CD95-ligand contributes to abdominal aortic aneurysm progression by modulating inflammation.

AIMS: Abdominal aortic aneurysm (AAA) is one of the number of diseases associated with a prominent inflammatory cell infiltration, matrix protein degradation, and smooth muscle cell apoptosis. CD95 is an inflammatory mediator and an apoptosis inducer. Previous studies have shown elevated expression of CD95 or CD95L in the aortic tissue of AAA patients. However, how the CD95L/CD95 contributes to aneurysm degeneration and whether blocking its signalling would be beneficial to disease progression remains largely unknown. In the present study, we sought to determine the role of CD95L and its downstream target, caspase 8, in AAA progression. METHODS AND RESULTS: By using the CaCl2 murine model of AAA, abdominal aortic aneurysms were induced in C57BL/6 mice. We found that both mRNA and protein levels of CD95L were increased in aneurysm tissue compared with NaCl-treated normal aortic tissue. To determine whether CD95L contributes directly to aneurysm formation, we used CD95L null (CD95L-/-) mice to examine their response to CaCl2 aneurysm induction. Six weeks after periaortic application of CaCl2, aortic diameters of CD95L-/- mice were significantly smaller compared to CaCl2-treated wild-type controls. Connective tissue staining of aortic sections from CaCl2-treated CD95L-/- mice showed minimal damage of medial elastic lamellae which was indistinguishable from the NaCl-treated sham control. Furthermore, CD95L deficiency attenuates macrophage and T cell infiltration into the aortic tissue. To study the role of CD95L in the myelogeous cells in AAA formation, we created chimaeric mice by infusing CD95L-/- bone marrow into sub-leathally irradiated wild-type mice (WT/CD95L-/-BM). As controls, wild-type bone marrow were infused into sub-leathally irradiated CD95L-/- mice (CD95L-/-/WTBM). WT/CD95L-/-BM mice were resistant to aneurysm formation compared to their controls. Inflammatory cell infiltration was blocked by the deletion of CD95L on myeloid cells. Western blot analysis showed the levels of caspase 8 in the aortas of CaCl2-treated wild-type mice were increased compared to NaCl-treated controls. CD95L deletion inhibited caspase 8 expression. Furthermore, a caspase 8-specific inhibitor was able to partially block aneurysm development in CaCl2-treated aneurysm models. CONCLUSION: These studies demonstrated that inflammatory cell infiltration during AAA formation is dependent on CD95L from myelogeous cells. Aneurysm inhibition by deletion of CD95L is mediated in part by down-regulation of caspase 8.

Endothelial cell injury is involved in atherosclerosis and lupus symptoms in gld.apoE- / - mice.

AIM: Cardiovascular complications related to atherosclerosis are major causes of morbidity and mortality in patients with systemic lupus erythematosus (SLE). However, the underlying mechanisms are not fully understood. Endothelial dysfunction has been identified as having involvement in pathogenesis of cardiovascular diseases and SLE. This study aims to evaluate endothelial cell injury in mice with the combination of lupus and atherosclerosis. METHODS: The mouse model of accelerated atherosclerosis in lupus (gld.apoE- / - mouse) was generated from apolipoprotein E-deficient (apoE- / - ) and Faslgld C57BL/6 mice. The lupus-like autoimmunity and atherosclerotic lesions were evaluated. The endothelial cell injury was determined. RESULTS: The results showed that the double-mutant gld.apoE- / - mice were generated. Spleens from 5-month-old gld.apoE- / - mice were significantly enlarged compared with wild-type mice (WT mice). The gld.apoE- / - mice produced high levels of total immunoglobulin G (IgG) and IgM and showed marked increase of IgG and C3 deposits in the glomeruli. The gld.apoE- / - mice displayed a pattern of glomerulonephritis typically found in SLE. The gld.apoE- / - mice have high levels of serum creatinine. The total cholesterol, low-density lipoprotein cholesterol and triglycerides were significantly increased, while high-density lipoprotein cholesterol decreased in the double-mutant mice. The circulating endothelial progenitor cells were significantly decreased. The serum levels of thrombomodulin and vascular cell adhesion molecule-1 were significantly elevated in gld.apoE- / - mice. The gld.apoE- / - mice simultaneously exhibited SLE and atherosclerosis characteristics. CONCLUSION: Our findings indicated that endothelial cell injury might be a biomarker for evaluating risks of cardiovascular disease in SLE and targeting endothelial cell dysfunction might prevent and treat atherosclerosis in SLE.

Association between Type I interferon and depletion and dysfunction of endothelial progenitor cells in C57BL/6 mice deficient in both apolipoprotein E and Fas ligand.

Patients with systemic lupus erythematosus (SLE) have a tremendously increased risk for cardiovascular disease (CVD), which could not be accounted in entirety by traditional Framingham risk factors. To study whether the accelerated atherosclerosis in SLE patients is mediated by type I interferon (IFN-I) through the regulation of endothelial progenitor cells (EPCs), we created a line of C57BL/6 mice with deficiency in both apolipoprotein E (ApoE-/-) and fas ligand (FasL-/-, gld.). As expected, the resultant gld. ApoE-/- mice exhibited both aggravated lupus-like disease and atherosclerosis under normal diet. Increased expression of IFN-I-stimulated genes (ISGs) was closely associated with depletion and dysfunction of EPCs, as well as with accelerated atherosclerotic lesion in gld. ApoE-/- mice. While only IFN-α instead of other interventions, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IRS423 and IRS661, impaired EPC function in vitro. Mechanistically, activation or inhibition of the TLR7/9 signaling could modulate EPC number and function in vivo. Decreased proliferation rate and increased apoptotic rate of EPCs induced by IFN-α might contribute to the results to a certain extent. Thus, our data suggest that excessive expression of IFN-I through the activation of TLR7/9 signaling may induce accelerated atherosclerosis in lupus through the depletion or dysfunction of EPCs, suggesting that targeting IFN-I might have potential therapeutic effects on both lupus disease and premature atherosclerosis in SLE patients.

Platelet-RBC interaction mediated by FasL/FasR induces procoagulant activity important for thrombosis.

Red blood cells (RBCs) influence rheology, and release ADP, ATP, and nitric oxide, suggesting a role for RBCs in hemostasis and thrombosis. Here, we provide evidence for a significant contribution of RBCs to thrombus formation. Anemic mice showed enhanced occlusion times upon injury of the carotid artery. A small population of RBCs was located to platelet thrombi and enhanced platelet activation by a direct cell contact via the FasL/FasR (CD95) pathway known to induce apoptosis. Activation of platelets in the presence of RBCs led to platelet FasL exposure that activated FasR on RBCs responsible for externalization of phosphatidylserine (PS) on the RBC membrane. Inhibition or genetic deletion of either FasL or FasR resulted in reduced PS exposure of RBCs and platelets, decreased thrombin generation, and reduced thrombus formation in vitro and protection against arterial thrombosis in vivo. Direct cell contacts between platelets and RBCs via FasL/FasR were shown after ligation of the inferior vena cava (IVC) and in surgical specimens of patients after thrombectomy. In a flow restriction model of the IVC, reduced thrombus formation was observed in FasL-/- mice. Taken together, our data reveal a significant contribution of RBCs to thrombosis by the FasL/FasR pathway.

Expression of Fas, FasL, caspase-8 and other factors of the extrinsic apoptotic pathway during the onset of interdigital tissue elimination.

Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.

Endothelial cell-derived CD95 ligand serves as a chemokine in induction of neutrophil slow rolling and adhesion.

Integrin activation is crucial for the regulation of leukocyte rolling, adhesion and trans-vessel migration during inflammation and occurs by engagement of myeloid cells through factors presented by inflamed vessels. However, endothelial-dependent mechanisms of myeloid cell recruitment are not fully understood. Here we show using an autoperfused flow chamber assay of whole blood neutrophils and intravital microscopy of the inflamed cremaster muscle that CD95 mediates leukocyte slow rolling, adhesion and transmigration upon binding of CD95-ligand (CD95L) that is presented by endothelial cells. In myeloid cells, CD95 triggers activation of Syk-Btk/PLCγ2/Rap1 signaling that ultimately leads to integrin activation. Excitingly, CD95-deficient myeloid cells exhibit impaired bacterial clearance in an animal model of sepsis induced by cecal ligation and puncture (CLP). Our data identify the cellular and molecular mechanisms underlying the chemoattractant effect of endothelial cell-derived CD95L in induction of neutrophil recruitment and support the use of therapeutic inhibition of CD95's activity in inflammatory diseases.

Fas Ligand Deficiency Impairs Tumor Immunity by Promoting an Accumulation of Monocytic Myeloid-Derived Suppressor Cells.

The Fas receptor ligand FasL regulates immune cell levels by inducing apoptosis of Fas receptor-positive cells. Here, we studied the impact of host FasL on tumor development in mice. Genetically targeting FasL in naïve mice increased myeloid cell populations, but, in marked contrast, it reduced the levels of myeloid-derived suppressor cells (MDSC) in mice bearing Lewis lung carcinoma tumors. Analysis of the MDSC subset distribution revealed that FasL deficiency skewed cell populations toward the M-MDSC subset, which displays a highly immunosuppressive activity. Furthermore, tumor-bearing mice that were FasL-deficient displayed an enhanced proportion of tumor-associated macrophages and regulatory T cells. Overall, the immunosuppressive environment produced by FasL targeting correlated with reduced survival of tumor-bearing mice. These results disclose a new role for FasL in modulating immunosuppressive cells.

Death induced by CD95 or CD95 ligand elimination.

CD95 (Fas/APO-1), when bound by its cognate ligand CD95L, induces cells to die by apoptosis. We now show that elimination of CD95 or CD95L results in a form of cell death that is independent of caspase-8, RIPK1/MLKL, and p53, is not inhibited by Bcl-xL expression, and preferentially affects cancer cells. All tumors that formed in mouse models of low-grade serous ovarian cancer or chemically induced liver cancer with tissue-specific deletion of CD95 still expressed CD95, suggesting that cancer cannot form in the absence of CD95. Death induced by CD95R/L elimination (DICE) is characterized by an increase in cell size, production of mitochondrial ROS, and DNA damage. It resembles a necrotic form of mitotic catastrophe. No single drug was found to completely block this form of cell death, and it could also not be blocked by the knockdown of a single gene, making it a promising way to kill cancer cells.

Cellular inhibitor of apoptosis protein cIAP2 protects against pulmonary tissue necrosis during influenza virus infection to promote host survival.

Cellular inhibitors of apoptosis proteins (cIAPs) are essential regulators of cell death and immunity. The corresponding contributions of IAPs to infectious disease outcomes are relatively unexplored. We find that mice deficient in cIAP2 exhibit increased susceptibility and mortality to influenza A virus infection. The lethality was not due to impaired antiviral immune functions, but rather because of death-receptor-induced programmed necrosis of airway epithelial cells that led to severe bronchiole epithelial degeneration, despite control of viral replication. Pharmacological inhibition of RIPK1 or genetic deletion of Ripk3, both kinases involved in programmed necrosis, rescued cIAP2-deficient mice from influenza-induced lethality. Genetic deletion of the death receptor agonists Fas ligand or TRAIL from the hematopoietic compartment also reversed the susceptibility of cIAP2-deficient mice. Thus, cIAP2-dependent antagonism of RIPK3-mediated programmed necrosis critically protects the host from influenza infection through maintenance of pulmonary tissue homeostasis rather than through pathogen control by the immune system.

Fas/FasL pathway participates in resolution of mucosal inflammatory response early during HSV-2 infection.

Apoptotic cell death is critical for maintaining integrity of the epithelia as well as for removal of the virus infected cells. We assessed the role of Fas/FasL-dependent pathway in apoptosis of genital epithelium during HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6, MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) mice and an in vitro model of HSV-2 infection in monocyte RAW 264.7 and keratinocyte 291.03C cell cultures and peritoneal macrophages. In contrast to keratinocyte in vitro cultures, HSV-2 infection of the monocytic cell cultures led to early induction of apoptosis. HSV-2 infection of peritoneal macrophages isolated from Fas- and FasL-deficient mice showed decreased activation of apoptosis, which could be further blocked by caspase-9 inhibitor. Infection of Fas and FasL-deficient mice increased the percentage of apoptotic cells and activation of caspase-9 in the vaginal tissue in comparison to C57BL6 wild type strain. Furthermore, Fas and FasL-deficient mice showed increased infiltration of neutrophiles in the vaginal mucosal epithelium at 3 and 7 day of infection in contrast to HSV-2 infected wild-type mice. Our results show that while the Fas/FasL pathway during HSV-2 infection of the vaginal epithelium plays an important role in controlling early local inflammatory response, mitochondrial apoptotic pathway also becomes activated by the inflammatory reaction.

The effect of mycophenolate mofetil on disease development in the gld.apoE (-/-) mouse model of accelerated atherosclerosis and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is characterized by autoantibody production and inflammatory disease involving multiple organs. Premature atherosclerosis is a common complication of SLE and results in substantial morbidity and mortality from cardiovascular disease (CVD). The reasons for the premature atherosclerosis in SLE are incompletely understood, although chronic inflammation is thought to play an important role. There is currently no known preventative treatment of premature atherosclerosis in SLE. Mycophenolate mofetil (MMF) is an immunosuppressive agent that is commonly used for treatment of patients with SLE. In order to study the impact of this drug on murine lupus disease including premature atherosclerosis development, we treated gld.apoE(-/-) mice, a model of SLE and accelerated atherosclerosis, with MMF. We maintained seven-week old gld.apoE(-/-) mice on a high cholesterol Western diet with or without MMF. After 12 weeks on diet, mice receiving MMF showed decreased atherosclerotic lesion area compared to the control group. MMF treatment also improved the lupus phenotype, indicated by a significant decrease circulating autoantibody levels and ameliorating lupus nephritis associated with this model. This data suggests that the effects of MMF on the immune system may not only be beneficial for lupus, but also for inflammation driving lupus-associated atherosclerosis.

Kupffer cells protect liver sinusoidal endothelial cells from Fas-dependent apoptosis in sepsis by down-regulating gp130.

Endothelial cell (EC) dysfunction is a key feature of multiple organ injury, the primary cause of fatality seen in critically ill patients. Although the development of EC dysfunction in the heart and lung is well studied in sepsis, it remains unclear in the liver. Herein, we report that liver sinusoidal ECs (LSECs; defined as CD146(+)CD45(-)) exhibit increased intercellular adhesion molecule-1 (CD54) and Fas in response to sepsis induced by cecal ligation and puncture (CLP). By using magnetically enriched LSEC (CD146(+)) populations, we show evidence of marked apoptosis, with a twofold decline in viable LSECs in CLP animals compared with sham controls. These changes and increased serum alanine aminotransferase levels were all mitigated in septic Fas(-/-) and Fas ligand(-/-) animals. Although we previously reported increased numbers of Fas ligand expressing CD8(+) T lymphocytes in the septic liver, CD8(+) T-cell deficiency did not reverse the onset of LSEC apoptosis/damage. However, Kupffer cell depletion with clodronate liposomes resulted in greater apoptosis and Fas expression after CLP and a decrease in glycoprotein 130 expression on LSECs, suggesting that STAT3 activation may protect these cells from injury. Our results document a critical role for death receptor-mediated LSEC injury and show the first evidence that Kupffer cells are essential to the viability of LSECs, which appears to be mediated through glycoprotein 130 expression in sepsis.

Graft-versus-host disease impairs vaccine responses through decreased CD4+ and CD8+ T cell proliferation and increased perforin-mediated CD8+ T cell apoptosis.

Tumor-targeted vaccines represent a strategy to enhance the graft-versus-leukemia effect after allogeneic blood and marrow transplantation (BMT). We have previously shown that graft-versus-host disease (GVHD) can negatively impact quantitative responses to vaccines. Using a minor histocompatibility Ag-mismatched BMT (B6 → B6 × C3H.SW) followed by adoptive transfer of HY-specific T cells and HY-expressing dendritic cells, we assessed whether GVHD induced by donor lymphocyte infusion (DLI) affects the persistence, proliferation, and survival of vaccine-responding, nonalloantigen reactive T cells. Both CD8(+) and CD4(+) HY-specific T cells undergo less vaccine-driven proliferation in allogeneic recipients with GVHD. Although vaccine-responding CD8(+) T cells show decreased IFN-γ and CD107a production, CD4(+) T cells exhibit increased programmed death 1 and T cell Ig mucin-like domain 3 expression. In addition, the degree of apoptosis in vaccine-responding CD8(+) T cells was higher in the presence of GVHD, but there was no difference in CD4(+) T cell apoptosis. Using Fas ligand-deficient or TRAIL-deficient DLI had no impact on apoptosis of HY-specific T cells. However, perforin-deficient alloreactive DLI induced significantly less apoptosis of vaccine-responding CD8(+) T cells and resulted in enhanced tumor protection. Thus, diminished vaccine responses during GVHD result from impaired proliferation of CD8(+) and CD4(+) T cells responding to vaccination, with an additional contribution from perforin-mediated CD8(+) T cell apoptosis. These results provide important insights toward optimizing vaccine responses after allogeneic BMT.

Autoimmune lymphoproliferative syndrome caused by a homozygous null FAS ligand (FASLG) mutation.

BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is characterized by chronic nonmalignant lymphoproliferation, accumulation of double-negative T cells, hypergammaglobulinemia G and A, and autoimmune cytopenia. OBJECTIVES: Although mostly associated with FAS mutations, different genetic defects leading to impaired apoptosis have been described in patients with ALPS, including the FAS ligand gene (FASLG) in rare cases. Here we report on the first case of complete FAS ligand deficiency caused by a homozygous null mutant. METHODS: Double-negative T-cell counts and plasma IL-10 and FAS ligand concentrations were determined as ALPS markers. The FASLG gene was sequenced, and its expression was analyzed by means of Western blotting. FAS ligand function was assessed based on reactivation-induced cell death. RESULTS: We describe a patient born to consanguineous parents who presented with a severe form of ALPS caused by FASLG deficiency. Although the clinical presentation was compatible with a homozygous FAS mutation, FAS-induced apoptosis was normal, and plasma FAS ligand levels were not detectable. This patient carries a homozygous, germline, single-base-pair deletion in FASLG exon 1, leading to a premature stop codon (F87fs x95) and a complete defect in FASLG expression. The healthy parents were each heterozygous for the mutation, confirming its recessive trait. CONCLUSION: FAS ligand deficiency should be screened in patients presenting with ALPS features but lacking the usual markers, including plasma soluble FAS ligand and an in vitro apoptotic defect. An activation-induced cell death test could help in discrimination.

Mice with mutations in Fas and Fas ligand demonstrate increased herpetic stromal keratitis following corneal infection with HSV-1.

HSV-1 infection of the cornea leads to a potentially blinding immunoinflammatory lesion of the cornea, termed herpetic stromal keratitis. It has also been shown that one of the factors limiting inflammation of the cornea is the presence of Fas ligand (FasL) on corneal epithelium and endothelium. In this study, the role played by FasL expression in the cornea following acute infection with HSV-1 was determined. Both BALB/c and C57BL/6 (B6) mice with HSV-1 infection were compared with their lpr and gld counterparts. Results indicated that mice bearing mutations in the Fas Ag (lpr) displayed the most severe disease, whereas the FasL-defective gld mouse displayed an intermediate phenotype. It was further demonstrated that increased disease was due to lack of Fas expression on bone marrow-derived cells. Of interest, although virus persisted slightly longer in the corneas of mice bearing lpr and gld mutations, the persistence of infectious virus in the trigeminal ganglia was the same for all strains infected. Further, B6 mice bearing lpr and gld mutations were also more resistant to virus-induced mortality than were wild-type B6 mice. Thus, neither disease nor mortality correlated with viral replication in these mice. Collectively, the findings indicate that the presence of FasL on the cornea restricts the entry of Fas(+) bone marrow-derived inflammatory cells and thus reduces the severity of HSK.

Destruction of lymphoid organ architecture and hepatitis caused by CD4+ T cells.

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.

Negative autoregulation by Fas stabilizes adult erythropoiesis and accelerates its stress response.

Erythropoiesis maintains a stable hematocrit and tissue oxygenation in the basal state, while mounting a stress response that accelerates red cell production in anemia, blood loss or high altitude. Thus, tissue hypoxia increases secretion of the hormone erythropoietin (Epo), stimulating an increase in erythroid progenitors and erythropoietic rate. Several cell divisions must elapse, however, before Epo-responsive progenitors mature into red cells. This inherent delay is expected to reduce the stability of erythropoiesis and to slow its response to stress. Here we identify a mechanism that helps to offset these effects. We recently showed that splenic early erythroblasts, 'EryA', negatively regulate their own survival by co-expressing the death receptor Fas, and its ligand, FasL. Here we studied mice mutant for either Fas or FasL, bred onto an immune-deficient background, in order to avoid an autoimmune syndrome associated with Fas deficiency. Mutant mice had a higher hematocrit, lower serum Epo, and an increased number of splenic erythroid progenitors, suggesting that Fas negatively regulates erythropoiesis at the level of the whole animal. In addition, Fas-mediated autoregulation stabilizes the size of the splenic early erythroblast pool, since mutant mice had a significantly more variable EryA pool than matched control mice. Unexpectedly, in spite of the loss of a negative regulator, the expansion of EryA and ProE progenitors in response to high Epo in vivo, as well as the increase in erythropoietic rate in mice injected with Epo or placed in a hypoxic environment, lagged significantly in the mutant mice. This suggests that Fas-mediated autoregulation accelerates the erythropoietic response to stress. Therefore, Fas-mediated negative autoregulation within splenic erythropoietic tissue optimizes key dynamic features in the operation of the erythropoietic network as a whole, helping to maintain erythroid homeostasis in the basal state, while accelerating the stress response.

Myeloid-derived suppressor cells express the death receptor Fas and apoptose in response to T cell-expressed FasL.

Myeloid-derived suppressor cells (MDSCs) inhibit adaptive and innate immunity and accumulate in the blood of persons with cancer, chronic inflammation, trauma, infection, and stress. Some of the factors inducing their accumulation are known; however, mechanisms regulating their turnover have not been identified. Mass spectrometry showed prominent expression of apoptosis pathway proteins, suggesting that MDSC turnover may be regulated by Fas-FasL-mediated apoptosis. This hypothesis was confirmed by showing that blood MDSCs induced by 3 mouse tumors were Fas(+) and apoptosed in response to Fas agonist in vitro and to activated FasL(+) T cells in vivo. FasL-deficient mice contained significantly more blood MDSCs than FasL(+/+) mice, and after removal of primary tumors MDSCs regressed in STAT6(-/-) and CD1(-/-) mice but not in STAT6(-/-)FasL(-/-) or CD1(-/-)FasL(-/-) mice. Fas(+) macrophages and dendritic cells did not apoptose in response to activated T cells, indicating that Fas-FasL regulation of myeloid cells was restricted to MDSCs. These results identify a new mechanism regulating MDSC levels in vivo and show a retaliatory relationship between T cells and MDSCs in that MDSCs suppress T-cell activation; however, once activated, T cells mediate MDSC apoptosis.

In vivo diabetogenic action of CD4+ T lymphocytes requires Fas expression and is independent of IL-1 and IL-18.

CD4(+) T lymphocytes are required to induce spontaneous autoimmune diabetes in the NOD mouse. Since pancreatic ß cells upregulate Fas expression upon exposure to pro-inflammatory cytokines, we studied whether the diabetogenic action of CD4(+) T lymphocytes depends on Fas expression on target cells. We assayed the diabetogenic capacity of NOD spleen CD4(+) T lymphocytes when adoptively transferred into a NOD mouse model combining: (i) Fas-deficiency, (ii) FasL-deficiency, and (iii) SCID mutation. We found that CD4(+) T lymphocytes require Fas expression in the recipients' target cells to induce diabetes. IL-1ß has been described as a key cytokine involved in Fas upregulation on mouse ß cells. We addressed whether CD4(+) T cells require IL-1ß to induce diabetes. We also studied spontaneous diabetes onset in NOD/IL-1 converting enzyme-deficient mice, in NOD/IL-1ß-deficient mice, and CD4(+) T-cell adoptively transferred diabetes into NOD/SCID IL-1ß-deficient mice. Neither IL-1ß nor IL-18 are required for either spontaneous or CD4(+) T-cell adoptively transferred diabetes. We conclude that CD4(+) T-cell-mediated ß-cell damage in autoimmune diabetes depends on Fas expression, but not on IL-1ß unveiling the existing redundancy regarding the cytokines involved in Fas upregulation on NOD ß cells in vivo.

Blocking Fas ligand on leukocytes attenuates kidney ischemia-reperfusion injury.

Inflammation contributes to the pathogenesis of ischemic acute kidney injury (AKI), and T cells mediate the early phase of ischemia-reperfusion injury (IRI). The Fas/Fas ligand (FasL) pathway modulates the balance of T cell subsets in the peripheral circulation as well as multiple inflammatory responses, suggesting that FasL may mediate ischemic AKI. Here, we induced bilateral renal IRI in mice bearing a loss-of-function mutation of FasL (the gld mutation) and in wild-type mice. Compared with wild-type mice, serum creatinine was lower in gld mice (1.4 ± 0.9 mg/dl versus 2.6 ± 0.4) at 24 hours after IRI (P<0.05). In addition, gld mice had fewer TNF-α-producing T lymphocytes in the kidneys and renal lymph nodes. Furthermore, pharmacologic blockade of FasL protected the kidneys of wild-type mice from IRI. Analysis of bone marrow chimeric mice suggested that the pathogenic effect of FasL involves leukocytes; reconstitution of wild-type mice with gld splenocytes attenuated IRI. In contrast, reconstitution of gld mice with wild-type splenocytes enhanced IRI. These data demonstrate that FasL, particularly on leukocytes, mediates ischemic AKI.