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There has been no previous systematic study of bullous skin diseases with granular basement membrane zone deposition exclusively of C3. In this study we collected 20 such patients, none of whom showed cutaneous vasculitis histopathologically. Oral dapsone and topical steroids were effective. Various serological tests detected no autoantibodies or autoantigens. Direct immunofluorescence for various complement components revealed deposition only of C3 and C5?C9, indicating that no known complement pathways were involved. Studies of in situ hybridization and micro-dissection with quantitative RT-PCR revealed a slight reduction in expression of C3 in patient epidermis. These patients may represent a new disease entity, for which we propose the term “granular C3 dermatosis”. The mechanism for granular C3 deposition in these patients is unknown, but it is possible that the condition is caused by autoantibodies to skin or aberrant C3 expression in epidermal keratinocytes., Acta Dermato Venereologica, 96(6), pp.748-753; 2016

Immune-mediated liver cell damage has been likely to occur in patients with chronic hepatitis C virus (HCV) infection. To clarify the T-cell clonality in patients with chronic hepatitis C, we analyzed T-cell receptor (TCR) Vβ chain messages expressed in peripheral blood mononuclear cells (PBMC) before and after treatment with interferon (IFN)-α. PBMC from 15 patients with chronic hepatitis C and from six healthy subjects were examined. The 15 patients were allocated to three groups based on their response to IFN-α treatment as follows: responders (group A, n=5), transient responders (group B, n=5), and non-responders (group C, n=5). Twenty-two TCR Vβ repertoires Vβ 1–Vβ 20 expressed in PBMC were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and single strand conformation polymorphism (SSCP). In responders, the mean total number of T-cell clones in 22 TCR Vβ repertoires was significantly decreased from 97.4±40.1 to 62.6±32.1 (P

The recent identification of two mannose-binding lectin-associated serine protease clones from Halocynthia roretzi, an ascidian, suggested the presence of a complement system in urochordates. To elucidate the structure and function of this possibly primitive complement system, we have isolated cDNA clones for ascidian C3 (AsC3) and purified AsC3 protein from body fluid. The deduced primary structure of AsC3 shows overall similarity to mammalian C3, including a typical thioester site with the His residue required for nucleophilic activation of the thioester. AsC3 has a two-subunit chain structure, and the α-chain is cleaved at a specific site near to the N terminus upon activation. Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and Ab against AsC3 inhibits this opsonic activity. These results indicate that the complement system played a pivotal role in innate immunity by enhancing phagocytosis before the emergence of the vertebrates and well ahead of the establishment of adaptive immunity, which is believed to have occurred at about the time of the appearance of cartilaginous fish.

症例は72歳,男性. 2003年11月亜急性甲状腺炎と診断されステロイドの内服を開始したが, 2004年1月ステロイド減量中に発熱し, 2月には右足背の筋力低下と両足背の痺れが出現し多発性単神経炎と診断された.感染症,脱髄性疾患,全身性血管炎は否定されNonsystemic vasculitic neuropathy(NSVN)と診断した.その後,アザチオプリンの追加投与により神経症状は改善した. NSVNは多臓器障害を欠き末梢神経障害が前景に立つ血管炎である. NSVNの治療はステロイドに免疫抑制薬を併用することが有用であると推測された.

The complement system, which consists of more than 30 plasma and cell surface proteins, is activated by three pathways: the classical, lectin, and alternative pathways, leading to the generation of opsonins and pathogen destruction. In the lectin pathway, mannose-binding lectin (MBL) and ficolins act as pattern recognition molecules for pathogens, resulting in the activation of MBL-associated serine proteases (MASPs: MASP-1, MASP-2, and MASP-3). Among these proteases, MASP-2 is a key enzyme that cleaves C4 and C2 to assemble a C3 convertase (C4b2a). However, the physiological function of MASP-1 and MASP-3 remains unclear. To investigate the roles of MASP-1 and MASP-3, we generated a MASP-1- and MASP-3-deficient (M1/3 KO) mouse model and found that the deficient mice lacked alternative pathway activation because factor D (Df) remained as a proenzyme in the serum. MASP-1 and MASP-3 were able to convert the proenzyme of Df to an active form in vitro. In addition, MASP-1 was able to activate MASP-2 and MASP-3 as C1r activates C1s. Thus, MASP-1 and MASP-3 seem to be involved in activation of both the lectin and alternative pathways.

Experimental pulmonary hypertension (PH) was induced by a single injection of monocrotaline (MCT), a pyrrolizidine alkaloid extracted from Crotalaria spectabilis. The effect of beraprost sodium, a stable prostacyclin analogue, on the development of MCT-induced PH in rats was studied. Chronic administration of beraprost sodium at a dose of 30 micrograms/kg/day initiated on the same day as MCT injection decreased the degree of PH determined by weight ratio of right ventricular free wall to that of left ventricle plus septum depending on the duration of administration. Although the injection of prostaglandin E1 (PGE1) at a dose of 200 micrograms/kg/day initiated 1 week after MCT injection did not decrease the degree of PH significantly, beraprost sodium administration at doses of 30 and 100 micrograms/kg/day decreased the degree of PH significantly. The cytoprotective effect of beraprost sodium against endothelial cell (EC) damage is believed to be involved in inhibiting development of PH in MCT-injected rats. The amounts of cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) produced by alveolar macrophages decreased in accordance with the inhibiting effect of beraprost sodium on development of PH, indicating that beraprost sodium inhibited the development of PH in MCT-injected rats not only through its effect of vasodilation and anti-platelet aggregation in pulmonary circulation but also through its antiinflammatory effects.

Peanut allergy is severe and persisting from childhood to adulthood. However, there is no effective prophylaxis or treatment for peanut allergy. Little is known to about the molecular process in the pathogenesis of peanuts allergy, especially in innate immunity. Thus we investigated the role of complement activation in murine peanut anaphylaxis. Complement component C3 deposition on peanut extract (PE) was evaluated using sera from wild-type (WT), mannose-binding lectin associated serine protease (MASP)-1/3 deficient, MASP-2 deficient, and C4 deficient mice. Sera from interferon regulatory factor-4 (IRF-4) deficient mice, which lack serum immunoglobulin, were also used. In anaphylaxis study, mice were pretreated with propranolol and a long-acting form of IL-4, and injected with PE. Mice were then assessed for plasma C3a levels and hypothermia shock by ELISA and rectal temperature measurement, respectively. C3 deposition on PE was abolished in immunoglobulin- and C4-deficient sera. No difference in C3 deposition levels were observed among WT, MASP-1/3 deficient and MASP-2 deficient sera. IgM, IgG2b, IgG3, C1q, and ficolin-A deposits were detected on PE. In anaphylaxis study, MASP-1/3 deficient mice showed elevation of plasma C3a levels similar to WT mice. However, they were significantly reduced in C4- and MASP-2-deficient mice compared to WT mice. Consistently, PE-induced anaphylactic shock was prevented in C4 deficient mice and partially in MASP-2 deficient mice. In conclusion, PE activates complement via both the lectin and classical pathways in vivo, and the complement activation contributes to hypothermia shock in mice.

The complement system can be activated by one of three pathways (classical, lectin and alternative) and provides important host defense, homeostasis and immune regulatory functions. However, the complement system also plays a pathogenic role in many autoimmune and inflammatory diseases, including systemic lupus erythematosus (SLE or lupus) (1). Systemic lupus erythematosus is the prototypic human autoimmune disease, and complement mediated injury is initiated by immune complexes (IC) formed by autoantibodies directed against a broad range of self-antigens (2, 3). The kidney is a major site of immune complex formation and/or deposition, and lupus nephritis is a major cause of morbidity and mortality, both in human SLE and in murine models. There is, however, an apparent dual role for complement in the development and progression of lupus, highlighted by the different roles of the classical and alternative complement pathways in the disease. Patients with homozygous deficiencies of early components of the classical pathway, such as C1 (C1q or C1r/s) or C4, have an increased incidence of lupus and lupus-like disease (>80%) (1, 4). On the other hand, homozygous deficiencies of C3, a protein that plays a pivotal role in all three complement activation pathways, is associated with membranoproliferative glomerulonephritis (5), but only rarely lupus (6). Studies in murine models also show a link between the classical pathway and autoimmune disease. Mice deficient in C1q have increased mortality and higher titers of autoantibodies, with 25% of C1q deficient mice developing glomerulonephritis with glomerular IC deposits and multiple apoptotic bodies (7). C1q and C4 deficiency are also associated with the development/acceleration of lupus-like disease in mice on an autoimmune background (8, 9), and C1q−/− and C4−/− mice (on 129/Sv × C57BL/6 background) exhibit an impaired ability to clear apoptotic cell bodies. Together, these data are consistent with the hypothesis that the classical pathway provides a protective role in the development of lupus via its role in the clearance of apoptotic cells that otherwise provide a source of autoantigens to fuel the disease process (10), although other hypotheses have been proposed (11). In contrast to the protective role of the classical pathway, there is strong evidence that the alternative pathway plays a key role in the development of lupus. MRL/lpr mice spontaneously develop an autoimmune syndrome similar to human SLE (12), and MRL/lpr mice deficient in either of the alternative pathway proteins fB or fD, are protected from renal disease (13, 14). In addition, various complement inhibitors are protective in murine models of lupus. Recombinant soluble forms of the mouse C3 inhibitor, Crry, provided protection against renal injury in MRL/lpr mice. These inhibitors, Crry-Ig (15), and CR2-Crry (16), also provided protection against skin/ear lesions and glomerular deposition of IC’s and C3. However, only the targeted inhibitor, CR2-Crry, reduced glomerular inflammation, mortality and autoantibody levels, and there were significantly increased levels of circulating IC in MRL/lpr mice treated with Crry-Ig compared to mice treated with CR2-Crry. These effects of CR2-Crry were observed in mice treated once a week with the inhibitor. These different outcomes may be related to the systemic vs. localized nature of complement inhibition by Crry-Ig and CR2-Crry, respectively. The CR2 moiety of the CR2-Crry fusion protein targets to iC3b, C3dg and C3d, cell-bound activation fragments of C3 that are deposited at sites of complement activation. Complement inhibition with an anti-C5 monoclonal antibody is also protective in the NZB/NZW F1 model of lupus (17). Significantly, however, whereas C3 inhibition and alternative pathway deficiency is protective in MRL/lpr mice, C3 deficiency is not. In fact, there is earlier and significantly greater albuminuria and increased glomerular IgG deposition in the C3−/− MRL/lpr mice compared to C3+/+ controls (18). Thus, total blockade of all complement pathways (as opposed to temporary and/or targeted blockade with inhibitors) was not protective and appeared to exacerbate disease. Collectively, the above studies suggest that selective inhibition of the alternative pathway will provide an effective therapeutic strategy for lupus, and that a targeted approach to complement inhibition has the potential to provide additional benefit with less immune suppression and toxicity. Here we report on the characterization of CR2-fH, a recently described targeted complement inhibitor that is specific for the alternative pathway, in treatment of disease in the MRL/lpr model of lupus. For clinical relevancy, treatment was begun after the onset of renal disease, and the effect of CR2-fH on immune modulation and disease progression was compared to the effect of CR2-Crry, an inhibitor of all complement pathways.

Complement plays a dual role in the progression of systemic lupus erythematosus since it has important protective functions, such as the clearance of immune complexes and apoptotic cells, but is also a mediator of renal inflammation. To investigate this balance in a clinically relevant setting, we investigated how targeted inhibition of all complement pathways vs. targeted inhibition of only the alternative pathway impacts immune and therapeutic outcomes in NZB/W F(1) mice. Following onset of proteinuria, mice were injected twice weekly with CR2-fH (inhibits alternative pathway), CR2-Crry (inhibits all pathways at C3 activation step), sCR2 (C3d targeting vehicle) or saline. Sera were analyzed every 2 weeks for anti-dsDNA antibody levels, and urinary albumin excretion was determined. Kidneys were collected for histological evaluation at 32 weeks. Compared to the control group, all CR2-fH, CR2-Crry and sCR2 treated groups showed significantly reduced serum anti-dsDNA antibody levels and strong trends towards reduced glomerular IgG deposition levels. Glomerular C3 deposition levels were also significantly reduced in all three-treated groups. However, significant reductions of disease activity (albuminuria and glomerulonephritis) were only seen in the CR2-fH treated group. These data highlight the dual role played by complement in the pathogenesis of lupus, and demonstrate a benefit of selectively inhibiting the alternative complement pathway, presumably because of protective contributions from the classical and/or lectin pathways. The sCR2 targeting moiety appears to be contributing to therapeutic outcome via modulation of autoimmunity. Furthermore, these results are largely consistent with our previous data using the MRL/lpr lupus model, thus broadening the significance of these findings.

Inhibitory oligodeoxynucleotides (ODNs), which are capable of blocking CpG-induced inflammation, have been anticipated to be beneficial therapeutic agents for autoimmune diseases. In this study, we show that GpC ODN, which inverted the cytosine guanine sequence of CpG motif to guanine cytosine sequence, is an inhibitory ODN. The inhibitory effects of GpC ODN on CpG ODN-induced immune activation were confirmed by cytokine assay using splenocytes from lupus-prone MRL-lpr/lpr mice. In vivo, injecting MRL-lpr/lpr mice with GpC ODN did not reduce the deposition of IgG and C3 in the glomeruli, the serum level of IL-12, the serum level of rheumatoid factors and anti-ds DNA antibody, or alter the composition of IgG isotypes of anti-ds DNA antibody. However, the mice in the GpC group showed less proteinuria, significantly lower blood urea nitrogen levels (BUN) and significantly prolonged survival. Our results suggest that inhibitory ODNs, such as GpC ODN, have the potential to become a treatment for autoimmune diseases, like lupus nephritis.

The complement inhibitor, Crry, which blocks both the classical and alternative pathways, alleviates CNS disease in the lupus model, MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mice. To understand the role of the alternative pathway, we studied mice deficient in a key alternative pathway protein, complement factor B (fB). Immune deposits (IgG and C3) were reduced in the brains of MRL/lpr fB-deficient (fB-/-MRL/lpr) compared to fB-sufficient (MRL/lpr) mice, indicating reduced complement activation. Reduced neutrophil infiltration (22% of MRL/lpr mice) and apoptosis (caspase-3 activity was reduced to 33% of MRL/lpr mice) in these mice indicates that the absence of the alternative pathway was neuroprotective. Furthermore, expression of phospho (p)-Akt (0.16+/-0.02 vs. 0.35+/-0.13, p

Ig class switch recombination (CSR) and somatic hypermutation serve to diversify antibody responses and are orchestrated by the activity of activation-induced cytidine deaminase and many proteins involved in DNA repair and genome surveillance. Msh5 , a gene encoded in the central MHC class III region, and its obligate heterodimerization partner Msh4 have a critical role in regulating meiotic homologous recombination and have not been implicated in CSR. Here, we show that MRL/ lpr mice carrying a congenic H-2 b/b MHC interval exhibit several abnormalities regarding CSR, including a profound deficiency of IgG3 in most mice and long microhomologies at Ig switch (S) joints. We found that Msh5 is expressed at low levels on the H-2 b haplotype and, importantly, a similar long S joint microhomology phenotype was observed in both Msh5 and Msh4 -null mice. We also present evidence that genetic variation in MSH5 is associated with IgA deficiency and common variable immune deficiency (CVID) in humans. One of the human MSH5 alleles identified contains two nonsynonymous polymorphisms, and the variant protein encoded by this allele shows impaired binding to MSH4. Similar to the mice, Ig S joints from CVID and IgA deficiency patients carrying disease-associated MSH5 alleles show increased donor/acceptor microhomology, involving pentameric DNA repeat sequences and lower mutation rates than controls. Our findings suggest that Msh4/5 heterodimers contribute to CSR and support a model whereby Msh4/5 promotes the resolution of DNA breaks with low or no terminal microhomology by a classical nonhomologous end-joining mechanism while possibly suppressing an alternative microhomology-mediated pathway.

We report a case of ulcerative colitis (UC) that occurred during the course of rheumatoid arthritis (RA). A 29-year-old woman with a 25-year history of RA was hospitalized for high fever, abdominal pain and hematochezia. Colonoscopy revealed erosive and reddish mucosa from the distal transverse colon to rectum. Histology revealed cryptitis (mainly caused by neutrophils), mild crypt abscess and goblet cell depletion. She was diagnosed with left-sided UC and treated with mesalamine enema. The abdominal symptoms and colonoscopic findings were greatly ameliorated. We conclude that the mesalamine enema was effective in this case of UC occurring during the course of RA.

Lupus nephritis is characterized by immune complex deposition and infiltration of inflammatory cells into the kidney including Ab-producing cells (AbPCs). Although AbPCs play a central role in the pathogenesis of immune complex glomerulonephritis in lupus, the specificity and pathogenic role of AbPCs infiltrating into the kidneys in lupus are poorly understood. To characterize AbPCs present in lupus kidneys, we isolated AbPCs from diseased MRL/MpJ-Faslpr (MRL/lpr) mouse kidneys. ELISPOT assays, using glomerular Ag (GA) extracts as Ag, demonstrated significant enhancement of anti-GA AbPCs in the kidneys as compared in peripheral blood or spleen of the same mouse. We isolated hybridomas with anti-GA specificity from MRL/lpr mouse kidneys. All the anti-GA mAbs had polyreactive binding to ssDNA, dsDNA, and IgG (i.e., rheumatoid factor), but not to histones or Sm. Sequence analysis of anti-GA Abs suggested the occurrence of somatic mutations and amino acid replacement in complementarity-determining regions with a high replacement to silent ratio resulting in charged amino acids. Intravenous administration of the monoclonal anti-GA Abs into BALB/c mice resulted in graded deposition in glomeruli paralleling their ELISA anti-GA reactivity. These results suggest that AbPCs infiltrating the kidneys in MRL/lpr mice accumulate as a result of Ag selection and likely play a pathologic role in lupus nephritis.

Terminal deoxynucleotidyl transferase (TdT) places non-template-coded nucleotides (N additions) in the VH CDR3 of T cell receptors and immunoglobulins. Amino acids coded for by N additions are important in autoantibody binding of dsDNA in lupus. We hypothesized that a genetic lack of TdT would modulate disease in lupus-prone mice. To test this hypothesis, we derived TdT-deficient MRL/lpr mice. Serum levels of anti-dsDNA antibodies and anti-dsDNA producing splenocytes were significantly lower in the TdT − versus TdT + littermates. Albuminuria, glomerular IgG deposition, and pathologic renal disease were significantly reduced in the TdT − mice. Sequence analysis of anti-dsDNA hybridomas derived from TdT − mice revealed a lack of N additions, short VH CDR3 segments, yet the presence of VH CDR3 arginines. Thus, the genetic absence of TdT reduces autoantibody production and clinical disease in MRL/lpr mice, confirming the importance of N additions in the autoimmune response in these mice.

Complement activation and tissue deposition of complement fragments occur during disease progression in lupus nephritis. Genetic deficiency of some complement components (e.g., Factor B) and infusion of complement inhibitors (e.g., Crry, anti-C5 Ab) protect against inflammatory renal disease. Paradoxically, genetic deficiencies of early components of the classical complement pathway (e.g., C1q, C4, and C2) are associated with an increased incidence of lupus in humans and lupus-like disease in murine knockout strains. Complement protein C3 is the converging point for activation of all three complement pathways and thus plays a critical role in biologic processes mediated by complement activation. To define the role of C3 in lupus nephritis, mice rendered C3 deficient by targeted deletion were backcrossed for eight generations to MRL/lpr mice, a mouse strain that spontaneously develops lupus-like disease. We derived homozygous knockout (C3−/−), heterozygous (C3+/−), and C3 wild-type (C3+/+) MRL/lpr mice. Serum levels of autoantibodies and circulating immune complexes were similar among the three groups. However, there was earlier and significantly greater albuminuria in the C3−/− mice compared with the other two groups. Glomerular IgG deposition was also significantly greater in the C3−/− mice than in the other two groups, although overall pathologic renal scores were similar. These results indicate that C3 and/or activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice and may in fact play a beneficial role via clearance of immune complexes.

We read with interest the recent article by Guikema et al. ([1][1]) where the authors concluded that Msh5 does not participate in Ig class switch recombination (CSR) in mice. This is clearly in contrast to our recent publication describing a role for Msh5 in CSR both in mice and humans ([2][2]), and

Progressive destruction of the intrahepatic bile ducts in patients with primary biliary cirrhosis (PBC) is thought to be mediated by cytotoxic T cells which recognize certain epitopes, such as the pyruvate dehydrogenase complex (PDC). To clarify the T-cell repertoire in PBC, we analyzed T-cell receptor (TCR) Vbeta-chain messages expressed in peripheral blood mononuclear cells (PBMCs) stimulated with PDC and in liver biopsy specimens. PBMCs from 12 PBC patients and 6 healthy controls were examined. The TCR Vbeta repertoires of unstimulated PBMCs and PBMCs stimulated with PDC purified from bovine heart were analyzed, using the reverse transcriptase-polymerase chain reaction (RT-PCR) and single-strand conformation polymorphism (SSCP). Liver biopsy specimens from 5 PBC patients were also analyzed. In the PBC patients, several different T-cell clones, some of which showed the same mobility, were evident in both the PDC-stimulated and unstimulated PBMCs, as demonstrated by SSCP analysis. In addition, TCR clonality of infiltrating lymphocytes in the liver was also observed in PBC patients, showing common clonal T-cell accumulation with that seen in PBMCs stimulated with PDC. These data indicate that common clonal T-cell accumulation specific for PDC may be present in both peripheral PBMCs and the liver of patients with PBC.

We have several concerns regarding the article by Degn et al. ([1][1]). In this article, the authors clearly showed that MASP-1 is essential for the activation of MASP-2 using deficient human serum in both MASP-1 and MASP-3. We are in agreement with this; however, we disagree with the following