Cytotoxic Granule Exocytosis Research Articles

Prevention of Hemophagocytosis by TNF-α and IL-6 Blockade in TLR9- and Nod1- Ligand Induced Hemophagocytic Syndrome.

Abstract 1351Poster Board I-373 IntroductionHemophagocytic syndrome (HPS, also referred as hemophagocytic lymphohistiocytosis; HLH) is a life-threatening condition caused by hyperinflammatory response. It can be classified into familial and acquired forms. Familial HPS is a disorder inherited in an autosomal recessive or X-linked manner. Several loci and genes have been implicated in the familial HPS, including perforin, or cytotoxic granule exocytosis genes. While, acquired (secondary) HPS is a sporadic syndrome occurring in association with systemic infection (virus, bacteria, protozoa, and fungi), underlying malignancy, or immunodeficient disorder. The central pathophysiologic abnormality in HPS is cytokine dysfunction, resulting in uncontrolled accumulation of activated T-lymphocytes and activated histiocytes (macrophages) in many organs. Several cytokines including interferon-γ, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10, IL-12, and soluble IL-2 receptor (sCD25), found at extremely high levels in the plasma of HPS patients. Treatment of hemophagocytic lymphohistiocytosis is based on epipodophyllotoxin derivatives, etoposide, corticosteroids, cyclosporin A, antithymocyte globulin (ATG), and, in selected patients, intrathecal methotrexate. Recently, anti-TNF-α therapy has been reported to prove patients with refractory HPS, however, there is not enough evidence. In this study, we established murine HPS model by intravenous injection of CpG oligodeoxynucleotide (ODN)-1668, a ligand for Toll-like receptor 9 (TLR9) and combination of CpG and iE-DAP, which is a ligand for Nod1, and investigated the role of TNF-α and other cytokine in HPS model. MethodsC57BL/6J mice were injected intravenously with 200 μg of CpG or combination of CpG and 100 μg of iE-DAP. The treated mice were assayed for temperature, spleen weight, and complete blood count. The serum cytokines levels (TNF-α and IL-6) and triglycerides were determined by ELISA and auto-analyzer, resectively. Hemophagocytosis was determined by flow cytometric (FCM) analysis in the peripheral blood and spleen. The spleen was subjected into immunohistochemistry staining and histological examination.For TNF-α and IL-6 blockade, the mice were treated with TNF receptor (TNFR)-Fc (etanercept) or combination of TNFR-Fc and anti-mouse IL-6 receptor (IL-6R) monoclonal antibody (MR16-1) by intravenous injection. ResultsCpG injected mouse displayed the typical HPS clinical criteria, such as, fever, cytopenia, splenomegaly, hemophagocytosis in the peripheral blood and spleen. Furthermore, elevations of inflammatory cytokines (TNF-α and IL-6) and triglyceride were detected in the serum of these mice. These clinical symptoms were not observed in CpG-injected TLR2−/−4−/−9−/− mice and LPS-injected wild type mice. Interestingly, combination challenge of CpG and iE-DAP led to synergistic induction of hemophagocytosis and enhanced production of TNF-α and IL-6 in the serum, however, single iE-DAP challenge did not induce HPS. FCM analysis revealed that monocyte-derived dendritic cells, which were CD11b, Ly6c, and F4/80, were mobilized and engulfed TER119+ erythrocyte in the peripheral blood and spleen after these challenge. Treatment of mice with TNFR-Fc partially prevented, and combination of TNFR-Fc and anti IL-6R mAb markedly suppressed hemophagocytosis in the peripheral blood and spleen.Conclusions: Based on these results, CpG or CpG + iE-DAP-induced mouse HPS model is valuable to study for cellular and molecular mechanism of HPS development, and potential system to apply preclinical research. Furthermore, prevention of both TNF-α and IL-6 might represent a novel therapy for HPS. DisclosuresNo relevant conflicts of interest to declare.

Munc18-2 deficiency causes familial hemophagocytic lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK cells

Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous autosomal recessive immune disorder characterized by the occurrence of uncontrolled activation of lymphocytes and macrophages infiltrating multiple organs. Disease-causing mutations in the perforin (PRF1; also known as FHL2), Munc13-4 (UNC13D; also known as FHL3), and syntaxin-11 (STX11; also known as FHL4) genes have been identified in individuals with FHL. These genes all encode proteins involved in the cytotoxic activity of lymphocytes. Here, we show that the gene encoding syntaxin-binding protein 2 (Munc18-2; official gene symbol STXBP2) is mutated in another subset of patients with FHL (designated by us as "FHL5"). Lymphoblasts isolated from these patients had strongly decreased STXBP2 protein expression, and NK cells exhibited impaired cytotoxic granule exocytosis, a defect that could be overcome by ectopic expression of wild-type STXBP2. Furthermore, we provide evidence that syntaxin-11 is the main partner of STXBP2 in lymphocytes, as its expression required the presence of STXBP2. Our work shows that STXBP2 deficiency causes FHL5. These data indicate that STXBP2 is required at a late step of the secretory pathway for the release of cytotoxic granules by binding syntaxin 11, another component of the intracellular membrane fusion machinery.

Hemophagocytic Lymphohistiocytosis: When the Immune System Runs Amok

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome in which an exaggerated but ineffective immune response leads to severe hyperinflammation. Key players in HLH are activated lymphocytes and histiocytes which infiltrate all organs and secrete large amounts of cytokines. Cardinal symptoms are prolonged fever, hepatosplenomegaly, cytopenias, and hemophagocytosis. Biochemical markers include elevated ferritin, triglycerides, and low fibrinogen. HLH occurs on the basis of various inherited and acquired immune defects. Impaired function of natural killer cells and cytotoxic T cells is shared by all forms of HLH. Nearly all genetic defects identified in inherited cases of HLH are either mutations in the perforin gene or in genes important for the exocytosis of cytotoxic granules. Cytotoxic granules contain perforin and granzymes which induce apoptosis upon entering the target cell. Additionally perforin is important for the down-regulation of the immune response. Acquired forms of HLH are found in association with infectious agents, in patients with autoimmune diseases, in malignant diseases, and in patients receiving immune suppression or after organ transplantation. - HLH is still difficult to diagnose and may be overlooked since initially it may masquerade as a normal infection. HLH should be considered when symptoms are more pronounced than usual and in case of progression. Suppression of the severe hyperinflammation can be achieved with immunosuppressive and immunomodulatory agents and cytostatic drugs. Patients with genetic HLH have to undergo stem cell transplantation for cure.

Rapid up-regulation and granule-independent transport of perforin to the immunological synapse define a novel mechanism of antigen-specific CD8+ T cell cytotoxic activity.

CTL are endowed with the ability to eliminate pathogens through perforin-mediated cytotoxic activity. The mechanism for perforin-mediated Ag-specific killing has been solely attributed to cytotoxic granule exocytosis from activated CD8(+) T cells. In this study, we redefine this mechanism, demonstrating that virus-specific CD8(+) T cells rapidly up-regulate perforin in response to stimulation temporally with IFN-gamma and CD107a expression. Following Ag-specific activation, newly synthesized perforin rapidly appears at the immunological synapse, both in association with and independent of cytotoxic granules, where it functions to promote cytotoxicity. Our work suggests a novel mechanism of CTL cytotoxicity and identifies a novel correlate of CD8(+) T cell-mediated immunity.

The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation

The granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties.

A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion

AbstractCytotoxic T lymphocytes (CTLs) and natural killer cells help control infections and tumors via a killing activity that is mediated by the release of cytotoxic granules. Granule secretion at the synapse formed between the CTL and the target cell leads to apoptosis of the latter. This process involves polarization of the CTL's secretory machinery and cytotoxic granules. The small GTPase Rab27a and the hMunc13-4 protein have been shown to be required for both granule maturation and granule docking and priming at the immunologic synapse. Using a tandem affinity purification technique, we identified a previously unknown hematopoietic form of Slp2a (Slp2a-hem) and determined that it is a specific effector of the active form of Rab27a. This interaction occurs in vivo in primary CTLs. We have shown that (1) Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs and (2) following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse, where it binds to the plasma membrane through its C2 domains. The overexpression of a dominant-negative form of Slp2a-hem markedly impaired exocytosis of cytotoxic granules—indicating that Slp2a is required for cytotoxic granule docking at the immunologic synapse.

Regulatory T Lymphocytes: Pivotal Components of the Host Antitumor Response

The role of host immunity in the development and progression of cancer has been a subject of speculation and considerable controversy for more than the last 50 years. Tumor-infiltrating lymphocytes (TILs), the primary immune component infiltrating solid tumors, are considered to be a manifestation of the host antitumor reaction. The report by Gao et al in this issue provides additional evidence for the prognostic significance of TILs in solid tumors in general and hepatocellular carcinoma (HCC) specifically. Historically, tumor-associated lymphoid infiltrates were categorized descriptively as brisk, nonbrisk, or absent, based on morphologic observation alone. The survival advantage of a brisk lymphocytic infiltrate in malignant melanoma has been demonstrated by Clark et al, and confirmed by others. However, there is accumulating evidence that the specific type of immune cells, rather than their sheer quantity, governs the host-versus-tumor immune response. With development of immunohistochemical and flow cytometry techniques, it has been demonstrated that the majority of TILs in solid tumors are of the CD3 T-cell phenotype. CD3 T cells can be stratified further into CD4 helper cells (including the Th1 and Th2 subtypes based on their cytokine profile), CD4 regulatory T cells (Tregs), previously designated as suppressor cells, and CD8 cytotoxic effector cells. Initial studies that set out to characterize the antitumor immune response were directed toward CD8 cytotoxic T lymphocytes (CTLs). CTLs destroy tumor cells via the triggering of apoptosis by means of cytotoxic granule exocytosis and/or the Fas/FasL receptor–mediated pathway. In the mid-1990s, antibodies that recognize cytotoxic molecules, including TIA-1, granzyme B, and perforin, became available for immunohistochemical studies. The number of CD8 CTLs expressing TIA-1 and/or granzyme B cytotoxic granules infiltrating tumors has been shown to be associated with improved prognosis in certain human neoplasms, such as Epstein-Barr virus–associated gastric cancer and carcinomas of the colon, breast, uterus, and esophagus. The role of CD4 T cells in the host antitumor response is an area of considerable debate. This subset may be considered as a double-edged immunologic sword, able to promote or inhibit tumor growth. CD4 T-helper cells are subdivided into Th1 cells, which induce cellular immunity, and Th2 cells, which elicit a humoral immune response. The Th1 response is associated with CTL activation and is considered to be beneficial for antitumor immunity. In contrast, a predominant Th2-like cytokine profile is associated with a metastasis-inclined microenvironment in HCC and other tumors. A subpopulation of CD4 T cells, Tregs, which accounts for 5% to 10% of all CD4 cells, is generating intense interest in tumor immunology, autoimmunity, and infectious disease. Historically, Tregs were first hypothesized as suppressive T cells in early 1971 by Gershon and Kondo, however, subsequent studies were hindered because of a lack of specific molecular markers and difficulties in their isolation and culture. The renaissance of these regulatory cells came a few years ago, when FOXP3, a member of the forkhead family of DNA transcription factors, was cloned. There is now considerable evidence that FOXP3 is a key control molecule for Treg development and function, and is an excellent marker for the study of Tregs. Recent studies have demonstrated that Tregs are beneficial for the prevention of autoimmune disease, such as type I diabetes, graft-versus-host disease, transplant rejection, and tissue destruction during infection. In contrast, Tregs seem to be a detrimental factor in the generation of host-versus-tumor immunity via suppression of tumorspecific effector T-cell responses and development of immune tolerance to neoplastic cells. The classic Treg is a thymus-derived CD4 CD25 FOXP3 T lymphocyte. In addition to these markers, Tregs express cell surface CTL-associated antigen 4 (CTLA-4), and the glucocorticoidinduced tumor necrosis factor alpha receptor and secrete immunosuppressive cytokines such as transforming growth factor beta and interleukin 10 (IL-10). Initially it was demonstrated that the number of CD4 CD25 T-cells were increased in several solid tumors and in certain hematologic malignancies. However, subsequent studies provided evidence that CD25, which is an IL-2 receptor subunit, is induced on activation in all T-cells, not just Tregs. Recently, there has been an sudden increase of immunohistochemical studies using FOXP3 as a more specific marker of Tregs. Increased numbers of FOXP3 Tregs infiltrating tumor cell nests have been demonstrated in ovarian, lung, breast, pancreatic, hepatocellular, head and neck, and anal carcinomas, and lymphomas. In most of the solid tumors studied, accumulation of FOXP3 Tregs predicts a striking reduction of patient survival; however, paradoxically, increased FOXP3 Tregs were found to be associated with improved prognosis in lymphoma patients. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 25 NUMBER 18 JUNE 2

TGF-beta1-induced expression of alphaE(CD103)beta7 integrin on Ag-specific tumor-infiltrating T lymphocytes promotes killing of E-cadherin-bearing tumor cells by triggering polarized cytotoxic granule exocytosis (50.22)

Abstract Interaction of intercellular adhesion molecule (ICAM)-1 with lymphocyte function-associated antigen (LFA)-1 enhances target cell-T cell adhesion and thus promotes TCR-mediated killing. In the absence of ICAM-1, frequently lost during tumor cell dissemination, we demonstrate that the interaction of the epithelial cell marker E-cadherin with αE(CD103)/1 _7 integrin, often expressed by tumor-infiltrating T lymphocytes (TIL), plays a major role for an effective cancer cell lysis. Indeed, while tumor-specific CD103+ TIL are able to kill E-cadherin+/ICAM− autologous tumor cells, CD103− PBL-derived counterparts are inefficient. Furthermore, tumor cell killing by TIL is abrogated by anti-CD103 mAb and RNA interference targeting E-cadherin. Confocal microscopy analysis demonstrated that αEβ7 is recruited at the immunological synapse and that its interaction with E-cadherin is essential for cytolytic granule polarization and subsequent exocytosis. Interestingly, pre-incubation of PBL-derived T cells with anti-CD3 mAb and TGF-β1 induced CD103 expression resulting in a dramatic potentiation of anti-tumor effector function. Thus, CD8+/CD103+ tumor-reactive T cells infiltrating epithelial tumors most likely play a major role in anti-cancer CTL response.

Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition

Granzymes are key components of the immune response that play important roles in eliminating host cells infected by intracellular pathogens. Several granzymes are potent inducers of cell death. However, whether granzymes use additional mechanisms to exert their antipathogen activity remains elusive. Here, we show that in adenovirus-infected cells in which granzyme B (gzmB) and downstream apoptosis pathways are inhibited, granzyme H (gzmH), an orphan granzyme without known function, directly cleaves the adenovirus DNA-binding protein (DBP), a viral component absolutely required for viral DNA replication. We directly addressed the functional consequences of the cleavage of the DBP by gzmH through the generation of a virus that encodes a gzmH-resistant DBP. This virus demonstrated that gzmH directly induces an important decay in viral DNA replication. Interestingly, gzmH also cleaves the adenovirus 100K assembly protein, a major inhibitor of gzmB, and relieves gzmB inhibition. These results provide the first evidence that granzymes can mediate antiviral activity through direct cleavage of viral substrates, and further suggest that different granzymes have synergistic functions to outflank viral defenses that block host antiviral activities.

Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4

Cytotoxic T lymphocytes and natural killer cells exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse. Rab27a and hMunc13-4 are critical effectors of the exocytosis of cytotoxic granules. Here we show that the cytotoxic function of lymphocytes requires the cooperation of two types of organelles: the lysosomal cytotoxic granule and the endosomal 'exocytic vesicle'. Independently of Rab27a, hMunc13-4 mediated the assembly of Rab11(+) recycling and Rab27(+) late endosomal vesicles, constituting a pool of vesicles destined for regulated exocytosis. It also primed cytotoxic granule fusion, possibly through interaction with active Rab27a. Cytotoxic T lymphocyte-target cell recognition induced rapid polarization of both types of organelles, which coalesced near the cell-cell contact area. Our data provide insight into the regulation of the generation and release of cytotoxic granules by effector cytotoxic T lymphocytes and natural killer cells.

Hemophagocytic lymphohistiocytosis and related disorders

The rate of diagnosis of hemophagocytic lymphohistiocytosis, a genetically heterogeneous and, frequently, rapidly fatal autosomal recessive disorder of immune regulation, is increasing worldwide. Awareness has grown through the Histiocyte Society and the publication of newly-recognized genetic causes. I summarize current knowledge regarding the pathophysiology, diagnosis and treatment of hemophagocytic lymphohistiocytosis. Genetic defects leading to life-threatening hemophagocytic syndromes have recently been described. Two autosomal recessive gene defects underlie 40-50% of primary (familial) cases worldwide: perforin, the major immune cytotoxic protein, and MUNC 13-4, a protein involved in exocytosis of perforin-bearing cytotoxic granules during apoptosis. Related autosomal recessive defects of secretory cytotoxic lysosomes - LYST 1 (Chediak-Higashi syndrome), Rab27A (Griscelli syndrome), and X-linked lymphoproliferative disorder - also carry a very high risk of fatal hemophagocytic lymphohistiocytosis. Concurrently, treatment protocols involving multiagent immunomodulatory therapy followed by allogeneic hematopoeitic cell transplantation have been tested. With immunomodulatory treatment, 75% of children with hemophagocytic lymphohistiocytosis are symptomatically improved after 2 months of therapy. Disease-free survival after allogeneic hematopoeitic cell transplantation currently ranges from 50 to 70%. Bench and clinical research have advanced understanding of the pathophysiology of hemophagocytic lymphohistiocytosis and related disorders, and significantly improved clinical outcomes during the past decade.

Nitric oxide inhibits exocytosis of cytolytic granules from lymphokine-activated killer cells.

NO inhibits cytotoxic T lymphocyte killing of target cells, although the precise mechanism is unknown. We hypothesized that NO decreases exocytosis of cytotoxic granules from activated lymphocytes. We now show that NO inhibits lymphokine-activated killer cell killing of K562 target cells. Exogenous and endogenous NO decreases the release of granzyme B, granzyme A, and perforin: all contents of cytotoxic granules. NO inhibits the signal transduction cascade initiated by cross-linking of the T cell receptor that leads to granule exocytosis. In particular, we found that NO decreases the expression of Ras, a critical signaling component within the exocytic pathway. Ectopic expression of Ras prevents NO inhibition of exocytosis. Our data suggest that Ras mediates NO inhibition of lymphocyte cytotoxicity and emphasize that alterations in the cellular redox state may regulate the exocytic signaling pathway.

Phospholipase C-γ2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells

AbstractPhospholipase C-γ2 (PLC-γ2) is a key component of signal transduction in leukocytes. In natural killer (NK) cells, PLC-γ2 is pivotal for cellular cytotoxicity; however, it is not known which steps of the cytolytic machinery it regulates. We found that PLC-γ2-deficient NK cells formed conjugates with target cells and polarized the microtubule-organizing center, but failed to secrete cytotoxic granules, due to defective calcium mobilization. Consequently, cytotoxicity was completely abrogated in PLC-γ2-deficient cells, regardless of whether targets expressed NKG2D ligands, missed self major histocompatibility complex (MHC) class I, or whether NK cells were stimulated with IL-2 and antibodies specific for NKR-P1C, CD16, CD244, Ly49D, and Ly49H. Defective secretion was specific to cytotoxic granules because release of IFN-γ on stimulation with IL-12 was normal. Plcg2-/- mice could not reject MHC class I-deficient lymphoma cells nor could they control CMV infection, but they effectively contained Listeria monocytogenes infection. Our results suggest that exocytosis of cytotoxic granules, but not cellular polarization toward targets, depends on intracellular calcium rise during NK cell cytotoxicity. In vivo, PLC-γ2 regulates selective facets of innate immunity because it is essential for NK cell responses to malignant and virally infected cells but not to bacterial infections.

Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients

Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder characteristics of this syndrome. So far, 3 Rab27a missense mutations have been identified. They open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the alpha 4 (Ala152Pro) or beta 5 (Leu130Pro) loop, observed in 2 of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within an invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding and GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.

Cellular and molecular pathophysiology of cutaneous drug reactions.

Hypersensitivity reactions to drugs can cause a variety of skin diseases like maculopapular, bullous and pustular eruptions. In recent years increasing evidence indicates the important role of T cells in these drug-induced skin diseases. Analysis of such drug-specific T cell clones has revealed that drugs can be recognized by alpha beta-T cell receptors, not only if bound covalently to peptides, but also if the drug binds in a rather labile way to the presenting major histocompatibility complex (MHC)-peptide. This presentation is sufficient to stimulate T cells. In maculopapular exanthema (MPE), histopathological analysis typically shows a dominant T cell infiltration together with a vacuolar interface dermatitis. Immunohistochemical studies demonstrate the presence of cytotoxic CD4+ and to a lesser degree of CD8+ T cells, which contain perforin and granzyme B. They are close to keratinocytes that show signs of cell destruction. Expression of Fas ligand is barely detectable, suggesting that cytotoxic granule exocytosis may be the dominant pathway leading to keratinocyte cell damage. While in MPE, the killing of cells seems to be predominantly mediated by CD4+ T cells, patients with bullous skin disease show a strong CD8+ T cell migration to the epidermis. This is probably due to a preferential presentation of the drug by MHC class I molecules, and a more extensive killing of cells that present drugs on MHC class I molecules. This might lead to bullous skin diseases. In addition to the presence of cytotoxic T cells, drug-specific T cells also orchestrate the inflammatory skin reaction through the release and induction of various cytokines [i.e. interleukin (IL)-5, IL-6, tumor necrosis factor-alpha and interferon-gamma] and chemokines (RANTES, eotaxin or IL-8). The increased expression of these mediators seems to contribute to the generation of tissue and blood eosinophilia, a hallmark of many drug-induced allergic reactions. However, in acute generalized exanthematous pustulosis (a peculiar form of drug allergy), neutrophils represent the predominant cell type within pustules, probably due to their recruitment by IL-8 secreting drug specific T cells and keratinocytes.

Immunohistology of drug-induced exanthema: clues to pathogenesis.

Hypersensitivity reactions to drugs can cause a variety of different skin disorders, the most frequent being maculopapular eruptions. In recent years increasing evidence has indicated the important involvement of T cells in this drug reaction. Histopathological changes typically show a dominant T-cell infiltration together with vacuolar interface dermatitis. Immunohistochemical studies demonstrate the presence of cytotoxic CD4+ and CD8+ T cells, which contain perforin and granzyme B, in close proximity to keratinocytes showing signs of cell destruction. Expression of Fas ligand is barely detectable, which suggests that cytotoxic granule exocytosis may be the dominant pathway leading to keratinocyte cell damage. In addition, drug-specific T cells may orchestrate the inflammatory skin reaction through the release and induction of various cytokines (i.e. IL-5, IL-6, TNF-alpha, IFN-gamma) and chemokines (i.e. regulated on activation, normal T-cell expressed and secreted; eotaxin). These mediators contribute to the generation of eosinophilia, which may amplify the underlying immune response through the release of further proinflammatory mediators in drug-induced maculopapular exanthema.

Functional dichotomy in natural killer cell signaling: Vav1-dependent and -independent mechanisms.

The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen-receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1-/- mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1-/- mice produced normal amounts of interferon (IFN)-gamma in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1-/- NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

Human natural killer cells mediate killing of intracellular Mycobacterium tuberculosis H37Rv via granule-independent mechanisms.

Despite the continued importance of tuberculosis as a world-wide threat to public health, little is known about the mechanisms used by human lymphocytes to contain and kill the intracellular pathogen Mycobacterium tuberculosis. We previously described an in vitro model of infection of human monocytes (MN) with virulent M. tuberculosis strain H37Rv in which the ability of peripheral blood lymphocytes to limit intracellular growth of the organism could be measured. In the current study, we determined that lymphocyte-mediated killing of intracellular M. tuberculosis occurs within the first 24 h of coculture with infected MN. Natural killer (NK) cells isolated from both purified protein derivative (PPD)-positive and PPD-negative subjects were capable of mediating this early killing of intracellular H37Rv. NK cell-mediated killing of intracellular M. tuberculosis was not associated with the production of gamma interferon. Transferred supernatants of cocultured NK cells and M. tuberculosis-infected MN could not mediate the killing of intracellular M. tuberculosis, and Transwell studies indicated that direct cell-to-cell contact was required for NK cells to mediate the killing of the organism. Killing was not dependent upon exocytosis of NK cell cytotoxic granules. NK cells induced apoptosis of mycobacterium-infected MN, but neither killing of intracellular M. tuberculosis by NK cells nor NK cell-induced apoptosis of infected MN was inhibited by blocking the interaction of FasL and Fas. Thus, human NK cells may mediate killing of intracellular M. tuberculosis via alternative apoptotic pathways.

Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome.

Griscelli syndrome (GS, MIM 214450), a rare, autosomal recessive disorder, results in pigmentary dilution of the skin and the hair, the presence of large clumps of pigment in hair shafts and an accumulation of melanosomes in melanocytes. Most patients also develop an uncontrolled T-lymphocyte and macrophage activation syndrome (known as haemophagocytic syndrome, HS), leading to death in the absence of bone-marrow transplantation. In contrast, early in life some GS patients show a severe neurological impairment without apparent immune abnormalities. We previously mapped the GS locus to chromosome 15q21 and found a mutation in a gene (MYO5A) encoding a molecular motor in two patients. Further linkage analysis suggested a second gene associated with GS was in the same chromosomal region. Homozygosity mapping in additional families narrowed the candidate region to a 3.1-cM interval between D15S1003 and D15S962. We detected mutations in RAB27A, which lies within this interval, in 16 patients with GS. Unlike MYO5A, the GTP-binding protein RAB27A appears to be involved in the control of the immune system, as all patients with RAB27A mutations, but none with the MYO5A mutation, developed HS. In addition, RAB27A-deficient T cells exhibited reduced cytotoxicity and cytolytic granule exocytosis, whereas MYO5A-defective T cells did not. RAB27A appears to be a key effector of cytotoxic granule exocytosis, a pathway essential for immune homeostasis.

CHRONIC LIVER ALLOGRAFT REJECTION IS NOT ASSOCIATED WITH CASPASE ACTIVATION

975 Previous studies have demonstrated correlations between acute liver allograft rejection and apoptosis of hepatocytes and bile duct cells. Furthermore, Fas ligand, perforin and the granzymes are upregulated during acute allograft rejection suggesting both the cytotoxic granule-exocytosis and the Fas-mediated pathways may be involved in the induction of apoptotic cell death. In recent studies, we have determined that apoptosis during acute liver allograft rejection involves activation of the caspase cascade. Taken together, these studies suggest a prominent role for apoptosis in the tissue damage that occurs during acute liver allograft rejection. To date, however, there have been few studies which directly examine apoptosis during chronic liver allograft rejection. To determine if caspase-dependent apoptosis occurs during chronic liver allograft rejection, RNA was isolated from liver tissue of patients with a diagnosis of chronic liver allograft rejection (n=5) and from liver tissue classified as normal (n=4), and analyzed by a specific and quantitative ribonuclease protection assay (RPA). Our data suggest that there are similar levels of the caspases 1 (ICE), 2, 5, 6, 7 and 9 in both chronic rejection and normal liver tissue. Interestingly, there is a marked decrease in the expression of both caspase 8 (FLICE) and caspase 3 (CPP-32) during chronic liver allograft rejection. Fas trimerization recruits the death-domain containing protein FADD, which in turn recruits caspase 8 and thus activates the cascade of caspases leading to apoptosis. Decreased expression of caspase 8 in chronic rejection may indicate that the Fas-associated death-inducing signaling complex is not active in this tissue. Caspase 3 can be directly activated by granzyme B and has been shown to induce the proteolysis of death substrates. Both RT-PCR and RPA analyses indicate similar expression of granzyme B in chronic rejection and normal liver. TUNEL staining for apoptotic cells of sections from acute and chronic liver allograft rejection and normal liver demonstrated abundant numbers of apoptotic hepatocytes (3-7 per high power field (HPF)) during acute rejection with only minimal numbers of apoptotic hepatocytes detected during chronic rejection (0-1/HPF) and in normal liver (0-1/HPF). In contrast, TUNEL staining of the endothelium, in both large and small vessels, was specifically detected in the chronic rejection specimens. Therefore, while the caspase cascade is prominent in acute rejection, our data suggest that apoptosis of endothelium, in chronic rejection is independent of caspase expression.