DOCK4 Regulation of Rho GTPases Mediates Pulmonary Vascular Barrier Function.

BACKGROUND: The vascular endothelium maintains tissue-fluid homeostasis by controlling the passage of large molecules and fluid between the blood and interstitial space. The interaction of catenins and the actin cytoskeleton with VE-cadherin (vascular endothelial cadherin) is the primary mechanism for stabilizing AJs (adherens junctions), thereby preventing lung vascular barrier disruption. Members of the Rho (Ras homology) family of GTPases and conventional GEFs (guanine exchange factors) of these GTPases have been demonstrated to play important roles in regulating endothelial permeability. Here, we evaluated the role of DOCK4 (dedicator of cytokinesis 4)-an unconventional Rho family GTPase GEF in vascular function. METHODS: We generated mice deficient in DOCK4' used DOCK4 silencing and reconstitution approaches in human pulmonary artery endothelial cells' used assays to evaluate protein localization, endothelial cell permeability, and small GTPase activation. RESULTS: Our data show that DOCK4-deficient mice are viable. However, these mice have hemorrhage selectively in the lung, incomplete smooth muscle cell coverage in pulmonary vessels, increased basal microvascular permeability, and impaired response to S1P (sphingosine-1-phosphate)-induced reversal of thrombin-induced permeability. Consistent with this, DOCK4 rapidly translocates to the cell periphery and associates with the detergent-insoluble fraction following S1P treatment, and its absence prevents S1P-induced Rac-1 activation and enhancement of barrier function. Moreover, DOCK4-silenced pulmonary artery endothelial cells exhibit enhanced basal permeability in vitro that is associated with enhanced Rho GTPase activation. CONCLUSIONS: Our findings indicate that DOCK4 maintains AJs necessary for lung vascular barrier function by establishing the normal balance between RhoA (Ras homolog family member A) and Rac-1-mediated actin cytoskeleton remodeling, a previously unappreciated function for the atypical GEF family of molecules. Our studies also identify S1P as a potential upstream regulator of DOCK4 activity.

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.

Interleukin-17 cytokines are critical in development of fatal lupus glomerulonephritis.

Systemic lupus erythematosus is a potentially fatal autoimmune disease. Although interleukin-17 (IL-17) has been linked to human lupus and mouse models of this disease, it has not been addressed whether this cytokine plays a critical role in fatal lupus pathology. Here we have demonstrated that increased production of IL-17 cytokines and their signaling via the adaptor protein CIKS (a.k.a. Traf3ip2, Act1) critically contributed to lethal pathology in an FcgammaR2b-deficient mouse model of lupus. Mice lacking IL-17 and especially those lacking CIKS showed greatly improved survival and were largely protected from development of glomerulonephritis. Importantly in this model, potential effects of IL-17 cytokines on antibody production could be distinguished from critical local contributions in kidneys, including recruitment of neutrophils and monocytes. These findings provide the proof of principle that signaling by IL-17 family cytokines mediated via CIKS presents promising therapeutic targets for the treatment of systemic lupus erythematosus, especially in cases with kidney involvement.

The many faces of Mac-1 in autoimmune disease.

Mac-1 (CD11b/CD18) is a ß2 integrin classically regarded as a pro-inflammatory molecule because of its ability to promote phagocyte cytotoxic functions and enhance the function of several effector molecules such as FcγR, uPAR, and CD14. Nevertheless, recent reports have revealed that Mac-1 also plays significant immunoregulatory roles, and genetic variants in ITGAM, the gene that encodes CD11b, confer risk for the autoimmune disease systemic lupus erythematosus (SLE). This has renewed interest in the physiological roles of this integrin and raised new questions on how its seemingly opposing biological functions may be regulated. Here, we provide an overview of the CD18 integrins and how their activation may be regulated as this may shed light on how the opposing roles of Mac-1 may be elicited. We then discuss studies that exemplify Mac-1's pro-inflammatory versus regulatory roles particularly in the context of IgG immune complex-mediated inflammation. This includes a detailed examination of molecular mechanisms that could explain the risk-conferring effect of rs1143679, a single nucleotide non-synonymous Mac-1 polymorphism associated with SLE.

Neutrophils in lupus nephritis.

PURPOSE OF REVIEW: Systemic lupus erythematosus (SLE) is a multiorgan autoimmune disease characterized by IgG-autoantibodies to nuclear antigens that can deposit in the kidney and trigger lupus nephritis. Neutrophils accumulate in the kidneys of patients with proliferative LUPUS NEPHRITIS and neutrophil products and a subset of granulocytes, called low-density granulocytes (LDG) may contribute to lupus nephritis pathogenesis. Here, we will discuss recent studies implicating neutrophils in the pathogenesis of human SLE nephritis and then examine studies that provide mechanistic insights into how these cells are recruited to the glomerulus following immune complex deposition and how their products may promote lupus nephritis. RECENT FINDINGS: SLE patients display unique blood transcriptional signatures linked to Type I interferon and myeloblast differentiation, which could help stratify lupus nephritis progression. Multiphoton intravital microscopy of kidney glomerular capillaries revealed a role for neutrophil FcγRs in the rapid capture of neutrophils following immune complex deposition. The view that reduced degradation of neutrophil extracellular traps (NETS) contributes to lupus nephritis progression, is now challenged by experimental data in lupus-prone mice that genetically fail to produce NETS but still are afflicted. SUMMARY: A greater understanding of the neutrophil dependent mechanisms that promote lupus nephritis may potentially inform on newer therapeutic options that target neutrophil accumulation and reactivity in the nephritic kidney.

Humanised effector-null FcγRIIA antibody inhibits immune complex-mediated proinflammatory responses.

OBJECTIVE: Immune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development. METHODS: VIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates. RESULTS: We generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies. CONCLUSIONS: VIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases.

The cerebral cavernous malformation proteins CCM2L and CCM2 prevent the activation of the MAP kinase MEKK3.

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe "big heart" and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.

AKAP9, a Regulator of Microtubule Dynamics, Contributes to Blood-Testis Barrier Function.

The blood-testis barrier (BTB), formed between adjacent Sertoli cells, undergoes extensive remodeling to facilitate the transport of preleptotene spermatocytes across the barrier from the basal to apical compartments of the seminiferous tubules for further development and maturation into spermatozoa. The actin cytoskeleton serves unique structural and supporting roles in this process, but little is known about the role of microtubules and their regulators during BTB restructuring. The large isoform of the cAMP-responsive scaffold protein AKAP9 regulates microtubule dynamics and nucleation at the Golgi. We found that conditional deletion of Akap9 in mice after the initial formation of the BTB at puberty leads to infertility. Akap9 deletion results in marked alterations in the organization of microtubules in Sertoli cells and a loss of barrier integrity despite a relatively intact, albeit more apically localized F-actin and BTB tight junctional proteins. These changes are accompanied by a loss of haploid spermatids due to impeded meiosis. The barrier, however, progressively reseals in older Akap9 null mice, which correlates with a reduction in germ cell apoptosis and a greater incidence of meiosis. However, spermiogenesis remains defective, suggesting additional roles for AKAP9 in this process. Together, our data suggest that AKAP9 and, by inference, the regulation of the microtubule network are critical for BTB function and subsequent germ cell development during spermatogenesis.

Human neutrophil Fcgamma receptors initiate and play specialized nonredundant roles in antibody-mediated inflammatory diseases.

Inflammation mediated by antibody-antigen complexes contributes to autoimmune diseases. Mice deficient in the common Fcgamma-chain are protected from IgG-mediated glomerulonephritis and the reverse passive Arthus (RPA) reaction and FcR-bearing macrophages, and mast cells have been assigned primary roles in these processes. Here we demonstrate that neutrophil-selective transgenic expression of the two uniquely human neutrophil Fc gamma receptors (FcgammaRs), FcgammaRIIA and FcgammaRIIIB, in Fcgamma-chain-deficient mice restored susceptibility to progressive glomerulonephritis and the cutaneous RPA reaction. FcgammaRIIIB and FcgammaRIIA mediated neutrophil accumulation, whereas FcgammaRIIA alone promoted organ injury. In a model of soluble immune complexes deposited within the vasculature, FcgammaRIIIB was responsible for neutrophil slow rolling and adhesion whereas in the cremaster RPA, induced by both vascular and tissue soluble immune complexes, FcgammaRIIA predominated. Thus, human FcgammaRs on neutrophils serve as molecular links between antibody and immunological disease, with FcgammaRIIA promoting tissue injury and FcgammaRIIIB and FcgammaRIIA displaying specialized context-dependent functions in neutrophil recruitment.

Caspase-8 modulates dectin-1 and complement receptor 3-driven IL-1ß production in response to ß-glucans and the fungal pathogen, Candida albicans.

Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1ß maturation and resistance to fungal dissemination in Candida albicans infection. ß-Glucans are major components of fungal cell walls that trigger IL-1ß secretion in both murine and human immune cells. In this study, we sought to determine the contribution of ß-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1ß production in response to ß-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating ß-glucan-induced IL-1ß processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting ß-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1ß maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1ß production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating ß-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between ß-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.

R-spondin3 prevents mesenteric ischemia/reperfusion-induced tissue damage by tightening endothelium and preventing vascular leakage.

Inflammation and vascular injury triggered by ischemia/reperfusion (I/R) represent a leading cause of morbidity and mortality in a number of clinical settings. Wnt and its homolog partners R-spondins, in addition to regulating embryonic development have recently been demonstrated to serve as wound-healing agents in inflammation-associated conditions. Here we ask whether R-spondins could prevent inflammation-associated tissue damage in ischemic disorders and thus investigate the role of R-spondin3 (R-spo3) in a mouse model of mesenteric I/R. We demonstrate that R-spo3 ameliorates mesenteric I/R-induced local intestinal as well as remote lung damage by suppressing local and systemic cytokine response and deposition of IgM and complement in intestinal tissues. We also show that decreased inflammatory response is accompanied by tightening of endothelial cell junctions and reduction in vascular leakage. We conclude that R-spo3 stabilizes endothelial junctions and inhibits vascular leakage during I/R and thereby mitigates the inflammatory events and associated tissue damage. Our findings uniquely demonstrate a protective effect of R-spo3 in I/R-related tissue injury and suggest a mechanism by which it may have these effects.

Dendritic Cells in Kidney Transplant Biopsy Samples Are Associated with T Cell Infiltration and Poor Allograft Survival.

Progress in long-term renal allograft survival continues to lag behind the progress in short-term transplant outcomes. Dendritic cells are the most efficient antigen-presenting cells, but surprisingly little attention has been paid to their presence in transplanted kidneys. We used dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin as a marker of dendritic cells in 105 allograft biopsy samples from 105 kidney transplant recipients. High dendritic cell density was associated with poor allograft survival independent of clinical variables. Moreover, high dendritic cell density correlated with greater T cell proliferation and poor outcomes in patients with high total inflammation scores, including inflammation in areas of tubular atrophy. We then explored the association between dendritic cells and histologic variables associated with poor prognosis. Multivariate analysis revealed an independent association between the densities of dendritic cells and T cells. In biopsy samples with high dendritic cell density, electron microscopy showed direct physical contact between infiltrating lymphocytes and cells that have the ultrastructural morphologic characteristics of dendritic cells. The origin of graft dendritic cells was sought in nine sex-mismatched recipients using XY fluorescence in situ hybridization. Whereas donor dendritic cells predominated initially, the majority of dendritic cells in late allograft biopsy samples were of recipient origin. Our data highlight the prognostic value of dendritic cell density in allograft biopsy samples, suggest a new role for these cells in shaping graft inflammation, and provide a rationale for targeting dendritic cell recruitment to promote long-term allograft survival.

TNF receptors: signaling pathways and contribution to renal dysfunction.

Tumor necrosis factor (TNF), initially reported to induce tumor cell apoptosis and cachexia, is now considered a central mediator of a broad range of biological activities from cell proliferation, cell death and differentiation to induction of inflammation and immune modulation. TNF exerts its biological responses via interaction with two cell surface receptors: TNFR1 and TNFR2. (TNFRs). These receptors trigger shared and distinct signaling pathways upon TNF binding, which in turn result in cellular outputs that may promote tissue injury on one hand but may also induce protective, beneficial responses. Yet the role of TNF and its receptors specifically in renal disease is still not well understood. This review describes the expression of the TNFRs, the signaling pathways induced by them and the biological responses of TNF and its receptors in various animal models of renal diseases, and discusses the current outcomes from use of TNF biologics and TNF biomarkers in renal disorders.

Endocytosis of soluble immune complexes leads to their clearance by FcγRIIIB but induces neutrophil extracellular traps via FcγRIIA in vivo.

Soluble immune complexes (ICs) are abundant in autoimmune diseases, yet neutrophil responses to these soluble humoral factors remain uncharacterized. Moreover, the individual role of the uniquely human FcγRIIA and glycophosphatidylinositol (GPI)-linked FcγRIIIB in IC-mediated inflammation is still debated. Here we exploited mice and cell lines expressing these human neutrophil FcγRs to demonstrate that FcγRIIIB alone, in the absence of its known signaling partners FcγRIIA and the integrin Mac-1, internalizes soluble ICs through a mechanism used by GPI-anchored receptors and fluid-phase endocytosis. FcγRIIA also uses this pathway. As shown by intravital microscopy, FcγRIIA but not FcγRIIIB-mediated neutrophil interactions with extravascular soluble ICs results in the formation of neutrophil extracellular traps (NETs) in tissues. Unexpectedly, in wild-type mice, IC-induced NETosis does not rely on the NADPH oxidase, myeloperoxidase, or neutrophil elastase. In the context of soluble ICs present primarily within vessels, FcγRIIIB-mediated neutrophil recruitment requires Mac-1 and is associated with the removal of intravascular IC deposits. Collectively, our studies assign a new role for FcγRIIIB in the removal of soluble ICs within the vasculature that may serve to maintain homeostasis, whereas FcγRIIA engagement of tissue soluble ICs generates NETs, a proinflammatory process linked to autoimmunity.

Cutting edge: protein phosphatase 2A confers susceptibility to autoimmune disease through an IL-17-dependent mechanism.

The contribution of individual molecular aberrations to the pathogenesis of systemic lupus erythematosus (SLE), an autoimmune disease that affects multiple organs, is often difficult to evaluate because of the presence of abundant confounding factors. To assess the effect of increased expression of the phosphatase protein phosphatase 2A (PP2A) in T cells, as recorded in SLE patients, we generated a transgenic mouse that overexpresses the PP2Ac subunit in T cells. The transgenic mouse displays a heightened susceptibility to immune-mediated glomerulonephritis in the absence of other immune defects. CD4(+) T cells produce increased amounts of IL-17 while the number of neutrophils in the peripheral blood is increased. IL-17 neutralization abrogated the development of glomerulonephritis. We conclude that increased PP2Ac expression participates in SLE pathogenesis by promoting inflammation through unchecked IL-17 production and facilitating the development of end-organ damage.

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.

AKAP9 regulation of microtubule dynamics promotes Epac1-induced endothelial barrier properties.

Adhesive forces at endothelial cell-cell borders maintain vascular integrity. cAMP enhances barrier properties and controls cellular processes through protein kinase A bound to A-kinase anchoring proteins (AKAPs). It also activates exchange protein directly activated by cAMP (Epac1), an exchange factor for Ras-related protein 1 (Rap1) GTPases that promotes cadherin- and integrin-mediated adhesion through effects on the actin cytoskeleton. We demonstrate that AKAP9 facilitates the microtubule polymerization rate in endothelial cells, interacts with Epac1, and is required for Epac1-stimulated microtubule growth. AKAP9 is not required for maintaining barrier properties under steady-state conditions. Rather, it is essential when the cell is challenged to make new adhesive contacts, as is the case when Epac activation enhances barrier function through a mechanism that, surprisingly, requires integrin adhesion at cell-cell contacts. In the present study, defects in Epac-induced responses in AKAP9-silenced cells were evident despite an intact Epac-induced increase in Rap activation, cortical actin, and vascular endothelial-cadherin adhesion. We describe a pathway that integrates Epac-mediated signals with AKAP9-dependent microtubule dynamics to coordinate integrins at lateral borders.

Circulating TNF receptors 1 and 2 predict ESRD in type 2 diabetes.

Levels of proinflammatory cytokines associate with risk for developing type 2 diabetes but whether chronic inflammation contributes to the development of diabetic complications, such as ESRD, is unknown. In the 1990s, we recruited 410 patients with type 2 diabetes for studies of diabetic nephropathy and recorded their characteristics at enrollment. During 12 years of follow-up, 59 patients developed ESRD (17 per 1000 patient-years) and 84 patients died without ESRD (24 per 1000 patient-years). Plasma markers of systemic inflammation, endothelial dysfunction, and the TNF pathway were measured in the study entry samples. Of the examined markers, only TNF receptors 1 and 2 (TNFR1 and TNFR2) associated with risk for ESRD. These two markers were highly correlated, but ESRD associated more strongly with TNFR1. The cumulative incidence of ESRD for patients in the highest TNFR1 quartile was 54% after 12 years but only 3% for the other quartiles (P<0.001). In Cox proportional hazard analyses, TNFR1 predicted risk for ESRD even after adjustment for clinical covariates such as urinary albumin excretion. Plasma concentration of TNFR1 outperformed all tested clinical variables with regard to predicting ESRD. Concentrations of TNFRs moderately associated with death unrelated to ESRD. In conclusion, elevated concentrations of circulating TNFRs in patients with type 2 diabetes at baseline are very strong predictors of the subsequent progression to ESRD in subjects with and without proteinuria.

Circulating TNF receptors 1 and 2 predict stage 3 CKD in type 1 diabetes.

Elevated plasma concentrations of TNF receptors 1 and 2 (TNFR1 and TNFR2) predict development of ESRD in patients with type 2 diabetes without proteinuria, suggesting these markers may contribute to the pathogenesis of renal decline. We investigated whether circulating markers of the TNF pathway determine GFR loss among patients with type 1 diabetes. We followed two cohorts comprising 628 patients with type 1 diabetes, normal renal function, and no proteinuria. Over 12 years, 69 patients developed estimated GFR less than 60 mL/min per 1.73 m(2) (16 per 1000 person-years). Concentrations of TNFR1 and TNFR2 were strongly associated with risk for early renal decline. Renal decline was associated only modestly with total TNFα concentration and appeared unrelated to free TNFα. The cumulative incidence of estimated GFR less than 60 mL/min per 1.73 m(2) for patients in the highest TNFR2 quartile was 60% after 12 years compared with 5%-19% in the remaining quartiles. In Cox proportional hazards analysis, patients with TNFR2 values in the highest quartile were threefold more likely to experience renal decline than patients in the other quartiles (hazard ratio, 3.0; 95% confidence interval, 1.7-5.5). The risk associated with high TNFR1 values was slightly less than that associated with high TNFR2 values. TNFR levels were unrelated to baseline free TNFα level and remained stable over long periods within an individual. In conclusion, early GFR loss in patients with type 1 diabetes without proteinuria is strongly associated with circulating TNF receptor levels but not TNFα levels (free or total).

Complement component C3 and complement receptor type 3 contribute to the phagocytosis and clearance of fibrillar Aß by microglia.

Complement components and their receptors are found within and around amyloid ß (Aß) cerebral plaques in Alzheimer's disease (AD). Microglia defend against pathogens through phagocytosis via complement component C3 and/or engagement of C3 cleavage product iC3b with complement receptor type 3 (CR3, Mac-1). Here, we provide direct evidence that C3 and Mac-1 mediate, in part, phagocytosis and clearance of fibrillar amyloid-ß (fAß) by murine microglia in vitro and in vivo. Microglia took up not only synthetic fAß(42) but also amyloid cores from patients with AD, transporting them to lysosomes in vitro. Fibrillar Aß(42) uptake was significantly attenuated by the deficiency or knockdown of C3 or Mac-1 and scavenger receptor class A ligands. In addition, C3 or Mac-1 knockdown combined with a scavenger receptor ligand, fucoidan, further attenuated fibrillar Aß(42) uptake by N9 microglia. Fluorescent fibrillar Aß(42) microinjected cortically was significantly higher in C3 and Mac-1 knockout mice compared with wild-type mice 5 days after surgery, indicating reduced clearance in vivo. Together, these results demonstrate that C3 and Mac-1 are involved in phagocytosis and clearance of fAß by microglia, providing support for a potential beneficial role for microglia and the complement system in AD pathogenesis. © 2012 Wiley Periodicals, Inc.