Hiroaki Komori

A Signal Adaptor SLAM-Associated Protein Regulates Spontaneous Autoimmunity and Fas-Dependent Lymphoproliferation in MRL-Faslpr Lupus Mice

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.

Simple Screening Method for Autoantigen Proteins Using the N-Terminal Biotinylated Protein Library Produced by Wheat Cell-Free Synthesis

Autoimmune diseases are a heterogeneous group of diseases characterized by immune reactions against either a major or a limited number of the bodies own autoantigens, causing inflammation and damage to tissues and organs. Thus, identification of autoantigens is an important first step to understanding autoimmune diseases. Here we demonstrate a simple screening method for identification of autoantigens reacting with patient serum antibodies by combination of an N-terminal biotinylated protein library (BPL), produced using a wheat cell-free protein production system, and a commercially available luminescence system. Optimization studies using well-characterized autoantigens showed specific interactions between N-terminal biotinylated proteins and antibody that were sensitively detected under homogeneous reaction conditions. In this optimized assay, 1 μL of the translation mixture expressing the biotinylated proteins produced significant luminescence signal by addition of diluted serum between 1:500 and 1:10 000 in 25 μL of reaction volume. For the BPL construction, 214 mouse genes, consisting of 103 well-known autoantigens and 111 genes in the mouse autoimmune susceptibility loci, and the sera of MRL/lpr mouse were used as an autoimmune model. By this screening method, 25 well-known autoantigens and 71 proteins in the loci were identified as autoantigen proteins specifically reacting with sera antibodies. Cross-referencing with the Gene Ontology Database, 26 and 38 of autoantigen proteins were predicted to have nuclear localization and identified as membrane and/or extracellular proteins. The immune reaction of six randomly selected proteins was confirmed by immunoprecipitation and/or immunoblot analyses. Interestingly, three autoantigen proteins were recognized by immunoprecipitation but not by immunoblot analysis. These results suggest that the BPL-based method could provide a simple system for screening of autoantigen proteins and would help with identification of autoantigen proteins reacting with antibodies that recognize folded proteins, rather than denatured or unfolded forms.

Role of SLAM-Associated Protein in the Pathogenesis of Autoimmune Diseases and Immunological Disorders

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 Vα14 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.

SYNOVIAL HEMANGIOMA OF THE KNEE JOINT IN A 3-YEAR-OLD GIRL

Synovial hemangioma has been reported to be relatively rare, and usually occurs in childhood and adolescence. However, there are a few reports of the disease in infants. In this report, we diagnosed synovial hemangioma in a 3-year-old girl who had swelling and pain in her left knee. Gadolinium-enhanced magnetic resonance imaging revealed abnormal intensity in an intra-articular lesion. We performed arthroscopy, and arrived at a final diagnosis based on a scopic biopsy. Synovial hemangioma should be considered as a possible diagnosis in infants with swelling and pain in the knee.

Aneurysms of the renal arteries associated with segmental arterial mediolysis in a case of polyarteritis nodosa

This is the first report of segmental arterial mediolysis (SAM) accompanied with polyarteritis nodosa (PN), and manifesting aneurysms of the renal arteries. A 73‐year‐old woman was admitted to hospital because of a high fever. Laboratory tests showed leukocytosis with increased CRP level in the serum. Myeloperoxidase‐anti‐neutrophil cytoplasmic antibody (MPO‐ANCA) and proteinase 3 (PR3)‐ANCA were negative. There were no signs indicating infection or malignancy. After admission renal function rapidly deteriorated. Treatment was then started with daily oral prednisolone and hemodialysis. On the 40th day of hospitalization the patient suddenly became comatose. Cranial CT showed a subarachnoid hemorrhage. The patient died and an autopsy was performed. The pathological findings showed necrotizing vasculitis of the small arteries in various organs, but not associated with that of arterioles or renal glomerular lesions, indicating PN. Unexpectedly, the segmental arteries of the bilateral kidneys showed vascular lesions of dissecting aneurysms, indicating SAM. This case indicates that SAM is one of the causes of aneurysms in PN and is clinically important when the clinical course of PN patients rapidly advances.

Genetic dissociation of dacryoadenitis and sialadenitis in a Sjögren's syndrome mouse model with common and different susceptibility gene loci.

PURPOSE Sjögrens syndrome (SS) is a systemic autoimmune disease in which the main lesions are dacryoadenitis and sialadenitis. It is unclear whether these lesions develop in a common genetic background. A quantitative trait locus (QTL) analysis was performed in the SS mouse model, MRL/MpJ-lpr/lpr (MRL/lpr), to identify the susceptibility loci to dacryoadenitis and sialadenitis and the association with both loci. METHODS MRL/lpr, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr x C3H/lpr) F1, and (MRL/lpr x C3H/lpr) F2 intercross mice were prepared, and the severity of dacryoadenitis and sialadenitis in individuals was quantified by histopathologic grading. In genomic DNA samples from the F2 mice, the polymorphic microsatellite markers highly associated with each lesion were determined as susceptibility loci. RESULTS QTLs with significant linkage for dacryoadenitis were mapped on chromosome 1 (the position of maximum logarithm of odds [LOD] score; 64.1 cM), designated Adacm1; chromosome 2 (88.4 cM), Adacm2; and chromosome 5 (63.9 cM), Adacm3. Those for sialadenitis were mapped on chromosome 1 (69.0 cM), Asm3, and chromosome 2 (65.3 cM and 82.1 cM), Asm4 and Asm5. Adacm1/Asm3 and Adacm2/Asm5 seemed to be a common chromosomal region, respectively. MRL-homozygous at Adacm1 and Adacm2 and at Asm3 and Asm5 manifested an additive effect on the development of dacryoadenitis and sialadenitis, respectively, whereas Adacm3 did not. CONCLUSIONS Dacryoadenitis and sialadenitis in MRL/lpr mice are under the control of common and different susceptibility loci, with an allelic combination that leads to regular variations in pathologic phenotypes.

Hexabromocyclododecanes in human adipose tissue from Japan

Environmental context. Contamination by hexabromocyclododecanes (HBCDs), a group of brominated flame retardants, is of great concern due to their bioaccumulative nature and toxic implications. HBCD consumption in Japan is the highest among Asian countries and is still growing. In this regard, human exposure to HBCDs is of great concern in Japan. We present here the results of our investigation on Japanese human adipose tissue using LC-MS/MS. HBCDs were detected in all the samples analysed, and were generally higher in men than in women. Abstract. Residue levels of hexabromocyclododecanes (HBCDs) were measured using LC-MS/MS in the Japanese human adipose tissue samples collected during 2003–2004 from Ehime prefecture (n = 26) and Kanto region (n = 9) of Japan. Concentrations of HBCDs (0.85–39 ng g–1 lipid) in the adipose tissue samples were 1–2 orders of magnitude lower than those of organochlorines (OCs). Regional differences between HBCD levels in Ehime prefecture and Kanto region were not significant. No age-dependent accumulation of HBCDs was observed. HBCD levels in men were significantly higher than those in women. Significant positive correlation between polybrominated diphenyl ethers (PBDEs) and HBCDs implies similar routes of exposure to these contaminants for Japanese citizens. α-HBCD was predominant among the three isomers, which is consistent with the other reports on bioaccumulation of α-HBCD in higher trophic animals.

Genomics of Vasculitis: Lessons from Mouse Models

A genome analysis of mouse models may shed some light on the complex clinicopathological manifestations of systemic vasculitis. In the study of susceptibility loci to vasculitis in MRL mouse models, we found that systemic vasculitis developed through the cumulative effect of multiple gene loci, each of which by itself did not have a significant effect in inducing the related phenotype, thus indicating a polygenic system. The mice developed vasculitis in an additive manner with a hierarchical effect. Some of the susceptibility loci seemed to be common to those in other collagen diseases. Moreover, the loci controlling tissue specificity of vasculitis were present. One of the positional candidate genes for vasculitis showed an allelic polymorphism in the coding region, thus possibly causing a qualitative difference in its function. As a result, a particular combination of polygenes with such an allelic polymorphism may thus play a critical role in leading the cascade reaction to develop vasculitis, and also a regular variation of systemic vasculitis. This is designated as the polygene network in systemic vasculitis. (J Jpn Coll Angiol, 2009, 49: 11-16).

An epistatic effect of the female specific loci on the development of autoimmune vasculitis and antinuclear autoantibody in murine lupus

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-Faslpr (MRL/lpr), C57BL/6-Faslpr (B6/lpr), (MRL/lpr×B6/lpr) F1, and MRL/lpr×(MRL/lpr×B6/lpr) F1 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.

Novel recombinant congenic mouse strain developing arthritis with enthesopathy

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 × (MRL/lpr × 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.