Endothelial TNF receptor 2 induces IRF1 transcription factor-dependent interferon-ß autocrine signaling to promote monocyte recruitment.

Endothelial-dependent mechanisms of mononuclear cell influx are not well understood. We showed that acute stimulation of murine microvascular endothelial cells expressing the tumor necrosis factor receptors TNFR1 and TNFR2 with the soluble cytokine TNF led to CXCR3 chemokine generation. The TNF receptors signaled through interferon regulatory factor-1 (IRF1) to induce interferon-ß (IFN-ß) and subsequent autocrine signaling via the type I IFN receptor and the transcription factor STAT1. Both TNFR2 and TNFR1 were required for IRF1-IFNß signaling and, in human endothelial cells TNFR2 expression alone induced IFN-ß signaling and monocyte recruitment. In vivo, TNFR1 was required for acute renal neutrophil and monocyte influx after systemic TNF treatment, whereas the TNFR2-IRF1-IFN-ß autocrine loop was essential only for macrophage accumulation. In a chronic model of proliferative nephritis, IRF1 and renal-expressed TNFR2 were essential for sustained macrophage accumulation. Thus, our data identify a pathway in endothelial cells that selectively recruits monocytes during a TNF-induced inflammatory response.

Human lupus serum induces neutrophil-mediated organ damage in mice that is enabled by Mac-1 deficiency.

Systemic lupus erythematosus (SLE) is a chronic, multiorgan inflammatory autoimmune disorder associated with high levels of circulating autoantibodies and immune complexes. We report that passive transfer of human SLE sera into mice expressing the uniquely human FcγRIIA and FcγRIIIB on neutrophils induces lupus nephritis and in some cases arthritis only when the mice additionally lack the CD18 integrin, Mac-1. The prevailing view is that Mac-1 on macrophages is responsible for immune complex clearance. However, disease permitted by the absence of Mac-1 is not related to enhanced renal immune complex deposition or in situ C1q/C3 complement activation and proceeds even in the absence of macrophages. Instead, disease is associated with increased FcγRIIA-induced neutrophil accumulation that is enabled by Mac-1 deficiency. Intravital microscopy in the cremasteric vasculature reveals that Mac-1 mitigates FcγRIIA-dependent neutrophil recruitment in response to deposited immune complexes. Our results provide direct evidence that human SLE immune complexes are pathogenic, demonstrate that neutrophils are primary mediators of end organ damage in a novel humanized lupus mouse model, and identify Mac-1 regulation of FcγRIIA-mediated neutrophil recruitment as a key step in development of target organ damage.

Impairment of the programmed cell death-1 pathway increases atherosclerotic lesion development and inflammation.

OBJECTIVE: Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals on interaction with its 2 ligands, PD-L1 and PD-L2. We studied the contribution of the PD-1 pathway to regulation of T cells that promote atherosclerotic lesion formation and inflammation. METHODS AND RESULTS: We show that compared with Ldlr-/- control mice, Pd1-/-Ldlr-/- mice developed larger lesions with more abundant CD4+ and CD8+ T cells and macrophages, accompanied by higher levels of serum tumor necrosis factor-α. Iliac lymph node T cells from Pd1-/-Ldlr-/- mice proliferated more to αCD3 or oxidized low-density lipoprotein stimulation compared with controls. CD8+ T cells from Pd1-/-Ldlr-/- mice displayed more cytotoxic activity compared with controls in vivo and in vitro. Administration of a blocking anti-PD-1 antibody increased lesional inflammation in hypercholesterolemic Ldlr-/- mice with more lesional T cells and more activated T cells in paraaortic lymph nodes. The changes in lesional T-cell content when PD-1 was absent or blocked were also observed in bone marrow chimeric Ldlr-/- mice lacking PD-L1 and PD-L2 on hematopoietic cells. CONCLUSIONS: PD-1 has an important role in downregulating proatherogenic T-cell responses, and blockade of this molecule for treatment of viral infections or cancer may increase risk of cardiovascular complications.

Statin-induced Krüppel-like factor 2 expression in human and mouse T cells reduces inflammatory and pathogenic responses.

The transcription factor Krüppel-like factor 2 (KLF2) is required for the quiescent and migratory properties of naive T cells. Statins, a class of HMG-CoA reductase inhibitors, display pleiotropic immunomodulatory effects that are independent of their lipid-lowering capacity and may be beneficial as therapeutic agents for T cell-mediated inflammatory diseases. Statins upregulate KLF2 expression in endothelial cells, and this activity is associated with an antiinflammatory phenotype. We therefore hypothesized that the immunomodulatory effects of statins are due, in part, to their direct effects on T cell KLF2 gene expression. Here we report that lipophilic statin treatment of mouse and human T cells increased expression of KLF2 through a HMG-CoA/prenylation-dependent pathway. Statins also diminished T cell proliferation and IFN-gamma expression. shRNA blockade of KLF2 expression in human T cells increased IFN-gamma expression and prevented statin-induced IFN-gamma reduction. In a mouse model of myocarditis induced by heart antigen-specific CD8+ T cells, both statin treatment of the T cells and retrovirally mediated overexpression of KLF2 in the T cells had similar ameliorating effects on disease induction. We conclude that statins reduce inflammatory functions and pathogenic activity of T cells through KLF2-dependent mechanisms, and this pathway may be a potential therapeutic target for cardiovascular diseases.

Mac-1 (CD11b/CD18) links inflammation and thrombosis after glomerular injury.

BACKGROUND: Inflammation and thrombosis coexist in several disorders. Although it is recognized that leukocytes may induce a procoagulant state at sites of inflammation, the critical molecular determinants of this process remain largely unknown. METHODS AND RESULTS: To examine mechanisms of inflammation-induced thrombosis, we developed a murine model of thrombotic glomerulonephritis (TGN), a known cause of acute renal failure in patients. This model, induced by lipopolysaccharide and antibody to the glomerular basement membrane, led to rapid glomerular neutrophil recruitment, thrombotic glomerular lesions with endothelial cell injury, and renal dysfunction. In mice immunodepleted of neutrophils or lacking the leukocyte-specific integrin Mac-1, neutrophil recruitment, endothelial injury, glomerular thrombosis, and acute renal failure were markedly attenuated despite the robust generation of renal cytokines. Neutrophil elastase is a likely effector of Mac-1 because its activity was reduced in Mac-1-deficient mice and the phenotype in mice deficient in Mac-1 or neutrophil elastase was similar. Platelets accumulated in glomerular capillaries within 4 hours of TGN before evidence of thrombosis. Platelet immunodepletion before TGN markedly exacerbated hematuria (hemorrhage), inflammation, and injury, whereas thrombocytopenic Mac-1-deficient mice remained resistant to disease, indicating that initial glomerular platelet deposition protects the vessel wall from neutrophil-mediated sequelae. The subsequent thrombosis relied on the interaction of Mac-1 on recruited neutrophils with glycoprotein Ibalpha on platelets as antibody-mediated disruption of this interaction attenuated TGN without affecting renal neutrophil accumulation. CONCLUSIONS: These observations establish Mac-1 on neutrophils as a critical molecular link between inflammation and thrombosis and suggest it as an attractive target for antithrombotic therapy.

The development of methodology for clinical measurement of 5-lipoxygenase pathway intermediates from human peripheral blood mononuclear cells.

Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B(4) (LTB(4)) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB(4), a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB(4) was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025-25ng/mL for LTB(4) and 0.1-25ng/mL for 5-HETE. The EIA method has a validated range covering 0.025-4ng/mL. When both assays were applied to analyze LTB(4) from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r=0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB(4) and 5-HETE measured by LC/MS/MS after ionophore stimulation (r=0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.

Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8+ T-cell mediated injury in the heart.

BACKGROUND: PD-L1 and PD-L2 are ligands for the inhibitory receptor programmed death-1 (PD-1), which is an important regulator of immune responses. PD-L1 is induced on cardiac endothelial cells under inflammatory conditions, but little is known about its role in regulating immune injury in the heart. METHODS AND RESULTS: Cytotoxic T-lymphocyte-mediated myocarditis was induced in mice, and the influence of PD-L1 signaling was studied with PD-L1/L2-deficient mice and blocking antibodies. During cytotoxic T-lymphocyte-induced myocarditis, the upregulation of PD-L1 on cardiac endothelia was dependent on T-cell-derived interferon-gamma, and blocking of interferon-gamma signaling worsened disease. Genetic deletion of both PD-1 ligands [PD-L1/2(-/-)], as well as treatment with PD-L1 blocking antibody, transformed transient myocarditis to lethal disease, in association with widespread polymorphonuclear leukocyte-rich microabscesses but without change in cytotoxic T-lymphocyte recruitment. PD-L1/2(-/-) mice reconstituted with bone marrow from wild-type mice remained susceptible to severe disease, which demonstrates that PD-L1 on non-bone marrow-derived cells confers the protective effect. Finally, depletion of polymorphonuclear leukocytes reversed the enhanced susceptibility to lethal myocarditis attributable to PD-L1 deficiency. CONCLUSIONS: Myocardial PD-L1, mainly localized on endothelium, is critical for control of immune-mediated cardiac injury and polymorphonuclear leukocyte inflammation.

CTLA-4 ablation and interleukin-12 driven differentiation synergistically augment cardiac pathogenicity of cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes contribute to viral and autoimmune myocarditis and cardiac allograft rejection. The role of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 as a negative regulator of CD4+ T cells is well defined, yet CTLA-4 regulation of CD8+ T cells is less clear. We studied CTLA-4 regulation of cytotoxic T lymphocytes in a transgenic model of CD8+ T-cell-mediated myocarditis. We generated CTLA-4(-/-) Rag 2(-/-) OT-1 mice, the CD8+ T cells of which express an ovalbumin (OVA) peptide-specific, class I major histocompatibility complex-restricted T-cell receptor. CTLA-4(-/-Tc12) OT-1 effectors, differentiated with interleukin-12 present, are hyperproliferative in vitro, compared with CTLA-4(+/+)Tc12 OT-1 controls. Transfer of low doses of CTLA-4(-/-Tc12) OT-1 cells to cMy-mOVA mice, which express OVA on cardiac myocytes, causes severe myocarditis, with 99% mortality, compared with no mortality after transfer of low doses of CTLA-4(+/+)Tc12 OT-1 cells. High doses of CTLA-4(+/+)Tc12 cells cause lethal myocarditis in cMy-mOVA mice, but high doses of CTLA-4(+/+)Tc0 CTL, generated without interleukin-12, are hypoproliferative within the cardiac-draining lymph node and do not significantly infiltrate the heart. In contrast, CTLA-4(-/-Tc0) cytotoxic T lymphocytes do proliferate in the cardiac-draining lymph node and diffusely infiltrate the heart. Nonetheless, high doses of CTLA-4(-/-Tc0) cells cause only limited tissue damage, and the disease is not lethal. These data show that CTLA-4 regulates myocarditic CD8+ T cell responses and that CTLA-4 deficiency partly overcomes a differentiation block that exists when naïve CD8+ T cells are stimulated without interleukin-12. Therefore, targeting CTLA-4 solely or in conjunction with interleukin-12 could influence effector CD8+ T cell responses in therapeutically beneficial ways.

Gene expression changes evoked in a venous segment exposed to arterial flow.

OBJECTIVE: This study was conducted to characterize the coordinated molecular changes evoked in the structure and composition of the wall of a venous segment when exposed to fistula flow. METHODS: An arteriovenous shunt was created in adult C57BL/6J mice. Remodeled veins and contralateral control jugular veins were isolated 7 days after surgery. Total RNA was isolated, linearly amplified, and the transcriptional profiles of this early adaptive response were obtained by microarray analysis. Histologic and immunohistochemical analyses were performed on remodeled veins and control veins isolated on days 1, 3, 5, and 7 after surgery to further examine distinct spatial and temporal aspects of this early process. RESULTS: There were 131 significantly upregulated and 165 downregulated genes in the remodeled vein compared with the control jugular vein. Genes involved in extracellular matrix reorganization were highly upregulated. Movat's pentachrome staining revealed ground substance on day 3 that was not observed on day 5. The appearance of elastin fibers was first observed on day 7. Morphometric analysis demonstrated maximum wall thickness on day 3. Immunohistochemical analysis revealed the presence of tenascin-C, thrombospondin, lysyl oxidase, and osteopontin in different cell types at different time points throughout the first week after surgery. CONCLUSION: Major changes in the organization of the extracellular matrix occur during the early response of venous remodeling. Elastin, tenascin-C, thrombospondin, lysyl oxidase, and osteopontin are expressed within the wall of the remodeling vein resulting in the de novo formation of an extracellular matrix scaffold that may be part of a critical adaptation program being evoked to allow the vessel to cope with its new biomechanical environment. CLINICAL RELEVANCE: The Kidney Dialysis Outcomes Quality Initiative has proposed the construction of arteriovenous fistulas as the primary vascular access for hemodialysis. As the vein is exposed to arterial flow, the vein wall dilates and a vascular remodeling process is triggered. With continued exposure, intimal hyperplasia occurs at the anastomosis that in many cases leads to failure. However, the molecular mechanisms by which the outflow vein remodels into a mature fistula remain incompletely understood. By investigating venous remodeling in a fistula model, candidate genes important for the remodeling process are discovered and their functional significance examined. Thus, the identification of relevant genes involved in this process should provide insight into arteriovenous fistula maturation and may suggest novel approaches for achieving higher patency rates.

Renal cell-expressed TNF receptor 2, not receptor 1, is essential for the development of glomerulonephritis.

TNF is essential for the development of glomerulonephritis, an immune-mediated disorder that is a major cause of renal failure worldwide. However, TNF has proinflammatory and immunosuppressive properties that may segregate at the level of the 2 TNF receptors (TNFRs), TNFR1 and TNFR2. TNFR1-deficient mice subjected to immune complex-mediated glomerulonephritis developed less proteinuria and glomerular injury, and fewer renal leukocyte infiltrates at early time points after disease induction, and this was associated with a reduced systemic immune response to nephrotoxic rabbit IgG. However, proteinuria and renal pathology were similar to those in wild-type controls at later time points, when lack of TNFR1 resulted in excessive renal T cell accumulation and an associated reduction in apoptosis of these cells. In sharp contrast, TNFR2-deficient mice were completely protected from glomerulonephritis at all time points, despite an intact systemic immune response. TNFR2 was induced on glomerular endothelial cells of nephritic kidneys, and TNFR2 expression on intrinsic cells, but not leukocytes, was essential for glomerulonephritis and glomerular complement deposition. Thus, TNFR1 promotes systemic immune responses and renal T cell death, while intrinsic cell TNFR2 plays a critical role in complement-dependent tissue injury. Therefore, therapeutic blockade specifically of TNFR2 may be a promising strategy in the treatment of immune-mediated glomerulonephritis.

T-bet deficiency reduces atherosclerosis and alters plaque antigen-specific immune responses.

The influence of the immune system on atherosclerosis involves both helper T (Th) cell and antibody responses to plaque antigens. These responses may have proatherogenic and protective effects. T-bet is a transcription factor required for Th1 differentiation and regulates the balance between Th1 and Th2 responses in inflammatory diseases. To clarify how helper T cell subset differentiation influences atherosclerosis, we compared lesion development and immune responses to plaque antigens in low-density lipoprotein receptor-deficient (Ldlr-/-) mice with or without functional T-bet genes. Atherosclerosis was significantly reduced in T-bet-deficient Ldlr-/- mice compared with Ldlr-/- controls, and the lesions that did develop in the absence of T-bet had less smooth muscle cell content. Furthermore, T-bet deficiency caused a Th2 switch in the response to the atherosclerosis-associated antigen heat shock protein-60, and a change in T-dependent isotypes of oxidized LDL-specific antibodies. Of particular significance, T-bet deficiency caused a >250% increase in the titer of E06 antibodies, which are known to be atheroprotective and whose production by B-1 B cells is enhanced by IL-5. These findings establish that T cell subset differentiation influences both T cell and antibody responses that modulate atherosclerosis, and validate the therapeutic goal of skewing T responses to atherosclerosis-associated antigens.

C1q governs deposition of circulating immune complexes and leukocyte Fcgamma receptors mediate subsequent neutrophil recruitment.

Inflammation induced by circulating immunoglobulin G-immune complexes (ICs) characterizes many immune-mediated diseases. In this work, the molecular requirements for the deposition of circulating ICs and subsequent acute leukocyte recruitment in mice were elucidated. We show that after intravenous injection, preformed soluble ICs are rapidly deposited in the postcapillary venules of the cremaster microcirculation, secondary to increased vascular permeability. This deposition is dependent on complement C1q. IC deposition is associated with leukocyte recruitment. Leukocyte rolling, which is mediated by P-selectin in the exteriorized cremaster muscle, is not further increased in response to ICs. In contrast, leukocyte rolling velocity is significantly decreased and leukocyte adhesion is significantly increased in the presence of ICs. The IC-mediated slow leukocyte rolling velocity and subsequent adhesion and emigration are dependent on Fcgamma receptors (FcgammaRs), particularly FcgammaRIII, with complement C3 and C5 having no detectable role. These studies suggest a regulatory mechanism of IC deposition and leukocyte trafficking in IC-mediated inflammation requiring C1q and FcgammaRs in sequential, noninteracting roles.

Early adaptive responses of the vascular wall during venous arterialization in mice.

Venous arterialization occurs when a vein segment is transposed as a bypass graft into the arterial circulation, resulting in a structural and functional reorganization of the vascular wall in response to the new local biomechanical environment. Although the anatomical changes of venous arterialization have been well characterized, the molecular mechanisms of vascular remodeling remain incompletely understood. Here, we present a novel model of venous arterialization in mice wherein the external jugular vein is connected to the common carotid artery. The hemodynamic characteristics of the arterialized vein, as assessed by ultrasound and magnetic resonance imaging, resemble features of the arterial circulation. Temporal analyses of the morphological changes in the venous segment at 1, 3, and 7 days after surgery demonstrate preservation of the endothelium at all time points and formation of multiple smooth muscle layers by day 7. Expression of endothelial E-selectin and VCAM-1 was documented at early time points, concomitant with the presence of neutrophils and monocytes/macrophages in the vascular wall. In addition, endothelium-dependent permeability was decreased in the arterialized vein when compared to the contralateral control vein. Thus, this novel mouse model of venous arterialization displays anatomical and cellular features present in other species, and should help to characterize the molecular mechanisms of this adaptive response of the vascular wall to changes in its biomechanical environment.

Neutrophils sustain pathogenic CD8+ T cell responses in the heart.

This study explores the influence of innate immunity on CD8(+) T-cell responses against heart tissue. Adoptive transfer of ovalbumin-specific CD8(+) effector T cells into CMy-mOva mice, which express ovalbumin in cardiac myocytes, results in a lethal acute myocarditis. The inflammatory infiltrate in the heart includes neutrophils as well as T cells. We used anti-Ly6G antibody to transiently deplete neutrophils at the time of onset of disease. By day 7 after receiving 5 x 10(5) CD8(+) effector T cells, 100% of control Ig-treated CMy-mOva mice had died, while 85% of anti-Ly6G-treated mice survived indefinitely. CD8(+) T-cell infiltration and tissue damage were present in both groups, but the disease was limited in the anti-Ly6G-treated mice, with a rapid disappearance of the adoptively transferred CD8(+) T cells within 11 days. Recovery occurred even though blood neutrophil counts began to rise 48 hours after the last anti-Ly6G treatment. Recovery was associated with a chronic CD4(+) cell infiltrate, and a rapid decline in expression of IFN-gamma and IP-10 mRNA in the myocardium. Neutrophil depletion did not effect survival of CMy-mOva mice that received 3 x 10(6) CD8(+) T cells. These data show that granulocytic inflammation sustains CD8(+) T-cell-mediated heart disease, which has important implications for the pathogenesis and treatment of acute myocarditis and allograft rejection.

Influence of interferon-gamma on the extent and phenotype of diet-induced atherosclerosis in the LDLR-deficient mouse.

OBJECTIVE: The aim of this study was to investigate the influence of interferon-gamma (IFN-gamma) on atherosclerosis in low density lipoprotein receptor (LDLR)-null mice. METHODS AND RESULTS: We cross-bred IFN-gamma-deficient mice with LDLR-null mice and analyzed lipoprotein profiles and atherosclerosis in the compound mutant progeny after 8 and 20 weeks on a cholesterol-enriched diet. IFN-gamma deficiency did not affect serum cholesterol levels or lipoprotein profiles, but it did affect the extent and phenotype of atherosclerosis. Atherosclerotic lesions in IFN-gamma-deficient mice were reduced by 75% in the aortic arch and by 46% in the descending aorta compared with control mice after 8 weeks on the diet. After 20 weeks, arch lesions were reduced by 43%, and descending aorta lesions were reduced by 65% in IFN-gamma-deficient mice compared with controls. At 8 weeks, percent lesional macrophage and smooth muscle content was significantly less in the IFN-gamma-deficient mice, but not at 20 weeks. Although there were fewer class II major histocompatibility complex-positive cells in the lesions of IFN-gamma-deficient animals compared with controls, class II major histocompatibility complex expression on endothelial cells overlying lesions persisted in the absence of IFN-gamma. CONCLUSIONS: These data provide direct evidence that IFN-gamma influences atherosclerosis development and phenotype in the LDLR-deficient mouse, independent of changes in blood lipoprotein profiles.

IL-12 is required for differentiation of pathogenic CD8+ T cell effectors that cause myocarditis.

Cardiac antigen-specific CD8(+) T cells are involved in the autoimmune component of human myocarditis. Here, we studied the differentiation and migration of pathogenic CD8(+) T cell effector cells in a new mouse model of autoimmune myocarditis. A transgenic mouse line was derived that expresses cardiac myocyte restricted membrane-bound ovalbumin (CMy-mOva). The endogenous adaptive immune system of CMy-mOva mice displays tolerance to ovalbumin. Adoptive transfer of naive CD8(+) T cells from the ovalbumin-specific T cell receptor-transgenic (TCR-transgenic) OT-I strain induces myocarditis in CMy-mOva mice only after subsequent inoculation with ovalbumin-expressing vesicular stomatitis virus (VSV-Ova). OT-I effector T cells derived in vitro in the presence or absence of IL-12 were adoptively transferred into CMy-mOva mice, and the consequences were compared. Although IL-12 was not required for the generation of cytolytic and IFN-gamma-producing effector T cells, only effectors primed in the presence of IL-12 infiltrated CMy-mOva hearts in significant numbers, causing lethal myocarditis. Furthermore, analysis of OT-I effectors collected from a mediastinal draining lymph node indicated that only effectors primed in vitro in the presence of IL-12 proliferated in vivo. These data demonstrate the importance of IL-12 in the differentiation of pathogenic CD8(+) T cells that can cause myocarditis.