The Journal of Experimental Medicine

Gasque, Philippe; Jones, Jane; Singhrao, Sim K.; Morgan, B.

doi: 10.1084/jem.187.4.451pmid: 9463395

The brain is an immunoprivileged organ isolated from the peripheral immune system. However, it has been shown that resident cells, notably astrocytes and microglia, can express numerous innate immune molecules, providing the capacity to generate a local antipathogen system. Perforin is a cytolytic protein present in the granules of cytotoxic T lymphocytes and natural killer cells. Expression in cells other than those of the hemopoetic lineage has not been described. We report here that fetal astrocytes in culture (passages 2 to 15), astrocytoma, and adult astrocytes expressed perforin. Reverse transcriptase polymerase chain reaction followed by Southern blot was carried out using multiple specific primers and all cDNAs were cloned and sequenced. Human fetal astrocyte perforin cDNA sequence was ∼100% identical to the reported perforin cDNA cloned from T cells. Western blot analysis using monoclonal and polyclonal antiperforin peptide antibodies revealed a protein of 65 kD in both human fetal astrocyte and rat natural killer cell lysates ( n = 4). Immunostaining followed by FACS ® and confocal and electron microscopy analysis revealed that perforin was expressed by 40–50% of glial fibrillary acidic protein positive cells present in the fetal brain culture ( n = 11). Perforin was not localized to granules in astrocytes but was present throughout the cytoplasm, probably in association with the endoplasmic reticulum. Perforin was not detected in normal adult brain tissue but was present in and around areas of inflammation (white and grey matter) in multiple sclerosis and neurodegenerative brains. Perforin-positive cells were identified as reactive astrocytes. These findings demonstrate that perforin expression is not unique to lymphoid cells and suggest that perforin produced by a subpopulation of astrocytes plays a role in inflammation in the brain. Footnotes The authors thank Dr. Gillian M. Griffiths for the generous gift of the monoclonal and polyclonal antiperforin antibodies and the human NK cell line YT and Jean Hopkins for her technical help. We also thank Dr. Jim W. Neal for normal and HD brains and helpful discussions. This work was supported by grants from the Medical Research Council (P. Gasque), the Wellcome Trust (B.P. Morgan), the Arthritis and Rheumatism Council (J. Jones), and the Welsh Scheme for the Development of Health and Social Research (S.K. Singhrao). Address correspondence to Dr. Philippe Gasque, University of Wales College of Medicine, Department of Medical Biochemistry, Brain Inflammation and Immunity Group (BIIG), Tenovus Building, Heath Park, Cardiff, CF4 4XX, UK. Phone: 44-1-222-744236; Fax: 44-1-222-744305; E-mail: [email protected] 1 Abbreviations used in this paper: AD, Alzheimer's disease; BCIP, 5-bromo-4-chloro-3indolyl phosphate; C4bp, C4B binding protein; CRP, C-reactive protein; DAB, diaminobenzidine; FH, complement factor H; GC, galactocerebroside; GFAP, glial fibrillary acidic protein; HD, Huntington's disease; MS, multiple sclerosis; NBT, nitroblue tetrazolium; NSE, neuron-specific enolase; PD, Pick's disease; RT, reverse transcriptase. Submitted: 20 June 1997 Revision received 1 December 1997

Alonzi, Tonino; Fattori, Elena; Lazzaro, Domenico; Costa, Patrizia; Probert, Lesley; Kollias, George; De Benedetti, Fabrizio; Poli, Valeria; Ciliberto, Gennaro

doi: 10.1084/jem.187.4.461pmid: 9463396

Interleukin-6 (IL-6) is overproduced in the joints of patients with rheumatoid arthritis (RA) and, based on its multiple stimulatory effects on cells of the immune system and on vascular endothelia, osteoclasts, and synovial fibroblasts, is believed to participate in the development and clinical manifestations of this disease. In this study we have analysed the effect of ablating cytokine production in two mouse models of arthritis: collagen-induced arthritis (CIA) in DBA/1J mice and the inflammatory polyarthritis of tumor necrosis factor α (TNF-α) transgenic mice. IL-6 was ablated by intercrossing an IL-6 null mutation into both arthritis-susceptible genetic backgrounds and disease development was monitored by measuring clinical, histological, and biochemical parameters. Two opposite responses were observed; while arthritis in TNF-α transgenic mice was not affected by inactivation of the IL-6 gene, DBA/1J, IL-6 −/− mice were completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints. These results are discussed in the light of the present knowledge of cytokine networks in chronic inflammatory disorders and suggest that IL-6 receptor antagonists might be beneficial for the treatment of RA. Footnotes We wish to thank Mr. M. Aquilina and Mrs. S. Germoni (Istituto Ricerche di Biologia Molecolare; IRBM) for animal care, H. Bazin for the generous gift of LO-DNP-57 antibody, and Drs. A. Nicosia, M. Sollazzo, and C. Toniatti, and Mrs. J. Clench (IRBM) for critically reading the manuscript. Address correspondence to Gennaro Ciliberto, IRBM P. Angeletti, Via Pontina Km 30,600, 00040 Pomezia, Rome, Italy. Phone: 39-6-91093205; Fax: 39-6-91093654; E-mail: [email protected] The present address of T. Alonzi and V. Poli is Department of Biochemistry, Wellcome Trust Building, University of Dundee, Dundee DD1 4HN, UK. 1 Abbreviations used in this paper: CII, type II collagen; CIA, collagen-induced arthritis; DIL, DBA/1J, IL-6; DIL-6, DBA/1J mice crossed with IL-6–deficient mice; gp, glycoprotein; RA, rheumatoid arthritis; sIL-6Rα, soluble IL-6 receptor subunit α; TIL, TNF, IL-6; TIL-6, TNF-α transgenic mice crossed with IL-6–deficient mice. Submitted: 15 September 1997 Revision received 26 November 1997

Tkachuk, Maria; Bolliger, Stephan; Ryffel, Bernhard; Pluschke, Gerd; Banks, Theresa A.; Herren, Suzanne; Gisler, Roland H.; Kosco-Vilbois, Marie H.

doi: 10.1084/jem.187.4.469pmid: 9463397

During immune responses the initial activation of B cells takes place in T cell zones of periarteriolar lymphoid sheaths (PALS) of the splenic white pulp. After initial activation, B cells migrate into the primary follicles and, in association with follicular dendritic cells (FDCs), undergo clonal expansion and differentiation giving rise to germinal centers (GCs). Peanut agglutinin binding (PNA + ) cells of the GC differentiate further into memory or plasma cells. Here we report that in tumor necrosis factor receptor 1–deficient mice (TNFR1 −/− ), the location of B cells was altered and that plasma cells were abnormally distributed in the splenic PALS. In contrast to lymphotoxin α–deficient mice (LTα −/− ), bone marrow or fetal liver transplantation did not correct the abnormal organization of the spleen, location of B cells, the lack of an FDC network, nor the antibody response in TNFR1 −/− mice. These results argue for a crucial role of TNFR1 expression on nonhematopoietic cells for the maintenance of the splenic architecture and proper B cell location. In addition, the lack in development of an FDC network after adoptive transfer suggests that either FDCs are not of bone marrow origin or that they depend on signals from nonhematopoietic cells for maturation. Footnotes We are grateful to L. Dudler, B. Kugelberg, N. Favre, Drs. W. Hein, and G. Bordmann for excellent technical support. We thank Drs. A. Rolink and S. Takeda for generously providing antibodies, and C. Hebert for the production of the photomicrographs. Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd. (Basel, Switzerland). Address correspondence to Marie H. Kosco-Vilbois, Geneva Biomedical Research Institute, 14 chemin des Aulx, Plan-les-Ouates, CH-1228, Switzerland. Phone: 041-022-706-9719, 706-9708; Fax: 041-022-794-6965; E-mail: [email protected] ; and Roland H. Gisler, Basel Institute for Immunology, Grenzacherstrasse 487, Basel, CH-4005, Switzerland. Phone: 041-061-605-1242; Fax: 041-061-605-1364; E-mail: [email protected] 1 Abbreviations used in this paper: BM, bone marrow; FDC, follicular dendritic cell; FL, fetal liver; GC, germinal center; LT-α, lymphotoxin α–deficient; PALS, periarteriolar lymphoid sheaths; PNA, peanut agglutinin; RT, room temperature; s, surface; SRBC, sheep red blood cell; WT, wild type. Submitted: 19 September 1997 Revision received 5 December 1997

Holmes, Margaret A.; Buss, Timothy N.; Foote, Jefferson

doi: 10.1084/jem.187.4.479pmid: 9463398

The crystal structure of the complex between hen egg lysozyme and the Fv fragment of a humanized antilysozyme antibody was determined to 2.7-Å resolution. The structure of the antigen combining site in the complex is nearly identical to that of the complexed form of the parent mouse antibody, D1.3. In contrast, the combining sites of the unliganded mouse and humanized antilysozymes show moderate conformational differences. This disparity suggests that a conformational readjustment process linked to antigen binding reverses adverse conformations in the complementarity determining regions that had been introduced by engineering these segments next to human framework regions in the humanized antibody. Footnotes We thank Steve Sheriff, Thomas B. Lavoie, and Greg Winter for suggestions on the manuscript. We gratefully acknowledge financial support from the Arnold and Mabel Beckman Foundation and the Department of the Army Breast Cancer Research Program (grant DAMD 17-97-1-7124). Address correspondence to Jefferson Foote, Division of Molecular Medicine, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, C3-168, PO Box 19024, Seattle, WA 98109-1024. Phone: 1-206-667-6720; Fax: 1-206-667-6524; E-mail: [email protected] 1 Abbreviations used in this paper: Cα, alpha carbon; CDR, complementarity determining region; Fv, dimer of immunoglobulin heavy and light chain variable domains; rms, root mean square. Submitted: 7 October 1997 Revision received 2 December 1997

Fan, Tao; Lu, Hang; Hu, He; Shi, Lianfa; McClarty, Grant A.; Nance, Dwight M.; Greenberg, Arnold H.; Zhong, Guangming

doi: 10.1084/jem.187.4.487pmid: 9463399

We report that chlamydiae, which are obligate intracellular bacterial pathogens, possess a novel antiapoptotic mechanism. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli including the kinase inhibitor staurosporine, the DNA-damaging agent etoposide, and several immunological apoptosis-inducing molecules such as tumor necrosis factor-α, Fas antibody, and granzyme B/perforin. The antiapoptotic activity was dependent on chlamydial but not host protein synthesis. These observations suggest that chlamydia may encode factors that interrupt many different host cell apoptotic pathways. We found that activation of the downstream caspase 3 and cleavage of poly (ADP-ribose) polymerase were inhibited in chlamydia-infected cells. Mitochondrial cytochrome c release into the cytosol induced by proapoptotic factors was also prevented by chlamydial infection. These observations suggest that chlamydial proteins may interrupt diverse apoptotic pathways by blocking mitochondrial cytochrome c release, a central step proposed to convert the upstream private pathways into an effector apoptotic pathway for amplification of downstream caspases. Thus, we have identified a chlamydial antiapoptosis mechanism(s) that will help define chlamydial pathogenesis and may also provide information about the central mechanisms regulating host cell apoptosis. Footnotes We thank Drs. Ronald N. Germain and Robert C. Brunham for helpful discussions and reading the manuscript. This work was supported by the Medical Research Council (MRC) of Canada. G. Zhong is the recipient of an MRC scholarship. Address correspondence to Guangming Zhong, Department of Medical Microbiology, University of Manitoba, 508-730 William Ave., Winnipeg, Manitoba, Canada R3E OW3. Phone: 204-789-3835; Fax: 204-789-3926; E-mail: [email protected] 1 Abbreviations used in this paper: EB, chlamydial infectious elementary body; ECL, enhanced chemiluminescence; MOI, multiplicity of infection; PARP, poly(ADP-ribose) polymerase; PT, permeability transition; RB, chlamydial noninfectious reticulate body. Submitted: 13 November 1997 Revision received 11 December 1997

Sibelius, Ulf; Hattar, Katja; Schenkel, Angelika; Noll, Thomas; Csernok, Elena; Gross, Wolfgang Ludwig; Mayet, Werner-Johannes; Piper, Hans-Michael; Seeger, Werner; Grimminger, Friedrich

doi: 10.1084/jem.187.4.497pmid: 9463400

Anti–neutrophil cytoplasmic antibodies (ANCAs) targeting proteinase 3 (PR3) have a high specifity for Wegener's granulomatosis (WG), and their role in activating leukocytes is well appreciated. In this study, we investigated the influence of PR3-ANCA and murine monoclonal antibodies on human umbilical vascular endothelial cells (HUVECs). Priming of HUVECs with tumor necrosis factor α induced endothelial upregulation of PR3 message and surface expression of this antigen, as measured by Cyto-ELISA, with a maximum occurrence after 2 h. Primed cells responded to low concentrations of both antibodies (25 ng–2.5 μg/ml), but not to control immunoglobulins, with pronounced, dose-dependent phosphoinositide hydrolysis, as assessed by accumulation of inositol phosphates. The signaling response peaked after 20 min, in parallel with the appearance of marked prostacyclin and platelet-activating factor synthesis. The F(ab) 2 fragment of ANCA was equally potent as ANCA itself. Disrupture of the endothelial F-actin content by botulinum C2 toxin to avoid antigen–antibody internalization did not affect the response. In addition to the metabolic events, anti-PR3 challenge, in the absence of plasma components, provoked delayed, dose-dependent increase in transendothelial protein leakage. We conclude that anti-PR3 antibodies are potent inductors of the preformed phosphoinositide hydrolysis–related signal tranduction pathway in human endothelial cells. Associated metabolic events and the loss of endothelial barrier properties suggest that anti-PR3–induced activation of endothelial cells may contribute to the pathogenetic sequelae of autoimmune vasculitis characterizing WG. Footnotes Address correspondence to Dr. F. Grimminger, Department of Internal Medicine, Justus-Liebig-University Giessen, D-35392 Giessen, FRG. Phone: 49-641-99-42500; Fax: 49-641-99-42509; E-mail: friedrich. [email protected] 1 Abbreviations used in this paper: ANCA, anti–neutrophil cytoplasmic antibodies; c-ANCA, cytoplasmic ANCA; HUVEC, human umbilical cord endothelial cell; IP, inositol phosphate; IPx, IP1, IP2, and IP3; MoAb-PR3, mAb to proteinase 3; PAF, platelet-activating factor; PR3, proteinase 3; WG, Wegener's granulomatosis. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 547). Submitted: 3 July 1997 Revision received 1 December 1997

Amigorena, Sebastian; Lankar, Danielle; Briken, Volker; Gapin, Laurent; Viguier, Mireille; Bonnerot, Christian

doi: 10.1084/jem.187.4.505pmid: 9463401

T cell receptors on CD4 + lymphocytes recognize antigen-derived peptides presented by major histocompatibility complex (MHC) class II molecules. A very limited set of peptides among those that may potentially bind MHC class II is actually presented to T lymphocytes. We here examine the role of two receptors mediating antigen internalization by antigen presenting cells, type IIb2 and type III receptors for IgG (FcγRIIb2 and FcγRIII, respectively), in the selection of peptides for presentation to T lymphocytes. B lymphoma cells expressing recombinant FcγRIIb2 or FcγRIII were used to assess the presentation of several epitopes from two different antigens. 4 out of the 11 epitopes tested were efficiently presented after antigen internalization through FcγRIIb2 and FcγRIII. In contrast, the 7 other epitopes were efficiently presented only when antigens were internalized through FcγRIII, but not through FcγRIIb2. The capacity to present these latter epitopes was transferred to a tail-less FcγRIIb2 by addition of the FcγRIII-associated γ chain cytoplasmic tail. Mutation of a single leucine residue at position 35 of the γ chain cytoplasmic tail resulted in the selective loss of presentation of these epitopes. Therefore, the nature of the receptor that mediates internalization determines the selection of epitopes presented to T lymphocytes within single protein antigens. Footnotes We are grateful to Drs. Ming-Zong Lai and Eli E. Sercarz for providing us with several anti-CI λ repressor and anti-HEL T cell hybridomas, to P. Benaroch and G. Langsley for critically reading the manuscript and to all members of the CJF-INSERM 95-01 for useful discussions. This work was supported by grants from the INSERM, Institut Curie, the Association de Recherche contre le Cancer (ARC), and the Ligue Nationale Contre le Cancer. L. Gapin was supported by ARC and Pasteur-Weizman fellowships and V. Briken by an E.C. fellowship. Address correspondence to Sebastian Amigorena, INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005, Paris, France. Phone: 33-01-42-34-63-89; Fax: 33-01-42-34-63-82; E-mail, [email protected] 1 Abbreviations used in this paper: Ii, invariant chain; ITAM, immunoreceptor tyrosine kinase activation motif; HEL, hen egg lysozome; HRP, horseradish, peroxidase; PTK, protein tyrosine kinase. Submitted: 15 September 1997 Revision received 2 December 1997

Kirtikara, Kanyawim; Morham, Scott G.; Raghow, Rajendra; Laulederkind, Stanley J. F.; Kanekura, Takuro; Goorha, Sarita; Ballou, Leslie R.

doi: 10.1084/jem.187.4.517pmid: 9463402

Prostaglandin E 2 (PGE 2 ) production in immortalized, nontransformed cells derived from wild-type, cyclooxygenase 1–deficient (COX-1 −/− ) or cyclooxygenase 2–deficient (COX-2 −/− ) mice was examined after treatment with interleukin (IL)-1β, tumor necrosis factor α, acidic fibroblast growth factor, and phorbol ester (phorbol myristate acetate). Compared with their wild-type counterparts, COX-1 −/− or COX-2 −/− cells exhibited substantially enhanced expression of the remaining functional COX gene. Furthermore, both basal and IL-1–induced expression of cytosolic phospholipase A 2 (cPLA 2 ), a key enzyme-regulating substrate mobilization for PGE 2 biosynthesis, was also more pronounced in both COX-1 −/− and COX-2 −/− cells. Thus, COX-1 −/− and COX-2 −/− cells have the ability to coordinate the upregulation of the alternate COX isozyme as well as cPLA 2 genes to overcome defects in prostaglandin biosynthetic machinery. The potential for cells to alter and thereby compensate for defects in the expression of specific genes such as COX has significant clinical implications given the central role of COX in a variety of disease processes and the widespread use of COX inhibitors as therapeutic agents. Footnotes This work was supported by research funds from the Department of Veterans Affairs (DVA), The Arthritis Foundation, and grants AR39166 and AR26034 from the National Institutes of Health (National Institute of Arthritis and Musculoskeletal and Skin Diseases). R. Raghow is a Career Scientist of the DVA. Address correspondence to Leslie R. Ballou, Department of Veterans Affairs Medical Center, Research Service (151), 1030 Jefferson Avenue, Memphis, TN 38104. Phone: 901-577-7283. Fax: 901-577-7273; E-mail: [email protected] 1 Abbreviations used in this paper: AA, arachidonic acid; COX, cyclooxygenase; cPLA, cytosolic phospholipase; FGF, fibroblast growth factor; PG, prostaglandin; RIA, radioimmunoassay. Submitted: 17 September 1997 Revision received 18 November 1997

Zeng, Defu; Dick, Michael; Cheng, Lirong; Amano, Masahiko; Dejbakhsh-Jones, Sussan; Huie, Philip; Sibley, Richard; Strober, Samuel

doi: 10.1084/jem.187.4.525pmid: 9463403

T cells with T cell receptor (TCR) transgenes that recognized CD1 on syngeneic B cells stimulated B cells to secrete immunoglobulins in vitro. The CD4 + , CD8 + , or CD4 − CD8 − T cells from the spleen of the TCR transgenic BALB/c donors induced lupus with anti–double stranded DNA antibodies, proteinuria, and immune complex glomerulonephritis in irradiated BALB/c nude mice reconstituted with nude bone marrow. Injection of purified CD4 − CD8 − T cells from the marrow of transgenic donors prevented the induction of lupus by the transgenic T cells. Transgenic T cells that induced lupus secreted large amounts of interferon (IFN)-γ and little interleukin (IL)-4, and those that prevented lupus secreted large amounts of IL-4 and little IFN-γ or IL-10. Footnotes This work was supported by a grant from The American Lupus Society, a research grant from the Arthritis Foundation, and a National Institutes of Health grant AI-40093. D. Zeng was supported in part by a fellowship from Activated Cell Therapy, Inc. We thank Drs. G. Rolink and B. Kotzin for advice concerning measurement of serum immunoglobulins, A. Mukhopadhyay for technical assistance, and V. Cleaver for preparation of the manuscript. Address correspondence to Samuel Strober, Division of Immunology and Rheumatology, Rm. S105B, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5111. Phone: 650-723-6500; Fax: 650-725-6104; E-mail: [email protected] 1 Abbreviations used in this paper: BCL, B cell lymphoma; BM, bone marrow; cGy, centiGray; DN, double-negative; ds, double-stranded; SP, single-positive. Submitted: 10 September 1997 Revision received 24 November 1997

Segal, Benjamin M.; Dwyer, Bonnie K.; Shevach, Ethan M.

doi: 10.1084/jem.187.4.537pmid: 9463404

Cells of the innate immune system secrete cytokines early in immune responses that guide maturing T helper (Th) cells along appropriate lineages. This study investigates the role of cytokine networks, bridging the innate and acquired immune systems, in the pathogenesis of an organ specific autoimmune disease. Experimental allergic encephalomyelitis (EAE), a demyelinating disease of the central nervous system, is widely used as an animal model for multiple sclerosis. We demonstrate that interleukin (IL)-12 is essential for the generation of the autoreactive Th1 cells that induce EAE, both in the presence and absence of interferon γ. The disease-promoting effects of IL-12 are antagonized by IL-10 produced by an antigen nonspecific CD4 + T cell which, in turn, is regulated by the endogenous production of IL-12. This unique immunoregulatory circuit appears to play a critical role in controlling Th cell differentiation and provides a mechanism by which microbial triggers of the innate immune system can modulate autoimmune disease. Footnotes The authors would like to thank D. Presky and M.K. Gately and J. Magram for generously providing anti– IL-12 antiserum and the IL-12 −/− mice, respectively, and B. Kelsall (Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases) for critical reading of the manuscript. B.K. Dwyer is a Research Scholar of the Howard Hughes Medical Institute. Address correspondence to Dr. Ethan M. Shevach, Laboratory of Immunology, NIAID, NIH, Bldg. 10, Rm. 11N311, 10 Center Dr. MSC 1892, Bethesda, MD 20892-1892. Phone: 301-496-6449; Fax: 301-496-0222; E-mail: [email protected] 1 Abbreviations used in this paper: CNS, central nervous system; EAE, experimental allergic encephalomyelitis; iNOS, inducible nitric oxide synthase; LTα, lymphotoxin-α; MBP, myelin basic protein. Submitted: 23 September 1997 Revision received 3 December 1997