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• W2004305328 abstract "Systemic sclerosis (SSc) is characterized by fibrosis and autoimmmunity. Peripheral blood B cells from SSc patients specifically overexpress CD19, a critical cell-surface signal transduction molecule in B cells. CD19 deficiency in B cells also attenuates skin fibrosis in the tight-skin (TSK/+) mouse, a genetic model for SSc. Herein we analyzed two transgenic mouse lines that overexpress CD19. Remarkably, 20% increase of CD19 expression in mice spontaneously induced SSc-specific anti-DNA topoisomerase I (topo I) antibody (Ab) production, which was further augmented by 200% overexpression. In TSK/+ mice overexpressing CD19, skin thickness did not increase, although anti-topo I Ab levels were significantly augmented, indicating that abnormal CD19 signaling influences autoimmunity in TSK/+ mice and also that anti-topo I Ab does not have a pathogenic role. The molecular mechanisms for abnormal CD19 signaling were further assessed. B-cell antigen receptor crosslinking induced exaggerated calcium responses and augmented activation of extracellular signal-regulated kinase in TSK/+ B cells. CD22 function was specifically impaired in TSK/+ B cells. Consistently, CD19, a major target of CD22-negative regulation, was hyperphosphorylated in TSK/+ B cells. These findings indicate that reduced inhibitory signal provided by CD22 results in abnormal activation of signaling pathways including CD19 in TSK/+ mice and also suggest that this disrupted B cell signaling contribute to specific autoantibody production. Systemic sclerosis (SSc) is characterized by fibrosis and autoimmmunity. Peripheral blood B cells from SSc patients specifically overexpress CD19, a critical cell-surface signal transduction molecule in B cells. CD19 deficiency in B cells also attenuates skin fibrosis in the tight-skin (TSK/+) mouse, a genetic model for SSc. Herein we analyzed two transgenic mouse lines that overexpress CD19. Remarkably, 20% increase of CD19 expression in mice spontaneously induced SSc-specific anti-DNA topoisomerase I (topo I) antibody (Ab) production, which was further augmented by 200% overexpression. In TSK/+ mice overexpressing CD19, skin thickness did not increase, although anti-topo I Ab levels were significantly augmented, indicating that abnormal CD19 signaling influences autoimmunity in TSK/+ mice and also that anti-topo I Ab does not have a pathogenic role. The molecular mechanisms for abnormal CD19 signaling were further assessed. B-cell antigen receptor crosslinking induced exaggerated calcium responses and augmented activation of extracellular signal-regulated kinase in TSK/+ B cells. CD22 function was specifically impaired in TSK/+ B cells. Consistently, CD19, a major target of CD22-negative regulation, was hyperphosphorylated in TSK/+ B cells. These findings indicate that reduced inhibitory signal provided by CD22 results in abnormal activation of signaling pathways including CD19 in TSK/+ mice and also suggest that this disrupted B cell signaling contribute to specific autoantibody production. Systemic sclerosis (SSc) is a multisystem disease characterized by fibrosis and autoimmunity.1LeRoy EC Black C Fleischmajer R Jablonska S Krieg T Medsger TAJ Rowell N Wollheim F Scleroderma (systemic sclerosis): classification, subsets and pathogenesis.J Rheumatol. 1988; 15: 202-205PubMed Google Scholar SSc patients develop excessive extracellular matrix deposition in the skin and other visceral organs, resulting in fibrotic changes.2Varga J Bashey RI Regulation of connective tissue synthesis in systemic sclerosis.Int Rev Immunol. 1995; 12: 187-199Crossref PubMed Scopus (58) Google Scholar The tight-skin (TSK) mouse is a genetic animal model for SSc, which was originally identified as a spontaneous mutation that results in increased synthesis and excessive accumulation of collagen and other extracellular matrix proteins in the skin and visceral organs.3Green MC Sweet HO Bunker LE Tight-skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton.Am J Pathol. 1976; 82: 493-512PubMed Google Scholar Although homozygous mice die in utero, heterozygous (TSK/+) mice survive, but develop cutaneous fibrosis resembling human SSc, if not identical.3Green MC Sweet HO Bunker LE Tight-skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton.Am J Pathol. 1976; 82: 493-512PubMed Google Scholar, 4Saito S Kasturi K Bona C Genetic and immunologic features associated with scleroderma-like syndrome of TSK mice.Curr Rheumatol Rep. 1999; 1: 34-37Crossref PubMed Scopus (19) Google Scholar TSK mutation is a tandem duplication within the gene encoding fibrillin 1, an extracellular matrix glycoprotein crucial for microfibril assembly.5Kielty CM Raghunath M Siracusa LD Sherratt MJ Peters R Shuttleworth CA Jimenez SA The tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils.J Cell Biol. 1998; 140: 1159-1166Crossref PubMed Scopus (103) Google Scholar TSK mutation causes a formation of the conformationally abnormal microfibrils, although it cannot account for all of the phenotypic abnormalities in TSK/+ mice. The autoimmune aspect is another prominent feature of SSc.6Reimer G Steen VD Penning CA Medsger TAJ Tan EM Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma).Arthritis Rheum. 1988; 31: 525-532Crossref PubMed Scopus (182) Google Scholar Antinuclear antibodies (Abs) are found in the sera from >95% of SSc patients, and the associations between some specific antinuclear Abs and clinical manifestations are well established.7Okano Y Antinuclear antibody in systemic sclerosis (scleroderma).Rheum Dis Clin North Am. 1996; 22: 709-735Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Autoantibody (autoAb) to DNA topoisomerase I (topo I) is detected most exclusively in SSc and correlated with diffuse cutaneous involvement, peripheral vascular disease, and pulmonary interstitial fibrosis.8Steen VD Powell DL Medsger TAJ Clinical correlations and prognosis based on serum autoantibodies in patients with systemic sclerosis.Arthritis Rheum. 1988; 31: 196-203Crossref PubMed Scopus (554) Google Scholar TSK mice also have a skewed humoral response and produce anti-topo I Abs.9Bona C Rothfield N Autoantibodies in scleroderma and tightskin mice.Curr Opin Immunol. 1994; 6: 931-937Crossref PubMed Scopus (38) Google Scholar Although the relationship between fibrosis and systemic autoimmunity is unclear in TSK/+ mice, we recently demonstrated that B-cell functional defects caused by the loss of CD19, a B-cell-specific cell-surface molecule, significantly decreased skin fibrosis in TSK/+ mice, suggesting that B cells play an important role in the pathogenesis of these disorders of humans and mice.10Saito E Fujimoto M Hasegawa M Komura K Hamaguchi Y Kaburagi Y Nagaoka T Takehara K Tedder TF Sato S CD19-dependent B lymphocyte signaling thresholds influence skin fibrosis and autoimmunity in the tight skin mice.J Clin Invest. 2002; 109: 1453-1462Crossref PubMed Scopus (231) Google Scholar Recent studies using animal models of autoimmune diseases have revealed that B cells play various important roles, including antigen presentation and cytokine production as well as pathogenic autoAb production. Elimination of B cells in the lupus-prone MRL/lpr mice results in a complete abrogation of nephritis, vasculitis, and skin disease, which is independent of Ab secretion.11Chan OT Hannum LG Haberman AM Madaio MP Shlomchik MJ A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus.J Exp Med. 1999; 189: 1639-1648Crossref PubMed Scopus (606) Google Scholar Pathogenic autoAb production is also important as K/BxN mice, a model for rheumatoid arthritis, have hyperactive B cells that produce arthritogenic autoAb.12Korganow AS Ji H Mangialaio S Duchatelle V Pelanda R Martin T Degott C Kikutani H Rajewsky K Pasquali JL Benoist C Mathis D From systemic T cell self-reactivity to organ-specific autoimmune disease via immunoglobulins.Immunity. 1999; 10: 451-461Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar Consistently, recent studies have shown that B cell depletion by anti-CD20 Ab is effective in treating patients with rheumatoid arthritis or systemic lupus erythematosus.13Edwards JC Cambridge G Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes.Rheumatology (Oxford). 2001; 40: 205-211Crossref PubMed Scopus (477) Google Scholar, 14Leandro MJ Edwards JC Cambridge G Ehrenstein MR Isenberg DA An open study of B lymphocyte depletion in systemic lupus erythematosus.Arthritis Rheum. 2002; 2002: 2673-2677Crossref Scopus (544) Google Scholar, 15De Vita S Zaja F Sacco S De Candia A Fanin R Ferraccioli G Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis: evidence for a pathogenetic role of B cells.Arthritis Rheum. 2002; 46: 2029-2033Crossref PubMed Scopus (230) Google Scholar Thus, B cells have recently attracted much attention as a crucial player in systemic autoimmune disorders.16Hasler P Zouali M B cell receptor signaling and autoimmunity.FASEB J. 2001; 15: 2085-2098Crossref PubMed Scopus (76) Google Scholar B cell fate and functions are primarily determined by signal transduction through a B-cell antigen receptor (BCR), which is further regulated by other cell-surface receptors that inform B cells of their extracellular microenvironment, including CD19, CD21, CD22, CD40, CD72, and FcγRIIB. Among them, the CD19/CD21 complex serves as a positive regulator that amplifies BCR signal transduction for a prompt and strong immune response. When CD19/CD21 is cross-linked with C3d fragment of activated C3, this allows B cells to respond antigens with 10- to 1000-fold lower concentration.17Carter RH Fearon DT CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes.Science. 1992; 256: 105-107Crossref PubMed Scopus (558) Google Scholar By contrast, negative regulators such as CD22 are crucial for terminating BCR signal transduction and thus for avoiding improper immune response such as against self-antigens.18Goodnow CC Pathways for self-tolerance and the treatment of autoimmune diseases.Lancet. 2001; 357: 2115-2121Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar We reported that circulating B cells from SSc patients overexpress CD19 by 20%. Strikingly, the current study revealed that this relatively small increase of CD19 expression in C57BL/6 mouse induced spontaneous production of SSc-specific anti-topo I autoAbs. Similarly, CD19 signaling was augmented in TSK/+ B cells that produced anti-topo I Abs. Therefore, we further assessed how B cell signaling was disturbed in TSK/+ mice. The present study showed that negative regulation provided by CD22 was disrupted in TSK/+ B cells, which resulted in abnormal activation of downstream signal transduction molecules including CD19. The results of this study may provide an important clue of how signaling pathways might be functionally disrupted in human patients with rheumatic diseases. TSK/+ (the C57BL/6 background) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Human CD19-transgenic (TG)-1 mice (C57BL/6 × B6/SJL), CD19TG-4 mice (C57BL/6 × B6/SJL), and CD22-deficient (CD22−/−) mice (C57BL/6 × 129) were described previously.19Zhou LJ Smith HM Waldschmidt TJ Schwarting R Daley J Tedder TF Tissue-specific expression of the human CD19 gene in transgenic mice inhibits antigen-independent B-lymphocyte development.Mol Cell Biol. 1994; 14: 3884-3894Crossref PubMed Scopus (30) Google Scholar, 20Sato S Miller AS Inaoki M Bock CB Jansen PJ Tang ML Tedder TF CD22 is both a positive and negative regulator of B lymphocyte antigen receptor signal transduction: altered signaling in CD22-deficient mice.Immunity. 1996; 5: 551-562Abstract Full Text PDF PubMed Scopus (391) Google Scholar CD19TG-1, CD19TG-4, and CD22−/− mice were backcrossed more than four generations onto the C57BL/6 background before use in these studies. CD19TG-1 and CD19TG-4 TSK/+ mice were generated through breedings of CD19TG+/−TSK/+ mice. Cell-surface CD19 and CD22 expressions were verified by two-color fluorescence cytometry. To verify the TSK/+ genotype, polymerase chain reaction amplification of a partially duplicated fibrillin 1 gene was performed using genomic DNA from each mouse as described.21McGaha T Saito S Phelps RG Gordon R Noben-Trauth N Paul WE Bona C Lack of skin fibrosis in tight skin (TSK) mice with targeted mutation in the interleukin-4R alpha and transforming growth factor-beta genes.J Invest Dermatol. 2001; 116: 136-143Crossref PubMed Scopus (114) Google Scholar Wild-type littermates generated from heterozygous matings were used appropriately as controls. All mice were housed in a specific pathogen-free barrier facility. Mice used in this study were 8 to 10 weeks of age. All studies and procedures were approved by the Animal Care Committee of International Medical Center of Japan and Kanazawa University School of Medicine. Monoclonal antibodies (mAbs) used included: anti-B220 (RA3-6B2; Beckman Coulter, Miami, FL); anti-CD19 (MB19.122Fujimoto M Bradney AP Poe JC Steeber DA Tedder TF Modulation of B lymphocyte antigen receptor signal transduction by a CD19/CD22 regulatory loop.Immunity. 1999; 11: 191-200Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar); anti-CD22 (MB22–123Sato S Hasegawa M Fujimoto M Tedder TF Takehara K Quantitative genetic variation in CD19 expression correlates with autoimmunity.J Immunol. 2000; 165: 6635-6643PubMed Google Scholar, Cy34; BD PharMingen, San Jose, CA); anti-I-A (M5/114.15.2, BD PharMingen); and anti-phosphotyrosine Abs (4G10; Upstate Biotechnology, Lake Placid, NY; PY99, Santa Cruz Biotechnology, Santa Cruz, CA). Antisera used included: fluorescein isothiocyanate (FITC)-conjugated anti-IgM (Southern Biotechnology Associations, Inc., Birmingham, AL); F(ab′)2 fragments of goat anti-mouse IgM (Cappel, ICN Biomedicals, Irvine, CA); anti-Lyn, anti-Syk, anti-PLCγ2, anti-ERK2, and anti-JNK1 (Santa Cruz Biotechnology); anti-SHP-1 (Upstate Biotechnology); anti-Akt, anti-phospho-Akt (Ser473), and anti-phosphoCD19 (Y513) (Cell Signaling, Beverly, MA); anti-active ERK and anti-active JNK Abs (Promega, Madison, WI). Rabbit anti-phosphoCD22 (Y762) was generated by immunizing phosphotyrosine peptide of the sequence. Immunofluorescence analysis was performed as described.22Fujimoto M Bradney AP Poe JC Steeber DA Tedder TF Modulation of B lymphocyte antigen receptor signal transduction by a CD19/CD22 regulatory loop.Immunity. 1999; 11: 191-200Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar Briefly, leukocytes from blood and spleen were stained at 4°C using predetermined optimal concentrations of Abs for 20 minutes. Blood erythrocytes were lysed after staining using the Whole Blood Immuno-Lyse kit (Beckman Coulter). Cells with the forward and side light scatter properties of lymphocytes were analyzed on an Epics Altra flow cytometer (Beckman Coulter). Positive and negative populations of cells were determined using unreactive isotype-matched Abs (Beckman Coulter) as controls for background staining. To stain phosphotyrosine intracellularly, cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA). Serum anti-topo I autoAb levels were determined with specific ELISA kits (Medical and Biological Laboratories, Nagoya, Japan). Briefly, 96-well microtiter plates coated with topo I were incubated with serum samples diluted 1:100. The bound Abs were detected with alkaline phosphatase-conjugated anti-mouse IgG or IgM Abs (Southern Biotechnology Associates, Inc.). Relative levels of autoAbs were determined for each group of mice using pooled serum samples. Sera were diluted at log intervals (1:10 to 1:105) and assessed for relative autoAb levels as above except the results were plotted as optical density versus dilution (log scale). The dilutions of sera giving half-maximal optical density values were determined by linear regression analysis, thus generating arbitrary unit per milliliter values for comparison between sets of sera. All skin sections were taken from the para-midline, lower back region (the same anatomical site to minimize regional variations in thickness) as full thickness sections extending down to the body wall musculature. Tissues were fixed in 10% formaldehyde solution for 24 hours and embedded in paraffin. Sections were stained with hematoxylin and eosin. Hypodermal thickness, which was defined as the thickness of a subcutaneous loose connective tissue layer (ie, the hypodermis or superficial fascia) beneath the panniculus carnosus, was measured for multiple transverse perpendicular sections using an ocular micrometer. The skin from male mice was generally thicker than that from female mice despite the presence or absence of TSK mutations (data not shown). Because similar results were obtained when male or female mice were analyzed separately, only data from female mice were presented for skin thickness and hydroxyproline content in this study. Splenic B cells were purified by removing T cells with anti-Thy1.2 Ab-coated magnetic beads (Dynal, Inc., Lake Success, NY) and resuspended in RPMI 1640 medium containing 5% fetal calf serum. Cells were stimulated with F(ab′)2 fragments of goat anti-mouse IgM Ab (40 μg/ml) at 37°C. To examine antigen-specific B cells, mice were immunized by an intraperitoneal injection with 50 μg of 2,4,6-trinitrophenol (TNP)-conjugated ficoll (Biosearch Technologies, Novato, CA) in phosphate-buffered saline with complete Freund's adjuvant (Difco, Detroit, MI) Splenic B cells were harvested on day 7, stimulated with FITC-conjugated TNP-ficoll (Biosearch Technologies), and analyzed by flow cytometry. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or subjected to immunoprecipitation, cells were lysed in buffer containing 1% Nonidet P-40, 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0), 1 mmol/L Na orthovanadate, 2 mmol/L ethylenediaminetetraacetic acid, 50 mmol/L NaF, and protease inhibitors. For immunoprecipitations, cell lysates were precleared twice by incubation with control Abs plus protein G-Sepharose beads (Amersham Pharmacia Biotech, Buckinghamshire, UK), followed by incubation with protein G-beads plus Abs to proteins of interest for 4 hours at 4°C. For CD19 immunoprecipitations, lysates were precleared with Affigel 10 beads (Bio-Rad Laboratories, Hercules, CA) conjugated with mouse IgA, then incubated with Affigel 10 beads bearing anti-CD19 Ab (MB19.1). Immunoprecipitated proteins were subjected to SDS-PAGE, and transferred onto nitrocellulose membranes for immunoblotting. The membranes were incubated with peroxidase-conjugated Abs. The blots were developed using an enhanced chemiluminescence kit (Pierce, Rockford, IL) and detected using Fluoro-S imager (Bio-Rad Laboratories). The blots were reprobed with Abs specific for the protein of interest. Band intensity was quantified using Quantity One software (Bio-Rad Laboratories). Spleen cells (107/ml) in RPMI 1640 medium containing 5% fetal calf serum and 10 mmol/L HEPES were loaded with 1 μmol/L indo-1-AM ester (Molecular Probes, Eugene, OR) at 37°C for 30 minutes. Cells were stained with FITC-conjugated anti-B220 for 15 minutes. The ratio of fluorescence (488/407 nm) of B220+ cells was determined using an Epics Altra flow cytometer (Beckman Coulter). Baseline fluorescence ratios were collected for 1 minute before F(ab′)2 anti-mouse IgM Ab (10 or 40 μg/ml) were added. Results were plotted as the fluorescence ratio at 15-second intervals with the background subtracted. Increased fluorescence ratios indicate increases in [Ca2+]i. All data are shown as mean values ± SEM. The Mann-Whitney U-test was used for determining the level of significance of differences between sample means and Bonferroni's test was used for multiple comparisons. We reported that circulating B cells from SSc patients overexpress CD19 by 20%.23Sato S Hasegawa M Fujimoto M Tedder TF Takehara K Quantitative genetic variation in CD19 expression correlates with autoimmunity.J Immunol. 2000; 165: 6635-6643PubMed Google Scholar To assess whether CD19 overexpression can induce SSc-specific autoAb production, we examined anti-topo I Ab production in transgenic mice that overexpress human CD19 specifically on B cells. Remarkably, 20% increase of CD19 expression in mice (CD19TG-4+/−) spontaneously induced IgG anti-topo I Ab production (Figure 1A). Furthermore, transgenic mice that overexpress 200% of endogenous CD19 levels (CD19TG-1+/+) showed prominent increase of both IgG and IgM anti-topo I autoAb levels in the sera (106- and 41-fold, P < 0.01, respectively; Figure 1A). Considering that CD19TG-1+/+ mice have approximately twofold higher Ig levels than wild-type mice, the autoAb levels were still significantly higher when serum Ig levels were normalized (data not shown). Thus, the anti-topo I Ab levels correlated with the CD19 expression levels in B cells, indicating that abnormal CD19 signaling pathway is closely associated with SSc-related autoimmunity including anti-topo I Ab production. TSK/+ mice have circulating anti-topo I Ab, and the loss of CD19 expression significantly decreases the autoAb titers.10Saito E Fujimoto M Hasegawa M Komura K Hamaguchi Y Kaburagi Y Nagaoka T Takehara K Tedder TF Sato S CD19-dependent B lymphocyte signaling thresholds influence skin fibrosis and autoimmunity in the tight skin mice.J Clin Invest. 2002; 109: 1453-1462Crossref PubMed Scopus (231) Google Scholar We examined whether CD19 overexpression further increased the autoAb levels in TSK/+ mice. Both IgG and IgM anti-topo I Ab levels significantly increased in both lines of CD19TG/TSK/+ mice compared with TSK/+ mice (Figure 1A). Furthermore, augmentation of anti-topo I levels paralleled with the CD19 density on B cell surface of these transgenic mice (Figure 1A). IgG anti-topo I levels were 7.9-fold higher (P < 0.01) in CD19TG-4+/−TSK/+ mice and 20-fold higher (P < 0.01) in CD19TG-1+/+TSK mice compared with TSK/+ mice (Figure 1A). Similarly, IgM anti-topo I levels in CD19TG-4+/TSK/+ mice and CD19TG-1+/+TSK mice were 2.7-fold (P < 0.05) and 12-fold higher (P < 0.01) than those in TSK/+ mice, respectively (Figure 1A). Therefore, CD19 overexpression enhanced anti-topo I Ab production in TSK/+ mice as well as C57BL/6 mice. Moreover, TSK genetic background in CD19TG-1+/+ and CD19TG-4+/− mice resulted in significantly augmented anti-topo I Ab production relative to CD19TG-1+/+ and CD19TG-4+/− mice, respectively (Figure 1A). Therefore, CD19 overexpression and TSK genetic background cooperatively contributed to anti-topo I Ab production. We previously reported that CD19 deficiency attenuates skin fibrosis in TSK/+ mice,10Saito E Fujimoto M Hasegawa M Komura K Hamaguchi Y Kaburagi Y Nagaoka T Takehara K Tedder TF Sato S CD19-dependent B lymphocyte signaling thresholds influence skin fibrosis and autoimmunity in the tight skin mice.J Clin Invest. 2002; 109: 1453-1462Crossref PubMed Scopus (231) Google Scholar raising a possibility that CD19 may play a direct role in the induction of fibrosis. Therefore, we examined skin thickness in CD19TG mice. However, histopathology of the full-thickness skin sections from CD19TG-1+/+ and CD19TG-4+/− mice showed normal hypodermal thickness compared with wild-type littermates (Figure 1B and data not shown). Skin content of hydroxyproline that is a modified amino acid uniquely found as a high percentage of collagen also showed no difference between CD19TG mice and wild-type littermates (data not shown). We then crossed CD19TG-1 and CD19TG-4 mice with TSK/+ mice to generate two TSK mouse lines that overexpress CD19 at different levels on B cells. However, hypodermal thickness and skin hydroxyproline content of TSK/+ mice were not altered by either level of CD19 overexpression (Figure 1B and data not shown). Collectively, although CD19 loss can decrease skin fibrosis of TSK/+ mice, CD19 overexpression did not increase skin fibrosis. Given that CD19 is a critical molecule that regulates B cell signaling, the finding that TSK/+ B cells exhibited the augmented anti-topo I Ab production that was enhanced by CD19 overexpression suggests that there are intrinsic signaling abnormalities of B cells in TSK/+ mice. Consistent with this, circulating B cells from TSK/+ mice expressed significantly lower levels of surface IgM (50 ± 3% decrease from wild-type mice, P < 0.05; Figure 2A), zref10 and higher levels of MHC class II (I-A) (30 ± 7% increase from wild-type mice, P < 0.05; Figure 2B) than those from wild-type littermates. By contrast, there was no significant difference in splenic B cells with the expression levels of IgM or I-A between TSK/+ and wild-type littermates. The phenotypes observed in circulating B cells form TSK/+ mice was equivalent to B cells that demonstrate augmented transmembrane signaling through the BCR complex, such as CD22−/− mice and mice overexpressing CD19.24Tedder TF Sato S Poe JC Fujimoto M CD19 and CD22 regulate a B lymphocyte signal transduction pathway that contributes to autoimmunity.Keio J Med. 2000; 49: 1-13Crossref PubMed Scopus (22) Google Scholar These mice also exhibit phenotypical abnormalities in circulating B cells, although the differences are usually mild in splenic B cells.22Fujimoto M Bradney AP Poe JC Steeber DA Tedder TF Modulation of B lymphocyte antigen receptor signal transduction by a CD19/CD22 regulatory loop.Immunity. 1999; 11: 191-200Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 25Sato S Steeber DA Jansen PJ Tedder TF CD19 expression levels regulate B lymphocyte development: human CD19 restores normal function in mice lacking endogenous CD19.J Immunol. 1997; 158: 4662-4669PubMed Google Scholar, 26Fujimoto M Poe JC Satterthwaite AB Wahl MI Witte ON Tedder TF Complementary roles for CD19 and Bruton's tyrosine kinase in B lymphocyte signal transduction.J Immunol. 2002; 168: 5465-5476PubMed Google Scholar These changes appeared distinct because TSK/+ B cells expressed other cell surface molecules, including CD19, CD21, CD22, CD40, and B220 at similar levels as those from wild-type littermates (data not shown). Therefore, TSK/+ B cells exhibited distinctive hyperreactive phenotype. Because B cell phenotype from TSK/+ mice suggested that TSK/+ B cells were hyperresponsive to transmembrane signals, an increase in the intracellular calcium concentration ([Ca2+]i) after BCR ligation was examined as an indicator of rapid B cell activation. Splenic B cells were freshly isolated from TSK/+ and wild-type littermates and were stimulated with F(ab′)2 fragments of anti-IgM Ab. TSK/+ B cells demonstrated significantly augmented rapid increase of [Ca2+]i as well as augmented sustained increase after IgM cross-linking when compared with wild-type B cells (Figure 3). In five experiments, maximum [Ca2+]i in TSK/+ B cells was 50% higher than wild-type B cells (P < 0.01). The kinetics of [Ca2+]i change was also slightly accelerated in TSK/+ B cells when compared with wild-type B cells. The augmented response in TSK/+ B cells did not result from a few exceptional B cells but represented most of B cells because the ratio of responding B cells from TSK/+ mice was equal to that of wild-type littermates (data not shown). Thus, BCR-induced [Ca2+]i response was augmented in TSK/+ B cells. Because TSK/+ B cells were likely to be hyperresponsive to BCR cross-linking, tyrosine phosphorylation of B-cell signaling molecules was assessed to determine which pathways were responsible for this alteration. Lyn and Syk are protein tyrosine kinases abundantly expressed in B cells and have crucial roles in [Ca2+]i responses.27Kurosaki T Genetic analysis of B cell antigen receptor signaling.Annu Rev Immunol. 1999; 17: 555-592Crossref PubMed Scopus (370) Google Scholar To assess Lyn and Syk phosphorylation, freshly isolated splenic B cells were stimulated with F(ab′)2 fragments of anti-IgM Ab and were lysed at different time points after BCR cross-linking. Lyn or Syk was immunoprecipitated from lysates, followed by SDS-PAGE and anti-phosphotyrosine immunoblotting. Anti-IgM ligation induced Lyn phosphorylation at similar levels between wild-type and TSK/+ B cells (Figure 4A). Also, Syk was comparably phosphorylated in TSK/+ B cells on BCR engagement (Figure 4B). Therefore, tyrosine phosphorylation of these protein tyrosine kinases was intact in TSK/+ B cells. Also, tyrosine kinase activities of Lyn and Syk determined by in vitro kinase assays were comparable between wild-type and TSK/+ B cells (data not shown). Signaling mediators were then assessed for tyrosine phosphorylation. SHIP and PLCγ2 also regulate [Ca2+]i response negatively and positively, respectively.27Kurosaki T Genetic analysis of B cell antigen receptor signaling.Annu Rev Immunol. 1999; 17: 555-592Crossref PubMed Scopus (370) Google Scholar However, despite the augmented [Ca2+]i response in TSK/+ B cells, SHIP phosphorylation was similar or slightly decreased in TSK/+ B cells (Figure 4C). BCR-induced phosphorylation of PLCγ2 was also similar or slightly had decreased levels in TSK/+ B cells (Figure 4D). Additionally, Shc was phosphorylated at wild-type level in TSK/+ B cells after BCR ligation (Figure 4E). Furthermore, phosphorylation level at tyrosine-317 of Shc was also similar between wild-type and TSK/+ B cells (data not shown). Therefore, these mediators were not responsible for augmented [Ca2+]i response in TSK/+ B cells. Because B cells from CD22−/− mice exhibit exaggerated [Ca2+]i response after BCR ligation,20Sato S Miller AS Inaoki M Bock CB Jansen PJ Tang ML Tedder TF CD22 is both a positive and negative regulato" @default.
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• W2004305328 title "B Lymphocyte Signaling Established by the CD19/CD22 Loop Regulates Autoimmunity in the Tight-Skin Mouse" @default.
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