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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 Fc? receptor type IIb (Fc?RIIb) is a major negative regulator of B cells and the lack of Fc?RIIb 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 Fc?RIIb deficient B6 mice. METHODS We established 2 lines of Fc?RIIb deficient B6 congenic mouse strains (KO1 and KO2) by selective backcrossing of the originally constructed Fc?RIIb deficient mice on a hybrid (129×B6) background into a B6 background. Although both lack Fc?RIIb 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.

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.

Heme oxygenase (HO) catalyzes oxidative breakdown of heme, and constitutes two isozymes, HO-1 and HO-2. Here, we explored the tissue-specific regulation of expression of HO-1 and HO-2 under hypoxemia. There was no significant change in the overall expression levels of HO-1 and HO-2 mRNAs and proteins in the lung during adaptation of C57BL/6 mice to normobaric hypoxia (10% O(2)). However, immunohistochemical analysis revealed the increased expression of HO-1 and HO-2 proteins after 28 days of normobaric hypoxia in the pulmonary venous myocardium that is the extension of the left atrial myocardium into pulmonary venous walls. Moreover, the expression of HO-2 protein was increased in the sub-endocardial myocardium of ventricles under hypoxia, while HO-1 protein level was increased in the full-thickness walls. Thus, hypoxemia induces expression of both HO-1 and HO-2 proteins in the myocardium. Using C57BL/6 mice lacking HO-2 (HO-2(-/-)), which manifest chronic hypoxemia, we also showed that the HO-1 protein level in the lung was similar between HO-2(-/-) mice and wild-type mice. Unexpectedly, HO-1 protein level was lower by 35% in the HO-2(-/-) mouse liver than the wild-type liver. These results indicate that the expression of HO-1 protein is regulated in a tissue-specific manner under hypoxemia.

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.

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.

Genomic recombination events, including VDJ recombination (VDJR) and class-switch recombination (CSR), are indispensable for the adaptation and progression of the acquired immune system. These processes are completed by orderly, temporal onsets of the gene rearrangements along with B-cell differentiation. The presence of various premature transcripts of immunoglobulin heavy chain (IgH) alleles has been demonstrated during B-cell ontogeny. These include D(H)-J(H) (DJ)-mu, J(H)-mu, and sterile transcripts of C(H). Since these transcripts can be detected during the onset of VDJR and CSR, their presence is believed to reflect a structural change in the genome, favoring VDJR and CSR. This report presents evidence of persistent DJ transcription and onset of CSR on an unproductive IgH allele in peripheral tissues. Nucleotide sequence analysis revealed that these transcripts showed DJ-gamma (Dgamma) configuration and that characteristics of the variable region were essentially the same as those of the DJ-mu transcript previously described. It was noted that the small intestine abundantly expresses Dgamma transcripts with gamma2b and gamma1 isotypes of the IgH constant region. The present findings indicate the onset of CSR preceding V(H) to DJ joining in an unproductive IgH allele of the peripheral B cell and the specificity for the gut-associated condition for B-cell differentiation in the small intestine.

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.

We have developed a method for the simultaneous estimation of the levels of the prostanoids 6-keto prostaglandin (PG) Flalpha, PGB2, PGD2, PGE2, PGF2(alpha), PGJ2, and thromboxane (TX) B2 in blood- or serum-containing medium using liquid chromatography-tandem mass spectrometry. These prostanoids and their deuterium derivatives, which were used as internal standards, were subjected to solid-phase extraction using Empore C18 HD disk cartridges and analyzed in the selected reaction-monitoring mode. A linear response curve starting at 10 pg of prostanoid/tube was observed for each prostanoid. The accuracy of the method was demonstrated with samples containing known amounts of the prostanoids. Furthermore, we used this method to analyze the prostanoids produced in mouse bone marrow-derived mast cells stimulated with arachidonic acid, which resulted in the production of PGD2, PGE2, PGF2alpha, and TXB2. The results suggest that this simultaneous quantification method is useful for the analysis of the production of biomedically important prostanoids.

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.

Fatty acids have a great influence on the process of lymphocyte apoptosis which is considered as a modulating factor of immune response in both humans and animals. However the mechanism underlying the function of fatty acids in the process of lymphocyte apoptosis is not fully understood. In this study we show that the appearance of adipocyte-type fatty acid binding protein (A-FABP) is induced upon administration of dexamethasone (DEX) in both in vivo and cultured lymphocytes, and its distinct nuclear localization occurs in close relation to the DEX-induced apoptosis process. In immuunohistochemistry of mouse spleen, A-FABP-immunoreactivity starts to occur 3 h after DEX stimulation, and it massively localizes in the nucleus 8 h after the treatment, while no A-FABP-immunoreactivity is discerned in the lymphocytes of normal as well as 24 h post-injection spleen. In the murine T-cell leukemia CTLL-2 cells, A-FABP-immunoreactivity is also induced in both of the cytoplasm and nucleus when the apoptosis is induced by IL-2 retrieval together with DEX treatment, while in the presence of IL-2 A-FABP-immunoreactivity is confined to the cytoplasm with DEX trreatment. On the other hand, A-FABP-immunoreactivity is not detected by IL-2 retrieval alone. The present findings altogether suggest that A-FABP and its ligands, fatty acids, play an important role in the process of apoptosis and the immune modulation induced by DEX.

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.

We have identified and characterized two mouse cDNAs in a mouse antigen-stimulated bone marrow-derived mast cell cDNA library, both of which encode type I transmembrane proteins. The genes were closely mapped in the distal region of mouse chromosome 11 and expressed not only in mast cells but also widely in leukocytes. The extracellular domains of their encoded proteins contain a single variable immunoglobulin (Ig) motif sharing about 90% identity with amino acids, showing that they comprise a pair of molecules and belong to the Ig superfamily. We named these molecules leukocyte mono-Ig-like receptor1 and 2 (LMIR1 and 2). The intracellular domain of LMIR1 contains several immunoreceptor tyrosine-based inhibition motifs (ITIMs). When cross-linked, the intracellular domain was tyrosine phosphorylated and capable of recruiting tyrosine phosphatases, SHP-1 and SHP-2 and inositol polyphosphate 5-phosphatase, SHIP. LMIR2, on the other hand, contains a short cytoplasmic tail and a characteristic transmembrane domain carrying two positively charged amino acids associated with three kinds of immunoreceptor tyrosine-based activation motif (ITAM)-bearing molecules, DAP10, DAP12, and FcRγ. These findings suggest that a new pair of ITIM/ITAM-bearing receptors, LMIR1 and 2, regulate mast cell-mediated inflammatory responses through yet to be defined ligand(s).

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.

A Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) plays an essential role in bone accrual and eye development. Here, we show that LRP5 is also required for normal cholesterol and glucose metabolism. The production of mice lacking LRP5 revealed that LRP5 deficiency led to increased plasma cholesterol levels in mice fed a high-fat diet, because of the decreased hepatic clearance of chylomicron remnants. In addition, when fed a normal diet, LRP5-deficient mice showed a markedly impaired glucose tolerance. The LRP5-deficient islets had a marked reduction in the levels of intracellular ATP and Ca 2+ in response to glucose, and thereby glucose-induced insulin secretion was decreased. The intracellular inositol 1,4,5-trisphosphate (IP3) production in response to glucose was also reduced in LRP5−/− islets. Real-time PCR analysis revealed a marked reduction of various transcripts for genes involved in glucose sensing in LRP5−/− islets. Furthermore, exposure of LRP5+/+ islets to Wnt-3a and Wnt-5a stimulates glucose-induced insulin secretion and this stimulation was blocked by the addition of a soluble form of Wnt receptor, secreted Frizzled-related protein-1. In contrast, LRP5-deficient islets lacked the Wnt-3a-stimulated insulin secretion. These data suggest that Wnt/LRP5 signaling contributes to the glucose-induced insulin secretion in the islets.

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.

Cardio-facio-cutaneous (CFC) syndrome is one of the 'RASopathies', a group of phenotypically overlapping syndromes caused by germline mutations that encode components of the RAS-MAPK pathway. Germline mutations in BRAF cause CFC syndrome, which is characterized by heart defects, distinctive facial features and ectodermal abnormalities. To define the pathogenesis and to develop a potential therapeutic approach in CFC syndrome, we here generated new knockin mice (here Braf(Q241R/+)) expressing the Braf Q241R mutation, which corresponds to the most frequent mutation in CFC syndrome, Q257R. Braf(Q241R/+) mice manifested embryonic/neonatal lethality, showing liver necrosis, edema and craniofacial abnormalities. Histological analysis revealed multiple heart defects, including cardiomegaly, enlarged cardiac valves, ventricular noncompaction and ventricular septal defects. Braf(Q241R/+) embryos also showed massively distended jugular lymphatic sacs and subcutaneous lymphatic vessels, demonstrating lymphatic defects in RASopathy knockin mice for the first time. Prenatal treatment with a MEK inhibitor, PD0325901, rescued the embryonic lethality with amelioration of craniofacial abnormalities and edema in Braf(Q241R/+) embryos. Unexpectedly, one surviving pup was obtained after treatment with a histone 3 demethylase inhibitor, GSK-J4, or NCDM-32b. Combination treatment with PD0325901 and GSK-J4 further increased the rescue from embryonic lethality, ameliorating enlarged cardiac valves. These results suggest that our new Braf knockin mice recapitulate major features of RASopathies and that epigenetic modulation as well as the inhibition of the ERK pathway will be a potential therapeutic strategy for the treatment of CFC syndrome.

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.

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.

Isolation and characterization were performed for cDNA encoding mouse testicular cell adhesion molecule-1 (TCAM-1) using 2908 bases coding for a protein having 548 amino acids (60 kDa). Mouse TCAM-1 protein was found to consist of seven domains for signal sequence, five immunoglobulin (Ig) domains, and the transmembrane plus cytoplasmic domain. TCAM-1 gene and the region linking it to growth hormone (GH) gene located downstream from the TCAM-1 gene were then analyzed. The mouse TCAM-1 gene was 11.6 kb in length with 8 exons; the same as for the 12.0 kb rat gene. The distance from the TCAM-1 to GH gene was 12.5 kb in the mouse genome, and 7.6 kb in the rat. By Northern hybridization, 3.1-kb TCAM-1 mRNA was detected in 17-day testis and would appear present in pachytene spermatocytes and round spermatids.Key words: mouse cell adhesion molecule, gene structure, expression.

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.

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.

Membrane growth factors that are processed to produce soluble ligands may function both as soluble factors and as membrane factors. The membrane growth factor Kit-ligand (KL), the ligand of the Kit receptor tyrosine kinase, is encoded at the Sl locus, and mice carrying Sl mutations have defects in hematopoiesis, gametogenesis, and melanogenesis. Two alternatively spliced KL transcripts encode two cell-associated KL protein products, KL-1 and KL-2. The KL-2 protein lacks the major proteolytic cleavage site for the generation of soluble KL, thus representing a more stable cell-associated form of KL. We investigated the consequences of exclusive expression of KL-2 in vivo . The KL gene in embryonic stem cells was modified and KL exon 6 was replaced with a PGKneoNTRtkpA cassette by homologous recombination, and mice carrying the Sl KL2 allele were obtained. Sl KL2 /Sl KL2 mice had only slightly reduced levels of soluble KL in their serum, suggesting that in vivo KL-2 may be processed to produce soluble KL-2S. The steady-state characteristics of the hematopoietic system and progenitor numbers were normal, and the mutant animals were not anemic. However, mast cell numbers in the skin and peritoneum were reduced and the mutant animals displayed increased sensitivity to sublethal doses of γ-irradiation. Therefore, KL-2 may substitute for KL-1 in most situations with the exception of the production of mast cells, and induced proteolytic cleavage of KL-1 to produce soluble KL may have a role in the regeneration of hematopoietic tissue after radiation injury.

Recently, we found a novel murine cell-surface glycoprotein, designated as p91, expressed mainly in myeloid cells such as macrophages and mast cells. The molecule has six immunoglobulin-like extracellular domains, a transmembrane segment, and a cytoplasmic tail containing four immunoreceptor tyrosine-based inhibition motif (ITIM) or ITIM-like sequences, resembling the structural features of human killer-cell inhibitory receptors (KIR). Here we show that p91 comprises a polymorphic gene family, harboring one potent inhibitory-type p91 and at least two other p91 genes. Tyrosine-phosphorylated, but not nonphosphorylated, synthetic peptides matching the third ITIM and the fourth ITIM-like sequences, respectively, found in the cytoplasmic portion of p91A, the sole inhibitory-type p91, were associated with the tyrosine phosphatases, SHP-1 and SHP-2. In addition, the phosphotyrosyl peptide matching the third ITIM sequence also bound the inositol 5-phosphatase, SHIP. These results support the notion that p91A may function as an inhibitory cell-surface molecule against cell activation. The p91 genes were shown to be clustered in the proximal region of mouse chromosome 7, a syntenic position of human chromosome 19 where the genes for the KIR family are found. A human cDNA clone cross-hybridizing to a murine p91 probe was isolated from a human spleen cDNA library, and was found to code for a molecule quite similar to members of the immunoglobulin-like transcript (or ILT) family. The gene was found to be located on human chromosome 19q13.3-13.4. These results establish the existence of a novel set of potent regulatory receptors in mouse and man, similar but different from the KIR family.

We have analyzed the molecules participating in the inhibitory function of gp49B1, a murine type I transmembrane glycoprotein expressed on mast cells and natural killer cells, as well as the chromosomal location of its gene. As assessed by SDS-polyacrylamide gel electrophoresis and immunoblot analysis, tyrosine-phosphorylated, but not nonphosphorylated, synthetic peptides matching each of the two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences found in the cytoplasmic portion of gp49B1 associated with the approximately 65-kDa tyrosine phosphatase SHP-1 and approximately 70-kDa SHP-2 derived from RBL-2H3 cells. In addition, the phosphotyrosyl peptide matching the second ITIM-like sequence also bound the approximately 145-kDa inositol polyphosphate 5-phosphatase SHIP. Thus, it has been strongly suggested that the inhibitory nature of gp49B involves the recruitment of SHP-1, SHP-2, and SHIP for the delivery of inhibitory signal to the cell interior upon phosphorylation of tyrosine residues in their ITIMs. The gp49B gene has been found to be in the juxtaposition of its cognate gene, gp49A. The gene pair was shown to locate in the B4 band of mouse chromosome 10. In this region, no conserved linkage homology to human chromosome 19, where the genes for killer cell inhibitory receptors are found, has been identified.

Cancers in the upper aerodigestive tract, including cancers of the tongue and the esophagus, are the third leading cause of cancer-related deaths in the world, and oxidative stress is well recognized as one of the major risk factors for carcinogenesis. The Keap1–Nrf2 system plays a critical role in cellular defense against oxidative stress, but little is known about its association with upper aerodigestive tract carcinogenesis. In this study, we examined whether loss of Nrf2-function exacerbates carcinogenesis by using an experimental carcinogenesis model that is induced by 4-nitroquinoline-1-oxide (4NQO). We found that Nrf2-knockout (Nrf2-KO) mice were more susceptible to 4NQO-induced tongue and esophageal carcinogenesis than wild-type mice, which suggests that Nrf2 is important for cancer prevention. We also examined how the suppression of Keap1 function or the induction of Nrf2 activity affected 4NQO carcinogenesis. Keap1-knockdown (Keap1-KD) mice were resistant to 4NQO-induced tongue and esophageal carcinogenesis. Importantly, no growth advantage was observed in tongue tumors in the Keap1-KD mice. These results show that the Keap1–Nrf2 system regulates an important defense mechanism against upper aerodigestive tract carcinogenesis. In addition to several important functions of Nrf2 that lead to cancer chemoprevention, we hypothesize that a mechanical defense of thickened keratin layers may also be a chemopreventive factor because thickened, stratified, squamous epithelium was found on the tongue of Keap1-KD mice. Cancer Prev Res; 6(2); 149–59. ©2012 AACR.

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.

Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. AMSH (associated molecule with the SH3 domain of STAM) is a deubiquitination enzyme that participates in the endosomal sorting of several cell-surface molecules. AMSH impairment results in missorted ubiquitinated cargoes in vitro and severe neurodegeneration in vivo, but it is not known how AMSH deficiency causes neuronal damage in the brain. Here, we demonstrate that AMSH−/− mice developed ubiquitinated protein accumulations as early as embryonic day 10 (E10), and that severe deposits were present in the brain at postnatal day 8 (P8) and P18. Interestingly, TDP-43 was found to accumulate and colocalize with glial marker-positive cells in the brain. Glutamate receptor and p62 accumulations were also found; these molecules colocalized with ubiquitinated aggregates in the brain. These data suggest that AMSH plays an important role in degrading ubiquitinated proteins and glutamate receptors in vivo. AMSH−/− mice provide an animal model for neurodegenerative diseases, which are commonly characterized by the generation of proteinaceous aggregates.

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.

Moyamoya disease (MMD) shows progressive cerebral angiopathy characterized by bilateral internal carotid artery stenosis and abnormal collateral vessels. Although ∼ 15% of MMD cases are familial, the MMD gene(s) remain unknown. A genome-wide association study of 785,720 single-nucleotide polymorphisms (SNPs) was performed, comparing 72 Japanese MMD patients with 45 Japanese controls and resulting in a strong association of chromosome 17q25-ter with MMD risk. This result was further confirmed by a locus-specific association study using 335 SNPs in the 17q25-ter region. A single haplotype consisting of seven SNPs at the RNF213 locus was tightly associated with MMD (P = 5.3 × 10(-10)). RNF213 encodes a really interesting new gene finger protein with an AAA ATPase domain and is abundantly expressed in spleen and leukocytes. An RNA in situ hybridization analysis of mouse tissues indicated that mature lymphocytes express higher levels of Rnf213 mRNA than their immature counterparts. Mutational analysis of RNF213 revealed a founder mutation, p.R4859K, in 95% of MMD families, 73% of non-familial MMD cases and 1.4% of controls; this mutation greatly increases the risk of MMD (P = 1.2 × 10(-43), odds ratio = 190.8, 95% confidence interval = 71.7-507.9). Three additional missense mutations were identified in the p.R4859K-negative patients. These results indicate that RNF213 is the first identified susceptibility gene for MMD.

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.

Hypoxemia is a common manifestation of various disorders and generates pressure overload to the heart. Here we analyzed the expression of lipocalin-type prostaglandin D synthase (L-PGDS) in the heart of C57BL/6 mice kept under normobaric hypoxia (10% O2) that generates hemodynamic stress. Northern and Western blot analyses revealed that the expression levels of L-PGDS mRNA and protein were significantly increased (> twofold) after 14 days of hypoxia, compared to the mice kept under normoxia. Immunohistochemical analysis indicated that L-PGDS was increased in the myocardium of auricles and ventricles and the pulmonary venous myocardium at 28 days of hypoxia. Moreover, using C57BL/6 mice lacking heme oxygenase-2 (HO-2(-/-)), a model of chronic hypoxemia, we showed that the expression level of L-PGDS protein was twofold higher in the heart than that of wild-type mouse. L-PGDS expression is induced in the myocardium under hypoxemia, which may reflect the adaptation to the hemodynamic stress.

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.

There is an increasing body of evidence that prostanoids modulate mast cell functions and contribute to the development of allergic inflammation. The present study aimed to identify an undetermined function of prostaglandin (PG) F(2alpha) in mast cell activation and the signaling mechanism involved in it. Simultaneous quantification of prostanoids by liquid chromatography/tandem mass spectrometry revealed the constitutive release of PGF(2alpha), thromboxane B(2), and 6-keto-PGF(1alpha) from bone marrow-derived mast cells (BMMCs). Upon activation of BMMCs by lipopolysaccharide, the cytokine production in BMMCs was enhanced when the culture was supplemented with PGF(2alpha). However, F prostanoid receptor-a selective receptor for PGF(2alpha)-was not detected in BMMCs. Further investigations performed using prostanoid receptor antagonists revealed an alternative mechanism wherein the receptors for PGE species-E prostanoid receptors-mediated the PGF(2alpha) signal in BMMCs. The present study provides an insight into a novel function of PGF(2alpha), i.e., an autocrine accelerator for mast cell activation.

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.

Spinal gene therapy is a promising option for treating various spinal-related disorders. Several previous studies using viral vectors reported successful transfer of therapeutic genes into the spinal nerve system. However, because of the considerable immunogenicity related to the use of viruses, non-viral gene transfer still needs to be developed. One possible approach is the combined use of ultrasound and echo-contrast microbubbles. The present study shows that this method can be applied for targeted intrathecal gene delivery. We intrathecally injected a mixture of plasmid-DNA encoded with luciferase and commercially available albumin microbubbles by needle puncture at the lower lumbar intervertebral space in mice. Subsequent percutaneous ultrasonication on the lumbar vertebrae significantly enhanced the luciferase expression, analyzed by imaging luciferin bioluminescence, in the dorsal meningeal cells at the insonated region. No apparent neurological damages were induced by the present spinal interventions. In addition to the general benefits of the combined use of ultrasound and microbubbles, our approach can offer some advantages specific to spinal gene transfection including minimal invasiveness of simple percutaneous dural puncture, targetability due to the limited access of ultrasound waves through anatomical apertures of the vertebrae, and possible paracrine delivery of therapeutic molecules to the spinal nerve system.

Fatty acids and their metabolites have recently been shown to modulate various functions of dendritic cells (DCs) including their differentiation and cytokine production, although the mechanisms underlying their cellular functions are not fully understood. In view of our previous finding that epidermal-type fatty acid binding protein (E-FABP) was exclusively expressed in splenic DCs among FABP family, we examined the phenotype of E-FABP-null mutant mice in order to elucidate the functional significance of E-FABP expression in DCs. Although E-FABP-null mutant mice showed no apparent abnormalities in the population density and subset distribution of DCs as well as the microscopic morphology in the spleen, DCs isolated from E-FABP-null spleen showed enhanced production of IL-12p70, a key cytokine for innate immune responses, in response to appropriate stimuli as compared with wild-type. In real-time PCR, the expression level of IL-12p35 mRNA after LPS stimuli was much higher in mutant DCs when compared with wild-type, while no apparent change of IL-12p40 mRNA level was detected. Phosphorylated forms of p38 mitogen-activated protein kinase (p38MAPK) and IkappaB-alpha, molecules critical for IL-12 production, were detected at higher levels in E-FABP-null-mutant DCs after LPS stimuli when compared with wild-type counterparts. Collectively, it is suggested that E-FABP may be a novel negative regulator of IL-12 production in DCs, and this regulation may be exerted via its involvement in the p38MAPK-mediated transcription of IL-12p35.

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.