 mRNA was ters, to neutropenia (56), and impairment of neutrophil anti- detected in promyelocytes and myelocytes/metamyelocytes fungal responses results in an increase of fungal burden (57). but not in bone marrow–segmented neutrophils. In agree- These data underline the essential role played by neutrophils ment with this result and a previous report (11), we observed in controlling A. fumigatus infection. We report that PTX3 that the promyelocytic cell line HL60 expresses PTX3 expressed by neutrophils is essential to control fungal growth mRNA. A previous study reported that PTX3 is expressed in vitro and in vivo. Innate and adaptive immunity are both only at the myelocyte stage (29), and on this basis localization essential for the development of a protective antifungal im- of PTX3 in specifi c granules was postulated, a hypothesis mune response. Generation of a Th1-oriented A. fumigatus– confi rmed by our results. Collectively, we can hypothesize specifi c immune response is associated with protection (58, 59). −/− that, in accordance with the targeting-by-timing hypothesis Injection of PTX3 in PTX3 mice favors the generation (the protein content into distinct granules is determined by of a protective Th1 anti-Aspergillus immune response (15). the time of their biosynthesis), PTX3 mRNA is expressed at Neutrophil-derived PTX3, in addition to DC-derived a late stage of promyelocyte diff erentiation and in myelo- PTX3, may be involved in the orientation of the immune cytes/metamyelocytes and that most PTX3 is synthesized at response toward a protective Th1 cell phenotype. Neutrophils, the myelocytes/metamyelocytes stage, a result in agreement an innate cell type without professional antigen- presenting with its preferential localization in secondary granules. functions, may participate in the orientation of specifi c anti- PTXs usually form multimers with a discoid arrangement microbial immune responses via the release of this preformed of fi ve subunits (3). PTX3 assembles as a decamer and can be soluble PRR. produced as 10–20 subunit multimer proteins (18). PTX3 in PTX3 is a long PTX conserved in evolution, with func- neutrophil granules is mainly in the monomer form, and mul- tional properties (e.g., complement activation and opsoniza- timeric forms are detected in the supernatants of activated tion) that qualify it as a functional ancestor of antibodies. This neutrophils. The formation of PTX3 multimers is not depen- fl uid-phase PRR binds diverse microbial agents, activates the dent on glycosylation (47). However, glycosylation appears classic pathway of complement, and facilitates ingestion by crucial for the release of neo-synthetized PTX3, as observed innate immunity cells (3, 17, 24, 28), including neutrophils in DCs (this study) and fi broblasts (48). These results show (Fig. 6 B). Gene-modifi ed mice unequivocally indicate that that human neutrophils contain a mature glycosylated form of PTX3 represents a nonredundant humoral amplifi cation loop PTX3 that assembles as multimers in the extracellular milieu. of the innate immune response to diverse microbial agents Myeloid, but not plasmacytoid, DCs and macrophages (3, 15, 19, 24). Neutrophils store a variety of constituents in are major producers of PTX3 (10). Over a period of 24 h, their granules, including adhesion receptors (e.g., CD11b DCs release 50 ng of PTX3 per 10 cells (10). We report and CD18), proteolytic enzymes (e.g., cathepsin G), eff ectors that neutrophils contain 24.9 ± 3.8 ng of this PRR per 10 and regulators of matrix degradation (e.g., gelatinase), and cells (n = 5). Upon stimulation, they release 25% of stored antimicrobial molecules (25–27). The results reported in this PTX3, with a part of it remaining cell associated, presumably study broaden the repertoire of eff ector molecules stored and with NETs. Given the abundance of neutrophils in the cir- released by neutrophils to include a humoral PRR with culation and in the early phases of infl ammatory reactions in functional properties of a predecessor of antibodies. PTX3- tissues, these cells represent a major source of PTX3 covering defi cient neutrophils have defective recognition, phagocy- a temporal window preceding gene expression–dependent tosis, and killing of conidia, completely restored by PTX3, production. Under conditions of tissue damage (e.g., myo- and this molecule is nonredundant for protection against cardial infarction) or infection (e.g., sepsis), PTX3 levels in- A. fumigatus by neutrophils. Thus, PTX3 stored in neutro- crease rapidly. For instance, in acute myocardial infarction phil granules amplifi es microbial recognition by neutrophils with ST elevation, PTX3 reaches a peak in 6–8 h, compared themselves as well as presumably by neighboring innate im- with 36–48 h for CRP (53). Under these conditions, high munity cells. PTX3 is an independent marker associated with death (54). The results reported in this paper shed new light on PTX3 MATERIALS AND METHODS elevations in pathological conditions and on their pathophys- Leukocyte purifi cation. Monocytes were isolated and diff erentiated iological implications. It is likely that rapid release of stored into DCs by a 5-d culture with 20 ng/ml IL-4 and 20 ng/ml GM-CSF PTX3 by activated neutrophils plays a role in the early phases − − (R&D Systems) (60). CD14 CD86 immature DCs were used. After of its elevation in pathology, preceding gene expression– Ficoll-Paque centrifugation, neutrophils were separated from erythrocytes by dependent production. 3% dextran (GE Healthcare) density gradient sedimentation. Purity, determined 800 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE by FACS analysis on forward scatter/side scatter parameters, was routinely FACS analysis. PTX3 expression was analyzed on fi xed (2% paraformalde- >98%. Spontaneous activation of purifi ed neutrophils was eva luated by ana- hyde) and permeabilized (0.1% saponin; Sigma-Aldrich) cells with anti- + low lyzing CD11b and l-selectin expression by FACS; only l-selectin CD11b PTX3 mAb (MNB4; Qbiogene) in PBS containing 0.01% saponin. Bound neutrophils were used. Neutrophils were also isolated from whole blood antibodies were revealed by PE-labeled anti–rat IgG mAb (BD Biosciences). collected from healthy donors using a two-step buoyant density centrifugation Isotype control mAb was obtained from BD Biosciences. Fluorescence was on a Ficoll gradient (61). analyzed using a cytofl uorometer (FACScan; BD Biosciences), and results Bone marrow cells (obtained from healthy donors) at diff erent myeloid are expressed in mean fl uorescence intensity values. diff erentiation stages were isolated as previously described (36). In brief, bone marrow cells were separated by density sedimentation on a discontinu- ELISA and Western blotting. PTX3 (53), MPO (sensitivity = 0.4 ng/ml; ous Percoll gradient (GE Healthcare) of 1.065 g/ml and 1.08 g/ml. Three HyCult Biotechnology), and total gelatinase (MMP-9, sensitivity = 30 pg/ml; bands of cells, numbered in order of decreasing density, were harvested: R&D Systems) were quantifi ed by ELISA. For Western blotting, proteins band 1 primarily contained segmented neutrophils, band 2 primarily con- (corresponding to 0.4 × 10 cells or 5 μl of supernatants) were electro- tained metamyelocytes and myelocytes, and band 3 contained promyelocytes phoretically separated on a 10% polyacrylamide gel in reducing conditions (36). The three populations were subjected to immunomagnetic depletion and transferred to a membrane (Immobilon; Millipore). After saturation, of nongranulocytic cells, as previously described (29) using MACS (Miltenyi membranes were incubated for 16 h at 4°C with 3 μg/ml anti-PTX3 pAbs Biotec). The purity of cell populations was assessed by microscopy and cell or mAbs (16B5) and with 1 μg/ml peroxydase-labeled anti–rabbit IgG anti- surface phenotype (29). Eosinophils and basophils were enriched by deple- body or peroxydase-labeled anti–rat IgG antibody (Biosource International). tion of magnetically labeled cells (MACS). Protein loading was verifi ed with an anti-actin pAb (Sigma-Aldrich) re- Blood and bone marrow samples were obtained with written informed vealed by the peroxydase-labeled anti–rabbit IgG antibody. Bound antibod- consent in accordance with the Angers University Hospital ethical com- ies were detected using the ECL system (GE Healthcare). mittee requirements. Confocal microscopy. Cytospins were fi xed with 4% paraformaldehyde Cell act ivation. 2 × 10 neutrophil cells/ml in RPMI 1640 (2% FCS) and permeabilized for 5 min with 0.2% Triton X-100 (Sigma-Aldrich) in were either nonstimulated or stimulated with 1–10 μg/ml E. coli, S. aureus, PBS, pH 7.4, before incubation for 1 h at 4°C with 10% normal goat or zymosan (all obtained from Invitrogen); 20 ng/ml IL-1β or TNFα (R&D serum (Sigma-Aldrich) and then for 2 h at 4°C with 0.5 μg/ml biotin- Systems); 1–10 ng/ml PMA; 0.01–1 μM ionomycine; 100 ng/ml LPS (from conjugated rat anti–human PTX3 mAb (1 μg/ml; MNB4), or with IgG2a E. coli serotype O55:B5; all purchased from Sigma-Aldrich); 500 ng/ml control mAb. Slides were incubated with streptavidin–Alexa Fluor 488 Pam3CSK4 (a TLR2 agonist); 5 μg/ml R848 (a TLR7/8 agonist), 2 μg/ml conjugate, followed by 1 μg/ml Hoechst 33258 or by 5 μg/ml propidium fl agellin (a TLR5 agonist; all obtained from InvivoGen); and 10 μg/ml poly iodide and RNase (Invitrogen). The following reagents were also used: (I:C) (a TLR3 agonist; Sigma-Aldrich). 2 × 10 DCs/ml were stimulated for rabbit anti-MPO pAb (Invitrogen), mouse antilactoferrin mAb (clone 8, 16, or 24 h with the stimuli indicated in the fi gures. PTX3 levels in super- NI25; Calbiochem), a rabbit antigelatinase pAb (Chemicon), and Alexa natants were quantifi ed by ELISA. Fluor 647–conjugated goat anti–rabbit and anti–mouse IgG secondary NET formation was induced as previously described (31). In brief, neutro- antibodies. In each step, cells were washed with 0.2% BSA/0.05% Tween 6 5 phils (400 μl of cells at 2 × 10 cells/ml and 8 × 10 cells/well) were seeded 20 in PBS, pH 7.4. on rounded glass coverslips treated with 1% poly-l-lysine (Sigma-Aldrich), To stain NETs, neutrophils were fi xed, blocked, and washed as de- allowed to settle, and treated for 40 min with 100 ng/ml IL-8 (PeproTech), scribed in the previous paragraph, without permeabilization. Cells were in- 2.5 ng/ml PMA, 100 ng/ml LPS, and A. fumigatus conidia at a 1:5 ratio. cubated with 1 μg/ml of biotin-conjugated PTX3 affi nity-purifi ed rabbit IgG, followed by streptavidin–Alexa Fluor 647 conjugate (Invitrogen). For Cell lysates. 10 cells were lysed in 10 mM Tris-HCl, pH 7.6, 5 mM DNA detection, Syto 13 (Invitrogen) plus RNase (Sigma-Aldrich) was used. EDTA, 1% Triton X-100, or Nonidet P40 (Sigma-Aldrich) plus protease Sections were mounted with a reagent (FluorSave; Calbiochem) and analyzed inhibitors (Roche Diagnostics). Lysates were centrifuged at 14,000 rpm for with a laser scanning confocal microscope (FluoView FV1000; Olympus). 15 min at 4°C to remove cellular debris. For N-glycosidase F treatment, Images (1,024 × 1,024 pixels) were acquired with an oil immersion objec- SDS (0.2% fi nal, vol/vol) was added to cell lysates or cell culture superna- tive (100× 1.4 NA Plan-Apochromat; Olympus). Diff erential interference tants before heating at 100°C for 5 min. Lysates were treated with 5 U/ml contrast (Nomarski technique) was also used. Overlay images were assem- N-glycosidase F (Roche Diagnostics) for 16 h at 37°C before analysis by bled, and ImarisColoc (version 4.2; Bitplane AG) software was used for Western blotting. quantitative colocalization and statistical analysis. Site-directed mutagenesis. The complete PTX3 cDNA was cloned into Analysis of PTX3 and MPO mRNA expression by RT-PCR. Total the pCDNA3.1+ vector (Invitrogen). The N220S substitution was intro- RNA from DCs and peripheral blood neutrophils was extracted using duced using a kit (QuickChange XL; Stratagene). CHO cells were trans- TRIzol reagent (Life Technologies). Total RNA from neutrophil precur- fected using Lipofectamine (Invitrogen). sors and HL60 (American Type Culture Collection) was purifi ed using the RNeasy mini kit (QIAGEN). cDNA was synthesized from 1 μg of total RNA using an oligo-dT primer and reverse transcriptase (GE Health- Subcellular fractionation. Subcellular fractionation of neutrophils was care). PCR amplifi cation was performed with an amount of cDNA corre- performed as previously described (62). In brief, fresh neutrophils were dis- sponding to 25 ng of the starting total RNA using specifi c oligonucleotides rupted by nitrogen cavitation (Parr Instruments Co.), and the resulting cavi- (PTX3, 5′-G T G C A G G G C T G G G C T G C C C G -3′ and 5′-G C C G C A C A- tates were centrifuged to eliminate pellet nuclei and the remaining intact G G T G G G T C C A C C -3′; MPO, 5′-C C T T C A T G T T C C G C C T G G A C A- cells (63). The postnuclear supernatant containing cytosol, granules, and A T C G -3′ and 5′-C G G A T C T C A T C C A C T G C A A T T T G G -3′). RNA light membranes was immediately separated by centrifugation on a three- integrity was assessed by GAPDH cDNA amplifi cation. The PCR products layer Percoll density gradient. 3-ml gradient fractions were collected after a were analyzed on a 1% agarose gel by electrophoresis and visualized with sonication, frozen, and thawed for two times and analyzed for PTX3 content ethidium bromide. and for localization of subcellular organelles by marker assays (62). An α-mannosidase functional assay was used for azurophil granules (64); gelatinase, lactoferrin, and albumin ELISA were used to identify, respectively, gelatinase Mouse neutrophils and phagocytosis assay. Neutrophils from C57BL/6 (tertiary) granules, lactoferrin (specifi c) granules, and secretory vesicles/ mice (Charles River Laboratories) were isolated from bone marrow by light membranes (40). Percoll step gradient (52, 65, and 75% Percoll). An enriched neutrophil JEM VOL. 204, April 16, 2007 801 population was recovered at the 65–75% interface. Purity was analyzed by R E F E R E N C E S morphology, Giemsa staining, and FACS using anti-Ly6G (clone 1A8; BD 1. Janeway, C.A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Biosciences), and antineutrophil mAb (clone 7/4; Serotec) was routinely Annu. Rev. Immunol. 20:197–216. −/− >96%. Cells were also isolated from wild-type and PTX3 total bone 2. Gordon, S. 2002. Pattern recognition receptors: doubling up for the innate immune response. Cell. 111:927–930. marrow (15, 65) or from the peritoneal cavity 4 h after a 1.5-ml 3% thiogly- 3. Garlanda, C., B. Bottazzi, A. Bastone, and A. Mantovani. 2005. Pentraxins collate (Difco) injection and plated at 5 × 10 cells/ml in a fi nal volume of at the crossroads between innate immunity, infl ammation, matrix depo- 0.5 ml RPMI 1640 with human serum in a 24-well plate. A. fumigatus co- 7 sition, and female fertility. Annu. Rev. Immunol. 23:337–366. nidia were added at 2.5 × 10 cells/well. After 15 or 45 min of incubation at 4. Szalai, A.J., A. Agrawal, T.J. Greenhough, and J.E. Volanakis. 1999. 37°C, phagocytosis was blocked by using NaF (fi nal concentration = 0.2 M). C-reactive protein: structural biology and host defense function. Clin. In some of the experiments, 50 μg/ml of recombinant PTX3 was added. Chem. Lab. Med. 37:265–270. Cytospins were stained with Diff Quick (Dade, Biomap). At least 200 neu- 5. Hirschfi eld, G.M., and M.B. Pepys. 2003. C-reactive protein and cardio- trophils per sample were counted under oil immersion microscopy (100× vascular disease: new insights from an old molecule. QJM. 96:793–807. objective). Results are expressed as a phagocytic index: (percentage of neu- 6. Bottazzi, B., A. Bastone, A. Doni, C. Garlanda, S. Valentino, L. Deban, trophils containing at least one conidia) × (mean number of conidia per V. Maina, A. Cotena, F. Moalli, L. Vago, et al. 2006. The long pen- positive cell). traxin PTX3 as a link among innate immunity, infl ammation, and fe- male fertility. J. Leukoc. Biol. 79:909–912. Infection. Mice were administered 150 mg/kg cyclophosphamide i.p. 2 d 7. Pepys, M.B., and M.L. Baltz. 1983. Acute phase proteins with special before the intranasal infection with 10 A. fumigatus conidia per 20 μl (15). reference to C-reactive protein and related proteins (pentaxis) and se- Mice received 10 cells per 500 μl of peritoneal neutrophils, splenic macro- rum amyloid A protein. Adv. Immunol. 34:141–212. phages, or CD4 T cells i.v. 3 h after the infection. Quantifi cation of fungal 8. Baumann, H., and J. Gauldie. 1994. The acute phase response. Immunol. growth in the lungs was done by the chitin assay (49), and results are ex- Today. 15:74–80. pressed as micrograms of glucosamine per pair of lungs. Peritoneal neutro- 9. Goodman, A.R., T. Cardozo, R. Abagyan, A. Altmeyer, H.G. phils were obtained 18 h after the i.p. injection of 1 ml of endotoxin-free Wisniewski, and J. Vilcek. 1996. Long pentraxins: an emerging group of 10% thioglycolate solution (Difco). Endotoxin was depleted from all solu- proteins with diverse functions. Cytokine Growth Factor Rev. 7:191–202. tions with Detoxi-Gel (Pierce Chemical Co.). Purity was >98%, as deter- 10. Doni, A., G. Peri, M. Chieppa, P. Allavena, F. Pasqualini, L. Vago, + + mined by Cytospin and FACS analysis (GR-1 and CD11b ). Macrophages L. Romani, C. Garlanda, and A. Mantovani. 2003. Production of the were obtained by 2 h of plastic adherence of spleen cells at 37C°. CD4 T soluble pattern recognition receptor PTX3 by myeloid, but not plasma- cells were purifi ed from spleens of mice using anti-CD4 magnetic Micro- cytoid, dendritic cells. Eur. J. Immunol. 33:2886–2893. Beads (Miltenyi Biotec). 11. Alles, V.V., B. Bottazzi, G. Peri, J. Golay, M. Introna, and A. Mantovani. 1994. Inducible expression of PTX3, a new member of the pentraxin Procedures involving animals and their care conformed to institutional family, in human mononuclear phagocytes. Blood. 84:3483–3493. guidelines in compliance with national (4D.L. N.116, G.U., suppl. 40, 18- 12. Breviario, F., E.M. d’Aniello, J. Golay, G. Peri, B. Bottazzi, A. 2-1992) and international (EEC Council Directive 86/609, OJ L 358,1,12- Bairoch, S. Saccone, R. Marzella, V. Predazzi, M. Rocchi, et al. 1992. 12-1987; National Institutes of Health Guide for the Care and Use of Interleukin-1-inducible genes in endothelial cells. Cloning of a new Laboratory Animals) law and policies. All eff orts were made to minimize the gene related to C-reactive protein and serum amyloid P component. number of animals used and their suff ering. J. Biol. Chem. 267:22190–22197. 13. Han, B., M. Mura, C.F. Andrade, D. Okutani, M. Lodyga, C.C. dos Statistical analysis. Statistical analysis was performed using the Student’s Santos, S. Keshavjee, M. Matthay, and M. Liu. 2005. TNFalpha- t test. induced long pentraxin PTX3 expression in human lung epithelial cells via JNK. J. Immunol. 175:8303–8311. Online supplemental material. Supplemental materials and methods de- 14. Vouret-Craviari, V., C. Matteucci, G. Peri, G. Poli, M. Introna, and A. scribes the binding assay and the analysis of PTX3, MPO, lactoferrin, and Mantovani. 1997. Expression of a long pentraxin, PTX3, by monocytes MMP-9 mRNA expression in HL60 cells and human bone marrow. Fig. exposed to the mycobacterial cell wall component lipoarabinomannan. S1 shows the constitutive expression of PTX3 (mRNA and protein) in the Infect. Immun. 65:1345–1350. human promyelocytic cell line HL60. Fig. S2 shows the relative content of 15. Garlanda, C., E. Hirsch, S. Bozza, A. Salustri, M. De Acetis, R. Nota, PTX3, as assessed by Western blotting, in neutrophils stimulated for 16 h with A. Maccagno, F. Riva, B. Bottazzi, G. Peri, et al. 2002. Non-redundant the indicated stimuli. Fig. S3 shows that the release of PTX3 by neutrophils role of the long pentraxin PTX3 in anti-fungal innate immune response. is not aff ected by tunicamycin, in contrast to PTX3 produced by activated Nature. 420:182–186. DCs. Fig. S4 shows PMN-released PTX3 binding to (A) immobilized C1q 16. Salustri, A., C. Garlanda, E. Hirsch, M. De Acetis, A. Maccagno, B. and OmpA and to (B) A. fumigatus conidia. Online supplemental material is Bottazzi, A. Doni, A. Bastone, G. Mantovani, P. Beck Peccoz, et al. 2004. available at http://www.jem.org/cgi/content/full/jem.20061301/DC1. PTX3 plays a key role in the organization of the cumulus oophorus extra- cellular matrix and in in vivo fertilization. Development. 131:1577–1586. We sincerely acknowledge Dr. Odile Blanchet and Ms. Irène Dobo for giving human 17. Nauta, A.J., B. Bottazzi, A. Mantovani, G. Salvatori, U. Kishore, W.J. Schwaeble, A.R. Gingras, S. Tzima, F. Vivanco, J. Egido, et al. 2003. bone marrow cells. Biochemical and functional characterization of the interaction between pentraxin 3 and C1q. Eur. J. Immunol. 33:465–473. S. Jaillon is supported by a grant from the Conseil Général du Maine et Loire. This 18. Bottazzi, B., V. Vouret-Craviari, A. Bastone, L. De Gioia, C. Matteucci, study is supported by the Institut National de la Santé et de la Recherche Médicale G. Peri, F. Spreafi co, M. Pausa, C. Dettorre, E. Gianazza, et al. 1997. (Avenir program), the Ministère de la Recherche (ACI program), Cancéropôle Grand- Multimer formation and ligand recognition by the long pentraxin PTX3. Ouest, the Sixth Research Framework Programme of the European Union (projects Similarities and diff erences with the short pentraxins C-reactive protein MUGEN LSHB-CT-2005-005203 and MUVAPRED), the Ministero dell’Istruzione, Università e della Ricerca (project FIRB), Telethon (grant GGP05095), the Fondazione and serum amyloid P component. J. Biol. Chem. 272:32817–32823. 19. Diniz, S.N., R. Nomizo, P.S. Cisalpino, M.M. Teixeira, G.D. Brown, CARIPLO (project Nobel), and the Italian Association for Cancer Research. G. Peri, A. Doni, C. Garlanda, and A. Mantovani are inventors in patent A. Mantovani, S. Gordon, L.F. Reis, and A.A. Dias. 2004. PTX3 func- tion as an opsonin for the dectin-1-dependent internalization of zymo- applications concerning PTX3. The authors have no other confl icting fi nancial interests. san by macrophages. J. Leukoc. Biol. 75:649–656. Submitted: 19 June 2006 20. Varani, S., J.A. Elvin, C. Yan, J. DeMayo, F.J. DeMayo, H.F. Horton, Accepted: 15 February 2007 M.C. Byrne, and M.M. Matzuk. 2002. Knockout of pentraxin 3, a 802 PTX3 IN NEUTROPHILS | Jaillon et al. ARTICLE downstream target of growth diff erentiation factor-9, causes female following exposure to proinfl ammatory mediators. J. Leukoc. Biol. 79: subfertility. Mol. Endocrinol. 16:1154–1167. 123–132. 21. Rovere, P., G. Peri, F. Fazzini, B. Bottazzi, A. Doni, A. Bondanza, V.S. 41. Dewald, B., U. Bretz, and M. Baggiolini. 1982. Release of gelatin- Zimmermann, C. Garlanda, U. Fascio, M.G. Sabbadini, et al. 2000. ase from a novel secretory compartment of human neutrophils. J. Clin. The long pentraxin PTX3 binds to apoptotic cells and regulates their Invest. 70:518–525. clearance by antigen-presenting dendritic cells. Blood. 96:4300–4306. 42. Mocsai, A., E. Ligeti, C.A. Lowell, and G. Berton. 1999. Adhesion- 22. Baruah, P., A. Propato, I.E. Dumitriu, P. Rovere-Querini, V. Russo, R. dependent degranulation of neutrophils requires the Src family kinases Fontana, D. Accapezzato, G. Peri, A. Mantovani, V. Barnaba, and A.A. Fgr and Hck. J. Immunol. 162:1120–1126. Manfredi. 2006. The pattern recognition receptor PTX3 is recruited 43. Colotta, F., F. Re, N. Polentarutti, S. Sozzani, and A. Mantovani. 1992. at the synapse between dying and dendritic cells, and edits the cross- Modulation of granulocyte survival and programmed cell death by cyto- presentation of self, viral, and tumor antigens. Blood. 107:151–158. kines and bacterial products. Blood. 80:2012–2020. 23. Souza, D.G., A.C. Soares, V. Pinho, H. Torloni, L.F. Reis, M.M. 44. Sabroe, I., R.C. Read, M.K. Whyte, D.H. Dockrell, S.N. Vogel, and Teixeira, and A.A. Dias. 2002. Increased mortality and infl ammation in S.K. Dower. 2003. Toll-like receptors in health and disease: complex tumor necrosis factor-stimulated gene-14 transgenic mice after ischemia questions remain. J. Immunol. 171:1630–1635. and reperfusion injury. Am. J. Pathol. 160:1755–1765. 45. Francois, S., J. El Benna, P.M. Dang, E. Pedruzzi, M.A. Gougerot- 24. Jeannin, P., B. Bottazzi, M. Sironi, A. Doni, M. Rusnati, M. Presta, V. Pocidalo, and C. Elbim. 2005. Inhibition of neutrophil apoptosis by Maina, G. Magistrelli, J.F. Haeuw, G. Hoeff el, et al. 2005. Complexity TLR agonists in whole blood: involvement of the phosphoinositide and complementarity of outer membrane protein A recognition by cel- 3-kinase/Akt and NF-kappaB signaling pathways, leading to increased lular and humoral innate immunity receptors. Immunity. 22:551–560. levels of Mcl-1, A1, and phosphorylated Bad. J. Immunol. 174:3633–3642. 25. Nathan, C. 2006. Neutrophils and immunity: challenges and opportu- 46. Hayashi, F., T.K. Means, and A.D. Luster. 2003. Toll-like receptors nities. Nat. Rev. Immunol. 6:173–182. stimulate human neutrophil function. Blood. 102:2660–2669. 26. Cassatella, M.A., S. Gasperini, and M.P. Russo. 1997. Cytokine expres- 47. Inforzato, A., G. Peri, A. Doni, C. Garlanda, A. Mantovani, A. Bastone, sion and release by neutrophils. Ann. N Y Acad. Sci. 832:233–242. A. Carpentieri, A. Amoresano, P. Pucci, A. Roos, et al. 2006. Structure 27. Segal, A.W. 2005. How neutrophils kill microbes. Annu. Rev. Immunol. and function of the long pentraxin PTX3 glycosidic moiety: fi ne-tuning 23:197–223. of the interaction with C1q and complement activation. Biochemistry. 28. Bottazzi, B., C. Garlanda, G. Salvatori, P. Jeannin, A. Manfredi, and A. 45:11540–11551. Mantovani. 2006. Pentraxins as a key component of innate immunity. 48. Lee, G.W., A.R. Goodman, T.H. Lee, and J. Vilcek. 1994. Relationship Curr. Opin. Immunol. 18:10–15. of TSG-14 protein to the pentraxin family of major acute phase proteins. 29. Theilgaard-Monch, K., L.C. Jacobsen, R. Borup, T. Rasmussen, M.D. J. Immunol. 153:3700–3707. Bjerregaard, F.C. Nielsen, J.B. Cowland, and N. Borregaard. 2005. The 49. Montagnoli, C., F. Fallarino, R. Gaziano, S. Bozza, S. Bellocchio, T. transcriptional program of terminal granulocytic diff erentiation. Blood. Zelante, W.P. Kurup, L. Pitzurra, P. Puccetti, and L. Romani. 2006. 105:1785–1796. Immunity and tolerance to Aspergillus involve functionally distinct regu- 30. Lominadze, G., D.W. Powell, G.C. Luerman, A.J. Link, R.A. Ward, and latory T cells and tryptophan catabolism. J. Immunol. 176:1712–1723. K.R. McLeish. 2005. Proteomic analysis of human neutrophil granules. 50. Sengelov, H., L. Kjeldsen, and N. Borregaard. 1993. Control of exocy- Mol. Cell. Proteomics. 4:1503–1521. tosis in early neutrophil activation. J. Immunol. 150:1535–1543. 31. Brinkmann, V., U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, 51. Sengelov, H., P. Follin, L. Kjeldsen, K. Lollike, C. Dahlgren, and N. D.S. Weiss, Y. Weinrauch, and A. Zychlinsky. 2004. Neutrophil extra- Borregaard. 1995. Mobilization of granules and secretory vesicles during cellular traps kill bacteria. Science. 303:1532–1535. in vivo exudation of human neutrophils. J. Immunol. 154:4157–4165. 32. Grossman, W.J., and T.J. Ley. 2004. Granzymes A and B are not ex- 52. Basile, A., A. Sica, E. D’Aniello, F. Breviario, G. Garrido, M. Castellano, pressed in human neutrophils. Blood. 104:906–907. A. Mantovani, and M. Introna. 1997. Characterization of the promoter 33. Metkar, S.S., and C.J. Froelich. 2004. Human neutrophils lack gran- for the human long pentraxin PTX3. J. Biol. Chem. 272:8172–8178. zyme A, granzyme B, and perforin. Blood. 104:905–906. 53. Peri, G., M. Introna, D. Corradi, G. Iacuitti, S. Signorini, F. Avanzini, 34. Wagner, C., C. Iking-Konert, B. Denefl eh, S. Stegmaier, F. Hug, and F. Pizzetti, A.P. Maggioni, T. Moccetti, M. Metra, et al. 2000. PTX3, G.M. Hansch. 2004. Granzyme B and perforin: constitutive expression a prototypic long pentraxin, is an early indicator of acute myocardial in human polymorphonuclear neutrophils. Blood. 103:1099–1104. infarction in man. Circulation. 102:636–641. 35. Doni, A., M. Michela, B. Bottazzi, G. Peri, S. Valentino, N. Polentarutti, 54. Latini, R., A.P. Maggioni, G. Peri, L. Gonzini, D. Lucci, P. Mocarelli, L. C. Garlanda, and A. Mantovani. 2006. Regulation of PTX3, a key Vago, F. Pasqualini, S. Signorini, D. Soldateschi, et al. 2004. Prognostic component of humoral innate immunity in human dendritic cells: signifi cance of the long pentraxin PTX3 in acute myocardial infarction. stimulation by IL-10 and inhibition by IFN-gamma. J. Leukoc. Biol. 79: Circulation. 110:2349–2354. 797–802. 55. Schaff ner, A., H. Douglas, and A. Braude. 1982. Selective protection 36. Cowland, J.B., and N. Borregaard. 1999. Isolation of neutrophil precur- against conidia by mononuclear and against mycelia by polymorphonu- sors from bone marrow for biochemical and transcriptional analysis. clear phagocytes in resistance to Aspergillus. Observations on these two J. Immunol. Methods. 232:191–200. lines of defense in vivo and in vitro with human and mouse phagocytes. 37. Devarajan, P., J.J. Johnston, S.S. Ginsberg, H.E. Van Wart, and N. J. Clin. Invest. 69:617–631. Berliner. 1992. Structure and expression of neutrophil gelatinase cDNA. 56. Gerson, S.L., G.H. Talbot, S. Hurwitz, B.L. Strom, E.J. Lusk, and P.A. Identity with type IV collagenase from HT1080 cells. J. Biol. Chem. Cassileth. 1984. Prolonged granulocytopenia: the major risk factor for 267:25228–25232. invasive pulmonary aspergillosis in patients with acute leukemia. Ann. 38. Johnston, J.J., P. Rintels, J. Chung, J. Sather, E.J. Benz Jr., and N. Intern. Med. 100:345–351. Berliner. 1992. Lactoferrin gene promoter: structural integrity and non- 57. Romani, L., and D.H. Howard. 1995. Mechanisms of resistance to fun- expression in HL60 cells. Blood. 79:2998–3006. gal infections. Curr. Opin. Immunol. 7:517–523. 39. Kjeldsen, L., D.F. Bainton, H. Sengelov, and N. Borregaard. 1993. 58. Cenci, E., S. Perito, K. Enssle, P. Mosci, J. Latge, L. Romani, and F. Structural and functional heterogeneity among peroxidase-negative Bistoni. 1997. Th1 and Th2 cytokines in mice with invasive aspergillosis. granules in human neutrophils: identifi cation of a distinct gelatinase- Infect. Immun. 65:564–570. containing granule subset by combined immunocytochemistry and sub- 59. Nagai, H., J. Guo, H. Choi, and V. Kurup. 1995. Interferon-gamma cellular fractionation. Blood. 82:3183–3191. and tumor necrosis factor-alpha protect mice from invasive aspergillosis. 40. Cassatella, M.A., V. Huber, F. Calzetti, D. Margotto, N. Tamassia, J. Infect. Dis. 172:1554–1560. G. Peri, A. Mantovani, L. Rivoltini, and C. Tecchio. 2006. Interferon- 60. Jeannin, P., T. Renno, L. Goetsch, I. Miconnet, J.P. Aubry, Y. activated neutrophils store a TNF-related apoptosis-inducing ligand Delneste, N. Herbault, T. Baussant, G. Magistrelli, C. Soulas, et al. (TRAIL/Apo-2 ligand) intracellular pool that is readily mobilizable 2000. OmpA targets dendritic cells, induces their maturation and JEM VOL. 204, April 16, 2007 803 delivers antigen into the MHC class I presentation pathway. Nat. gamma, and GM-CSF and the necessity to overcome the action of Immunol. 1:502–509. endogenous proteases. Biochemistry. 37:13165–13173. 61. Bourke, E., D. Bosisio, J. Golay, N. Polentarutti, and A. Mantovani. 2003. 64. De Togni, P., G. Cabrini, and F. Di Virgilio. 1984. Cyclic AMP inhi- The Toll-like receptor repertoire of human B lymphocytes: inducible bition of fMet-Leu-Phe-dependent metabolic responses in human and selective expression of TLR9 and TLR10 in normal and trans- neutrophils is not due to its eff ects on cytosolic Ca2+. Biochem. J. formed cells. Blood. 102:956–963. 224:629–635. 62. Kjeldsen, L., H. Sengelov, and N. Borregaard. 1999. Subcellular frac- 65. Pruijt, J.F., P. Verzaal, R. van Os, E.J. de Kruijf, M.L. van Schie, A. tionation of human neutrophils on Percoll density gradients. J. Immunol. Mantovani, A. Vecchi, I.J. Lindley, R. Willemze, S. Starckx, et al. 2002. Methods. 232:131–143. Neutrophils are indispensable for hematopoietic stem cell mobili- 63. McDonald, P.P., C. Bovolenta, and M.A. Cassatella. 1998. Activation zation induced by interleukin-8 in mice. Proc. Natl. Acad. Sci. USA. of distinct transcription factors in neutrophils by bacterial LPS, interferon- 99:6228–6233. 804 PTX3 IN NEUTROPHILS | Jaillon et al.

Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Apr 16, 2007

There are no references for this article.