Cell death, clearance and immunity in the skeletal muscle.

The skeletal muscle is an immunologically unique tissue. Leukocytes, virtually absent in physiological conditions, are quickly recruited into the tissue upon injury and persist during regeneration. Apoptosis, necrosis and autophagy coexist in the injured/regenerating muscles, including those of patients with neuromuscular disorders, such as inflammatory myopathies, dystrophies, metabolic and mitochondrial myopathies and drug-induced myopathies. Macrophages are able to alter their function in response to microenvironment conditions and as a consequence coordinate changes within the tissue from the early injury throughout regeneration and eventual healing, and regulate the activation and the function of stem cells. Early after injury, classically activated macrophages ('M1') dominate the picture. Alternatively activated M2 macrophages predominate during resolution phases and regulate the termination of the inflammatory responses. The dynamic M1/M2 transition is increasingly felt to be the key to the homeostasis of the muscle. Recognition and clearance of debris originating from damaged myofibers and from dying stem/progenitor cells, stromal cells and leukocytes are fundamental actions of macrophages. Clearance of apoptotic cells and M1/M2 transition are causally connected and represent limiting steps for muscle healing. The accumulation of apoptotic cells, which reflects their defective clearance, has been demonstrated in various tissues to prompt autoimmunity against intracellular autoantigens. In the muscle, in the presence of type I interferon, apoptotic myoblasts indeed cause the production of autoantibodies, lymphocyte infiltration and continuous cycles of muscle injury and regeneration, mimicking human inflammatory myopathies. The clearance of apoptotic cells thus modulates the homeostatic response of the skeletal muscle to injury. Conversely, defects in the process may have deleterious local effects, guiding maladaptive tissue remodeling with collagen and fat accumulation and promoting autoimmunity itself. There is strong promise for novel treatments based on new knowledge of cell death, clearance and immunity in the muscle.

Vessel-associated myogenic precursors control macrophage activation and clearance of apoptotic cells.

Swift and regulated clearance of apoptotic cells prevents the accumulation of cell remnants in injured tissues and contributes to the shift of macrophages towards alternatively activated reparatory cells that sustain wound healing. Environmental signals, most of which are unknown, in turn control the efficiency of the clearance of apoptotic cells and as such determine whether tissues eventually heal. In this study we show that vessel-associated stem cells (mesoangioblasts) specifically modulate the expression of genes involved in the clearance of apoptotic cells and in macrophage alternative activation, including those of scavenger receptors and of molecules that bridge dying cells and phagocytes. Mesoangioblasts, but not immortalized myoblasts or neural precursor cells, enhance CD163 membrane expression in vitro as assessed by flow cytometry, indicating that the effect is specific. Mesoangioblasts transplanted in acutely or chronically injured skeletal muscles determine the expansion of the population of CD163(+) infiltrating macrophages and increase the extent of CD163 expression. Conversely, macrophages challenged with mesoangioblasts engulf significantly better apoptotic cells in vitro. Collectively, the data reveal a feed-forward loop between macrophages and vessel-associated stem cells, which has implications for the skeletal muscle homeostatic response to sterile injury and for diseases in which homeostasis is jeopardized, including muscle dystrophies and inflammatory myopathies.

Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps.

BACKGROUND: Increasing evidence implicates both platelets and neutrophils in the formation, stabilization, and growth of peripheral and coronary thrombi. Neutrophil extracellular traps (NETs) play a key role. The early events in the deregulated cross-talk between platelets and neutrophils are poorly characterized. OBJECTIVES: To identify at the molecular level the mechanism through which platelets induce the generation of NETs in sterile conditions. PATIENTS/METHODS: The presence of NETs was determined in 26 thrombi from patients with acute myocardial infarction by immunohistochemistry and immunofluorescence and markers of NETs assessed in the plasma. In vitro NET generation was studied in static and in physiological flow conditions. RESULTS: Coronary thrombi mainly consist of activated platelets, neutrophils, and NETs in close proximity of platelets. Activated platelets commit neutrophils to NET generation. The event abates in the presence of competitive antagonists of the high mobility group box 1 (HMGB1) protein. Hmgb1(-/-) platelets fail to elicit NETs, whereas the HMGB1 alone commits neutrophils to NET generation. Integrity of the HMGB1 receptor, Receptor for Advanced Glycation End products (RAGE), is required for NET formation, as assessed using pharmacologic and genetic tools. Exposure to HMGB1 prevents depletion of mitochondrial potential, induces autophagosome formation, and prolongs neutrophil survival. These metabolic effects are caused by the activation of autophagy. Blockade of the autophagic flux reverts platelet HMGB1-elicited NET generation. CONCLUSIONS: Activated platelets present HMGB1 to neutrophils and commit them to autophagy and NET generation. This chain of events may be responsible for some types of thromboinflammatory lesions and indicates novel paths for molecular intervention.

Cardiometabolic and immune factors associated with increased common carotid artery intima-media thickness and cardiovascular disease in patients with systemic lupus erythematosus.

BACKGROUND AND AIM: Patients with systemic lupus erythematosus (SLE) have a higher prevalence of subclinical atherosclerosis and higher risk of cardiovascular (CV) events compared to the general population. The relative contribution of CV-, immune- and disease-related risk factors to accelerated atherogenesis in SLE is unclear. METHODS AND RESULTS: Fifty SLE patients with long-lasting disease (mean age 44 ± 10 years, 86% female) and 50 sex- and age-matched control subjects were studied. Common carotid artery intima-media thickness (CCA-IMT) was used as a surrogate marker of atherosclerosis. We evaluated traditional and immune- and disease-related factors, assessed multiple T-cell subsets by 10-parameter-eight-colour polychromatic flow cytometry and addressed the effect of pharmacological therapies on CCA-IMT. In SLE patients, among several cardiometabolic risk factors, only high-density lipoprotein levels (HDL) and their adenosine triphosphate-binding cassette transporter 1 (ABCA-1)-dependent cholesterol efflux capacity were markedly reduced (p < 0.01), whereas the CCA-IMT was significantly increased (p = 0.03) compared to controls. CCA-IMT correlated with systolic blood pressure, low-density lipoprotein (LDL) cholesterol and body mass index (BMI), but not with disease activity and duration. The activated CD4(+)HLA-DR(+) and CCR5(+) T-cell subsets were expanded in SLE patients. Patients under hydroxychloroquine (HCQ) therapy showed lower CCA-IMT (0.62 ± 0.08 vs. 0.68 ± 0.10 mm; p = 0.03) and better risk-factor profile and presented reduced circulating pro-atherogenic effector memory T-cell subsets and a parallel increased percentage of naïve T-cell subsets. CONCLUSION: HDL represents the main metabolic parameter altered in SLE patients. The increased CCA-IMT in SLE patients may represent the net result of a process in which 'classic' CV risk factors give a continuous contribution, together with immunological factors (CD4(+)HLA-DR(+) T cells) which, on the contrary, could contribute through flares of activity of various degrees over time. Patients under HCQ therapy present a modified metabolic profile, a reduced T-cell activation associated with decreased subclinical atherosclerosis.

Accumulation of plasma nucleosomes upon treatment with anti-tumour necrosis factor-alpha antibodies.

OBJECTIVE: Patients undergoing anti-tumour necrosis factor-alpha (TNF-alpha) treatment often develop autoantibodies. Apoptotic cell antigens are a potential initiating stimulus for autoantibodies. Our goal was to verify whether anti-cytokine therapy causes the release of nucleosomes, a major autoantigen generated during cell death. DESIGN: Laboratory research study with comparison group. SETTING: Clinical Immunology Unit and Lab, H San Raffaele University Hospital, Italy. SUBJECTS: Eleven healthy controls and 87 rheumatic patients were studied, including 51 with rheumatoid arthritis (RA) and 33 patients with systemic lupus erythematosus (SLE). INTERVENTIONS: Vein blood samples were taken via the antecubital vein. Blood was retrieved from 11 patients before and after injection of anti-TNF-alpha humanized antibodies. Nucleosomes were measured with an enzyme-linked immunosorbent assay. Cell death induced by anti-TNF-alpha antibodies and by the soluble cytokine was assessed in vitro. MAIN OUTCOME MEASURES: Nucleosome level by treatment. RESULTS: Enzyme-linked immunosorbent assay effectively detected nucleosomes either released by dying cells in vitro or circulating in the plasma. SLE but not RA patients had circulating nucleosomes at the steady state. Eight of 11 patients had significantly higher levels of plasma nucleosomes after infliximab. Minute amounts of TNF-alpha enabled infliximab to induce cell death in vitro. CONCLUSIONS: The accumulation of nucleosomes possibly fosters the development of autoantibodies in subjects with appropriate genetic backgrounds.

The disposal of dying cells in living tissues.

Cells continuously die and disappear from the midst of living tissues. However, some of their constituents survive. DNA is horizontally transferred to phagocytic cells, and apoptotic cell antigens shape the immune repertoire. When massive apoptosis occurs, which overwhelms tissue scavenger cells, or when the function of phagocytes abates, dying cells escape clearance in vivo. Remnant dying cells come to phagocytes disguised: factors capable to envelop their membranes pervade the entire organism, or are generated in given tissues. Some are constitutively present, while other are generated during early or late phases of the inflammatory response, possibly to face the further burden of the dead inflammatory cells. This camouflage influences the disposal of the corpses: decoying molecules either bridge the corpse to the phagocyte or hide it. Furthermore, factors associated to the plasma membrane of the apoptotic cell shape the signals the phagocyte releases in situ. Finally, molecules contained or released by the dying cell alter the apprehension by the phagocyte of its prey, influencing its immunogenicity.

Inhibition of caspases maintains the antineoplastic function of gammadelta T cells repeatedly challenged with lymphoma cells.

T lymphocytes recognizing tumor antigens eventually undergo anergy or Fas-mediated death. V gamma9/V delta2+ T cells recognize poorly characterized ligand moieties on human B-cell lymphomas. Here we show that gammadelta T cells, a model for the study of activation-induced apoptosis, activate on repeated in vitro antigen-recognition caspase 3 and 8 and dramatically down-regulate their cytotoxic and secretory function. Caspase hindrance enhanced gammadelta T cell survival and sustained the killing of neoplastic cells and the release of IFN-gamma and tumor necrosis factor alpha. Caspases of tumor-specific T cells represent a candidate target to complement adoptive immunotherapy strategies.

Cytokine secretion associated with the clearance of apoptotic bodies in renal cell carcinoma patients.

The factors determining the outcome of immunotherapy in metastatic renal cell carcinoma (RCC) patients remain elusive. Macrophages from normal donors that phagocytose apoptotic cells secrete the immunosuppressive cytokine IL-10 in vitro. Conversely, IL-10 genetic deletion enhances the immunogenicity of apoptotic tumor cells in vivo. Elevated pre-treatment levels of IL-10 are associated with an unfavorable outcome of RCC. We examined whether the ability to release IL-10 by macrophages from RCC patients that phagocytosed apoptotic cells correlated with the outcome of immunotherapy. To this aim, we derived macrophages from 30 patients with metastatic RCC and from 21 healthy subjects (11 sex- and age-matched healthy controls and 10 younger donors). Patients either had a clinical response after immunotherapy, with a median survival after treatment of more than 18 months (n = 16), or were beginning immunotherapy after diagnosis of metastatic disease (n = 14). Macrophages from responding patients challenged with apoptotic cells released significantly less IL-10 than controls (p = 0.0075) and recently diagnosed patients (p = 0.0198), as ascertained by a 2-sided Student's t-test. This was not because macrophages from responding patients lost the ability to secrete IL-10, because antibody opsonization of apoptotic cells rescued IL-10 secretion. In contrast, macrophages from all groups of donors released similar amounts of TNF-alpha. The failure in IL-10 secretion by engulfing macrophages of responding subjects may exalt the immunogenicity of dying tumor cells, contributing to the success of immunotherapy.

Anti-beta2 glycoprotein I antibodies cause inflammation and recruit dendritic cells in platelet clearance.

Scavenger phagocytes are mostly responsible for the in vivo clearance of activated or senescent platelets. In contrast to other particulate substrates, the phagocytosis of platelets does not incite proinflammatory responses in vivo. This study assessed the contribution of macrophages and dendritic cells (DCs) to the clearance of activated platelets. Furthermore, we verified whether antibodies against the beta2 Glycoprotein I (beta2GPI), which bind to activated platelets, influence the phenomenon. DCs did not per se intemalise activated platelets. In contrast, macrophages efficiently phagocytosed platelets. In agreement with the uneventful nature of the clearance of platelets in vivo, phagocytosing macrophages did not release IL-1beta, TNF-alpha, or IL-10, beta2GPI bound to activated platelets and was required for their recognition by anti-beta2GPI antibodies. DCs internalised platelets opsonised by anti-beta2GPI antibodies. The phagocytosis of opsonised platelets determined the release of TNF-alpha and IL-1beta by DCs and macrophages. Phagocytosing macrophages, but not DCs, secreted the antiinflammatory cytokine IL-10. We conclude that anti-beta2GPI antibodies cause inflammation during platelet clearance and shuttle platelet antigens to antigen presenting DCs.

PTX3 in small-vessel vasculitides: an independent indicator of disease activity produced at sites of inflammation.

OBJECTIVE: To verify whether the prototypical long pentraxin PTX3 represents an indicator of the activity of small-vessel vasculitis. METHODS: Concentrations of PTX3, a pentraxin induced in endothelium by cytokines, were measured by enzyme-linked immunosorbent assay in the sera of 43 patients with Churg-Strauss syndrome, Wegener's granulomatosis, and microscopic polyangiitis. PTX3 was also measured in the sera of 28 patients with systemic lupus erythematosus (SLE), 22 with rheumatoid arthritis, and 16 with CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). Serum concentrations of C-reactive protein (CRP) were measured by immunoturbidimetry. The cells involved in PTX3 production in vivo were identified in skin biopsy samples. RESULTS: Patients with active vasculitis had significantly higher concentrations of PTX3 than did those with quiescent disease (P < 0.001). PTX3 levels in the latter group were similar to those in healthy controls. PTX3 levels were higher in patients with untreated vasculitis and lower in patients who underwent immunosuppressive treatments (P < 0.005). In contrast, patients with active SLE had negligible levels of the pentraxin. PTX3 levels did not correlate with CRP levels in vasculitis patients. Endothelial cells produced PTX3 in active skin lesions. CONCLUSION: PTX3 represents a novel acute-phase reactant produced at sites of active vasculitis.

Caspase inhibition reveals functional cooperation between p55- and p75-TNF receptors in cell necrosis.

TNF-induced caspase activation is critically involved in both apoptosis and protection from cell necrosis. We have investigated the roles of the p55- and p75-TNF receptors (TNFR1 and TNFR2) in the induction of mouse L-M cell death in the presence of a caspase inhibitor (zVAD-fmk) and a transcription inhibitor (actinomycin D), i.e. under conditions in which protective pathways requiring caspase activation and protein synthesis were blocked. Cytometric analysis after TNF treatment showed that apoptosis was inhibited, while necrosis was highly activated. In contrast, apoptosis was observed in cells treated with TNF and actinomycin D alone. Stimulation of TNFR1 was sufficient to induce either cell necrosis or apoptosis, even when we blocked endogenous TNF with an anti-murine TNF antibody. Experiments based on the use of receptor-agonist and antagonist antibodies also showed that TNFR2 contributes to cell necrosis and apoptosis. Simultaneous stimulation of TNFR2 and TNFR1 with specific agonists indicated that TNFR2 functionally cooperates with TNFR1 to potentiate the response indirectly, by inducing endogenous TNF cytotoxicity. Caspase inhibitors enhanced the cytotoxic effect of endogenous TNF, suggesting that TNFR2 modulation can regulate the global necrotic response to TNF. TNFR2 modulation could play an important role in determining the response to TNF in pathophysiological conditions characterized by caspase down-regulation and local TNF production.

The long pentraxin PTX3 binds to apoptotic cells and regulates their clearance by antigen-presenting dendritic cells.

Pentraxins are acute-phase proteins produced in vivo during inflammatory reactions. Classical short pentraxins, C-reactive protein, and serum amyloid P component are generated in the liver in response to interleukin (IL)-6. The long pentraxin PTX3 is produced in tissues under the control of primary proinflammatory signals, such as lipopolysaccharide, IL-1 beta, and tumor necrosis factor-alpha, which also promote maturation of dendritic cells (DCs). Cell death commonly occurs during inflammatory reactions. In this study, it is shown that PTX3 specifically binds to dying cells. The binding was dose dependent and saturable. Recognition was restricted to extranuclear membrane domains and to a chronological window after UV irradiation or after CD95 cross-linking-induced or spontaneous cell death in vitro. PTX3 bound to necrotic cells to a lesser extent. Human DCs failed to internalize dying cells in the presence of PTX3, while they took up normally soluble or inert particulate substrates. These results suggest that PTX3 sequesters cell remnants from antigen-presenting cells, possibly contributing to preventing the onset of autoimmune reactions in inflamed tissues. (Blood. 2000;96:4300-4306)

Anti-beta2 glycoprotein I antibodies prevent the De-activation of platelets and sustain their phagocytic clearance.

Exposure to phosphatidylserine (PS) tags dying and senescent cells for removal and identifies activated platelets. In this study we followed the fate of PS-exposing platelets in the presence of antibodies purified from Systemic Lupus Erythematosus (SLE) and primary Anti-phospholipid Syndrome (APS) patients' sera by beta2GPI affinity chromatography. Thrombin-activated platelets exposed PS and associated to beta2GPI. Both events were required for recognition by antibodies. Human monocyte-derived macrophages phagocytosed activated platelets only. Each macrophage internalized an average of 3.16+/-0.2 platelets after 60 min at 37 degrees C. Phagocytosis did not increase after longer incubations (4.65+/-0.26 platelets internalized by each macrophage after 300 min). Recognition of platelets by anti-beta2GPI antibodies significantly increased phagocytosis (P< 0.01). Upon withdrawal of thrombin, platelets downregulated PS (PS exposure t(1/2): 242 min) and the ability to be recognized by macrophages. Purified beta2GPI bound to PS-exposing platelets (association t(1/2): 250 min). Phosphatidyl serine exposure and beta2GPI association had virtually identical kinetics. Antibody binding prolonged the exposure of the beta2GPI/PS complex (t(1/2): >1200 min). The ability to phagocytose opsonized platelets was accordingly sustained (5.3+/-0.2 opsonized platelets were internalized by each macrophage after 60 min and 9.4+/-0.3 after 300 min). Anti-beta2GPI antibodies therefore poise activated platelets in a PS-exposing status, preventing the recycling of their function and favoring their phagocytic clearance.

Engagement of CD30 shapes the secretion of cytokines by human gamma delta T cells.

CD30 is a member of the TNF receptor superfamily, previously shown to be expressed on Hodgkin's lymphoma cells and on normal activated lymphocytes. We here show that CD30 is highly expressed on recently activated human gamma delta T cells. Elevated surface levels of this molecule persisted in long-term cultures of gamma delta cells, without further cell stimulation. CD30 acted as a co-stimulus in gamma delta T cells by potentiating the intracellular Ca(2+) fluxes induced by CD3 cross-linking. The engagement of CD30 enhanced the expression of several cytokines induced upon CD3 stimulation such as IL-4 and IFN-gamma but not IL-10. The CC chemokines RANTES and macrophage inflammatory protein-1beta were constitutively expressed and not affected by stimulation. The inducible expression of the neutrophil chemoattractant IL-8 was enhanced by CD30 co-stimulation, as well as that of the CC chemokines I-309 and MDC, whereas the secretion of the monocyte chemotactic protein-1 was not detected. Triggering of CD30 may therefore modulate the expression of several cytokines released by gamma delta cells; the expression of its physiologic ligand by APC and neutrophils at the site of infection may contribute to determine the outcome of an immune response.

Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines.

Broad spectrum caspase inhibitors have been found to reduce neurodegeneration caused by cerebral ischemia. We studied whether blockade of group I caspases, mainly caspase-1, using the inhibitor Ac-YVAD.cmk reduced infarct volume and produced prolonged neuroprotection. Ac-YVAD.cmk (300 ng/rat) was injected intracerebroventricularly 10 min after permanent middle cerebral artery occlusion in the rat. Drug treatment induced a significant reduction of infarct volume not only 24 hr after ischemia (total damage, percentage of hemisphere volume: control, 41.1 +/- 2.3%; treated, 26.5 +/- 2.1%; p < 0.05) but also 6 d later (total damage: control, 30.6 +/- 2.2%; treated, 23.0 +/- 2.2%; p < 0.05). Ac-YVAD. cmk treatment resulted in a reduction not only of caspase-1 (control, 100 +/- 20.3%; treated, 3.4 +/- 10.4%; p < 0.01) but also of caspase-3 (control, 100 +/- 30.3%; treated, 13.2 +/- 9.5%; p < 0.05) activity at 24 hr and led to a parallel decrease of apoptosis as measured by nucleosome quantitation (control, 100 +/- 11.8%; treated, 47 +/- 5.9%; p < 0.05). Six days after treatment no differences in these parameters could be detected between control and treated animals. Likewise, brain levels of the proinflammatory cytokines IL-1beta and TNF-alpha were reduced at 24 hr (39.5 +/- 23.7 and 51.9 +/- 10.3% of control, respectively) but not at 6 d. Other cytokines, IL-10, MCP-1, MIP-2, and the gaseous mediator nitric oxide, were not modified by the treatment. These findings indicate that blockade of caspase-1-like activity induces a long-lasting neuroprotective effect that, in our experimental conditions, takes place in the early stages of damage progression. Finally, this effect is achieved by interfering with both apoptotic and inflammatory mechanisms.

Nitric oxide inhibits the tumor necrosis factor alpha -regulated endocytosis of human dendritic cells in a cyclic GMP-dependent way.

Tumor necrosis factor-alpha (TNFalpha)-induced maturation of dendritic cells (DC), with down-regulation of their endocytic ability, has been reported to be mediated by the accumulation of the lipid messenger ceramide. We have now studied the effects and mechanisms of action of NO on endocytosis, investigated with fluorescein isothiocyanate-labeled dextran using human monocyte-derived DC, both immature and after treatment with TNFalpha. Exposure of DC to NO, released by either bystander phagocytes or NO donors, reversed the inhibition of endocytosis induced by TNFalpha. The intracellular accumulation of ceramide induced by TNFalpha was also inhibited by NO. In addition, NO was found to exert an inhibitory effect downstream of the TNFalpha-triggered ceramide accumulation, because NO donors reversed the inhibition of endocytosis induced by the cell-permeant C(2)-ceramide. These effects of NO were mimicked by the membrane-permeant cyclic GMP analogue, 8-Br cyclic GMP, and prevented by inhibition of the soluble guanylyl cyclase. At variance with rodents, the inducible isoform of the NO synthase was expressed neither in immature human DC nor after cell treatment with TNFalpha, interferon-gamma, and lipopolysaccharide, suggesting that regulation of these cells depends on exogenous NO. NO, working through cyclic GMP, might therefore prolong the ability of human DC to internalize antigens at the site of inflammation and thus modulate the initial steps leading to antigen-specific immune responses.

In vivo administration of GM-CSF promotes the clearance of apoptotic cells: effects on monocytes and polymorphonuclear leukocytes.

The clearance of apoptotic cells is crucial to avoid chronic inflammation and autoimmunity. Little is known about the factors that regulate it in vivo. We show that granulocyte-macrophage colony-stimulating factor (GM-CSF) administration to carcinoma patients confers to their leukocytes a significantly higher ability to phagocytose apoptotic cells than before (P < 0.005). GM-CSF increased the concentration of monocytes and polymorphonuclear leukocytes in the peripheral blood and activated circulating polymorphonuclear leukocytes. Both effects abated early after treatment, whereas phagocytosis of apoptotic cells was still significantly higher after 18 days compared with basal values (P < 0.005 and P < 0.025 for monocytes and polymorphonuclear leukocytes, respectively). On in vitro phagocytosis of apoptotic cells monocytes, but not polymorphonuclear leukocytes, up-regulated MHC class II membrane expression. These findings are consistent with the possibility that GM-CSF endows both scavenger and antigen-presenting leukocytes with the ability to internalize apoptotic tumor cells.

Delayed clearance of apoptotic lymphoma cells allows cross-presentation of intracellular antigens by mature dendritic cells.

Single cells are deleted from the midst of living tissue during normal turnover and embryogenesis. This event is not associated with inflammation or autoimmunity. Little is known of the clearance of apoptotic cells during dangerous situations, accompanied by extensive cell death and tissue damage: when macrophages are overwhelmed by apoptotic cells, other phagocytes, including immature dendritic cells (DCs), may become involved. DCs efficiently present antigens derived from the processing of internalized apoptotic bodies to class I- and class II-restricted T cells. Antigen presentation results either in T cell activation or in their functional blockade. The outcome is influenced by pro-inflammatory maturative signals: efficient T cell cross-priming requires fully mature DCs. Here we discuss in vitro data suggesting that the number of apoptotic cells that die at a given time influences DC maturation and therefore their ability to uptake antigens from apoptotic cells and cross-activate T lymphocytes.

Dendritic cell presentation of antigens from apoptotic cells in a proinflammatory context: role of opsonizing anti-beta2-glycoprotein I antibodies.

OBJECTIVE: To verify whether opsonization of apoptotic cells skews the outcome of apoptotic cell antigen presentation by dendritic cells (DCs). METHODS: RMA cells, which were engineered with a mutant ovalbumin (OVA) protein and were devoid of the leader secretory sequence (OVA-RMA), underwent ultraviolet irradiation to induce apoptosis. Binding of anti-beta2-glycoprotein I antibodies (anti-beta2GPI) and phagocytosis of apoptotic cells were assessed by flow cytometry and confocal microscopy. Presentation of processing antigens and major histocompatibility complex (MHC) class II-restricted or MHC class I-restricted antigens was assessed using OVA-specific T cell hybridomas. RESULTS: Anti-beta2GPI facilitated presentation of epitopes from internalized apoptotic cells to MHC class II-restricted, but not to class I-restricted, T lymphocytes. DCs challenged with supernatants of apoptotic cells did not activate OVA-specific T cells, making it unlikely that anti-beta2GPI complexed with antigen released from dying cells plays a role in antigen presentation. DCs challenged with low numbers of anti-beta2GPI-opsonized apoptotic cells secreted interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-10 in an autocrine/paracrine manner. CONCLUSION: Opsonization influences the outcome of the disposal of low numbers of apoptotic cells by DCs. This implies that soluble factors bound to apoptotic cells modulate their immunogenicity.

Generation of nitric oxide by the inducible nitric oxide synthase protects gamma delta T cells from Mycobacterium tuberculosis-induced apoptosis.

Gamma delta T cells are early recruited into mycobacterial lesions. Upon microbial Ag recognition, gamma delta cells secrete cytokines and chemokines and undergo apoptosis via CD95/CD95 ligand (CD95L) interaction, possibly influencing the outcome of infection and the characteristics of the disease. In this paper we show that activated phagocytes acquire, upon challenge with Mycobacterium tuberculosis, the ability to inhibit M. tuberculosis-induced gamma delta cell apoptosis. Apoptosis protection was due to NO because it correlated with NO synthase (NOS)-2 induction and activity in scavenger cells and was abrogated by NOS inhibitors. Furthermore, the NO donor S-nitrosoacetylpenicillamine mimicked the effect of enzyme induction. NO left unaffected the expression of CD95 and CD95L, suggesting interference with an event ensuing CD95/CD95L interaction. NO was found to interfere with the intracellular accumulation of ceramide and the activation of caspases, which were involved in gamma delta T cells apoptosis after M. tuberculosis recognition. We propose that NO generated by infected macrophages determines the life span and therefore the function of lymphocytes at the infection site, thus linking innate and adaptive immunity.