The Journal of Experimental Medicine

Poltoratsky, Vladimir; Goodman, Myron F.; Scharff, Matthew D.

doi: 10.1084/jem.192.10.F27pmid: 11085756

Heibein, Jeffrey A.; Goping, Ing Swie; Barry, Michele; Pinkoski, Michael J.; Shore, Gordon C.; Green, Douglas R.; Bleackley, R. Chris

doi: 10.1084/jem.192.10.1391pmid: 11085742

Cytotoxic T lymphocytes (CTLs) destroy target cells through a mechanism involving the exocytosis of cytolytic granule components including granzyme B (grB) and perforin, which have been shown to induce apoptosis through caspase activation. However, grB has also been linked with caspase-independent disruption of mitochondrial function. We show here that cytochrome c release requires the direct proteolytic cleavage of Bid by grB to generate a 14-kD grB-truncated product (gtBid) that translocates to mitochondria. In turn, gtBid recruits Bax to mitochondria through a caspase-independent mechanism where it becomes integrated into the membrane and induces cytochrome c release. Our results provide evidence for a new pathway by which CTLs inflict damage and explain the caspase-independent mechanism of mitochondrial dysfunction. cytotoxic T lymphocyte granzyme B Bcl-2 Bid Bax Footnotes Abbreviations used in this paper: Ad, adenovirus; DISC, death-inducing signaling complex; grB, granzyme B; HRP, horseradish peroxidase; t, truncated. Submitted: 11 August 2000 Revision requested 18 September 2000 Accepted: 23 September 2000

Sutton, Vivien R.; Davis, Joanne E.; Cancilla, Michael; Johnstone, Ricky W.; Ruefli, Astrid A.; Sedelies, Karin; Browne, Kylie A.; Trapani, Joseph A.

doi: 10.1084/jem.192.10.1403pmid: 11085743

The essential upstream steps in granzyme B–mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B–resistant Bcl-2–overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B–treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B. granzyme B Bid apoptosis Bcl-2 perforin Footnotes Abbreviations used in this paper: Apaf-1, apoptotic protease-activating factor 1; IAP, inhibitor of apoptosis proteins; PLO, pneumococcal pneumolysin; t, truncated; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling. Submitted: 17 July 2000 Revision requested 14 September 2000 Accepted: 18 September 2000

Persson, Kristin; Mörgelin, Matthias; Lindbom, Lennart; Alm, Per; Björck, Lars; Herwald, Heiko

doi: 10.1084/jem.192.10.1415pmid: 11085744

Vascular damage induced by trauma, inflammation, or infection results in an alteration of the endothelium from a nonactivated to a procoagulant, vasoconstrictive, and proinflammatory state, and can lead to life-threatening complications. Here we report that activation of the contact system by Salmonella leads to massive infiltration of red blood cells and fibrin deposition in the lungs of infected rats. These pulmonary lesions were prevented when the infected animals were treated with H-D-Pro-Phe-Arg-chloromethylketone, an inhibitor of coagulation factor XII and plasma kallikrein, suggesting that inhibition of contact system activation could be used therapeutically in severe infectious disease. bradykinin factor XII high molecular weight kininogen plasma kallikrein protease inhibitor Footnotes K. Persson and M. Mörgelin contributed equally to this work. Abbreviations used in this paper: aPTT, activated partial thromboplastin time; F, factor; HK, H-kininogen; PK, plasma kallikrein; PT, prothrombin. Submitted: 4 April 2000 Revision requested 14 September 2000 Accepted: 19 September 2000

Gretz, J. Elizabeth; Norbury, Christopher C.; Anderson, Arthur O.; Proudfoot, Amanda E.I.; Shaw, Stephen

doi: 10.1084/jem.192.10.1425pmid: 11085745

Lymph-borne, soluble factors (e.g., chemokines and others) influence lymphocyte recirculation and endothelial phenotype at high endothelial venules (HEVs) in lymph node cortex. Yet the route lymph-borne soluble molecules travel from the subcapsular sinus to the HEVs is unclear. Therefore, we injected subcutaneously into mice and rats a wide variety of fluorophore-labeled, soluble molecules and examined their distribution in the draining lymph nodes. Rather than percolating throughout the draining lymph node, all molecules, including microbial lipopolysaccharide, were very visible in the subcapsular and medullary sinuses but were largely excluded from the cortical lymphocyte microenvironments. Exclusion prevailed even during the acute lymph node enlargement accompanying viral infection. However, low molecular mass (MW) molecules, including chemokines, did gain entry into the cortex, but in a very defined manner. Low MW, fluorophore-labeled molecules highlighted the subcapsular sinus, the reticular fibers, and the abluminal and luminal surfaces of the associated HEVs. These low MW molecules were in the fibers of the reticular network, a meshwork of collagen fibers ensheathed by fibroblastic reticular cells that connects the subcapsular sinus floor and the HEVs by intertwining with their basement membranes. Thus, low MW, lymph-borne molecules, including chemokines, traveled rapidly from the subcapsular sinus to the HEVs using the reticular network as a conduit. reticular network mouse rat lymphocyte recirculation antigen Footnotes Abbreviations used in this paper: GAG, glycosaminoglycan; HEL, hen egg lysozyme; HEV, high endothelial venule; HRP, horseradish peroxidase; LCA, lens culinaris agglutinin; MIP, macrophage inflammatory protein; MR, hydrodynamic radius; PSA, pisum sativum agglutinin; RANTES, regulated upon activation, normal T cell expressed and secreted; SLC, secondary lymphoid tissue chemoattractant; TEM, transmission electron microscopy; WGA, wheat germ agglutinin. Portions of this work have appeared in abstract form (1997. FASEB J. 11:687). Submitted: 31 May 2000 Revision requested 6 September 2000 Accepted: 23 September 2000

Biedermann, Tilo; Kneilling, Manfred; Mailhammer, Reinhard; Maier, Konrad; Sander, Christian A.; Kollias, George; Kunkel, Steven L.; Hültner, Lothar; Röcken, Martin

doi: 10.1084/jem.192.10.1441pmid: 11085746

Polymorphonuclear leukocytes (PMNs) characterize the pathology of T cell–mediated autoimmune diseases and delayed-type hypersensitivity reactions (DTHRs) in the skin, joints, and gut, but are absent in T cell–mediated autoimmune diseases of the brain or pancreas. All of these reactions are mediated by interferon γ–producing type 1 T cells and produce a similar pattern of cytokines. Thus, the cells and mediators responsible for the PMN recruitment into skin, joints, or gut during DTHRs remain unknown. Analyzing hapten-induced DTHRs of the skin, we found that mast cells determine the T cell–dependent PMN recruitment through two mediators, tumor necrosis factor (TNF) and the CXC chemokine macrophage inflammatory protein 2 (MIP-2), the functional analogue of human interleukin 8. Extractable MIP-2 protein was abundant during DTHRs in and around mast cells of wild-type (WT) mice but absent in mast cell–deficient WBB6F 1 - Kit W / Kit W- v ( Kit W / Kit W -v ) mice. T cell–dependent PMN recruitment was reduced >60% by anti–MIP-2 antibodies and >80% in mast cell–deficient Kit W / Kit W -v mice. Mast cells from WT mice efficiently restored DTHRs and MIP-2–dependent PMN recruitment in Kit W / Kit W - v mice, whereas mast cells from TNF −/ − mice did not. Thus, mast cell–derived TNF and MIP-2 ultimately determine the pattern of infiltrating cells during T cell–mediated DTHRs. chemokines inflammation type 1 T cells cytokines autoimmune disease Footnotes Abbreviations used in this paper: BMMC, bone marrow–derived mast cell; CHSR, contact hypersensitivity reaction; DTHR, delayed-type hypersensitivity reaction; EAE, experimental allergic encephalomyelitis; IP, IFN-γ inducible protein; KC, cytokine-induced neutrophil chemoattractant; MIP, macrophage inflammatory protein; MPO, myeloperoxidase; TNCB, trinitrochlorobenzene; WT, wild-type. Submitted: 10 May 2000 Revision requested 14 August 2000 Accepted: 25 September 2000

Batten, Marcel; Groom, Joanna; Cachero, Teresa G.; Qian, Fang; Schneider, Pascal; Tschopp, Jurg; Browning, Jeffrey L.; Mackay, Fabienne

doi: 10.1084/jem.192.10.1453pmid: 11085747

B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance. B cell maturation autoimmunity transitional B lymphocyte spleen antigen receptor Footnotes Abbreviations used in this paper: ANOVA, analysis of variance; BAFF, B cell activation factor from the TNF family; BCMA, B cell maturation antigen; BCR, B cell receptor; BM, bone marrow; FSC, forward light scatter; HSA, heat stable antigen; MLN, mesenteric LN; MZ, marginal zone; PI, propidium iodine; PLN, peripheral LN; SSC, side light scatter; T1, transitional immature B cell type 1; T2, transitional immature B cell type 2; TACI, transmembrane activator and calcium-modulating and cyclophilin ligand interactor; Tg, transgenic. Submitted: 4 August 2000 Revision requested 7 September 2000 Accepted: 6 October 2000

Kim, Dongku; Mebius, Reina E.; MacMicking, John D.; Jung, Steffen; Cupedo, Tom; Castellanos, Yaneth; Rho, Jaerang; Wong, Brian R.; Josien, Regis; Kim, Nacksung; Rennert, Paul D.; Choi, Yongwon

doi: 10.1084/jem.192.10.1467pmid: 11085748

Proper lymph node (LN) development requires tumor necrosis factor–related activation-induced cytokine (TRANCE) expression. Here we demonstrate that the defective LN development in TRANCE −/ − mice correlates with a significant reduction in lymphotoxin (LT)αβ + α 4 β 7 + CD45 + CD4 + CD3 − cells and their failure to form clusters in rudimentary mesenteric LNs. Transgenic TRANCE overexpression in TRANCE −/ − mice results in selective restoration of this cell population into clusters, and results in full LN development. Transgenic TRANCE-mediated restoration of LN development requires LTαβ expression on CD45 + CD4 + CD3 − cells, as LNs could not be induced in LTα −/ − mice. LTα −/ − mice also showed defects in the fate of CD45 + CD4 + CD3 − cells similar to TRANCE −/ − mice. Thus, we propose that both TRANCE and LTαβ regulate the colonization and cluster formation by CD45 + CD4 + CD3 − cells in developing LNs, the degree of which appears to correlate with the state of LN organogenesis. TRANCE lymphotoxin tumor necrosis factor lymph node organogenesis Footnotes Abbreviations used in this paper: CLN, cervical LN; GC, germinal center; HPRT, 5′-hypoxanthine ribosyltransferase; LT, lymphotoxin; MAdCAM, mucosal addressin cell adhesion molecule; MLN, mesenteric LN; PLN, peripheral LN; PP, Peyer's patch; rMLN; rudiments of MLN; TRANCE, TNF-related activation-induced cytokine; WT, wild-type. D. Kim and R.E. Mebius contributed equally to this work. Submitted: 1 August 2000 Revision requested 26 September 2000 Accepted: 6 October 2000

Giannola, Diane M.; Shlomchik, Warren D.; Jegathesan, Mithila; Liebowitz, David; Abrams, Charles S.; Kadesch, Tom; Dancis, Andrew; Emerson, Stephen G.

doi: 10.1084/jem.192.10.1479pmid: 11085749

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5′ promoter. Culture of highly purified human CD34 + bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3–10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20–50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element HXRE-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34 + cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation. homeobox genes self-renewal stem cells USF transcription Footnotes Abbreviations used in this paper: DMSO, dimethyl sulfoxide; D/N, dominant negative; EGFP, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay; HOX, homeobox; HLH, helix loop helix; HUCS, human umbilical cord serum; HXRE, HOX response element; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MITF, microphthalmia transcription factor; PI3K, phosphatidylinositol 3-kinase; RACE, rapid amplification of cDNA ends; SCF, stem cell factor; TPO, thrombopoietin; USF, upstream stimulatory factor. Submitted: 21 June 2000 Revision requested 7 September 2000 Accepted: 15 September 2000

Kawamura, Tatsuyoshi; Cohen, Sandra S.; Borris, Debra L.; Aquilino, Elisabeth A.; Glushakova, Svetlana; Margolis, Leonid B.; Orenstein, Jan M.; Offord, Robin E.; Neurath, A. Robert; Blauvelt, Andrew

doi: 10.1084/jem.192.10.1491pmid: 11085750

Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1 Ba-L infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08–4.77%). HIV-1–infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1–1 μg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1 Ba-L (an R5 HIV-1 strain) more efficiently infected LC–T cell cocultures when compared with HIV-1 IIIB (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1. AIDS dendritic cells cellulose acetate phthalate aminooxypentane-RANTES transmission Footnotes Abbreviations used in this paper: AOP, aminooxypentane; CAP, cellulose acetate phthalate; LCs, Langerhans cells; PHA, phytohemagglutinin; RANTES, regulated upon activation, normal T cell expressed and secreted. T. Kawamura and S.S. Cohen contributed equally to this work. Submitted: 21 July 2000 Revision requested 28 September 2000 Accepted: 6 October 2000