The Journal of Experimental Medicine

Estess, Pila; Nandi, Animesh; Mohamadzadeh, Mansour; Siegelman, Mark H.

doi: 10.1084/jem.190.1.9pmid: 10429666

T cell recruitment to extralymphoid tissues is fundamental to the initiation and perpetuation of the inflammatory state during immune and autoimmune responses. Interleukin (IL)-15 is a proinflammatory cytokine whose described functions largely overlap with those of IL-2. The latter is attributable in large part to its binding of the heterotrimeric receptor that contains the β and γ chains of the IL-2R in combination with an unique IL-15Rα chain. However, unlike IL-2, IL-15 and its receptor have a wide tissue and cell type distribution, including endothelial cells. Here, we examine the effect of IL-15 on hyaluronan expression by endothelial cells, and investigate its role in vivo in promoting the extravasation of antigen-activated T cells through a CD44-dependent pathway. The expression of hyaluronan on primary endothelial cells and microvascular endothelial cell lines is induced by IL-15, whereas IL-2 has no such activity. Moreover, intraperitoneal administration of IL-15 or TNF-α in the absence of other exogenous proinflammatory stimuli allows the extravasation of superantigen-stimulated T cells into this site in vivo in a CD44-dependent manner. T cell recruitment induced by IL-15 requires expression of an intact IL-2Rβ chain, indicating that IL-15 operates in this context through the traditional IL-15R. The results suggest that IL-15 can regulate endothelial cell function and thereby enables a CD44-initiated adhesion pathway that facilitates entry of activated T lymphocytes into inflammatory sites. They further demonstrate a novel role for IL-15 (distinct from any of IL-2) in regulating microvascular endothelial cell adhesive function, help to understand the role of IL-15R expression on endothelium, and further support a central position for this cytokine in orchestrating multiple sequential aspects of T cell effector function and therefore chronic inflammatory processes. CD44 endothelial cell hyaluronate interleukin 15 lymphocyte adhesion Footnotes 1used in this paper: bPG, bovine proteoglycan; CFDA-SE, 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester; EC, endothelial cell; HA, hyaluronan; HUVEC, human umbilical vein endothelial cell; ICAM, intracellular adhesion molecule; MLN, mesenteric lymph node; RT-PCR, reverse transcription polymerase chain reaction; SA-PE, phycoerythrin-labeled Streptavidin; SEB, Staphylococcal enterotoxin B; VCAM, vascular cell adhesion molecule Submitted: 27 January 1999 Revision requested 26 April 1999 Accepted: 10 May 1999

Kim, Nayun; Bozek, Grazyna; Lo, James C.; Storb, Ursula

doi: 10.1084/jem.190.1.21pmid: 10429667

Somatic hypermutation of Ig genes is probably dependent on transcription of the target gene via a mutator factor associated with the RNA polymerase (Storb, U., E.L. Klotz, J. Hackett, Jr., K. Kage, G. Bozek, and T.E. Martin. 1998. J. Exp. Med. 188:689–698). It is also probable that some form of DNA repair is involved in the mutation process. It was shown that the nucleotide excision repair proteins were not required, nor were mismatch repair (MMR) proteins. However, certain changes in mutation patterns and frequency of point mutations were observed in Msh2 (MutS homologue) and Pms2 (MutL homologue) MMR-deficient mice (for review see Kim, N., and U. Storb. 1998. J. Exp. Med. 187:1729–1733). These data were obtained from endogenous immunoglobulin (Ig) genes and were presumably influenced by selection of B cells whose Ig genes had undergone certain mutations. In this study, we have analyzed somatic hypermutation in two MutL types of MMR deficiencies, Pms2 and Mlh1. The mutation target was a nonselectable Ig-κ gene with an artificial insert in the V region. We found that both Pms2- and Mlh1-deficient mice can somatically hypermutate the Ig test gene at approximately twofold reduced frequencies. Furthermore, highly mutated sequences are almost absent. Together with the finding of genome instability in the germinal center B cells, these observations support the conclusion, previously reached for Msh2 mice, that MMR-deficient B cells undergoing somatic hypermutation have a short life span. Pms2- and Mlh1-deficient mice also resemble Msh2-deficient mice with respect to preferential targeting of G and C nucleotides. Thus, it appears that the different MMR proteins do not have unique functions with respect to somatic hypermutation. Several intrinsic characteristics of somatic hypermutation remain unaltered in the MMR-deficient mice: a preference for targeting A over T, a strand bias, mutational hot spots, and hypermutability of the artificial insert are all seen in the unselectable Ig gene. This implies that the MMR proteins are not required for and most likely are not involved in the primary step of introducing the mutations. Instead, they are recruited to repair certain somatic point mutations, presumably soon after these are created. DNA mismatch repair immunoglobulin genes somatic hypermutation transgenic mice Footnotes 1used in this paper: GC, germinal center; MMR, mismatch repair; NER, nucleotide excision repair; SSR, simple sequence repeat Submitted: 29 January 1999 Revision requested 21 April 1999 Accepted: 6 May 1999

Wild, Martin K.; Cambiaggi, Anna; Brown, Marion H.; Davies, Elizabeth A.; Ohno, Hiroshi; Saito, Takashi; van der Merwe, P. Anton

doi: 10.1084/jem.190.1.31pmid: 10429668

The T cell antigen receptor (TCR) and its ligand peptide–major histocompatibility complex (MHC) are small (∼7 nm) compared with other abundant cell surface molecules such as integrins, CD43, and CD45 (23–50 nm). We have proposed that molecules at the T cell/antigen-presenting cell (APC) interface segregate according to size, with small “accessory” molecules (e.g., CD2, CD4, CD8, CD28, and CD154) contributing to the formation of a close-contact zone, within which the TCR engages peptide–MHC, and from which large molecules are excluded (Davis, S.J., and P.A. van der Merwe. 1996. Immunol . Today. 17:177–187). One prediction of this model is that increasing the size of these small accessory molecules will disrupt their function. Here, we test this prediction by varying the dimensions of the CD2 ligand, CD48, and examining how this affects T cell antigen recognition. Although the interaction of CD2 on T cells with wild-type or shortened forms of CD48 on APCs enhances T cell antigen recognition, the interaction of CD2 with elongated forms of CD48 is strongly inhibitory. Further experiments indicated that elongation of the CD2/CD48 complex inhibited TCR engagement of peptide–MHC, presumably by preventing the formation of sufficiently intimate contacts at the T cell/APC interface. These findings demonstrate the importance of small size in CD2/CD48 function, and support the hypothesis that T cell antigen recognition requires segregation of cell surface molecules according to size. T cell receptor CD2 CD48 T cell activation plasma membrane Footnotes 1used in this paper: CHO, Chinese hamster ovary; GPI, glycosyl phosphatidylinositol; ICAM, intercellular adhesion molecule; MCC, moth cytochrome c Submitted: 3 February 1999 Revision requested 5 May 1999 Accepted: 6 May 1999

Godin, Isabelle; Garcia-Porrero, Juan Antonio; Dieterlen-Lièvre, Françoise; Cumano, Ana

doi: 10.1084/jem.190.1.43pmid: 10429669

In the mouse embryo, the generation of candidate progenitors for long-lasting hemopoiesis has been reported in the paraaortic splanchnopleura (P-Sp)/aorta-gonad-mesonephros (AGM) region. Here, we address the following question: can the P-Sp/AGM environment support hemopoietic differentiation as well as generate stem cells, and, conversely, are other sites where hemopoietic differentiation occurs capable of generating stem cells? Although P-Sp/AGM generates de novo hemopoietic stem cells between 9.5 and 12.5 days post coitus (dpc), we show here that it does not support hemopoietic differentiation. Among mesoderm-derived sites, spleen and omentum were shown to be colonized by exogenous cells in the same fashion as the fetal liver. Cells colonizing the spleen were multipotent and pursued their evolution to committed progenitors in this organ. In contrast, the omentum, which was colonized by lymphoid-committed progenitors that did not expand, cannot be considered as a hemopoietic organ. From these data, stem cell generation appears incompatible with hemopoietic activity. At the peak of hemopoietic progenitor production in the P-Sp/AGM, between 10.5 and 11.5 dpc, multipotent cells were found at the exceptional frequency of 1 out of 12 total cells and 1 out of 4 AA4.1 + cells. Thus, progenitors within this region constitute a pool of undifferentiated hemopoietic cells readily accessible for characterization. aorta-gonad-mesonephros spleen omentum hemopoiesis reconstitution Footnotes 1used in this paper: AGM, aorta-gonad-mesonephros; BFU-E, burst-forming unit–erythroid; CFC, colony-forming cell; CFU-S, colony-forming unit–splenic; dpc, day(s) post coitus; Epo, erythropoietin; FTOC, fetal thymic organ culture; HSC, hemopoietic stem cell; KL, c-Kit ligand; LTR, long-term reconstitution; PeC, peritoneal cavity; P-Sp, paraaortic splanchnopleura; YS, yolk sac Submitted: 15 February 1999 Revision requested 15 April 1999 Accepted: 27 April 1999

Miller, Carla; Ragheb, Jack A.; Schwartz, Ronald H.

doi: 10.1084/jem.190.1.53pmid: 10429670

Recombinant-activating gene 2 (RAG-2 −/− ) T cell receptor–transgenic mice repeatedly injected with the superantigen staphylococcal enterotoxin A entered a tolerant state in which splenic CD4 + T cells produced little interleukin (IL)-2, interferon γ, or IL-4. This state resulted from a combination of both clonal anergy and cytokine-mediated immunosuppression. The anergy persisted for at least 3 wk and could be distinguished from the suppression by a decrease in IL-2 production per cell, a block in the activation of early response kinases, and a failure to be reversed with anti–transforming growth factor (TGF)-β. Full suppression lasted for only 1 wk and involved both IL-10 and TGF-β, but required additional unknown molecules for optimal effect. These experiments show that complex in vivo interactions of multiple peripheral tolerance mechanisms can now be dissected at both the cellular and molecular levels. CD4 + T cell receptor transgenic cells early response kinase activation interleukin 2 interleukin 10 transforming growth factor β Footnotes 1used in this paper: AP, alkaline phosphatase; CT, threshold cycle number; ERK, early response kinase; E/R, 50% EHAA plus 50% RPMI 1640; 6-FAM, 6-carboxyfluorescein; MAP, mitogen-activated protein; PCC, pigeon cytochrome c; RAG-2−/−, recombinant activating gene 2 knockout; RT, reverse transcriptase; SEA, staphylococcal enterotoxin A Submitted: 18 March 1999 Accepted: 11 May 1999

Kishimoto, Hidehiro; Sprent, Jonathan

doi: 10.1084/jem.190.1.65pmid: 10429671

Repeated attempts to show that costimulation for negative selection is controlled by a single cell surface molecule have been unsuccessful. Thus, negative selection may involve multiple cell surface molecules acting in consort. In support of this idea, we show here that at least three cell surface molecules, namely CD28, CD5, and CD43, contribute to Fas-independent negative selection of the tolerance-susceptible population of heat-stable antigen (HSA) hi CD4 + 8 − cells found in the medulla. The costimulatory function of these three molecules can be blocked by certain cytokines, IL-4 and IL-7, and coinjecting these cytokines with antigen in vivo abolishes negative selection; Fas-dependent negative selection, however, is maintained. The results suggest that efficient negative selection requires the combined functions of at least four cell surface molecules: CD28, CD5, CD43, and Fas. thymus tolerance costimulatory signals lymphokines medulla Footnotes 1used in this paper: HSA, heat-stable antigen; Sags, superantigens; SEB, staphylococcal enterotoxin; TUNEL, Tdt-mediated dUTP-biotin nick-end labeling Submitted: 25 March 1999 Revision requested 7 May 1999 Accepted: 10 May 1999

Loder, By Florienne; Mutschler, Bettina; Ray, Robert J.; Paige, Christopher J.; Sideras, Paschalis; Torres, Raul; Lamers, Marinus C.; Carsetti, Rita

doi: 10.1084/jem.190.1.75pmid: 10429672

Only mature B lymphocytes can enter the lymphoid follicles of spleen and lymph nodes and thus efficiently participate in the immune response. Mature, long-lived B lymphocytes derive from short-lived precursors generated in the bone marrow. We show that selection into the mature pool is an active process and takes place in the spleen. Two populations of splenic B cells were identified as precursors for mature B cells. Transitional B cells of type 1 (T1) are recent immigrants from the bone marrow. They develop into the transitional B cells of type 2 (T2), which are cycling and found exclusively in the primary follicles of the spleen. Mature B cells can be generated from T1 or T2 B cells. B cell development transitional B cells spleen CD45 Bruton's tyrosine kinase Footnotes 1used in this paper: BCR, B cell antigen receptor; BrdU, bromodeoxyuridine; Btk, Bruton's tyrosine kinase; HSA, heat-stable antigen; MAP, mitogen-activated protein; MZ, marginal zone; PALS, periarteriolar lymphoid sheath; PH, plekstrin homology; PKC, protein kinase C; T1, transitional type 1; T2, transitional type 2 Submitted: 16 November 1998 Revision requested 22 April 1999 Accepted: 5 May 1999

Choudhri, Tanvir F.; Hoh, Brian L.; Prestigiacomo, Charles J.; Huang, Judy; Kim, Louis J.; Schmidt, Ann Marie; Kisiel, Walter; Connolly, E. Sander; Pinsky, David J.

doi: 10.1084/jem.190.1.91pmid: 10429673

Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa–dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 μg/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111 In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary. Factor IX thrombosis anticoagulation intrinsic coagulation pathway cerebral ischemia Footnotes 1used in this paper: APTT, activated partial thromboplastin time; CBF, cerebral blood flow; Factor IXai, active site–blocked Factor IXa; ICH, intracerebral hemorrhage; MCAO, middle cerebral artery occlusion; MCCT, modified cephalin clotting time; tPA, tissue plasminogen activator; TTC, triphenyl tetrazolium chloride Submitted: 10 December 1998 Revision requested 26 March 1999 Accepted: 27 April 1999

Zhao, Jian; Ben, Li-Hong; Wu, Ya-Lan; Hu, Wei; Ling, Kun; Xin, Shun-Mei; Nie, Hui-Ling; Ma, Lan; Pei, Gang

doi: 10.1084/jem.190.1.101pmid: 10429674

Trichosanthin (TCS), an active protein component isolated from a traditional Chinese medicinal herb Trichosanthes kirilowii , has been shown to inhibit HIV infection and has been applied in clinical treatment of AIDS. The recent development that chemokines and chemokine receptors play important roles in HIV infection led us to investigate the possible functional interaction of TCS with chemokines and their receptors. This study demonstrated that TCS greatly enhanced both RANTES (regulated upon activation, normal T cell expressed and secreted)– and stromal cell–derived factor (SDF)-1α–stimulated chemotaxis (EC 50 ≅ 1 nM) in leukocytes (THP-1, Jurkat, and peripheral blood lymphocyte cells) and activation of pertussis toxin–sensitive G proteins (EC 50 ≅ 20 nM). TCS also significantly augmented chemokine-stimulated activation of chemokine receptors CCR5 and CXCR4 as well as CCR1, CCR2B, CCR3, and CCR4 transiently expressed in HEK293 cells. A mutant TCS with 4,000-fold lower ribosome-inactivating activity showed similar augmentation activity as wild-type TCS. Moreover, flow cytometry demonstrated that the specific association of TCS to the cell membranes required the presence of chemokine receptors, and laser confocal microscopy reveals that TCS was colocalized with chemokine receptors on the membranes. The results from TCS-Sepharose pull-down and TCS and chemokine receptor coimmunoprecipitation and cross-linking experiments demonstrated association of TCS with CCR5. Thus, our data clearly demonstrated that TCS synergizes activities of chemokines to stimulate chemotaxis and G protein activation, and the effects of TCS are likely to be mediated through its interaction with chemokine receptors. chemokine receptors trichosanthin G proteins chemotaxis HIV Footnotes 1used in this paper: DSS, disuccinimidyl suberate; FBS, fetal bovine serum; GPCR, G protein–coupled receptor; HA, hemagglutinin; HEK, human embryonic kidney; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; RANTES, regulated upon activation, normal T cell expressed and secreted; RIP, ribosome-inactivating protein; SDF, stromal cell–derived factor; TCS, trichosanthin Submitted: 13 January 1999 Revision requested 21 April 1999 Accepted: 6 May 1999