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The Journal of Immunology, 2006, 176: 395-400.
Copyright © 2006 by The American Association of Immunologists

A Signal Adaptor SLAM-Associated Protein Regulates Spontaneous Autoimmunity and Fas-Dependent Lymphoproliferation in MRL-Faslpr Lupus Mice1

Hiroaki Komori*, Hiroshi Furukawa{dagger}, Shiro Mori§, Mitsuko R. Ito*, Miho Terada*, Ming-Cai Zhang{dagger}, Naoto Ishii, Nobuhiro Sakuma{ddagger}, Masato Nose* and Masao Ono2,{dagger}

* Department of Pathology, Ehime University School of Medicine, Toon, Japan; {dagger} Department of Pathology and {ddagger} Department of Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan; and § Department of Oro-Maxillofacial Surgical Science and Department of Clinical Laboratory, Tohoku University Hospital, Sendai, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 x 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Spontaneous and targeted mutations in the mouse germline have proven an essential function of the gene mutated with evidence for ultimate significance or existence of redundancy in the in vivo context. The spontaneous mutation of the Fas gene that encodes a membrane receptor to mediate apoptosis signal generated a famous autoimmune strain of mice, currently designated MRL.Faslpr (MRL/lpr) (1, 2). This strain exhibits remarkable lymphadenopathy and systemic lupus erythematosus-like manifestations, including autoantibody production, glomerulonephritis, systemic vasculitis, and so forth (1, 3, 4, 5). The lymphadenopathy is known to develop due to accumulation of unique CD4nul.CD8nul.B220+ T cell subsets (lpr-T) in lymphoid organs. This subset origin is not fully understood. The impact of Fas deficiency clearly demonstrates the physiological role of Fas in prevention of autoimmune responses in vivo.

It has been postulated that a lpr-dependent cell lineage plays a critical role in pathogenesis of autoimmune disease in MRL/lpr; however, this cell lineage remains elucidative. Several studies have shown that removal of the thymus from neonate MRL/lpr results in marked suppression of lymphadenopathy and autoantibody production in adulthood, suggesting that the pathogenic cell lineage in MRL/lpr is thymus dependent (6, 7, 8). There are another lpr congenic strains, C57BL/6.Faslpr and C3H.Faslpr (C3H/lpr). Importantly, these strains display little pathological manifestations with reduced autoantibody production (9, 10). These findings indicate that lpr is necessary but not sufficient for autoimmune onset, and the other genetic factors are necessarily involved in pathogenesis of autoimmune diseases in MRL/lpr. We have been interested in the regulatory mechanism for the development of pathogenic cell lineage by autoimmune predisposed genetic factors.

We previously observed an expansion of abnormal mice in our breeding colony of the MRL/lpr strain. Notably, the pathological traits of MRL/lpr such as early death of autoimmune disease and remarkable lymphadenopathy were highly attenuated in those abnormal mice. We predicted that a new mutation occurred in MRL/lpr and caused regression of autoimmune phenotypes in the mutant mice. In this study, we describe genetic and pathological characterizations of the new mutant strain. The present data indicate an essential role of the small adaptor protein, signaling lymphocyte activation molecule (SLAM)3 -associated protein (SAP), in the development of autoimmunity and lymphadenopathy in MRL/lpr. Now an active role of the SLAM family receptor in autoimmunity is underscored.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

MRL/lpr were purchased from Charles River Breeding Laboratories. C3H/lpr, MRL/+, and the mutant MRL.Faslpr (MRLrpl) were bred under specific pathogen-free conditions in the Integrated Center of Science, Ehime University, or the Animal Research Institute of Tohoku University Graduate School of Medicine. The F1 and F2 mice were generated from female MRLrpl and male C3H/lpr. In all animal experiments in this study, we observed the Tohoku University guideline or the Ehime University guideline for animal experimentation.

Histological examinations

At 18- to 20-wk-old, mice were killed under ether anesthesia. Organs were immersed to fix in 10% Formalin in 0.01 M phosphate buffer (pH 7.2) and embedded in paraffin. Sections were stained with H&E, elastica-Masson’s trichrome, and periodic acid-Schiff. Evaluation of glomerulonephritis or renal vasculitis was performed with light microscopic examination. Pathological scores for glomerulonephritis and vasculitis were determined according to the pathological criteria as defined previously (11). Briefly, the vasculitis score was defined as the highest score of all arterial lesions observed. Each score denotes normal as 0, destruction of external elastic lamina as 1, and severe lesion of 1 with intimal thickening as 2. The glomerulonephritis score was defined as an average of scores given from 50 glomeruli. Each score denotes normal as 0, cell proliferation or infiltration as 1, membranoproliferation, lobulation, or hyaline deposition as 2, and crescent formation or global hyalinosis as 3.

Serological examinations

Antinuclear titer was measured with mouse normal hepatocytes derived from 6-wk-old C57BL/6J mice. Nuclear samples were prepared by stamping minced liver on a glass slide, drying immediately, and fixing in ice-cold acetone for 5 min. After masking nuclear preparation with 10% normal goat serum, serial dilutions of serum samples were incubated on nuclear samples slide for 2 h at 4°C. Washed three times with PBS, nuclear samples were further incubated with secondary Ab of FITC-conjugated anti-mouse IgG, {gamma}-chain specific (Zymed Laboratories) or anti-mouse IgM, µ-chain specific (Zymed Laboratories). The titer index of the antinuclear Ab was defined with titration of maximal serum dilution to visualize the fluorescent nuclear signal: 0, no staining with 1 x 102 dilution; 1, faintly stained with 1 x 102 dilution; 2, stained with 1 x 102 dilution; 3, stained with 1 x 103 dilution; 4, stained with 1 x 104 dilution; 5, stained with 3 x 104 dilution; and 6, stained with 9 x 104 dilution. Anti-DNA titers and rheumatoid factors (RF) were measured with ELISA as described previously (12). Briefly, exonuclease-treated calf thymus DNA fragments were fixed on the bottom surfaces of protamine-treated 96-flat well plates. Sample dilutions (2 x 102) were incubated on this plate at 4°C for 12 h, washed, and incubated with alkaline phosphatase-conjugated goat anti-mouse whole IgG (Sigma-Aldrich), followed by coloring for OD measurement at 490 nm. For RF measurement, human recombinant Fc protein was used as Ag. To determine serum Ig concentration, the single radial immunodiffusion assay was performed with goat anti-mouse IgG (H + L) (Jackson ImmunoResearch Laboratories) or rabbit anti-mouse IgM (Zymed Laboratories) as described previously (13). We also examined a standard serum to give a standard curve for Ig concentrations. Blood urea nitrogen (BUN) was measured with standard reagents (Nittobo Medical) and a Hitachi 7350-E instrument (Hitachi).

FACS analysis

FITC- or PE-conjugated Abs to Thy1.2, CD45R/B220, and Fas (Jo-2) were purchased from BD Pharmingen. Immunostainings with these Abs were processed for thymic or splenic cells (106 cell) incubated in diluted fluorescence-conjugated Ab at 4°C for 30 min, then washed twice in 1% BSA/PBS. Small lymphocytic cells were gated in and analyzed.

Positional cloning of the mutant gene

We weighed a spleen or two lymph nodes of bilateral axillary regions in a 20-wk-old mouse. We selected 60 mice from 176 F2 male mice based on lymph node weight, 40 mice with the heaviest lymph nodes and 20 with the lightest. Using 59 polymorphic microsatellite markers as listed below, we determined genotypes of the 60 mice based on simple strand length polymorphism of genomic PCR products. Association between genotype and phenotype (lymphadenopathy) was statistically estimated by the {chi}2 test. The mutation was surveyed by mRNA sequencing for the positional candidate genes. Sequence primers for SAP transcript were GCAACAGCAGCAGCAAAGT and TGTCTCGCAGCATAGCTT. Microsatellite markers used in this study were D1Mit(276, 46, 423, 291), D2Mit(522, 305, 22), D3Mit(203, 101, 88), D4Mit(271, 306, 259), D5Mit(74, 259, 33), D6Mit(223, 115, 15,), D7Mit(229, 215, 334), D8Mit(65, 112,), D9Mit(128, 182), D10Mit(222, 96), D11Mit(71, 41, 214), D12Mit(136, 4, 141), D13Mit(39, 60, 171), D14Mit(113, 195), D15Mit(98, 189), D16Mit(42, 94, 165), D17Mit(264, 127, 155), D18Mit(12, 51), D19Mit(60, 89), and DXMit(89, 48, 192, 22, 159, 91, 73, 79), in which numbers in parenthesis individually denote the numerical designations of markers.

Western blot analysis

Samples were prepared from thymus and spleen of the mutant or wild-type MRL/lpr male mice. Cell pellet equivalent to 107 cells was dissolved in 100 µl of radioimmunoprecipitation assay buffer, and its supernatant was mixed with an equal volume of SDS-PAGE reducing sample buffer, and then boiled for 2 min. Western blot was performed with 15% SDS-PAGE followed by electric transfer to polyvinylidene difluoride membrane (Millipore), and probing SAP or Src homology 2-containing protein tyrosine phosphatase 1 (SHP-1) with rabbit polyclonal anti-SAP or anti-SHP1 (Santa Cruz Biotechnology), respectively. HRP-conjugated anti-rabbit IgG (Amersham Pharmacia) was used as a secondary Ab for detection with ECL plus (Amersham Pharmacia).

Detection of the mutant allele by RFLP

Primers for genomic PCR were mSAP-1, GA GAAGCTCTTACTCGGTA, and mSAP-2, CCACTACCACGAGATATACT. Underlined nucleotide is mismatched to the SAP germline sequence, which creates a KpnI recognition site in the PCR products of wild-type SAP origin. PCR was performed under the conditions of 94°C for 2 min followed by 26 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. PCR products were incubated at 37°C for 1 h with KpnI (New England Biolabs) and separated on a 4% agarose gel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
A new mutation in MRL/lpr caused suppression of autoimmune traits and lymphadenopathy

We previously found an unusual group of MRL/lpr mice in our breeding colony. When compared with the other line of the MRL/lpr strain that was newly purchased from an animal provider, the unusual MRL/lpr mice were grossly characterized by marked improvement of sickness and lymphadenopathy. Hereafter, we termed the new line of MRL/lpr wild type. Because these phenotypic changes were readily inherited by their offspring in our closed breeding colony, we predicted a new spontaneous mutation that caused the phenotypic changes in the MRL/lpr strain. Microscopic examination of the kidney showed marked improvement of glomerulonephritis and renal vasculitis in the mutant MRL/lpr (Fig. 1A). The impact of mutation on arthritis or sialoadenitis was not presently concluded because of low incidence of these traits in the wild-type MRL/lpr. Lymphadenopathy of spleen and lymph nodes was significantly suppressed in the mutant MRL/lpr (Fig. 1B). The pathological indexes for glomerulonephritis and vasculitis and the weights of spleen and axillary lymph nodes were significantly different between the wild-type and mutant MRL/lpr strains (Table I). The significant decrease in blood urea nitrogen supported improvement of the glomerular function in the mutant MRL/lpr (Table I).



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  		FIGURE 1. Regression of lpr-related traits in the mutant MRL/lprrpl mice. A, Micrographs of glomerular lesion in the mutant (rpl) and wild-type MRL/lpr (periodic acid-Schiff; original magnification, x200). Asterisks and arrow indicate crescent formations and hyaline deposition in glomeruli, respectively. B, Photographs of the isolated lymphoid tissues from MRL/+, the mutant (rpl), and wild-type MRL/lpr. Spleen (upper), right and left axillary lymph nodes (middle), and mesenteric lymph node (lower) are shown.

 

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  		Table I. changes of phenotypes by the rpl mutationa

 
In flow cytometric analysis, both of the mutant and wild-type MRL/lpr were defective in Fas expression on thymocytes or spleen cells, indicating that lpr mutation was unchanged in the mutant MRL/lpr (Fig. 2A). Hereafter, we will temporally use rpl (regression of phenotype associated with lpr) or MRLrpl as the designation for the mutant gene and mice, respectively. Immunostaining with B (B220)- and T lymphocyte (Thy-1)-specific markers, by which lpr-T cells are sorted in double-positive fraction, showed a striking reduction of the lpr-T cell number in the spleen and lymph nodes of MRLrpl (Fig. 2B). The change of lpr-T in MRLrpl was consistent; however, the small number of lpr-T cells was detectable in some MRLrpl, suggesting that rpl links to the development of lpr-T.



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  		FIGURE 2. Fas expression of thymocytes and the emergence of lpr-dependent abnormal T cells in the spleen. A, Defect in Fas expression of thymocytes was shown in the mutant MRL/lpr (rpl, black). Histogram of wild-type MRL/lpr (gray) or MRL/+ indicates a null or normal expression level of Fas, respectively. B, Representative profile of the spleen shown by double immunostaining of Thy1 and B220 Abs. The lpr-dependent abnormal T cells are sorted in double-positive fractions. Mean value and SD of double-positive fractions of three mice are indicated in each graph.

 
Hypergammoglobulinemia and autoantibody production are characteristic of the wild-type MRL/lpr. We examined these autoimmune-associated traits in MRLrpl. Total Ig concentrations and serum titers to nuclear Ag (antinuclear autoantibody (ANA)), dsDNA (anti-dsDNA), or IgG constant regions (RF) were determined. Notably, all of these traits were significantly reduced in MRLrpl (Table I and Fig. 3). However, IgG ANA was highly detected in MRLrpl (Fig. 3).



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  		FIGURE 3. Titer of ANA in the male mutant MRL/lpr (rpl), male wild-type MRL/lpr, or male MRL/+ mice. ANA titer was determined for serially diluted serum with staining nuclei prepared from normal mouse hepatocytes as described in Materials and Methods. Double asterisks indicate a statistical significance (the Mann-Whitney U test, p < 0.01) of the difference between the two groups.

 
Positional cloning of the mutant gene

To identify rpl, we mapped linkage locus to the lymph node phenotype of MRLrpl using the (MRLrpl x C3H/lpr)F2 mice. Since the lymphadenopathy in C3H/lpr was prominent as well as in MRL/lpr, the lymph node phenotype was predicted to link simply to rpl without influence of any C3H/lpr locus in the F2 mice. In contrast, other rpl-associated phenotypes such as tissue damages and serological abnormalities could be influenced by some C3H/lpr loci as previously demonstrated (14, 15). In the (MRLrpl x C3H/lpr)F2 mice, the possibility of epistatic effects from C3H-derived genes should be considered. Accordingly, we chose the lymph node phenotype as an rpl trait in this linkage analysis.

In the F1 mice, a significant difference in lymph node weight was evident between gender groups, suggesting an X chromosome-linked trait (Fig. 4A). Therefore, we analyzed only the male F2 mice in the subsequent linkage analysis. The 60 mice consisting of the 40 with prominent lymphadenopathy and the 20 with no lymphadenopathy were subjected to genome-wide screening. The results indicated three neighboring markers, DXMit89, DXMit48, and DXMit192, significantly linked to the phenotype, suggesting a single linkage locus on the X chromosome (Fig. 4B). Complete concordance of phenotypes and genotypes was observed at DXMit48. Six and two discordant cases were observed at DXMit89 and DXMit192, respectively, suggesting that rpl was located near DXMit48. We then selected positional candidate genes near this marker on the basis of information from the public genome database. In this selection, we took into account the immunological or apoptosis-related function for candidate genes. Extensive and careful sequence analyses of candidate genes located near DXMit48 proved an adenyl nucleotide insertion at the 21st codon of the SAP transcript. This mutation was confirmed in genomic DNA samples prepared from MRLrpl using the RFLP method as described in Materials and Methods (data not shown). It was found that the insertion was located in the first exon and potentially caused abnormal translation (Fig. 5A). However, Western blot analysis with anti-SAP Ab proved a defect in the expression of the mutant SAP in thymocytes (Fig. 5B).



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  		FIGURE 4. Inheritance mode of a rpl-related trait and its linkage to genetic markers. A, Axillary lymph node weight of the mutant MRL/lpr (rpl) and C3H/lpr and the F1 and F2 mice at 18 to 20 wk old are shown. Dots over a bar denote the samples weighed >2 g. B, Suppression of lymph node swelling closely links to DXMit48, which is located at 14.2 cM on the X chromosome. The number of mice with three marker haplotypes are indicated.

 


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  		FIGURE 5. SAP mutation and deletion of protein in the mutant MRL/lpr. A, Deduced amino acid sequence of the mutant SAP. An adenyl nucleotide insertion at the 21st codon of the SAP transcript causes abnormal translation. Hyphens denote the same residue as the normal SAP sequence shown in the top line. Residues marked by asterisks indicate the region facing the Fyn-SH3 structure (40 ). B, Western blot analysis for the detection of SAP protein in thymocytes. Rabbit polyclonal anti-SAP or anti-SHP-1 Ab was used for primary Ab. The results from anti-SHP-1 blot estimate the comparable load of protein for each lane.

 
Association of rpl mutation with regression of autoimmune diseases and lymphadenopathy

We have mapped and identified rpl as a linkage mutation to regression of lymphadenopathy. We finally examined a causal contribution of rpl to regression of autoimmune disease in (MRLrpl x C3H/lpr)F2 mice (Table II). The SAP mutation was determined by RFLP as described in Materials and Methods. Although the incidence of glomerulonephritis and renal vasculitis was reduced in the F2 mice by the resistant effect of C3H allele as previously demonstrated (14, 15), the result statistically proved significant association of rpl with regression of glomerulonephritis and renal vasculitis. Although it remains formally possible that the other gene closely linked to SAP critical for the rpl phenotype, the present findings suggest that a deletion of SAP causes regression of autoimmune diseases in MRLrpl.


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  		Table II. Associations of SAP with the onset of pathological traits in (MRL/ lprrplx C3H/lpr)F2male mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Deleterious germline mutation of the SAP gene is known to cause the onset of X-linked lymphoproliferative syndrome (XLP), a congenital immunodeficiency in humans (16). XLP patients are highly susceptible to EBV infection and endure invariable clinical courses of EBV-related diseases, including acquired immunodeficiency, infectious mononucleosis, lymphoid hyperplasia, and neoplasia. A number of studies on SAP-deficient mice and XLP patients have proven that SAP is not required for the development of conventional T and B cells, but is important in activation of B, NK, CTL, and Th2 cells in immune responses (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28). Recently, SAP was shown to be essential for the development of NKT cells in mouse and humans (29, 30, 31). From lessons of studies on SAP-deficient mice and XLP patients, SAP function seems pleiotropic but is central to the host immune response to infection and persistence of EBV. In contrast, since the mouse is not susceptible to EBV, SAP-deficient mice never endure human EBV-associated-like diseases. But notably, SAP-deficient mice were found to be insensitive to induced autoimmune models for collagen-induced arthritis and experimental allergic encephalomyelitis (32). Together with our spontaneous autoimmune case, it is emphasized that SAP has an important role in mediating the signal for autoimmune response in the mouse. The role of SAP in human autoimmune diseases is now highly interesting. Currently, accumulating evidence may further provide an insight into the commonality between autoimmune response and protective response to EBV.

MRLrpl mice may defect in the development of NKT cells as previously shown in other SAP-deficient mice and XLP patients. Now a possibility arises regarding correlation of NKT cell function and autoimmune diseases. However, previous studies clearly demonstrated anti-autoimmune activity of NKT cells (33, 34). It is unlikely that the defect of NKT cells simply results in suppressions of autoimmunity and lpr-T cell development in MRLrpl.

Studies using thymectomized MRL/lpr demonstrated that the lpr-dependent cell lineages responsible for autoantibody production and lymphadenopathy were thymus dependent (6, 7, 8). Importantly, removal of the thymus from neonate MRL/lpr mice resulted in marked suppression of autoantibody production and lymphadenopathy, whereas the thymectomy delayed after 6 wk had no effect on these traits. These findings indicate critical function of neonate thymus and significance of the postthymic compartment in the development of the lpr-dependent cell lineages. Other studies using TCR-transgenic mice, in which clonal diversity of TCR{alpha}{beta} was highly limited, showed that the TCR-transgenic mice had no lymphadenopathy, but still retained comparable autoantibody production, suggesting that the development of lpr-dependent cell lineage for autoantibody production is independent of a signal through TCR{alpha}{beta} and that the cell lineages responsible for autoantibody production and lymphadenopathy were distinct (35, 36, 37). The present study provides a new insight into SAP-dependent regulation for the development of the two lpr-dependent lineages. Upstream events of SAP on this concern are now underscored.

SAP has two functional domains for protein interaction. The first domain is a Src homology (SH) 2, which serves for binding to a unique class of cytoplasmic tyrosine-based motifs presented in SLAM family receptors (38, 39, 40, 41, 42). There is so far no other class of receptor than the SLAM family for a target of the SAP-SH2 domain. The second domain of SAP recently has been proven to bind to and activate Fyn tyrosine kinase (43, 44, 45). Latour et al. (44) showed that SAP was unable to bind to the other src family kinases, including Lck, Fgr, Lyn, Hck, Yes, or Src, suggesting that the downstream signal of SAP is highly dependent on Fyn. Accordingly, our data further indicate a profound role of the SLAM family receptor(s) and Fyn as well as SAP in spontaneous autoimmunity and lymphadenopathy in MRL/lpr.

Genetic studies of mouse autoimmune models have frequently led to the conclusion of significant linkage of the distal region of chromosome 1 to autoantibody production or autoimmune diseases (46, 47, 48). The human syntenic region to this mouse linkage locus was also proven to be associated with human autoimmune diseases. Wakeland and colleagues (48) achieved a fine mapping of this locus by analyzing interval congenic strains of mice (48) and recently suggested high candidacy of the SLAM family receptor gene(s) for a lupus-predisposed genetic factor (49). They showed polymorphic characters for some SLAM family receptors, notably for CD229, CD244, and Ly108/NTB-A. Our data support a contribution of genetic polymorphisms of the SLAM family receptor gene(s) to lupus susceptibility in a gain-of-function manner.

Katagiri et al. (50) showed unusual activation of Fyn kinase in lpr-T cells derived from MRL/lpr, suggesting a role for Fyn in survival of lpr-T cells. Other studies showed that Fyn deficiency in MRL/lpr resulted in a marked reduction of lymphadenopathy and autoantibody production, suggesting the requirement of Fyn for the lpr-T cell development and autoantibody production (51, 52). The phenotypes of Fyn-deficient MRL/lpr seem largely paralleled to those of SAP-deficient MRL/lpr. Overall findings provide a possible understanding that SAP-dependent Fyn activation is a main path for autoimmunity and lymphadenopathy in MRL/lpr.

There has been a long-standing question on what are the pathogenic cell lineages associated with the onset of autoimmunity in MRL/lpr. MRLrpl proved an essential role of SAP in the development of the pathogenic cell lineages. Now, a further question arises on what SLAM family receptor(s) acts toward autoimmune onset upstream of SAP. And it should be an important question whether SAP is generally prerequisite to autoimmunity in the other model. The SAP-deficient strain will be useful to address these questions. Any answer must help to understand the pathogenic mechanism and a new therapeutic target for autoimmune diseases.


   Acknowledgments
 
We thank Dr. Noemi Nagy and Dr. Toshiyuki Takai for their advice; Dr. Shin-ichi Fujimaki, Yoshie Tsuji, and Ai Mori for their technical assistance; and the members of the Integrated Center for Sciences, Ehime University for animal breeding assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (Grant 16390113; to M.O.). Back

2 Address correspondence and reprint requests to Dr. Masao Ono, Department of Pathology, Tohoku University Graduate School of Medicine, 2-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan. E-mail address: onomasao{at}mail.tains.tohoku.ac.jp Back

3 Abbreviations used in this paper: SLAM, signaling lymphocyte activation molecule; SAP, SLAM-associated protein; XLP, X-linked lymphoproliferative disease; ANA, antinuclear autoantibody; RF, rheumatoid factor; BUN, blood urea nitrogen; SHP1, Src homology region 2 domain-containing phosphatase 1; SH, Src homology. Back

Received for publication July 20, 2005. Accepted for publication October 26, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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