Search

SH2D1A, also known as signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), is an adaptor protein. Recently, it was reported that SAP deficient mice were protected from systemic lupus erythematosus (SLE). In this study, we postulated SH2D1A gene to be a candidate susceptibility gene for SLE and analyzed its association with SLE. A case-control association study was conducted on 5 tag single nucleotide polymorphisms (SNPs) in SH2D1A region in 506 Japanese female SLE patients and 330 healthy female controls. The luciferase assay was performed to determine the functional role of the SNP associated with SLE. One SNP in the intron 2, rs2049995, showed association with SLE ( p = 0.0110, odds ratio (OR) 1.97, 95% confidence interval (CI) 1.16–3.34, under the dominant model). The association of rs2049995 seemed to be stronger in the subset with the age of onset less than 20 years ( p = 0.0067, OR 2.65, 95% CI 1.28–5.46). Functional evaluation of rs2049995 showed that reporter gene activity was increased 1.9-fold for the susceptible allele compared with the resistant allele. An intronic SNP of SH2D1A is associated with SLE.

Although costimulatory molecules have been shown to play crucial roles in the immune response, their involvement in the pathogenesis of Sjögren's syndrome is incompletely understood. In this study, we evaluated the relationship between the severity of spontaneous Sjögren's syndrome-like autoimmune sialadenitis in MRL/MpJ-lpr/lpr (MRL-Fas(lpr)) mice and the expression of 6 costimulatory molecules that play important roles in the immune response: CD80, CD86, OX40 ligand (OX40L), 4-1BB ligand (4-1BBL), glucocorticoid-induced TNF receptor-related protein ligand (GITRL), and B cell-activating factor of the tumor necrosis factor family (BAFF). Expression of the costimulatory molecules in the submandibular salivary glands of age-matched autoimmune MRL-Fas(lpr) mice and non-autoimmune MRL/MpJ-+/+(MRL/+) and C3H/HeJ-lpr/lpr (C3H-Fas(lpr)) mice was examined immunohistochemically and scored on a scale of 0 to 3. The severity of sialadenitis was evaluated histologically and scored on a scale of 0 to 3. We found that all of the costimulatory molecules were expressed in duct epithelial cells of salivary glands from MRL-Fas(lpr) mice, whereas immunoreactivity was absent or weak in the MRL/+ mice. The staining intensity for all 6 costimulatory molecules was significantly higher in the MRL-Fas(lpr) than in the MRL/+ mice. Partial correlation analysis was performed to assess the degree of association between costimulatory molecule staining scores and disease scores, which clearly revealed a significant correlation for only GITRL and 4-1BBL. These molecules showed negligible immunoreactivity in the submandibular glands of C3H-Fas(lpr) mice, suggesting that their expression was independent of the Fas(lpr) mutation. In conclusion, the expression of GITRL and 4-1BBL in salivary gland duct epithelial cells is associated with background genes in the MRL strain, but not with the Fas(lpr) mutation itself, and contributes significantly to the pathogenesis of autoimmune sialadenitis in MRL-Fas(lpr) mice. These results suggest that GITRL and 4-1BBL may be effective targets for the development of therapies for Sjögren's syndrome.

Objective Fcγ receptor type IIb (FcγRIIb) is a major negative regulator of B cells, and the lack of FcγRIIb expression has been reported to induce systemic lupus erythematosus (SLE) in mice of the C57BL/6 (B6) genetic background. The 129 strain–derived Sle16 locus on the telomeric region of chromosome 1 including polymorphic Fcgr2b confers the predisposition to systemic autoimmunity when present on the B6 background. We undertook this study to examine the effect of the Sle16 locus on autoimmune disease in FcγRIIb-deficient B6 mice. Methods We established 2 lines of FcγRIIb-deficient B6 congenic mouse strains (KO1 and KO2) by selective backcrossing of the originally constructed FcγRIIb-deficient mice on a hybrid (129 × B6) background into a B6 background. Although both lack FcγRIIb expression, the KO1 and KO2 strains carry different lengths of the 129 strain–derived telomeric chromosome 1 segment flanked to the null-mutated Fcgr2b gene; the KO1 strain carries a 129 strain–derived ∼6.3-Mb interval distal from the null-mutated Fcgr2b gene within the Sle16 locus, while this interval in the KO2 strain is of B6 origin. Results Unexpectedly, both strains failed to develop SLE; instead, the KO1 strain, but not the KO2 strain, spontaneously developed severe rheumatoid arthritis (RA) with an incidence reaching >90% at age 12 months. Conclusion The current study shows evidence that the epistatic interaction between the Fcgr2b-null mutation and a polymorphic gene(s) in the 129 strain–derived interval located in the distal Sle16 locus contributes to RA susceptibility in a new mouse model with the B6 genetic background, although the participation of other genetic polymorphisms cannot be totally excluded.

A cumulative effect of the susceptibility genes with polymorphic alleles may be responsible for rheumatoid arthritis (RA). The objective of this study was to clarify whether susceptibility to RA is under the control of common allelic loci between two different RA models induced by extrinsic and intrinsic factors, collagen-induced arthritis (CIA) in DBA/1 mice and arthritis in MRL/Mp (MRL) mice associated with the Fas deficient mutant gene, Fas(lpr), respectively. CIA was examined in mice of parental DBA/1 and MRL, (MRL x DBA/1) F1 and (MRL x DBA/1) F2 progenies. In genome-wide screening of the severity in the F2 using microsatellite markers, significant linkage was observed on chromosomes 5 and 17 at map position of D5Mit259 and H-2, respectively, associated with DBA/1 alleles, while there was no loci associated with arthritis of MRL-Fas(lpr) mice previously identified. In a quantitative trait locus (QTL) analysis, the locus on chromosome 5 showed the highest peak at map position 35 cM (LOD score 6.0). This study may indicate that the arthritis induced by extrinsic and intrinsic factors is under the control of a different combination of susceptibility genes with common and different alleles, possibly simulating the genetic heterogeneity of RA.

Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is an adaptor molecule containing a Src homology 2 (SH2) domain. SAP is expressed in T cells and natural killer (NK) cells and binds to the cytoplasmic domains of SLAM family receptors, resulting in the subsequent recruitment of Fyn. The SAP (SH2D1A) gene is located on the X chromosome and is responsible for X-linked lymphoproliferative disease, characterized by higher susceptibility to Epstein-Barr virus infection. The SAP-mediated signal is not only essential for the development of NKT cells, i.e. unconventional CD1d-restricted T cells with invariant Valpha14 T cell receptors, but also for the regulation of the function of NK cells and conventional T cells. The role of SAP-mediated signaling in the induction of autoimmune diseases has been analyzed using animal models such as lupus, hepatitis, and graft-versus-host disease and is considered important in their pathogenesis in humans. In this review we highlight the current findings on SAP-mediated signaling in hematopoietic cells and discuss its importance in autoimmune diseases and immunological disorders.

Problem statement: Mast cells develop from bone marrow-derived progenitor cells and are distributed in the skin or mucosa where they play proinflammatory roles in the first line of defense. Since some tumors in humans and experimental animals exhibited infiltration of increased mast cells, we investigated the contribution of mast cells to the override of tumor rejection. Approach: MRL/N-1 cells are malignant fibrous histiocytoma-like cells established from the spleen of a Fas ligand (FasL)-deficient MRL/Mp-FasLgld/gld (MRL/gld) mouse and are implantable in Fas-deficient MRL/Mp-Faslpr/lpr (MRL/lpr) mice. MRL/N-1 cells were implanted in MRL/gld, MRL/lpr and MRL/+mice after antibody treatments or with mast cells or macrophages and the tumor growth was observed. Results: MRL/N-1 cells were rejected by Fas-intact syngeneic MRL/+ mice in CD8+ T cell-mediated manner. This rejection was inhibited by the co-implanted mast cells. MRL/N-1 cells transfected with FasL were rejected by MRL/+ and MRL/gld mice. Conclusion: Mast cells abrogate the rejection of MRL/N-1 tumor cells and that this tumor rejection is mediated by CD8+ T cells and dependent on host Fas-FasL axis.

The cysteinyl leukotrienes (LTs), LTC(4), LTD(4), and LTE(4), are potent inflammatory mediators and are involved in allergic reactions, such as bronchoconstriction, eosinophilic inflammation, and allergic cell proliferation. The present study aimed to elucidate the role of constitutively produced cysteinyl LTs in mast cell activation. We used a newly developed quantification method based on mass spectrometry to detect cysteinyl LTs in the cultured medium of mouse bone marrow-derived mast cells (BMMCs), which were obtained by interleukin (IL)-3-conditioned culture of mouse bone marrow. BMMCs were stimulated with immunoglobulin (Ig) E and antigen (IgE/Ag) or lipopolysaccharide for 1 or 24 h. This new quantification method revealed that unstimulated BMMCs produced and secreted LTB4 and LTE4 after 24 h of incubation. The treatment of unstimulated BMMCs for 2 h with montelukast, an antagonist of a cysteinyl LT receptor, CysLT1, resulted in the suppression of a downstream signaling event of this receptor, i.e., the decrease in phosphorylation of extracellular responsive kinases. Thus, cysteinyl LTs constitutively simulate BMMCs through the CysLT1 receptor in an autocrine manner. Treatment of BMMCs for 3 weeks with montelukast, which caused long-term inhibition of the autocrine cyteinyl LT-derived signal, significantly attenuated the IgE/Ag-dependent degranulation, as judged by the decrease in the release of beta-hexosaminidase, an enzyme contained in the granules, whereas the production of cytokines, such as IL-6 and tumor necrosis factor-alpha, were largely unaffected. In conclusion, an autocrine signal derived from constitutively produced cysteinyl LTs predisposes mast cells to the degranulation upon allergic stimulation.

Pathogens are sensed by Toll-like receptors (TLRs) expressed in leukocytes in the innate immune system. However, excess stimulation of TLR pathways is supposed to be connected with provocation of autoimmunity. We show that paired immunoglobulin (Ig)-like receptor B (PIR-B), an immunoreceptor tyrosine-based inhibitory motif–harboring receptor for major histocompatibility class I molecules, on relatively primitive B cells, B-1 cells, suppresses TLR9 signaling via Bruton's tyrosine kinase (Btk) dephosphorylation, which leads to attenuated activation of nuclear factor κB p65RelA but not p38 or Erk, and blocks the production of natural IgM antibodies, including anti-IgG Fc autoantibodies, particularly rheumatoid factor. The autoantibody production in PIR-B–deficient (Pirb−/−) mice was further augmented in combination with the Faslpr mutation, which might be linked to the development of autoimmune glomerulonephritis. These results show the critical link between TLR9-mediated sensing and a simultaneously evoked, PIR-B–mediated inhibitory circuit with a Btk intersection in B-1 cells, and suggest a novel way toward preventing pathogenic natural autoantibody production.

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs(flox/flox);SynI-cre mice. Notably, the hrs(flox/flox);SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs(flox/flox);SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.

Invariant natural killer T (iNKT) cells are a special subset of alphabeta T cells with invariant TCR, which recognize alpha-galactosylceramide (alpha-GalCer) presented by CD1d. In addition to signals through the invariant TCR upon stimulation with alpha-GalCer, costimulatory signals, such as signals through CD28 and OX40, are indispensable for full activation of iNKT cells. In this study, we investigated the functions of a well-known costimulatory molecule, glucocorticoid-induced TNF receptor (GITR), on Ag-induced iNKT cell activation. Unexpectedly, engagement of GITR by agonistic mAb DTA-1 suppressed proliferation and cytokine production of iNKT cells upon alpha-GalCer stimulation. In addition, GITR signals in iNKT cells during only the Ag-priming phase was sufficient to inhibit the iNKT cell activation. Consistent with these results, the GITR-deficient iNKT cells showed enhanced proliferation and increased cytokine production upon alpha-GalCer stimulation both in vitro and in vivo. Furthermore, the in vivo administration of alpha-GalCer suppressed tumor metastasis more efficiently in GITR-deficient mice than in wild-type mice. Collectively, GITR plays a co-inhibitory role in Ag-induced iNKT cell activation.

There has been increasing evidence for the involvement of fatty acid-binding proteins (FABPs) in the cytokine production of macrophages and dendritic cells probably through the control of cellular lipid metabolism and signal transduction. Since mast cells (MCs) are recently shown to be involved in immune response through modification of cytokine production, it is possible that some FABPs could also be involved in the immune response of MCs. In this study, we found that epidermal-type FABP (E-FABP) was expressed in murine bone marrow-derived MCs (BMMCs). Using BMMCs from genetically E-FABP-null mutated mice, we demonstrated that E-FABP in BMMCs plays a key role in the production of TNF-alpha following lipopolysaccharide (LPS) stimulation. In the in vivo septic peritonitis model (cecal ligation and puncture model), E-FABP-null mice showed a significantly increased mortality compared to wild-type mice. However, no significant difference in antigen-induced cytokine production was observed between wild-type and E-FABP-null BMMCs, and systemic anaphylaxis was equally induced in vivo in both wild-type and E-FABP-null mice. These results suggest that E-FABP is specifically involved in the LPS-induced cytokine production of MCs, and could play a role in the host-defense against bacterial infection, possibly through regulation of TNF-alpha production.

Based on the hypothesis that the complex pathological and immunological manifestations of rheumatoid arthritis (RA) and the related diseases are under the control of multiple gene loci with allelic polymorphism, a recombinant congenic mouse strain was prepared between an MRL/Mp-lpr/lpr (MRL/lpr) strain, which develops arthritis resembling RA, and a non-arthritic strain C3H/HeJ-lpr/lpr (C3H/lpr). In MRL/lpr x (MRL/lpr x C3H/lpr) F1 mice, the mice developing severe arthritis were selected based on joint swelling to further continue intercrosses, and then an McH-lpr/lpr-RA1 (McH/lpr-RA1) strain was established and its histopathological phenotypes of joints and autoimmune traits were analyzed. Arthritis in McH/lpr-RA1 mice developed at a higher incidence by 20 weeks of age, compared with that in the MRL/lpr mice, who had severe synovitis (ankle, 60.3%; knee, 65.1%), and also fibrous and fibrocartilaginous lesions of articular ligamenta resembling enthesopathy (ankle, 79.4%; knee, 38.1%), resulting in ankylosis. The lymphoproliferative disorder was less, and serum levels of IgG and IgG autoantibodies including anti-dsDNA and rheumatoid factor were lower than those of both MRL/lpr and C3H/lpr strains. McH/lpr-RA1 mice may provide a new insight into the study of RA regarding the common genomic spectrum of seronegative RA and enthesopathy.

Aging readily affects immune system under the influence of environmental and/or intrinsic factors while accelerating the development of various immune disorders including autoimmune diseases. Little is known about molecular and cellular mechanisms connecting between immune senescence and development of autoimmune diseases. Here, we first show strain-specific and aging-sensitive onset of B-cell abnormality in a lupus-prone MRL/Mp.Fas(lpr) (MRL/lpr) strain of mice. This abnormality was characterized by the regression of B lymphopoiesis in the bone marrow of this strain. We next examined the association between the B-cell regression and onset of autoimmune diseases in aged (MRL/lpr x C3H/He.Fas(lpr)) F2 mice, in which pathologic phenotypes, such as glomerulonephritis, vasculitis, sialoadenitis and arthritis, variously developed. We also searched whole genome to identify genetic loci linked to the B-cell regression by using the same F2 mice. The B-cell regression manifested in the spleen of F2 mice was retrospectively evaluated by reverse transcriptase-based PCR quantification. The results demonstrated that the onset of autoimmune diseases in the F2 mice was not associated with the aging-sensitive B-cell regression. The genetic study identified a significant locus responsible for the B-cell regression in the vicinity of D5Mit233 (29 cM). This is first evidence for the presence of a genetic locus that affects B lymphopoiesis in an aging-sensitive manner.

Sex-related differences (SrD) are a general characteristic of human autoimmune diseases. There is an increasing body of evidence that suggests a link between sex-related hormones and autoimmune onsets. Here, through a genetic approach using a lupus mouse model, we attempted to show the involvement of genetic factors in the development of SrD in autoimmune diseases. Using MRL/lpr x (MRL/lpr x C57BL/6.Fas(lpr))F1 (MBN2) mice, the whole genome was searched to identify linkage loci to autoimmune phenotypes inherited from a lupus MRL/Mp.Fas(lpr) (MRL/lpr) strain of mice, which exhibits glomerulonephritis, splenomegaly and antinuclear autoantibody. The genome-wide association study confirmed four linkage loci on chromosomes 4, 7, 13, and 17. Furthermore, differential analyses performed using male and female groups of MBN2 mice revealed that two loci located on chromosomes 4 (41-72 cM, MRL/lpr allele) and 7 (4-21 cM, B6/lpr allele) were male specific and suppressed autoimmune phenotypes. Notably, the sum effect of the two loci adequately explained a range of SrD developed in the MBN2 mice. Our present findings suggest the presence of a male-predominant mechanism underlying the development of SrD in autoimmunity, depending on the effects of autosomal loci under an undefined male-specific condition.

Background: The incidence and characteristics of spontaneous ankylosis in the ankle of specific F 1 mice descended from two Fas -deficient strains were reported. Here the coincidence of synovial proliferation and ankylosis in the descendent F 2 mice is reported. Aim: To clarify whether the two distinct manifestations are genetically different. Methods: An arthropathic group of mice (MCF 2 ) were bred by intercrossing MRL/Mp. Fas lpr - sap – /sap – and C3H/He. Fas lpr mice. All mice were killed by bleeding under anaesthesia when they were 6 months old. Pathological grades for synovial proliferation were determined by microscopical examination. To obtain a linkage locus, the whole genome of male MCF 2 mice was scanned by using 73 microsatellite markers. Results: Synovial proliferation was equally observed in male and female MCF 2 mice. No correlation was observed between the grades of synovial proliferation and the ankylosis occurring in the MCF 2 mice. A suggestive susceptibility locus was shown in the middle of chromosome 11. This locus was an MRL allele with a recessive inheritance mode. Conclusion: The pathogenic mechanisms of synovial proliferation and ankylosis are genetically different. The present locus is overlapped with some loci associated with rheumatoid arthritis and with others associated with experimental arthritides.

Objective: To identify the genetic loci regulating the incidence and severity of renal autoimmune vasculitis developed in murine lupus. Methods: Vasculitis of renal arteries was histopathologically evaluated in MRL/Mp- Fas lpr (MRL/lpr), C57BL/6- Fas lpr (B6/lpr), (MRL/lpr×B6/lpr) F 1 , and MRL/lpr×(MRL/lpr×B6/lpr) F 1 backcross mice. Using genomic DNA samples of the backcross mice, genome-wide scans, association studies, and linkage analyses were carried out based on genotypes of polymorphic microsatellite markers. Correlations of vasculitis grade and levels of various autoantibodies were also evaluated. Results: Two recessive susceptibility loci of the MRL allele were identified on chromosomes 4 and 1, which had previously been defined as the autoimmune related loci termed Arvm1 and Sle-1/Nba2 , respectively. The former was epistatic to the latter in a female specific manner. The titre of antinuclear autoantibody (ANA) in IgG class, but not ANA in IgM class or anti-dsDNA in either IgG or IgM class, correlated significantly with vasculitis grade. Conclusions: The present loci have been reported in previous studies using a different set of murine strains, suggesting that they are of importance in the development of autoimmune vasculitis in murine models. The concomitance of autoimmune vasculitis and IgG ANA suggests a shared genetic factor regulating these traits.

Objective Crescent formation in the renal glomerulus is a typical manifestation of progressive glomerulopathy associated with fatal renal failure; therefore, its prevention is of clinical importance. Little is known about the pathogenic mechanism for crescent formation. This study was undertaken in an attempt to identify the events that are critical for crescent formation in immune complex crescentic glomerulonephritis (CGN) by analyzing a novel mutant strain of mice. Methods A spontaneous mutant strain of mice was isolated from the autoimmune-prone strain EOD, which stably develops fatal CGN. The mutant phenotypes were assessed histopathologically, hematologically, and immunologically. The mutation was searched for with positional cloning using microsatellite markers. Results Compared with wild-type EOD (WT-EOD) mice, mutant EOD (mut-EOD) mice showed marked improvement in CGN in conjunction with an improvement in spontaneous mortality. In WT-EOD mice, an inverse correlation between blood urea nitrogen concentration and blood platelet count and massive accumulation of platelets in the glomerulus were evident, suggesting that an accumulation of platelets in the glomerulus contributes to the progression of CGN. The mutant platelets showed an abnormal aggregation in response to collagen and thrombin, associated with a bleeding tendency in mut-EOD mice. Genetic analysis revealed a deleterious mutation in the cappuccino gene (cno), which encodes a protein that belongs to a complex called the biogenesis of lysosome-related organelle complex 1 and is profoundly involved in platelet function. Morphologic examination revealed a partial defect in dense body formation in the δ-granule of platelets. Conclusion The present findings suggest that platelet functions have a critical role in crescent formation in autoimmune GN.

Background: The spontaneous onset of macroscopic arthropathy in the ankle of the particular F 1 mice descended from two Fas -deficient strains of mice; a mutant substrain of MRL/Mp. Fas lpr (MRL/rpl) and C3H/He. Fas lpr (C3H/lpr) was recently observed. Aim: To histopathologically characterise and genetically interpret the unique inheritance mode of disease in this arthropathy model. Methods: MRL/rpl, C3H/lpr, (MRL/rpl × C3H/lpr; MC) F 1 , (C3H/lpr × MRL/rpl; CM) F 1 and MCF 2 mice were bred under specific pathogen-free conditions. Histopathological grade of arthropathy was determined at 6 months by examination under a light microscope. To search for a linkage locus to the arthropathy, the whole genome of selected 48 male MCF 2 mice with 71 polymorphic microsatellite markers was scanned, followed by quantitative trait locus analysis. Results: The incidence of microscopically defined arthropathy in the male and female MCF 1 groups was 100% and 19.4%, respectively. No incidence was observed in the parental strains, MRL/rpl and C3H/lpr, and in CMF 1 mice. In the MCF 1 mice, the arthropathy mainly affected the ankle joints and was histopathologically characterised by marked entheseal proliferation with chondrocytic differentiation and ossification in the ankle joints, the manifestations similar to ankylosing enthesitis reported previously. An MRL/rpl-derived autosomal dominant susceptibility locus was mapped in the distal of D7Mit68 (60 cM) to the ankylosis onset. Conclusion: The MCF 1 mice stably develop spontaneous ankylosing disorders in the ankle, with a male predominance. The unique inheritance mode of ankylosis is possibly interpreted by the genetic interaction between the autosomal dominant locus and a Y-linked locus.

Potentially, autoimmune diseases develop from a combination of multiple genes with allelic polymorphisms. An MRL/Mp-Fas(lpr) (/) (lpr) (MRL/lpr) strain of mice develops autoimmune diseases, including lupus nephritis, but another lpr strain, C3H/HeJ-Fas(lpr) (/) (lpr) (C3H/lpr) does not. This indicates that MRL polymorphic genes are involved in the development of the diseases. By quantitative trait loci (QTL) analysis using 527 of the (MRL/lpr x C3H/lpr)F(2) mice, we identified a novel locus for susceptibility to lupus nephritis at map position D5Mit115 on chromosome 5, the same alias of the osteopontin (Opn) gene (LOD score =4.0), susceptible in the MRL allele. In functional analyses of the MRL and C3H Opn alleles using synthetic osteopontin (OPN) made with a new method "cell-free system" with wheat germ ribosomes, the MRL-OPN induced higher expression and production of immunoglobulins as well as cytokines including TNF-alpha, IL-1beta and IFN-gamma in splenocytes and/or macrophages than that of the C3H allele. These findings suggest that allelic polymorphism of OPN causes the functional differences in antibody production and macrophage activation between MRL and C3H strains, possibly involved in the development of lupus nephritis.

MRL/MpTn-gld/gld (MRL/gld) mice, which are deficient in a functional Fas ligand (FasL), spontaneously develop autoimmune diseases involving both lethal glomerulonephritis and systemic arteritis, while MRL/Mp-+/+ (MRL/+) and C3H/HeJ-gld/gld (C3H/gld) do not. To determine the cells responsible for the development of glomerulonephritis and arteritis, we transferred bone marrow cells from MRL/gld mice to undiseased MHC-compatible gld/gld or +/+ mice. In bone marrow irradiation chimeras, MRL/gld bone marrow cells were transferred to lethally irradiated MRL/+ or C3H/HeJ-+/+ (C3H/+) mice, and both recipients developed glomerulonephritis associated with hypergammaglobulinemia without causing graft-versus-host (GVH)-like diseases. However, a striking difference between them was that MRL/+ recipients developed arteritis, but C3H/+ recipients did not. In bone marrow mixed chimeras formed by transferring MRL/gld bone marrow cells to unirradiated mice, the MRL/gld bone marrow cells induced glomerulonephritis in C3H/gld mice, but not in C3H/+ and MRL/+ mice. These results indicate that bone marrow cells from MRL/gld mice can cause glomerulonephritis in mice, even in those with a C3H background, possibly if they survive longer by escaping from Fas-mediated apoptosis, while the development of arteritis requires the MRL genetic background in the re-cipients. This is the first report of the transfer of arteritis in lupus mice to undiseased recipients.

Objective To clarify the mode of inheritance of the tissue distribution of vasculitis in MRL/Mp-lpr/lpr (MRL/lpr) lupus-prone mice and to identify the susceptibility loci. Methods Vasculitis in individual MRL/lpr, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr × C3H/lpr)F1, and (MRL/lpr × C3H/lpr)F2 intercross mice was analyzed by histopathologic grading of main branches of the aorta and of medium-sized arteries in the lower limbs. Genomic DNA samples from F2 intercross mice were examined by simple sequence-length polymorphism analysis, and the polymorphic microsatellite markers highly associated with vasculitis in each tissue were determined as vasculitis susceptibility loci. Results A susceptibility locus with significant linkage to vasculitis of main branches of the aorta was mapped on chromosome 4 at D4Mit213 (map position 13.3cM) selectively in males, while vasculitis of medium-sized arteries in the lower limbs was mapped to different chromosomes: at D8Mit31 on chromosome 8 (map position 33.0) selectively in females and at D5Mit36 on chromosome 5 (map position 65.0). All of these were different from the previously defined loci governing susceptibility to vasculitis involving the kidneys. Conclusion Systemic vasculitis in MRL/lpr mice is genetically controlled with cumulative effects of multiple gene loci, each of which has tissue specificity.

Ankylosis is a major pathological manifestation of spondyloarthropathy. The aim of this study was to evaluate the effects of anti-IL-17 therapy on spontaneous ankylosing enthesitis in mice. In this study, we used male DBA/1 mice as a spontaneous ankylosis model. Serum IL-17 concentrations were determined using enzyme-linked immunosorbent assay. Male DBA/1 mice from different litters were mixed and caged together preceding the treatment at 10 weeks (wk) of age (prophylaxis) or 21 wk of age (intervention). Treatment with anti-IL-17 antibodies or saline was initiated after caging in groups of mice and administered weekly. The onset of tarsal ankylosis was assessed by ankle swelling and histopathological examination. Pathological changes and mRNA expression levels were assessed in joints and ears obtained at the experimental end-point. We found that circulating IL-17 increased with the onset of ankylosis in male DBA/1 mice, coinciding with the onset of dermatitis. The symptoms of dermatitis corresponded to the pathological characteristics of psoriasis: acanthosis with mild hyperkeratosis, scaling, epidermal microabscess formation and augmented expression of K16, S100A8 and S100A9. Prophylactic administration of anti-IL-17 antibodies significantly prevented the development of both ankylosis and dermatitis in male DBA/1 mice caged together. On the other hand, administration of anti-IL-17 antibodies after disease onset had a lesser but significant effect on ankylosis progression but did not affect dermatitis progression. In conclusion, IL-17 is a key mediator in the pathogenic process of tarsal ankylosis and psoriasis-like dermatitis in male DBA/1 mice caged together. Thus, IL-17 is a potential therapeutic target in ankylosing enthesitis and psoriasis in humans.

Pathogenic effects of reactive oxygen species (ROS) in the salivary glands of patients with Sjogren's syndrome have been demonstrated. Epigallocatechin gallate (EGCG), which is a catechin derivative and exhibits potent antioxidant activity, has been reported to ameliorate autoimmune sialadenitis in a murine model, but the mechanism underlying its protective action remains to be investigated. Herein, we examined the effects of EGCG administration to MRL/MpJ-lpr/lpr (MRL-Fas(lpr)) mice on disease severity of autoimmune sialadenitis and protein expression levels of 11 sialadenitis-related molecules - heme oxygenase-1 (HO-1) (antioxidant); thymidine glycol (marker of DNA damage); gp91phox/NADPH oxidase 2 (prooxidant); single-stranded DNA (ssDNA) and cleaved caspase 3 (apoptotic cell markers); p53 and Bax (proapoptotic molecules); Bcl-2 (antiapoptotic molecule); SSA/Ro, SSB/La, and Ifi202 (autoantigens). In EGCG-treated mice, the severity of sialadenitis was substantially decreased. Expression levels of thymidine glycol, gp91phox, ssDNA, cleaved caspase 3, p53, Bax, SSA/Ro, SSB/La, and Ifi202 in duct epithelial cells of salivary glands from EGCG-treated mice were reduced, whereas HO-1 and Bcl-2 were overexpressed. Results of correlation analysis among sialadenitis severity and 11 sialadenitis related-molecules, and those of partial correlation analysis between apoptotic related-molecules and sialadenitis severity or HO-1 suggested that the consecutive pathogenic cycle including activated autoimmune reactions, ROS synthesis, DNA damage and p53-dependent apoptosis was associated with the pathogenesis of autoimmune sialadenitis in MRL-Fas(lpr) mice. Overexpression of HO-1 and Bcl-2 mediated by EGCG blocked this pathogenic cycle, subsequently resulting in the inhibition of ROS-mediated DNA damage and apoptosis, and protected salivary gland tissues from oxidative stress. Clinically, green tea catechin may have therapeutic efficacy for Sjogren's syndrome.

Clones for murine paired immunoglobulin-like receptors (PIR) were first isolated as those coding for type I transmembrane glycoproteins with six immunoglobulin-like domains homologous to human Fc alphaR, bovine Fc gamma2R, and other related receptors. However, they turned out to bind neither IgA nor other immunoglobulins in the case of the ectopic expression on COS-1 fibroblastic cells. PIR-A and B are expressed on a wide variety of cells in the murine immune system, such as in B cells, mast cells, macrophages, and dendritic cells, mostly in a pairwise fashion. PIR-A requires homodimeric Fc receptor common gamma chain, which harbors an immunoreceptor tyrosine-based activation motif, for its efficient cell surface expression and for the delivery of activation signaling. In contrast, PIR-B contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic portion and inhibits receptor-mediated activation signaling in vitro upon engagement with other activating-type receptors such as the antigen receptor on B cells and the high affinity Fc receptor for IgE on mast cells. ITIMs of PIR-B on macrophages and B cells have been shown to be constitutively phosphorylated in their tyrosine residues. Although the ligand for PIR still remains unknown, the transgenics and the gene-targeted mice will provide us with valuable information on their physiological roles in the immune regulation.

The Src family kinase Lyn initiates intracellular signal transduction by associating with a variety of immune receptors such as antigen receptor on B cells and high-affinity Fc receptor (FcR) for immunoglobulin Ig(E) (FcepsilonRI) on mast cells. Involvement of Lyn in the IgE-mediated immediate-type hypersensitivity is well documented, but the physiological significance of Lyn in IgG-dependent, type III low-affinity FcR for IgG (FcgammaRIII)-mediated responses is largely unknown. In this study, we generated a double-mutant mouse strain deficient in both type II FcR for IgG (FcgammaRIIB) and Lyn to exclude any involvement of inhibitory signaling by FcgammaRIIB, which otherwise downregulates FcgammaRIII-mediated cellular responses. FcgammaRIIB-deficient but Lyn-sufficient mice served as controls. The Lyn deficiency attenuated IgG-mediated systemic anaphylaxis in vivo, and significantly reduced calcium mobilization and degranulation responses of bone marrow-derived mast cells (BMMCs) in vitro. However, we found that either interleukin 4 or tumor necrosis factor alpha release by BMMCs was comparable to that from Lyn-deficient and control mice, and the reverse-passive Arthus reaction was equally induced in both mutant mice, indicating that Lyn is not involved in the onset of the IgG-mediated, FcgammaRIII-dependent late phase responses of mast cells. These findings provide us with insight into distinct signaling mechanisms in mast cells underlying the development of diverse pathologies as well as a therapeutic potential for selective treatment of allergic disorders.

The combination of hemorrhagic pneumonitis and rapidly progressive glomerulonephritis is a characteristic feature of Goodpasture's syndrome (GPS), an autoimmune disease resulting from the interaction of pathogenic anti–collagen type IV (C-IV) antibodies with alveolar and glomerular basement membranes. Lack of a suitable animal model for this fatal disease has hampered both a basic understanding of its etiology and the development of therapeutic strategies. We now report a novel model for GPS using mice deficient in a central regulatory receptor for immunoglobulin (Ig)G antibody expression and function, the type IIB Fc receptor for IgG (FcγRIIB). Mutant mice immunized with bovine C-IV reproducibly develop massive pulmonary hemorrhage with neutrophil and macrophage infiltration and crescentic glomerulonephritis. The distinctive linear, ribbon-like deposition of IgG immune complex seen in GPS was observed along the glomerular and tubulointerstitial membranes of diseased animals. These results highlight the role of FcγRIIB in maintaining tolerance and suggest that it may play a role in the pathogenesis of human GPS.

The Kit and PDGFRa receptor tyrosine kinases are encoded in close proximity at the murine white spotting (W) and patch (Ph) loci. Whereas W mutations affect hematopoiesis, melanogenesis, and gametogenesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. The W(sh), W(57), and Ph mutations diminish Kit expression in certain cell types such as mast cells and enhance it in others. The W(sh), W(57), and Ph mutations arose from deletions and inversions affecting sequences in between the Kit and PDGFRa genes. We have determined the precise location of the breakpoint of the W(sh) inversion and the endpoints of the W(57) deletion upstream of the Kit transcription start site and examined the effect of these mutations on Kit expression in mast cells and hematopoietic stem cells and lineage progenitors. Our results indicate that positive elements controlling Kit expression in mast cells mapping in between -23 and -154 kb from the transcription start site can be dissociated from negative elements controlling Kit misexpression during embryonic development in the vicinity of the PDGFRa gene. In addition, we have identified two clusters of hypersensitive sites in mast cells at -23 -28 kb and -147 -154 kb from the Kit gene transcription start site. Analysis of these hypersensitive sites in mutant mast cells indicates a role for HS4-6 in Kit expression in mast cells. These findings provide a molecular basis for the phenotype of these Kit expression mutations and they provide insight into the complex mechanisms governing the regulation of Kit expression.

It is widely accepted that immunoglobulin (Ig)E triggers immediate hypersensitivity responses by activating a cognate high-affinity receptor, FcepsilonRI, leading to mast cell degranulation with release of vasoactive and proinflammatory mediators. This apparent specificity, however, is complicated by the ability of IgE to bind with low affinity to Fc receptors for IgG, FcgammaRII and III. We have addressed the in vivo significance of this interaction by studying IgE-mediated passive systemic anaphylaxis in FcgammaR-deficient mice. Mice deficient in the inhibitory receptor for IgG, FcgammaRIIB, display enhanced IgE-mediated anaphylactic responses, whereas mice deficient in an IgG activation receptor, FcgammaRIII, display a corresponding attenuation of IgE-mediated responses. Thus, in addition to modulating IgG-triggered hypersensitivity responses, FcgammaRII and III on mast cells are potent regulators of IgE-mediated responses and reveal the existence of a regulatory pathway for IgE triggering of effector cells through IgG Fc receptors that could contribute to the etiology of the atopic response.

Autoimmune diseases, like rheumatoid arthritis, result from a dysregulation of the immune response culminating in hyperactivation of effector cells leading to immune-mediated injury. To maintain an appropriate immune response and prevent the emergence of autoimmune disease, activation signals must be regulated by inhibitory pathways. Biochemical and genetic studies indicate that the type IIB low-affinity receptor for immunoglobulin (Ig)G (FcgammaRIIB) inhibits cellular activation triggered through antibody or immune complexes and may be an important component in preventing the emergence of autoimmunity. To investigate the role of FcgammaRIIB in the development of type II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans, we have examined its contribution in determining the susceptibility to CIA in the nonpermissive H-2(b) haplotype. H-2(b) mice immunized with bovine CII do not develop appreciable disease. In contrast, immunization of the FcgammaRIIB-deficient, H-2(b) mice with bovine CII induced CIA at an incidence of 42.2%. The maximal arthritis index of the FcgammaRIIB-deficient mice developing CIA (6.9 +/- 3.6) was comparable to that of DBA/1 mice (8.6 +/- 1.9), an H-2(q) strain susceptible for CIA induction. IgG1, IgG2a, and IgG2b antibody responses against CII were elevated in the FcgammaRIIB-deficient animals, especially in those mice showing arthritis, but less pronounced than DBA/1 mice. Histological examinations of the arthritic paws from FcgammaRIIB-deficient mice revealed that cartilage was destroyed and bone was focally eroded in association with marked lymphocyte and monocyte/macrophage infiltration, very similar to the pathologic findings observed in DBA/1 mice. These results indicate that a nonpermissive H-2(b) haplotype can be rendered permissive to CIA induction through deletion of FcgammaRIIB, suggesting that FcgammaRIIB plays a critical role in suppressing the induction of CIA.

Autoantibodies and immune complexes are major pathogenic factors in autoimmune injury, responsible for initiation of the inflammatory cascade and its resulting tissue damage. This activation results from the interaction of immunoglobulin (Ig)G Fc receptors containing an activation motif (ITAM) with immune complexes (ICs) and cytotoxic autoantibodies which initiates and propagates an inflammatory response. In vitro, this pathway can be interrupted by coligation to FcgammaRIIB, an IgG Fc receptor containing an inhibitory motif (ITIM). In this report, we describe the in vivo consequences of FcgammaRII deficiency in the inflammatory response using a mouse model of IC alveolitis. At subthreshold concentrations of ICs that fail to elicit inflammatory responses in wild-type mice, FcgammaRII-deficient mice developed robust inflammatory responses characterized by increased hemorrhage, edema, and neutrophil infiltration. Bronchoalveolar fluids from FcgammaRII-/- stimulated mice contain higher levels of tumor necrosis factor and chemotactic activity, suggesting that FcgammaRII deficiency lowers the threshold of IC stimulation of resident cells such as the alveolar macrophage. In contrast, complement- and complement receptor-deficient mice develop normal inflammatory responses to suprathreshold levels of ICs, while FcRgamma-/- mice are completely protected from inflammatory injury. An inhibitory role for FcgammaRII on macrophages is demonstrated by analysis of FcgammaRII-/- macrophages which show greater phagocytic and calcium flux responses upon FcgammaRIII engagement. These data reveal contrasting roles for the cellular receptors for IgG on inflammatory cells, providing a regulatory mechanism for setting thresholds for IC sensitivity based on the ratio of ITIM to ITAM FcgammaR expression. Exploiting the FcgammaRII inhibitory pathway could thus provide a new therapeutic approach for modulating antibody-triggered inflammation.

The ζ family includes ζ, η, and FcεRIγ (Fcγ). Dimers of the ζ family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking ζ/η chains, T cell development is impaired, yet low numbers of CD4+ and CD8+ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a ζ family dimer can promote T cell maturation, or that in the absence of ζ/η, Fcγ serves as a subunit in TCR complexes. To elucidate the role of ζ family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only ζ/η or Fcγ. The data reveal that surface complexes that are expressed in the absence of ζ family dimers are capable of transducing signals required for α/β–T cell development. Strikingly, T cells generated in both ζ/η−/− and ζ/η−/−–Fcγ−/− mice exhibit a memory phenotype and elaborate interferon γ. Finally, examination of different T cell populations reveals that ζ/η and Fcγ have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of ζ family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.

Humoral and cellular immune responses communicate with each other via Fc receptors (FcR) expressed on various hematopoietic cells. Recent studies on several FcR knockout mice demonstrated pivotal roles of an IgG/FcγR system in the regulation of immune responses and the onset of hypersensitivity. The γ subunit of FcR is an essential component of the complex and is required for both receptor assembly and signal transduction. FcR γ chain-deficient mice have lost the functional expression of FceRI, FcγRI, and FcγRIII and are unable to mount several types of hypersensitive reactions, including the skin Arthus reaction. In contrast, FcγRII-deficient mice exhibit augmented humoral immune responses and IgG-mediated anaphylaxis reactions. Thus, the regulatory system of murine hypersensitive responses involves both positive and negative signaling through FcR. In B cells, FcγRIIb modulates membrane Ig-induced Ca 2+ mobilization by inhibiting Ca 2+ influx through phosphorylation of its immunoreceptor tyrosine-based inhibition motif and recruitment of cytoplasmic phosphatases. Elucidation of the detailed mechanisms of negative regulatory signaling in the inflammatory effector cells by FcγRIIb as well as several groups of potent inhibitory molecules expressed on such cells should be valuable in the development of novel therapeutic procedures for allergic disorders.

Acute necrotizing encephalopathy (ANE) is characterized by symmetrical brain necrosis, suggested to be due to breakdown of the blood–brain barrier (BBB). We experienced a rare case of ANE complicated with systemic lupus erythematosus (SLE), and found that the patient’s serum (V10-5) had binding activity to human umbilical vein endothelial cells (HUVECs). By SARF (Serological identification system for Autoantigens using a Retroviral vector and Flow cytometry) method using V10-5 IgG, a clone bound to V10-5 IgG was isolated. This cell clone was integrated with cDNA identical to EphB2, which plays critical roles in neuronal cells and endothelial cells. HUVECs and human brain microvascular endothelial cells expressed EphB2 and the V10-5 IgG bound specifically to EphB2-transfected cells. Anti-EphB2 antibody was not detected in other SLE patients without ANE. In this report, we identified EphB2 as a novel autoantigen, and anti-EphB2 antibody may define a novel group of brain disorders. Anti-EphB2 antibody can interfere not only with endothelial cells including those of the BBB (acting as an anti-endothelial cell antibody), but also neuronal cells (acting as an anti-neuronal antibody) if the BBB has been breached. Future studies should determine the clinical prevalence and specificity of anti-EphB2 antibody, and the molecular mechanisms by which anti-EphB2 antibody mediates neuronal and vascular pathological lesions.

Autoantibodies against integral membrane proteins are usually pathogenic. Although anti-endothelial cell antibodies (AECAs) are considered to be critical, especially for vascular lesions in collagen diseases, most molecules identified as autoantigens for AECAs are localized within the cell and not expressed on the cell surface. For identification of autoantigens, proteomics and expression library analyses have been performed for many years with some success. To specifically target cell-surface molecules in identification of autoantigens, we constructed a serological identification system for autoantigens using a retroviral vector and flow cytometry (SARF). Here, we present an overview of recent research in AECAs and their target molecules and discuss the principle and the application of SARF. Using SARF, we successfully identified three different membrane proteins: fibronectin leucine-rich transmembrane protein 2 (FLRT2) from patients with systemic lupus erythematosus (SLE), intercellular adhesion molecule 1 (ICAM-1) from a patient with rheumatoid arthritis, and Pk (Gb3/CD77) from an SLE patient with hemolytic anemia, as targets for AECAs. SARF is useful for specific identification of autoantigens expressed on the cell surface, and identification of such interactions of the cell-surface autoantigens and pathogenic autoantibodies may enable the development of more specific intervention strategies in autoimmune diseases.

SUMMARY In order to investigate the genetic origin of nephritogenic antibodies in MRL/Mp-lpr/lpr (MRL/lpr) lupus mice, we isolated the germ-line heavy chain variable region (VH) gene corresponding to the nephritogenic antibody, B1, derived from an unmanipulated MRL/lpr mouse. Injection of this antibody into C.B-17/Icr-scid/scid mice resulted in the generation of wire loop-like glomerular lesions resembling those of lupus nephritis. Nucleotide sequences of this germ-line VH gene showed no replacement mutation in the VH region of the B1 antibody. Furthermore, this gene was identical to that found in the C3H/HeJ-lpr/lpr strain of mice. Our results suggest that germ-line VH genes can encode nephritogenic antibodies without somatic mutation, even in a mouse strain not prone to lupus.

Background Prolyl isomerase Pin1 may be involved in innate immunity against microbial infection, but the mechanism how Pin1 controls the innate immunity is poorly understood. Methodology/Principal Findings Injection of lipopolysaccharide (LPS) into the mice induces inflammatory pulmonary disorder and sometimes the serious damages lead to death. Comparing to the wild-type (WT) mice, the Pin1−/− mice showed more serious damages in lung and the lower survival rate after the LPS injection. We compared the levels of typical inflammatory cytokines. Pin1−/− mice overreacted to the LPS injection to produce inflammatory cytokines, especially IL-6 more than WT mice. We showed that Pin1 binds phosphorylated PU.1 and they localize together in a nucleus. These results suggest that Pin1 controls the transcriptional activity of PU.1 and suppresses overreaction of macrophage that causes serious damages in lung. Conclusions/Significance Pin1 may protect the mice from serious inflammation by LPS injection by attenuating the increase of IL-6 transcription of the mouse macrophages.

Concanavalin A (Con A)-induced hepatitis is a T-cell-mediated murine experimental model of autoimmune hepatitis. Mice lacking Valpha14 NKT cells were found to be less sensitive to this hepatitis and the MRL/Mp-Fas(lpr/lpr) (MRL/lpr; i.e. Fas deficient) mice were also less sensitive. We report herein that MRL/Mp-Fas(lpr/lpr)-Sap(rpl/-) (MRL/lpr/rpl) mice lack Valpha14 NKT cells and are deficient in the Fas antigen but sensitive to Con A-induced hepatitis. The signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is an adaptor molecule containing a Src homology 2 (SH2) domain. We previously reported new mutant mice found among MRL/lpr mice and revealed that SAP deficiency led to the regression of autoimmune phenotypes in mutant MRL/lpr/rpl mice. It was also revealed that CD4(+) and CD8(+) T cells were effector cells and that blockade of 2B4, one of the SLAM family receptors, inhibited the induction of hepatitis in MRL/lpr/rpl mice. These data suggest that signals mediated by molecules other than SAP from 2B4 in T cells played important roles in the induction of hepatitis in MRL/lpr/rpl mice.

Desmosomal cadherins are essential cell adhesion molecules expressed in the epidermis. We identified a mutation of a cadherin superfamily member, namely, desmoglein 4 (Dsg4), in early onset of death (EOD) hage mice with hypotrichosis. The mutation was induced by the insertion of an early transposon II-β into intron 8 of Dsg4. Mast cell hyperplasia was observed in the skin of EOD hage mice. The abnormally expanded population of lpr T cells, i.e., CD4−CD8−B220+Thy1.2+ αβT cells, in the splenocytes of EOD mice was reduced in EOD hage mice. Therefore, it was suspected that the long-living mutant EOD hage mice were selected from lupus-prone EOD mice because of their immunological immaturity. These findings clearly indicate that Dsg4 is an important molecule for the formation of hair follicles and hypothesize that unorganized hyperplastic hair follicles in anagen due to the Dsg4 mutation provide niches for mast cell precursors in the skin.

Some molecules originally shown to be critical for immune responses, such as the major histocompatibility complex (MHC) class I molecules, CD3ζ, and semaphorin 7A (3, 8, 15, 23), also serve neuronal functions. Based on studies of mutant mice, CD3ζ proved critical for the development of lateral geniculate nucleus (LGN) and long-term synaptic plasticity in the adult hippocampus (3, 8). In the immune system, CD3 subunits are expressed on T cells. The T-cell receptor (TCR)-CD3 complex recognizing specific antigens bound to MHC present on antigen-presenting cells (APCs) is composed of a TCR heterodimer and CD3 polypeptides organized as dimers. The cell-cell interaction between APCs and T cells is known as an immunological synapse (5) in the mature immune system. In αβ T cells, when the TCR interacts with the antigen/MHC complex, it transmits information to a signal-transducing complex consisting of two CD3 subunit dimers, CD3ɛ-CD3γ and CD3ɛ-CD3δ, and the CD3ζ-CD3ζ homodimer (10). Among CD3 subunits, CD3ζ is a crucial subunit having three immunoreceptor tyrosine-based activation motifs (ITAMs), whereas the remaining subunits have one ITAM (25). Tyrosine residues within these motifs are phosphorylated by src family tyrosine kinases, and then Src homology 2-containing proteins, including the tyrosine kinase ZAP70, participate in signaling (13). The signaling in γδ TCRs is different from that in αβ TCRs. Most γδ TCRs lack CD3δ, and signal transduction by γδ TCR is superior to that by αβ TCR, as measured by its ability to induce calcium mobilization, extracellular signal-regulated kinase activation, and cellular proliferation (6). FcγRIIB is a low-affinity membrane receptor for immune complexes broadly distributed on hematopoietic cells, such as B cells, mast cells, basophils, macrophages, eosinophils, neutrophils, dendritic cells, and Langerhans cells. FcγRIIB negatively regulates B-cell receptor-induced signaling in B cells via the inhibitory immunoreceptor tyrosine-based inhibition motif in its cytoplasmic domain (24, 30). Coengagement of the B-cell receptor and FcγRIIB results in the tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibition motif and the recruitment of SHIP. SHIP, by hydrolyzing PIP3, causes the dissociation of Brutons tyrosine kinase from the membrane and the inhibition of calcium influx into the cell (29). Although the functional significance of FcγRIIB has been elucidated in hematopoietic cells, the physiological roles of FcγRIIB have not been explored in the nervous system. The cerebellum is a key region operating motor learning and motor coordination. Cerebellar functions are regulated by coordinated neural networks. There are two major types of inputs to the cerebellum: climbing fibers (CFs) and mossy fibers. CFs are the axons of neurons located in the inferior olive. They enter the cerebellum and establish two branches, one to the deep nuclei and one to the Purkinje cells (PCs) of the cerebellar cortex. Mossy fibers synapse with the claw-like dendrites of the granule cells (GCs) in the cerebellar cortex. The GCs in turn communicate with the PC dendrites via their long parallel fiber (PF) axons. PC axons are the sole efferents from the cerebellar cortex. Here, we found an unexpected common functional significance of CD3 and FcγRIIB in the cerebellum. Both CD3ɛ and FcγRIIB are located on Purkinje cells. CD3ɛ-deficient mice and FcγRIIB-deficient mice shared impaired development of Purkinje neurons, enhanced paired-pulse facilitation (PPF) of parallel fiber-Purkinje cell synapses, and poor rotarod performance at high speed.

Background— Recent studies have suggested that endogenous erythropoietin (Epo) plays an important role in the mobilization of bone marrow–derived endothelial progenitor cells (EPCs). However, it remains to be elucidated whether the Epo system exerts protective effects on pulmonary hypertension (PH), a fatal disorder encountered in cardiovascular medicine. Methods and Results— A mouse model of hypoxia-induced PH was used for study. We evaluated right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in mice lacking the Epo receptor (EpoR) in nonerythroid lineages (EpoR −/− rescued mice) after 3 weeks of exposure to hypoxia. Those mice lack EpoR in the cardiovascular system but not in the hematopoietic system. The development of PH and pulmonary vascular remodeling were accelerated in EpoR −/− rescued mice compared with wild-type mice. The mobilization of EPCs and their recruitment to the pulmonary endothelium were significantly impaired in EpoR −/− rescued mice. By contrast, reconstitution of the bone marrow with wild-type bone marrow cells ameliorated PH in the EpoR −/− rescued mice. Hypoxia enhanced the expression of EpoR on pulmonary endothelial cells in wild-type but not EpoR −/− rescued mice. Finally, hypoxia activated endothelial nitric oxide synthase in the lungs in wild-type mice but not in EpoR −/− rescued mice. Conclusions— These results indicate that the endogenous Epo/EpoR system plays an important role in the recruitment of EPCs and prevents the development of PH during chronic hypoxia in mice in vivo, suggesting the therapeutic importance of the system for the treatment of PH.

Autoantibody production and lymphadenopathy are common features of systemic autoimmune disease. Targeted or spontaneous mutations in the mouse germline have generated many autoimmune models with these features. Importantly, the models have provided evidence for the gene function in prevention of autoimmunity, suggesting an indispensable role for the gene in normal immune response and homeostasis. We describe here pathological and genetic characterizations of a new mutant strain of mice, the mutation of which spontaneously occurred in the Fas-deficient strain, MRL/Mp.Faslpr (MRL/lpr). MRL/lpr is known to stably exhibit systemic lupus erythematosus-like diseases. However, the mutant mice barely displayed autoimmune phenotypes, though the original defect in Fas expression was unchanged. Linkage analysis using (mutant MRL/lpr × C3H/lpr)F2 mice demonstrated a nucleotide insertion that caused loss of expression of small adaptor protein, signaling lymphocyte activation molecule (SLAM)-associated protein (SAP). SAP is known to be a downstream molecule of SLAM family receptors and to mediate the activation signal for tyrosine kinase Fyn. Recent studies have shown pleiotropic roles of SAP in T, B, and NK cell activations and NKT cell development. The present study will provide evidence for an essential role for SAP in the development of autoimmune diseases, autoantibodies, and lymphadenopathy in MRL/lpr lupus mice.

A role for dendritic cells (DCs) has been emphasized in the onset of acute graft-versus-host disease (GVHD). We have made efforts to develop a new strategy for suppression of DC functions with a chemical compound in the treatment of acute GVHD. We here describe the immunological characterization of the new chemical compound NK026680. It was found that NK026680 significantly suppressed (1) expression of CD83, CD86, and major histocompatibility complex (MHC) class I and II antigens on human monocyte-derived DCs, (2) excretion of interleukin-12p40 on activation of monocyte-derived DCs, (3) allogeneic responses of human and mouse T cells and (4) mortality in mice with acute GVHD evoked across MHC class I or II. The beneficial effect of NK026680 administered orally was without any recognizable adverse effects. Early intervention in acute GVHD was required for this effect, indicating that an early event in acute GVHD is a critical target of NK026680. We propose the use of NK026680 as a prophylactic for acute GVHD.

Objective To analyze the influence of the genetic background of an arthritis-prone strain of mice, MRL, on the spontaneous development of arthropathy in DBA/1 mice, which histopathologically resembles enthesopathy in humans, and to clarify the strain-specific gene loci and their interactions that confer susceptibility to arthropathy. Methods MRL, DBA/1, (MRL × DBA/1)F1, and (MRL × DBA/1)F2 intercross mice were prepared, and the severity and onset of arthropathy of the ankle joints in individual mice were quantified (0–3 and 0–5 scale, respectively). A genome-wide scan of 271 male F2 intercross mice with polymorphic microsatellite markers was performed. Results Only male DBA/1, (MRL × DBA/1)F1, and (MRL × DBA/1)F2 mice developed arthropathy. The macroscopic and histopathologic findings of arthropathy in the F2 mice were similar to those in the parental DBA/1 mice, but the onset was significantly earlier. In the quantitative trait locus analysis of male F2 mice, 1 susceptibility locus for both the severity and early onset of the disease in the region of an MRL allele, Amd1, was located at marker D10Mit259 (map position 40.0 cM), which was common to 1 of the sialadenitis susceptibility loci in MRL mice, Asm1. Another susceptibility locus for the severity and early onset of arthropathy in the region of a DBA allele, Amd2, was located at D3Mit46 (29.5 cM). These loci manifested an additive effect on the development of arthropathy. Conclusion Arthropathy in DBA/1 mice is under the control of an allelic combination of gene loci, one of which is common to the locus for sialadenitis in MRL/MpJ-lpr/lpr mice.

Reported here is the cloning of the gene for the beta-chain (Ig beta) of the chicken B-cell receptor for antigen. A cDNA library made from B lymphocyte-derived DT40 cells was first screened with a beta-chain cDNA clone from rat. A full-length clone (1556 bp) was obtained encoding a 226-amino-acid beta-chain having 40% sequence identity with the highly conserved beta-chains present in mammals and producing mRNA of 1.7 kb. Clones containing the full B29 gene encoding chicken Ig beta were isolated and fully sequenced. As in mammals, the chicken B29 gene consists of six exons. The chicken B29 gene is somewhat larger (4336 bp) than that of rat (3.1 kb) and human (3.6 kb). Genome collinearity was found in the region of the chicken B29 gene. Genes for potential skeletal muscle sodium channel (2.2 kb upstream) and for growth hormone (2.3 kb downstream) were found in the same order and transcriptional orientation as in mammals, though the genes are more closely positioned in the chicken.

Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B–mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5′-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B–mediated inhibition requires redundant functions of SHP-1 and SHP-2.

The Kit ligand (KL)/Kit receptor pair functions in hematopoiesis, gametogenesis, and melanogenesis. KL is encoded at the murine steel (Sl) locus and encodes a membrane growth factor which may be proteolytically processed to produce soluble KL. The membrane-associated form of KL is critical in mediating Kit function in vivo. Evidence for a role of cytoplasmic domain sequences of KL comes from the Sl17H mutation, a splice site mutation that replaces the cytoplasmic domain with extraneous amino acids. Using deletion mutants and the Sl17H allele, we have investigated the role of the cytoplasmic domain sequences of KL in biosynthetic processing and cell surface presentation. The normal KL protein products are processed for cell surface expression, where they form dimers. Both Sl17H and the cytoplasmic deletion mutants of KL were processed to the cell surface; however, the rate of transport and protein stability were affected by the mutations. Deletion of cytoplasmic domain sequences of KL did not affect dimerization of KL. In contrast, dimerization of the Sl17H protein was reduced substantially. In addition, we have characterized the hematopoietic cell compartment in Sl17H mutant mice. The Sl17H mutation has only minor effects on hematopoiesis. Tissue and peritoneal mast cell numbers were reduced in mutant mice as well as in myeloid progenitors. Interestingly, long-term bone marrow cultures from Sl17H mice did not sustain the long-term production of hematopoietic cells. In addition, homing of normal hematopoietic progenitors to the spleen of irradiated Sl17H/Sl17H recipient mice was diminished in transplantation experiments, providing evidence for a role of Kit in homing or lodging. These results demonstrate that the membrane forms of KL exist as homodimers on the cell surface and that dimerization may play an important role in KL/Kit-mediated juxtacrine signaling.

Despite its widespread distribution on both lymphoid and myeloid cells, the biological role of the low-affinity immunoglobulin-G receptor, Fc gamma RII, is not fully understood. Defects in this receptor or its signalling pathway in B cells result in perturbations in immune-complex-mediated feedback inhibition of antibody production. We now report that Fc gamma RII-deficient animals display elevated immunoglobulin levels in response to both thymus-dependent and thymus-independent antigens. Additionally, the effector arm of the allergic response is perturbed in these mice. Mast cells from Fc gamma RII-/- are highly sensitive to IgG-triggered degranulation, in contrast to their wild-type counterparts. Fc gamma RII-deficient mice demonstrate an enhanced passive cutaneous analphylaxis reaction, the result of a decreased threshold for mast-cell activation by Fc gamma RIII cross-linking. These results demonstrate that Fc gamma RII acts as a general negative regulator of immune-complex-triggered activation in vivo for both the afferent and efferent limbs of the immune response. Exploiting this property offers new therapeutic opportunities for the treatment of allergic and autoimmune disorders.

A simple method based on the polymerase chain reaction (PCR) was developed for detecting the relative mRNA amounts of the four mouse IgG subclasses in total RNA samples and is described in this report. The main features of this method are, first, cDNA amplification including V H through the constant region(C H 2 domain) of each IgG subclass with a set of consensus PCR primers(V H 1BACK and 32 P-labeled Cγ32), and secondly, cleavage of the amplified DNA fragments with Bam HI and Xho I endonucleases which act at distinct cleavage sites in the constant region of each IgG subclass. The radioactive intensities of the different sized fragments separated on polyacrylamide gel were used to show the relative amounts of IgG subclasses at the RNA level. This method provides clear detection of each IgG subclass using RNA samples from tissues in which Ig-producing cells are rare.

Introduction Anti-endothelial cell antibodies (AECAs) are thought to be critical for vasculitides in collagen diseases, but most were directed against molecules localized within the cell and not expressed on the cell surface. To clarify the pathogenic roles of AECAs, we constructed a retroviral vector system for identification of autoantigens expressed on the endothelial cell surface. Methods AECA activity in sera from patients with collagen diseases was measured with flow cytometry by using human umbilical vein endothelial cells (HUVECs). A cDNA library of HUVECs was retrovirally transfected into a rat myeloma cell line, from which AECA-positive clones were sorted with flow cytometry. cDNA of the cells was analyzed to identify an autoantigen, and then the clinical characteristics and the functional significance of the autoantibody were evaluated. Results Two distinct AECA-positive clones were isolated by using serum immunoglobulin G (IgG) from a patient with systemic lupus erythematosus (SLE). Both clones were identical to cDNA of fibronectin leucine-rich transmembrane protein 2 (FLRT2). HUVECs expressed FLRT2 and the prototype AECA IgG bound specifically to FLRT2-transfected cells. Anti-FLRT2 antibody activity accounted for 21.4% of AECAs in SLE. Furthermore, anti-FLRT2 antibody induced complement-dependent cytotoxicity against FLRT2-expressing cells. Conclusions We identified the membrane protein FLRT2 as a novel autoantigen of AECAs in SLE patients by using the retroviral vector system. Anti-FLRT2 antibody has the potential to induce direct endothelial cell cytotoxicity in about 10% of SLE patients and could be a novel molecular target for intervention. Identification of such a cell-surface target for AECAs may reveal a comprehensive mechanism of vascular injury in collagen diseases.