The Journal of Experimental Medicine

Shiff, Steven J.; Rigas, Basil

doi: 10.1084/jem.190.4.445pmid: 10449515

Zhang, Xinping; Morham, Scott G.; Langenbach, Robert; Young, Donald A.

doi: 10.1084/jem.190.4.451pmid: 10449516

In this study, we use primary embryonic fibroblasts derived from cyclooxygenase-deficient transgenic embryos to further investigate the role of the two cyclooxygenases, cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2), in the process of neoplastic transformation. Cells with either, neither, or both of the cyclooxygenases were transformed by Ha- ras and/or SV40. Our results show that when a cyclooxygenase enzyme is present, the transformed cells have marked increases in COX-2 and/or COX-1 expression. Nevertheless, each type of cell, deficient in either or both cyclooxygenases, can be readily transformed at almost equal efficiency. Different nonsteroidal antiinflammatory drugs (NSAIDs) were used to examine their possible antineoplastic effects on the transformed cells, which have various levels of expression of COX-1 or COX-2. Our results show that NSAIDs suppress the colony formation in soft agar in a dosage-dependent manner in the absence of the cyclooxygenase(s). Thymidine incorporation and apoptosis analyses further demonstrate that the NSAIDs are effective in the cyclooxygenase-null cells. Our findings with cyclooxygenase knockout cells confirm recent reports that some of the antiproliferative and antineoplastic effects of NSAIDs are independent of the inhibition of either COX-1 or COX-2. They also show that transformation is independent of the status of cyclooxygenase expression, suggesting that the involvement of the cyclooxygenases in tumorigenesis may occur at later steps. cyclooxygenase nonsteroidal antiinflammatory drugs neoplastic transformation transgenic knockout cells ras Footnotes 1used in this paper: COX, cyclooxygenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NSAID, nonsteroidal antiinflammatory drug; TPA, 12-o-tetradecanoylphorbol 13-acetate Submitted: 27 January 1999 Revision requested 21 June 1999 Accepted: 22 June 1999

Watanabe, Norihiko; Nisitani, Sazuku; Ikuta, Koichi; Suzuki, Misao; Chiba, Tsutomu; Honjo, Tasuku

doi: 10.1084/jem.190.4.461pmid: 10449517

Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains. Although various degrees of suppression of endogenous Ig expression are observed in Ig transgenic (Tg) mice, it was not clear whether this difference is due to different onsets of Tg expression or to different levels of Tg expression, which are obviously affected by integration sites of the transgene. In this study we generated antierythrocyte antibody Tg mice that carry tandem joined H and L chain transgenes (H+L) and confirmed that homozygosity of the transgene loci enhances the level of transgene expression as compared with heterozygosity. Suppression of endogenous H and L chain gene expression was stronger in homozygous than in heterozygous Tg mice. Similar results were obtained in control Tg mice carrying the H chain only. These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion. In addition, despite the same B cell receptor specificity, the size of Tg autoreactive B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous mice, although the number of the Tg B-2 cell subset decreased in the spleen and bone marrow of homozygous Tg mice as compared with heterozygous Tg mice. By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset. These results suggest that the size of the B-1 cell subset in the Tg mice may depend on strength of signals through B cell receptors triggered by self-antigens. transgenic lines homozygosity flow cytometry anti-RBC antibody Footnotes 1used in this paper: BCR, B cell receptor; H+L mice, tandem joined H and L chain transgenic mice; H×L mice, double transgenic mice with H and L chain transgenes; Id, idiotype; MFI, mean fluorescence intensity; SA, streptavidin; Tg, transgenic S. Nisitani's present address is Howard Hughes Medical Institute, University of California, Los Angeles, 5-720 MRL, 675 Circle Dr. South, Box 951662, Los Angeles, CA 90095-1662. Submitted: 10 February 1999 Revision requested 16 June 1999 Accepted: 17 June 1999

Lam, Kong-Peng; Rajewsky, Klaus

doi: 10.1084/jem.190.4.471pmid: 10449518

Mice expressing the immunoglobulin (Ig) heavy (H) chain variable (V) region from a rearranged V H 12 gene inserted into the IgH locus generate predominantly B-1 cells, whereas expression of two other V H region transgenes (V H B1-8 and V H glD42) leads to the almost exclusive generation of conventional, or B-2, cells. To determine the developmental potential of B cells bearing two distinct B cell antigen receptors (BCRs), one favoring B-1 and the other favoring B-2 cell development, we crossed V H 12 insertion mice with mice bearing either V H B1-8 or V H glD42. B cells coexpressing V H 12 and one of the other V H genes are readily detected in the double IgH insertion mice, and are of the B-2 phenotype. In mice coexpressing V H 12, V H B1-8 and a transgenic κ chain able to pair with both H chains, double H chain–expressing B-2 cells, and B-1 cells that have lost V H B1-8 are generated, whereas V H B1-8 single producers are undetectable. These data suggest that B-1 but not B-2 cells are selected by antigenic stimuli in whose delivery BCR specificity and surface density are of critical importance. B-1 cells B-2 cells immunoglobulin heavy chain allelic inclusion gene targeting Footnotes 1used in this paper: BCR, B cell receptor; Id, idiotype; PtC, phosphatidyl choline; tg, transgenic Submitted: 14 April 1999 Revision requested 4 June 1999 Accepted: 10 June 1999

Poulin, Jean-François; Viswanathan, Mohan N.; Harris, Jeffrey M.; Komanduri, Krishna V.; Wieder, Eric; Ringuette, Nancy; Jenkins, Morgan; McCune, Joseph M.; Sékaly, Rafick-Pierre

doi: 10.1084/jem.190.4.479pmid: 10449519

The understanding of human thymic function and evaluation of its contribution to T cell homeostasis are matters of great importance. Here we report the development of a novel assay to quantitate the frequency and diversity of recent thymic emigrants (RTEs) in the peripheral blood of humans. Such cells were defined by the presence of T cell receptor (TCR) rearrangement deletion circles (DCs), episomal byproducts of TCR-β V(D)J rearrangement. DCs were detected in T cells in the thymus, cord blood, and adult peripheral blood. In the peripheral blood of adults aged 22 to 76 years, their frequency was highest in the CD4 + CD45RA + CD62L + subpopulation of naive T cells. TCR DCs were also observed in other subpopulations of peripheral blood T cells, including those with the CD4 + CD45RO − CD62L + and CD4 + CD45RO + CD62L + phenotypes. RTEs were observed to have more than one Vβ rearrangement, suggesting that replenishment of the repertoire in the adult is at least oligoclonal. These results demonstrate that the normal adult thymus continues to contribute, even in older individuals, a diverse set of new T cells to the peripheral circulation. thymus T cell receptor deletion circles naive T cells immune reconstitution Footnotes 1used in this paper: CBMCs, cord blood mononuclear cells; DCs, deletion circles; DCF, deletion circle frequency; RAG, recombination activating gene; RSS, recombination signal sequences; RTEs, recent thymic emigrants; SP, single positive Submitted: 19 November 1998 Revision requested 4 June 1999 Accepted: 11 June 1999

Woerly, Gaëtane; Roger, Nadine; Loiseau, Sylvie; Dombrowicz, David; Capron, André; Capron, Monique

doi: 10.1084/jem.190.4.487pmid: 10449520

Eosinophils are the source of various immunoregulatory cytokines, but the membrane molecules involved in their secretion have not been clearly identified. Here we show that peripheral blood eosinophils from hypereosinophilic patients could express membrane CD86 but not CD80. The T cell costimulatory molecule CD28 is also detected on the eosinophil surface. CD28 ligation but not CD86 ligation resulted in interleukin (IL)-2 and interferon (IFN)-γ secretion by eosinophils, whereas IL-4, IL-5, and IL-10 were not detected. In contrast to T cells requiring two signals for effective stimulation, CD28 ligation alone was sufficient for optimal eosinophil activation. Eosinophil-derived IL-2 and IFN-γ were biologically active, as supernatants from anti-CD28–treated cells were able to induce CTLL-2 proliferation and major histocompatibility complex class II expression on the colon carcinoma cell line Colo 205, respectively. Addition of secretory immunoglobulin (Ig)A–anti-IgA complexes, which could induce the release of IL-10, very significantly inhibited both CD28-mediated IL-2 and IFN-γ release. These results suggest that the release of type 1 (IFN-γ and IL-2) versus type 2 cytokines by eosinophils is not only differential but also dependent on cross-regulatory signals. They confirm that through activation of costimulatory molecules, eosinophils could function as an immunoregulatory cell involved in the release of both type 1 and type 2 cytokines. eosinophils CD28 CD86 type 1 cytokines secretory IgA Footnotes 1used in this paper: APAAP, alkaline phosphatase anti-alkaline phosphatase; HRP, horseradish peroxidase; LDH, lactate dehydrogenase Submitted: 17 December 1998 Revision requested 6 May 1999 Accepted: 15 June 1999

Ward, Alister C.; van Aesch, Yvette M.; Gits, Judith; Schelen, Anita M.; de Koning, John P.; van Leeuwen, Daphne; Freedman, Melvin H.; Touw, Ivo P.

doi: 10.1084/jem.190.4.497pmid: 10449521

Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand–receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness. cytokine receptor signal transduction cell survival structure activity relationship Footnotes 1used in this paper: CRH, cytokine receptor homologous; RT, reverse transcription; SCN, severe congenital neutropenia; STAT, signal transducer and activator of transcription Submitted: 1 February 1999 Revision requested 8 June 1999 Accepted: 15 June 1999

Vasselon, Thierry; Hailman, Eric; Thieringer, Rolf; Detmers, Patricia A.

doi: 10.1084/jem.190.4.509pmid: 10449522

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14–EGFP) was used to follow trafficking of mCD14 and BODIPY–LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex–like structure. mCD14–EGFP was functional in mediating binding of and responses to LPS. BODIPY–LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14–EGFP on the cell surface. However, within 5–10 min, the BODIPY–LPS distributed to intracellular vesicles that did not contain mCD14–EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS. enhanced green fluorescent protein U373 cells intracellular trafficking Footnotes 1used in this paper: BODIPY, boron dipyrromethane; DAF, decay accelerating factor; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GPI, glycosylphosphatidyl inositol; LBP, LPS binding protein; m, membrane-bound; PI-PLC, phosphatidyl inositol phospholipase C; s, soluble; SP, signal peptide; TLR, Toll-like receptor A preliminary version of this work was presented at the Fifth Conference of the International Endotoxin Society, Santa Fe, NM, September 12–15, 1998. Le Grand, C.B., N. Lamping, T. Sugiyama, S.D. Wright, and R. Thieringer, manuscript submitted for publication. Submitted: 12 February 1999 Revision requested 11 June 1999 Accepted: 22 June 1999

Thieblemont, Nathalie; Wright, Samuel D.

doi: 10.1084/jem.190.4.523pmid: 10449523

Addition of lipopolysaccharide (LPS) to cells in the form of LPS–soluble (s)CD14 complexes induces strong cellular responses. During this process, LPS is delivered from sCD14 to the plasma membrane, and the cell-associated LPS is then rapidly transported to an intracellular site. This transport appears to be important for certain cellular responses to LPS, as drugs that block transport also inhibit signaling and cells from LPS-hyporesponsive C3H/HeJ mice fail to exhibit this transport. To identify the intracellular destination of fluorescently labeled LPS after its delivery from sCD14 into cells, we have made simultaneous observations of different organelles using fluorescent vital dyes or probes. Endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus were labeled using Texas red (TR)–dextran, LysoTracker™ Red DND-99, DiOC6(3), and boron dipyrromethane (BODIPY)–ceramide, respectively. After 30 min, LPS did not colocalize with endosomes, lysosomes, or endoplasmic reticulum in polymorphonuclear leukocytes, although some LPS-positive vesicles overlapped with the endosomal marker, fluorescent dextran. On the other hand, LPS did appear to colocalize with two markers of the Golgi apparatus, BODIPY–ceramide and TRITC (tetramethylrhodamine isothiocyanate)–labeled cholera toxin B subunit. We further confirmed the localization of LPS in the Golgi apparatus using an epithelial cell line, HeLa, which responds to LPS–sCD14 complexes in a CD14-dependent fashion: BODIPY–LPS was internalized and colocalized with fluorescently labeled Golgi apparatus probes in live HeLa cells. Morphological disruption of the Golgi apparatus in brefeldin A–treated HeLa cells caused intracellular redistribution of fluorescent LPS. These results are consistent with the Golgi apparatus being the primary delivery site of monomeric LPS. lipopolysaccharide endosomes Golgi apparatus retrograde transport Footnotes 1used in this paper: BODIPY, boron dipyrromethane; CTB, cholera toxin B subunit; DF, defatted; ER, endoplasmic reticulum; HSA, human serum albumin; LBP, LPS binding protein; Lpsd, LPS hyporesponsive; s, soluble; SCAP, SREBP (sterol regulatory element–binding proteins) cleavage–activating protein; Tf, transferrin; TGN, trans-Golgi network; TLR, Toll-like receptor; TR, Texas red; TRITC, tetramethylrhodamine isothiocyanate Submitted: 12 February 1999 Revision requested 11 June 1999 Accepted: 22 June 1999

Song, Xiao-yu; Zeng, Li; Jin, Wenwen; Thompson, John; Mizel, Diane E.; Lei, Ke-jian; Billinghurst, R.C.; Poole, A. Robin; Wahl, Sharon M.

doi: 10.1084/jem.190.4.535pmid: 10449524

Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor α and nuclear factor κB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases. inflammation cartilage resorption serine protease inhibitor Footnotes 1used in this paper: AI, severity of arthritis; NF, nuclear factor; PM, peritoneal macrophage; SCW, streptococcal cell wall; SLPI, secretory leukocyte protease inhibitor Submitted: 19 May 1999 Revision requested 24 June 1999 Accepted: 25 June 1999