Deposition Of IgG Immune Complexes Research Articles

Fangji Huangqi decoction ameliorates membranous nephropathy through the upregulation of BNIP3-mediated mitophagy

Ethnopharmacological relevanceFangji Huangqi Decoction (FJHQ), a traditional Chinese medicinal formula outlined in Zhang Zhongjing's “Jin Gui Yao Lue” during the Han Dynasty, is often used to treat conditions characterized by symptoms like edema and dysuria, including membranous nephropathy (MN). Despite its proven clinical effectiveness, the exact mechanisms through which FJHQ acts on MN remain elusive. Aim of the studyThis study aimed to investigate whether FJHQ enhances BNIP3-mediated mitophagy in podocytes by promoting BNIP3 expression and whether this improvement leads to the amelioration of MN. Materials and methodsIn this study, by establishing passive Heymann nephritis (PHN) rats, an experimental rat model of MN induced by sheep anti-rat Fx1A serum, we evaluated the effects of FJHQ in vivo. In vitro experiments were carried out by treating primary podocytes with experimental rat serum. Furthermore, the potential mechanism by which FJHQ acts through BNIP3 was further examined by transfecting primary podocytes with the siRNA of BNIP3 or the corresponding control vector. ResultsAfter 4 weeks, significant kidney damage was observed in the rats in the model group, comparatively, FJHQ markedly decreased urine volume, 24-h urinary protein, blood urea nitrogen (BUN), creatinine (Scr), and increased serum total albumin (ALB). Histology showed that FJHQ caused significant improvements in glomerular hyperplasia, and IgG immune complex deposition in MN rats. JC-1 fluorescence labelling and flow cytometry analysis showed that FJHQ could significantly increase mitochondrial membrane potential in vivo. In the mitochondria of MN model rats, FJHQ was able to down-regulate the expression of P62 and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I, according to Western blot and immunofluorescence studies. Furthermore, FJHQ has been shown to significantly up-regulate mitochondrial membrane potential, down-regulate P62 expression in mitochondria, and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I in mitochondria at the cellular level. After the administration of the autophagy inhibitor chloroquine, the serum of rats treated with FJHQ further increased the expression of LC3 II/LC3 I in primary podocytes, showing higher autophagy flow. After the interference of BNIP3 in podocytes, the effect of FJHQ on mitochondrial membrane potential and autophagy-related proteins almost disappeared. ConclusionFJHQ enhanced mitophagy in podocytes by promoting the expression of BNIP3, thereby contributing to the amelioration of MN. This work reveals the possible underlying mechanism by which FJHQ improves MN and provides a new avenue for MN treatment.

IgG immune complex-induced acute lung injury is ameliorated by cAMP via down-regulation of C/EBP- and AP-1-mediated transcriptions

BackgroundAcute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are life threatening pulmonary diseases, and we are now lack of effective therapeutic methods. Inflammatory responses are essential for initiating ALI/ARDS. Thus, ameliorating inflammatory reaction might be beneficial for treatment of the disease. There are increasing data that phosphodiesterase-4 (PDE4)-selective inhibitors, which may elevate cellular cyclic adenosine 3′, 5′-monophosphate (cAMP) level, could suppress inflammation. However, whether they could be used to treat IgG immune complex (IgG-IC)-associated ALI has not been determined.MethodsALI is induced by treating mice with airway deposition of IgG immune complexes. Cellular cAMP concentrations are elevated by treating mice or macrophages with Rolipram/Roflumilast. The degree of pulmonary injury is reflected by lung permeability, leukocyte accumulation, histological change and expressions of pro-inflammatory mediators. 6-Bnz-cAMP and H-89 are used to regulate protein kinase A (PKA) activity, and 8-pCPT-2′-O-Me-cAMP is applied to activate exchange proteins directly activated by cAMP (Epac). Gene expressions are analyzed by real-time PCR, ELISA or Western blot. CCAAT/enhancer binding protein (C/EBP) and activation protein 1 (AP-1) transcription activities are estimated by measuring the luciferase productions.ResultsIgG-IC-induced ALI is attenuated by the PDE4-selective inhibitor, which is due to reduced expressions of cytokine and chemokines. Interestingly, we find that cAMP downstream effector molecule PKA but not Epac is involved in negative regulation of IgG-IC-mediated pro-inflammatory mediators’ productions. Mechanistically, activation of cAMP-PKA signal axis leads to inactivation of MAPK pathway, resulting in a decrease in C/EBP- and AP-1-mediated transcriptions of pro-inflammatory mediators.ConclusionsOur data demonstrate, for the first time, that cAMP-PKA signal is involved in down-regulation of IgG-IC-associated inflammatory responses via down-regulating MAPK activation, which is critical for transcriptional activities of C/EBP and AP-1. Collectively, our experiments provide theoretical base for the potential application of PDE4-selective inhibitor to clinic for treatment of IgG-IC-related acute lung injury.

Jianpi Qushi Heluo Formula alleviates renal damages in Passive Hemann nephritis in rats by upregulating Parkin-mediated mitochondrial autophagy

Jianpi Qushi Heluo Formula (JQHF) is an empirical traditional Chinese medicine prescription for treating Membranous Nephropathy (MN) clinically in China. The therapeutic effect of JQHF has been reported in our previous studies. However, the exact mechanism is still unknown. In this study, by establishing an experimental rat model of MN induced by Sheep anti-rat Fx1A serum, we evaluated the effects of JQHF and Tetrandrine (TET), and Benazepril was used as a positive control. As an autophagy agonist, TET is one of the most active components in JQHF. After 4 weeks, significant kidney damage was observed in the rats in the Model group; comparatively, JQHF markedly decreased 24 h urinary protein, Total Cholesterol (TC), and increased serum total Albumin (ALB). Histology showed that JQHF caused significant improvements in glomerular hyperplasia, renal tubular damage, IgG immune complex deposition, and the ultrastructure of mitochondria in MN rats. Flow cytometry analysis showed that treatment with JQHF reduced the level of reactive oxygen species and apoptosis rate, and upregulated mitochondrial membrane potential. Western blot analysis demonstrated that JQHF could protect against mitochondrial dysfunction and apoptosis by upregulating the expression of PINK1, Mitochondrial Parkin, and LC3-II/I, downregulating the expression of Cytoplasmic Parkin, P62, Cytochrome c, and Caspase-3 in the kidneys of MN rats. From images of co-immunofluorescence, it is observed significantly increase in the co-localization of PINK1 and Parkin, as well as LC3 and mitochondria. Similarly, TET treatment significantly upregulated the mitochondrial autophagy and reduced apoptosis in rats after 4 weeks compared with the model group. Comparatively, the ability of JQHF to alleviate renal damage was significantly higher than those of Benazepril and TET. It was demonstrated that JQHF could delay pathology damage to the kidney and hold back from the progression of MN by inhibiting apoptosis and upregulating the mitochondrial autophagy by PINK1/Parkin pathways.

SHIP-1 Deficiency in AID+ B Cells Leads to the Impaired Function of B10 Cells with Spontaneous Autoimmunity.

Unlike conventional B cells, regulatory B cells exhibit immunosuppressive functions to downregulate inflammation via IL-10 production. However, the molecular mechanism regulating the production of IL-10 is not fully understood. In this study, we report the finding that activation-induced cytidine deaminase (AID) is highly upregulated in the IL-10-competent B cell (B10) cell from Innp5dfl/flAicdaCre/+ mice, whereas the 5' inositol phosphatase SHIP-1 is downregulated. Notably, SHIP-1 deficiency in AID+ B cells leads to a reduction in cell count and impaired IL-10 production by B10 cells. Furthermore, the Innp5dfl/flAicdaCre/+ mouse model shows B cell-dependent autoimmune lupus-like phenotypes, such as elevated IgG serum Abs, formation of spontaneous germinal centers, production of anti-dsDNA and anti-nuclear Abs, and the obvious deposition of IgG immune complexes in the kidney with age. We observe that these lupus-like phenotypes can be reversed by the adoptive transfer of B10 cells from control Innp5dfl/fl mice, but not from the Innp5dfl/flAicdaCre/+ mice. This finding highlights the importance of defective B10 cells in Innp5dfl/flAicdaCre/+ mice. Whereas p-Akt is significantly upregulated, MAPK and AP-1 activation is impaired in B10 cells from Innp5dfl/flAicdaCre/+ mice, resulting in the reduced production of IL-10. These results show that SHIP-1 is required for the maintenance of B10 cells and production of IL-10, and collectively suggests that SHIP-1 could be a new potential therapeutic target for the treatment of autoimmune diseases.

Partial protection from lupus-like disease in B-cell specific IFNAR-deficient B6.Nba2 mice

Abstract A majority of patients presenting with systemic lupus erythematosus (SLE) have increased serum concentrations of interferon alpha (IFNα). IFNα has previously been shown to affect lymphocyte activities such as B cell differentiation and T cell maintenance, but whether IFNα signaling specifically in B cells is required for disease pathology remains unknown. In order to examine the role of IFNα stimulation on B cells, we backcrossed mice lacking the IFNα receptor on B cells, Mb1.cre IFNARflx/flx, with the B6.Nba2 lupus mouse model. B6.Nba2.BΔIFNAR mice were evaluated alongside littermate control mice for splenomegaly, abnormal spleen subset distribution, IgG immune-complex deposition in the kidney glomeruli. Serum total IgM, total IgG and anti-chromatin IgG levels were determined monthly. BΔIFNAR mice display normal IgM and IgG levels, but decreased serum antinuclear autoantibody levels (p<0.05), while deposition of IgG immune complexes in kidney glomeruli was similar to that of control mice. Both spleen weight and total splenocyte number were found to be significantly decreased in BΔIFNAR mice (p<0.05). Analyses of spleen cell subsets at 4 months of age revealed a trend towards decreased plasma cells and GC B cells in spleens of BΔIFNAR mice. Despite IFNAR being removed from B cells only, we observed a shift in the balance of effector/memory to naïve CD4 T cells in BΔIFNAR mice. In summary, we found that B6.Nba2.BΔIFNAR mice were partly protected from lupus pathology including splenomegaly, autoantibody production and B cell hyperactivation. Most surprisingly, T cell activation appeared to be diminished in B6.Nba2.BΔIFNAR mice, suggesting that B cells play a central role as both APCs and ASCs in the B6.Nba2 mouse model of lupus.

Protective Role of Rho Guanosine Diphosphate Dissociation Inhibitor, Ly-GDI, in Pulmonary Alveolitis.

Growing evidences indicate that Ly-GDI, an inhibitory protein of Rho GTPases, plays an essential role in regulating actin cytoskeletal alteration which is indispensible for the process such as phagocytosis. However, the role of Ly-GDI in inflammation remains largely unknown. In the current study, we found that Ly-GDI expression was significantly decreased in the IgG immune complex-injured lungs. To determine if Ly-GDI might regulate the lung inflammatory response, we constructed adenovirus vectors that could mediate ectopic expression of Ly-GDI (Adeno-Ly-GDI). In vivo mouse lung expression of Ly-GDI resulted in a significant attenuation of IgG immune complex-induced lung injury, which was due to the decreased pulmonary permeability and lung inflammatory cells, especially neutrophil accumulation. Upon IgG immune complex deposition, mice with Ly-GDI over-expression in the lungs produced significant less inflammatory mediators (TNF-α, IL-6, MCP-1, and MIP-1α) in bronchoalveolar lavage fluid when compared control mice receiving airway injection of Adeno-GFP. Mechanically, IgG immune complex-induced NF-κB activity was markedly suppressed by Ly-GDI in both alveolar macrophages and lungs as measured by luciferase assay and electrophoretic mobility shift assay. These findings suggest that Ly-GDI is a critical regulator of inflammatory injury after deposition of IgG immune complexes and that it negatively regulates the lung NF-κB activity.

Protective Role of Rho Guanosine Diphosphate (GDP)‐Dissociation Inhibitor Ly‐GDI in Acute Pulmonary Alveolitis

Ly‐GDI is an inhibitory protein of Rho GTPases. Growing evidences indicate that Ly‐GDI plays an essential role in regulating actin cytoskeletal alteration which is indispensible for the process such as phagocytosis. However, the role of Ly‐GDI in inflammation remains largely unknown. In the current study, we found that Ly‐GDI expression was significantly decreased in the IgG immune complex‐injured lungs. To determine if Ly‐GDI might regulate the lung inflammatory response, we constructed adenovirus vectors that could mediate ectopic expression of Ly‐GDI (Adeno‐Ly‐GDI). In vivo mouse lung expression of Ly‐GDI resulted in a significant attenuation of IgG immune complex‐induced lung injury, which was due to the decreased pulmonary permeability and lung inflammatory cells, especially neutrophil accumulation. Upon IgG immune complex deposition, mice with Ly‐GDI over‐expression in the lungs produced significant less inflammatory mediators (TNF‐α, IL‐6, MCP‐1, and MIP‐1α) in bronchoalveolar lavage fluid when compared control mice receiving airway injection of Adeno‐GFP. Mechanically, IgG immune complex‐induced NF‐κB activity was markedly suppressed by Ly‐GDI in both alveolar macrophages and lungs as measured by luciferase assay and electrophoretic mobility shift assay. These findings suggest that Ly‐GDI is a critical regulator of inflammatory injury after deposition of IgG immune complexes and that it negatively regulates the lung NF‐kB activity.

Sialic acid-binding immunoglobulin-type lectin H-positive plasmacytoid dendritic cells drive spontaneous lupus-like disease development in B6.Nba2 mice.

Patients with systemic lupus erythematosus (SLE) often present with elevated levels of interferon-α (IFNα) in serum. Plasmacytoid dendritic cells (pDCs) have been suggested to be the primary source of IFNα in SLE due to their capacity to produce high levels of IFNα. During viral infection, a subset of pDCs expressing sialic acid-binding immunoglobulin-type lectin H (Siglec H) produces the majority of pDC-derived IFNα. The aim of this study was to provide evidence that Siglec H-positive pDCs are pathogenic in the IFNα-dependent B6.Nba2 mouse model of lupus. B6.Nba2 blood dendritic cell antigen 2 (BDCA-2)-diphtheria toxin receptor (DTR)-transgenic (Tg) mice were treated intraperitoneally with DT 3 times weekly starting at 4 weeks or 12 weeks of age and analyzed at 12 weeks and 18 weeks of age, respectively. Lupus-like disease development was measured by the presence of elevated levels of autoantibodies in serum (as determined by enzyme-linked immunosorbent assay), increased expression of IFN-inducible genes (as determined by real-time reverse transcription-polymerase chain reaction), increased IgG immune complex deposition in kidney glomeruli (as determined by immunofluorescence staining), spontaneous lymphocyte activation, and differentiation of B cells into antibody-producing plasma cells (as determined by flow cytometry). B6.Nba2 mice in which Siglec H-positive pDCs were depleted for 6-8 weeks displayed reduced levels of IFNα-induced gene transcripts and decreased anti-chromatin autoantibody levels in serum, and significantly fewer activated splenic T cells and B cells, germinal center B cells, follicular helper T cells, and splenic plasma cells. In 18-week-old mice, IgG immune complex deposition in kidney glomeruli was similarly reduced. The development of lupus-like disease in congenic B6.Nba2 mice depends on Siglec H-positive pDCs. We suggest that depletion of Siglec H-positive pDCs represents a novel cellular target in SLE.

The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity

Tyrosine phosphorylation of signaling molecules that mediate B cell activation in response to various stimuli is tightly regulated by protein tyrosine phosphatases (PTPs). PTP1B is a ubiquitously expressed tyrosine phosphatase with well-characterized functions in metabolic signaling pathways. We show here that PTP1B negatively regulates CD40, B cell activating factor receptor (BAFF-R), and TLR4 signaling in B cells. Specifically, PTP1B counteracts p38 mitogen-activated protein kinase (MAPK) activation by directly dephosphorylating Tyr(182) of this kinase. Mice with a B cell-specific PTP1B deficiency show increased T cell-dependent immune responses and elevated total serum IgG. Furthermore, aged animals develop systemic autoimmunity with elevated serum anti-dsDNA, spontaneous germinal centers in the spleen, and deposition of IgG immune complexes and C3 in the kidney. In a clinical setting, we observed that B cells of rheumatoid arthritis patients have significantly reduced PTP1B expression. Our data suggest that PTP1B plays an important role in the control of B cell activation and the maintenance of immunological tolerance.

Persistent Neutrophil Dysfunction and Suppression of Acute Lung Injury in Mice following Cecal Ligation and Puncture Sepsis

Sepsis, both in humans and in rodents, is associated with persistent immunosuppression accompanied by defects in innate immunity during the acute phase of sepsis. Mice were rendered septic by cecal ligation and puncture (CLP) followed by the induction of acute lung injury, employing distal airway deposition of IgG immune complexes, in order to quantitatively evaluate innate immune responses following the induction of sepsis. Suppression of innate immune responses in the lung occurred as early as 12 h after CLP and up to 21 days thereafter. The mechanism of innate immune defects included a reduced leak of albumin into the lungs together with reduced levels of tumor necrosis factor in bronchoalveolar lavage fluids and increased levels of interleukin-10 that were persistent. Bone marrow-derived neutrophils (polymorphonuclear neutrophils; PMNs) from CLP mice also had reduced levels of the activation marker CD11b and a depressed respiratory burst following stimulation in vitro. These results were not observed in mice with endotoxemia, where the innate inflammatory response was preserved. However, sustained lymphopenia was present in both models, suggesting differential regulation of innate and adaptive immunity in the two sepsis models. These data indicate that CLP induced a prolonged suppression of inflammatory responses both in the lung and systemically, as defined by bone marrow-derived PMN dysfunction.

Zonulin as prehaptoglobin2 regulates lung permeability and activates the complement system

Zonulin is a protein involved in the regulation of tight junctions (TJ) in epithelial or endothelial cells. Zonulin is known to affect TJ in gut epithelial cells, but little is known about its influences in other organs. Prehaptoglobin2 has been identified as zonulin and is related to serine proteases (MASPs, C1qrs) that activate the complement system. The current study focused on the role of zonulin in development of acute lung injury (ALI) in C57BL/6 male mice following intrapulmonary deposition of IgG immune complexes. A zonulin antagonist (AT-1001) and a related peptide with permeability agonist activities (AT-1002) were employed and given intratracheally or intravenously. Also, zonulin was blocked in lung with a neutralizing antibody. In a dose-dependent manner, AT-1001 or zonulin neutralizing antibody attenuated the intensity of ALI (as quantitated by albumin leak, neutrophil accumulation, and proinflammatory cytokines). A similar pattern was found using the bacterial lipopolysaccharide model of ALI. Using confocal microscopy on sections of injured lungs, staining patterns for TJ proteins were discontinuous, reduced, and fragmented. As expected, the leak of blood products into the alveolar space confirmed the passage of 3 and 20 kDa dextran, and albumin. In contrast to AT-1001, application of the zonulin agonist AT-1002 intensified ALI. Zonulin both in vitro and in vivo induced generation of complement C3a and C5a. Collectively, these data suggest that zonulin facilitates development of ALI both by enhancing albumin leak and complement activation as well as increased buildup of neutrophils and cytokines during development of ALI.

Curcumin attenuates lupus nephritis upon interaction with regulatory T cells in New Zealand Black/White mice

Curcumin has been used in Asian traditional medicine for its medicinal properties. Recent studies have demonstrated that curcumin has antioxidant, anti-tumour and anti-inflammatory activities. The aim of the present study is to investigate the effects of curcumin on established lupus nephritis (LN) in New Zealand Black/White (NZB/W) F1 female mice, in particular, its interaction with regulatory T (Treg) cells. Starting at 18 weeks of age, mice were fed a standard diet or a diet containing 1 % curcumin until the end of the study. The proteinuria level and the serum levels of IgG1, IgG2a and anti-double-stranded DNA (dsDNA) IgG antibodies were measured. Additionally, IgG immune complex deposition in the glomeruli and renal inflammation were compared between curcumin-treated mice and control mice. Curcumin decreased the proteinuria level and serum levels of IgG1, IgG2a and anti-dsDNA IgG antibodies in NZB/W F1 female mice. IgG immune complex deposition in the glomeruli was reduced in curcumin-treated mice. Furthermore, renal inflammation was also decreased after curcumin treatment. Interestingly, these therapeutic effects of curcumin disappeared after Treg depletion by anti-CD25 antibody injection. Curcumin exerted a protective effect against LN in NZB/W F1 mice. We speculate that the protective effects of curcumin in LN may involve, at least in part, its interaction with Treg cells.

Critical Role for CCAAT/Enhancer-Binding Protein β in Immune Complex-Induced Acute Lung Injury

C/EBPs, particularly C/EBPβ and C/EBPδ, are known to participate in the regulation of many genes associated with inflammation. However, very little is known regarding the activation and functions of C/EBPβ and C/EBPδ in acute lung inflammation and injury. In this study, we show that both C/EBPβ and C/EBPδ activation are triggered in lungs and in alveolar macrophages following intrapulmonary deposition of IgG immune complexes. We further show that mice carrying a targeted deletion of the C/EBPβ gene displayed significant attenuation of the permeability index (lung vascular leak of albumin), lung neutrophil accumulation (myeloperoxidase activity), total number of WBCs, and neutrophils in bronchoalveolar lavage fluids compared with wild-type mice. Moreover, the mutant mice expressed considerably less TNF-α, IL-6, and CXC/CC chemokine and soluble ICAM-1 proteins in bronchoalveolar lavage fluids, and corresponding mRNAs in the IgG immune complex-injured lung, compared with wild-type mice. These phenotypes were associated with a significant reduction in morphological lung injury. In contrast, C/EBPδ deficiency had no effect on IgG immune complex-induced lung injury. IgG immune complex-stimulated C/EBPβ-deficient alveolar macrophages released significantly less TNF-α, IL-6, MIP-2, keratinocyte cell-derived chemokine, and MIP-1α compared with wild-type cells. Similar decreases in IgG immune complex-induced inflammatory mediator production were observed following small interfering RNA ablation of C/EBPβ in a murine alveolar macrophage cell line. These findings implicate C/EBPβ as a critical regulator of IgG immune complex-induced inflammatory responses and injury in the lung.

Therapeutic Effect of the Impatiens balsamina Linne Extract on the Membranous Nephropathy

Objective : Membranous nephropathy(MN) is one of the most common causes of nephrotic syndrome in adults. MN has been defined as granular subepithelial deposition of IgG immune complexes along the glomerular basement membrane(GBM). However, there is not a satisfactory treatment for MN. We aimed to identify the effect of Impatiens balsamina Linne(IBL) treatment on cationic bovine serum albumin(cBSA)-induced MN in a mouse model. Methods : Mice were divided into 4 groups. The normal group was injected with saline. The Control group was treated with cBSA(50 mg/kg i.p) only. The third group IBL-100, was treated with cBSA(50 mg/kg, i.p) and IBL(100 mg/kg, p.o). The fourth group IBL-400, was treated with cBSA(50 mg/kg, i.p) and IBL (400 mg/kg, p.o). After cBSA and IBL treatment for 6 weeks, we measured change of body weight, proteinuria, serum albumin, total cholesterol, triglyceride, BUN, creatinine, IgA, IgM, IgG, TNF-, IL-6 and IL- levels. The morphologic changes of renal glomeruli were also observed with a light microscope. Results : The level of proteinuria significantly decreased and serum albumin increased in groups treated with cBSA and IBL extract compared with the control. The levels of serum triglyceride, BUN, IgG, TNF-, IL- significantly decreased in both IBL groups. In histological findings of kidney tissue, thickening of GBM decreased in both IBL groups. Conclusions : This study shows that IBL might be effective for treatment of acute stage MN. More clinical data and studies are to be done for efficient application.

Attenuation of IgG immune complex‐induced acute lung injury by silencing C5aR in lung epithelial cells

Acute lung injury (ALI) in mouse lung occurs after distal airway deposition of IgG immune complexes (IgGICs), resulting in a breakdown of the vascular-airway barrier, causing intrapulmonary edema, hemorrhage, and accumulation of neutrophils [polymorphonuclear leukocytes (PMNs)] in the alveolar compartment, these changes being complement (C5a) and C5a receptor (C5aR) dependent. In this ALI model, C5aR expression (protein) was found to occur on upper (bronchial) and lower (alveolar) airway epithelial cells. An adenovirus construct (siRNA) was used to silence mRNA for C5aR in the lung. Under such conditions, C5aR protein was markedly reduced on lung epithelial cells, resulting in much reduced leakage of albumin into the lung, diminished buildup of PMNs, and lower levels of proinflammatory mediators in bronchoalveolar lavage fluids. These studies indicate that bronchial and alveolar epithelial cell C5aR is up-regulated and greatly contributes to inflammation and injury in the lung. The use of siRNA administered into the airways avoids systemic suppression of C5aR, which might compromise innate immunity. It is possible that such an intervention might be employed in humans with ALI or acute respiratory distress syndrome as well as in upper-airway inflammatory diseases, such as chronic obstructive pulmonary disease and asthma, where there is evidence for complement activation and buildup of PMNs.

Asbestos-Induced Autoimmunity in C57Bl/6 Mice

Environmental impacts on autoimmunity have significant public health implications. Epidemiological studies have shown associations between exposure to airborne silicates, such as crystalline silica or asbestos, and autoimmunity, but the etiology remains unclear. The purpose of this study was to test the hypothesis that asbestos could lead to a specific pattern of autoantibodies and pathology indicative of systemic autoimmune disease (SAID). Female C57Bl/6 mice were instilled intratracheally with 2 doses × 60 μ g/mouse of amphibole asbestos (tremolite), wollastonite (a non-fibrogenic control fiber), or saline alone. Serum samples were collected and urine was checked for protein bi-weekly for 7 months. By 26 weeks, the asbestos-instilled animals had a significantly higher frequency of positive anti-nuclear antibody (ANA) tests compared to wollastonite and saline groups. The majority of positive ANAs showed homogeneous or combined homogeneous/speckled patterns, and tested positive for antibodies to dsDNA and SSA/Ro 52. Serum isotyping showed no significant changes in IgM, IgA, or IgG subclasses. However, there was an overall decrease in the mean IgG serum concentration in asbestos-instilled mice. IgG immune complex deposition was demonstrated in the kidneys of asbestos-instilled mice, with evidence of glomerular and tubule abnormalities suggestive of glomerulonephritis. Flow cytometry demonstrated moderate changes in the percentages of CD25+ T-suppressor cells and B1a B-cells in the superficial cervical lymph nodes of the asbestos-instilled mice. These data demonstrate that asbestos leads to immunologic changes consistent with the development of autoimmunity. This study provides a non-autoimmune prone murine model for use in future elucidation of mechanisms involved in asbestos-induced autoimmune disease.

Adenoviral-Mediated Overexpression of SOCS3 Enhances IgG Immune Complex-Induced Acute Lung Injury

The lung inflammatory response caused by intratracheal deposition of IgG immune complexes (IC) includes the production of IL-6, which signals through activation of STAT transcription factors. Recently, suppressor of cytokine signaling 3 (SOCS3) has been shown to be a key negative regulator of IL-6/gp130/Jak/STAT3 signal transduction. Although SOCS3 has been implicated in several inflammatory diseases, very little is known regarding its activation and its function in the lung during acute inflammation. Our previous study showed that IL-6/STAT3 activation was triggered in lungs after intrapulmonary deposition of IgG IC in rats. In the current study, we sought to determine whether SOCS3 is playing a regulatory role in the lung inflammatory response. SOCS3 induction occurred during development of inflammation in the IgG IC model of lung injury. Overexpression of SOCS3 in lung using a recombinant adenovirus encoding murine SOCS3 resulted in substantial increases in lung vascular permeability and lung myeloperoxidase, together with enhanced levels of TNF-alpha, MIP-2, and keratinocyte-activated cytokine in bronchoalveolar lavage fluids. SOCS3 overexpression in lungs led to overproduction of bronchoalveolar lavage IL-6, but not IL-10, in this inflammatory model. We further show that activation of STAT3 was inhibited by SOCS3 overexpression as well as by anti-IL-6 treatment during IgG IC-induced lung injury, as determined by EMSA. In vitro, SOCS3 overexpression abrogated IL-6-induced activation of STAT3 in lung epithelial cells. These findings suggest SOCS3 is an important regulator of lung inflammatory injury after deposition of IgG IC.

Generation of C5a in the absence of C3: a new complement activation pathway

Complement-mediated tissue injury in humans occurs upon deposition of immune complexes, such as in autoimmune diseases and acute respiratory distress syndrome. Acute lung inflammatory injury in wild-type and C3-/- mice after deposition of IgG immune complexes was of equivalent intensity and was C5a dependent, but injury was greatly attenuated in Hc-/- mice (Hc encodes C5). Injury in lungs of C3-/- mice and C5a levels in bronchoalveolar lavage (BAL) fluids from these mice were greatly reduced in the presence of antithrombin III (ATIII) or hirudin but were not reduced in similarly treated C3+/+ mice. Plasma from C3-/- mice contained threefold higher levels of thrombin activity compared to plasma from C3+/+ mice. There were higher levels of F2 mRNA (encoding prothrombin) as well as prothrombin and thrombin protein in liver of C3-/- mice compared to C3+/+ mice. A potent solid-phase C5 convertase was generated using plasma from either C3+/+ or C3-/- mice. Human C5 incubated with thrombin generated C5a that was biologically active. These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation.

Relationship of Acute Lung Inflammatory Injury to Fas/FasL System

There is mounting evidence that apoptosis plays a significant role in tissue damage during acute lung injury. To evaluate the role of the apoptosis mediators Fas and FasL in acute lung injury, Fas (lpr)- or FasL (gld)-deficient and wild-type mice were challenged with intrapulmonary deposition of IgG immune complexes. Lung injury parameters ((125)I-albumin leak, accumulation of myeloperoxidase, and wet lung weights) were measured and found to be consistently reduced in both lpr and gld mice. In wild-type mice, lung injury was associated with a marked increase in Fas protein in lung. Inflamed lungs of wild-type mice showed striking evidence of activated caspase-3, which was much diminished in inflamed lungs from lpr mice. Intratracheal administration of a monoclonal Fas-activating antibody (Jo2) in wild-type mice induced MIP-2 and KC production in bronchoalveolar lavage fluids, and a murine alveolar macrophage cell line (MH-S) showed significantly increased MIP-2 production after incubation with this antibody. Bronchoalveolar lavage fluid content of MIP-2 and KC was substantially reduced in lpr mice after lung injury when compared to levels in wild-type mice. These data suggest that the Fas/FasL system regulates the acute lung inflammatory response by positively affecting CXC-chemokine production, ultimately leading to enhanced neutrophil influx and tissue damage.

Complement Factor H Limits Immune Complex Deposition and Prevents Inflammation and Scarring in Glomeruli of Mice with Chronic Serum Sickness

Factor H is the major complement regulator in plasma. Abnormalities in factor H have been implicated in membranoproliferative glomerulonephritis in both humans and experimental animals. It has been shown that factor H on rodent platelets functions analogously to human erythrocyte complement receptor 1 in its role to traffic immune complexes to the mononuclear phagocyte system. C57BL/6 factor H-deficient mice (Cfh(-/-)) and wild-type (wt) controls were immunized daily for 5 wk with heterologous apoferritin to study the chronic serum sickness GN model. Immunizations were started in 6- to 8-wk-old mice, which was before the development of spontaneous membranoproliferative glomerulonephritis in some Cfh(-/-) animals. Glomerular deposition of IgG immune complexes in glomeruli was qualitatively and quantitatively increased in Cfh(-/-) mice compared with wt mice. Consistent with the increase in glomerular immune complexes and possibly because of alternative pathway complement activation, Cfh(-/-) mice had increased glomerular C3 deposition. Wt mice developed no glomerular pathology. In contrast, Cfh(-/-) mice developed diffuse proliferative GN with focal crescents and glomerulosclerosis. In addition, there was significantly increased expression of collagen IV, fibronectin, and laminin mRNA in Cfh(-/-) glomeruli. These data show a role for platelet-associated factor H to process immune complexes and limit their accumulation in glomeruli. Once deposited in glomeruli, excessive complement activation can lead to glomerular inflammation and the rapid development of a scarring phenotype.