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Expression Correlates with AutoimmunityQuantitative Genetic Variation in CD19

Thomas F. Tedder and Kazuhiko TakeharaShinichi Sato, Minoru Hasegawa, Manabu Fujimoto,

http://www.jimmunol.org/content/165/11/6635doi: 10.4049/jimmunol.165.11.6635

2000; 165:6635-6643; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Quantitative Genetic Variation in CD19 Expression Correlateswith Autoimmunity 1

Shinichi Sato,2* Minoru Hasegawa,2* Manabu Fujimoto, † Thomas F. Tedder,3† andKazuhiko Takehara*

Signaling thresholds influence the balance between humoral immunity and autoimmunity. Cell surface CD19 regulates intrinsicand Ag receptor-induced B lymphocyte signaling thresholds, and transgenic mice that overexpress CD19 by 3-fold generatespontaneous autoantibodies in a genetic background not associated with autoimmunity. To quantify the extent that geneticallydetermined differences in expression of a single cell surface molecule can influence autoantibody production, we have assessedautoimmunity in a C57BL/6-transgenic mouse line with subtle 15–29% increases in CD19 cell surface expression (CD19 trans-genic). Antinuclear Abs, especially anti-spindle pole Abs, rheumatoid factor, and autoantibodies for ssDNA, dsDNA, and histonewere produced in these transgenic mice, but not littermate controls. This demonstrates that small changes in CD19 expression caninduce autoantibody production. Remarkably, similar changes in CD19 expression were found on B cells from patients withsystemic sclerosis, a multisystem disorder of connective tissue with autoantibody production. CD19 density on blood B cells fromsystemic sclerosis patients was significantly (;20%) higher compared with normal individuals, whereas CD20, CD22, and CD40expression were normal. These results suggest that modest changes in the expression or function of regulatory molecules such asCD19 may shift the balance between tolerance and immunity to autoimmunity. Thereby autoimmune disease may result from acollection of subtle multigenic alterations that could include incremental density changes in cell surface signaling molecules.TheJournal of Immunology,2000, 165: 6635–6643.

H umoral immune responses and the production of auto-antibodies are regulated in part by signaling through Bcell Ag receptors. Autoimmunity and immune responses

are further regulated or “fine tuned” by signal transduction mole-cules that amplify or inhibit Ag receptor signaling during re-sponses to self and foreign Ags (1, 2). These regulatory moleculesinclude a subset of functionally interrelated cell surface receptors,such as CD19, CD21, and CD22, and their intracellular signalingcomponents including Lyn, Btk, Vav, and the SHP1 protein ty-rosine phosphatase (2, 3). Significant alterations in function or ex-pression of these molecules can predispose for autoantibody pro-duction. For example, transgenic mice that overexpress CD19 by3-fold exhibit loss of tolerance and generate spontaneous autoan-tibodies (4, 5). Lyn-deficient mice exhibit glomerulonephritis dueto the presence of immune complexes containing autoantibodies(6, 7). Motheaten viable (mev/mev) mice with SHP1 mutationsdemonstrate elevated levels of spontaneous autoantibodies, hyper-gammaglobulinemia, and tissue deposition of immune complexes(8). Thus, these “response regulatory” molecules of B cell signal

transduction may play critical roles in autoantibody production. Inaddition, unidentified polygenic variations present in a variety ofmouse strains regulate Ag receptor signaling and the generation ofautoreactive B cell clones (9). However, the extent that individualgenetic alterations or subtle alterations in the functions of thesemolecules can quantitatively influence the development of auto-immunity remains relatively unknown. Therefore, in this study, wehave assessed the extent to which small alterations in cell surfaceCD19 expression can bias mice toward autoantibody production.

CD19 is a B cell-specific member of the Ig superfamily ex-pressed by early pre-B cells from the time of heavy chain rear-rangement until plasma cell differentiation. The cell surface den-sity of CD19 is tightly regulated during B cell differentiation,particularly in mice (10, 11). After B cell maturation, cellular ac-tivation induced by various stimuli, such as anti-IgM Abs, LPS,and IL-4, does not affect CD19 expression in either mice or hu-mans (4, 12). Nonetheless, the B1 subset of mouse B cells ex-presses CD19 at levels 60% higher than conventional B cells (4).Mouse lines that overexpress CD19 have been generated by the Bcell-specific expression of a human CD19 (hCD19)4 transgene(13). Since hCD19 and mouse CD19 (mCD19) are functionallyequivalent in vivo when expressed at comparable site densities(11), these different mouse lines express overall CD19 at variouscell surface densities. In these mice, CD19 expression levels cor-relate directly with altered B cell function, B cell hyperactivity,and autoantibody production (4, 11). Dose-dependent changes in Bcell development and function resulting from CD19 overexpres-sion in vivo presumably result from the fact that the cytoplasmicdomain of CD19 is a central regulatory component of B cells uponwhich multiple signaling pathways converge (2). Perhaps mostimportant, CD19 regulates a Src family protein tyrosine kinase

*Department of Dermatology, Kanazawa University School of Medicine, Kanazawa,Japan; and†Department of Immunology, Duke University Medical Center, Durham,NC 27710

Received for publication April 19, 2000. Accepted for publication September11, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a grant-in-aid from the Ministry of Education, Science,and Culture of Japan (to S.S.), by Uehara Memorial Foundation (to S.S.), by a grantfor basic dermatological research from Shiseido Co. (to S.S.), by a grant from LydiaO’Leary Memorial Foundation (to S.S.), and by National Institutes of Health GrantsCA81776 and CA54464 (to T.F.T.).2 S.S. and M.H. contributed equally to this study and share first authorship.3 Address correspondence and reprint requests to Dr. Thomas F. Tedder, Departmentof Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710.E-mail address: thomas.tedder@duke.edu

4 Abbreviations used in this paper: hCD19, human CD19; mCD19, mouse CD19;ANA, antinuclear Ab; hCD19TG, hCD19-transgenic mice; RF, rheumatoid factor;RNP, RNA polymerase; SLE, systemic lupus erythematosus; SSc, systemic sclerosis.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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activation loop in resting and Ag receptor-stimulated B cells thatestablishes basal signaling thresholds (14, 15).

Since previous “gene titration” studies in mice have shown that2- or 3-fold increases in CD19 expression can predispose mice toautoantibody production, we assessed whether more subtlechanges in CD19 expression could alter B cell homeostasis. Re-markably, a genetically determined quantitative increase in CD19expression by 15–29% induced autoantibody production in micethat are otherwise genetically wild-type. CD19 expression was also20% higher on B cells from autoimmune patients with systemicsclerosis (SSc) compared with healthy individuals. Therefore, it ispossible that modest alterations in CD19 function or expressioncontribute to the development of autoimmunity. Moreover, similarsubtle alterations in the expression or function of other importantregulatory molecules may predispose to autoimmune susceptibilityin other syndromes.

Materials and MethodsMice

hCD19-transgenic (TG)-1 (C57BL/63 B6/SJL) mice and hCD19TG-4(C57BL/6 3 B6/SJL) mice were described previously (13). hCD19TG-1(C57BL/6 3 B6/SJL) mice were backcrossed with C57BL/6 mice for ei-ther 7 or 12 generations before use in these studies. The hCD19TG-1 linebackcrossed with C57BL/6 mice for seven generations overexpressesCD19 by 2.6-fold (4, 11). However, the hCD19TG-1 line backcrossed withC57BL/6 mice for 12 generations expressed hCD19 at levels similar tohuman blood B cells and thereby only overexpressed CD19 by 2-fold (datanot shown). The molecular basis for the decrease in hCD19 expression inthe hCD19TG-1 line is unknown. Reduced hCD19 expression could haveresulted from a decrease in the number of hCD19 gene copies in this lineof mice or subtle genetic changes. Nonetheless, hCD19TG-1 mice thatwere backcrossed with C57BL/6 mice for 12 generations and overexpressCD19 by 2-fold were used as positive controls for the current experimentsunless noted otherwise. Wild-type littermates generated from breedings ofhemizygous transgenic mice were used as negative controls. Results withwild-type littermates of hCD19TG-1 and hCD19TG-4 mice were similarand were therefore pooled. All mice were between 2 and 3 mo of age whenused for this study. Mice were housed in a specific pathogen-free barrierfacility. All mice were regularly checked for infections, pathogens, andparasites by clinical veterinarians. All tests have been negative for.3years. All studies and procedures were approved by the Committee onAnimal Experimentation of Kanazawa University School of Medicine andthe Animal Care and Use Committee of Duke University.

Flow cytometric analysis

Abs used in these studies included the anti-mCD19 mAbs, rat IgG2a clone6D5 (Caltag, Burlingame, CA), and mouse IgA clone MB19-1 (4). Anti-human Abs used in this study included PE- or FITC-conjugated anti-hCD19(B4), anti-CD20 (HRC20), anti-CD21 (B2), anti-CD22 (B3), anti-CD40 (MAB89; Coulter, Miami, FL), and anti-hCD19 mAbs (mouse IgG1clone SJ25-C1; Caltag). For immunofluorescence staining, fresh heparin-ized whole blood samples were placed on ice immediately after collection.Blood samples (50ml) were stained at 4°C using predetermined saturatingconcentrations of the test mAb for 20 min as previously described (11, 16).Blood erythrocytes were lysed after staining using the Coulter WholeBlood Immuno-Lyse kit as detailed by the manufacturer (Coulter). Cellswere analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose,CA). Five thousand cells with the forward and side light scatter propertiesof mononuclear cells were analyzed for each sample, with fluorescenceintensity shown on a four-decade log scale. Fluorescence contours areshown as 50% log density plots. Positive and negative populations of cellswere determined using unreactive isotype-matched mAbs (Coulter) as con-trols for background staining. Background levels of staining were delin-eated using gates positioned to include 98% of the control cells.

CD19 expression density assessment

Cell surface densities of hCD19 and mCD19 were determined by stainingblood lymphocytes using PE-conjugated anti-mCD19 (6D5) and anti-hCD19 (SJ25-C1) mAbs. The two Ab preparations had fluorochrome:Abmolar ratios of 1.0. After direct immunofluorescence staining and flowcytometry analysis, the number of PE molecules bound on the surface ofCD191 lymphocytes was determined using the QuantiBRITE PE Fluores-cence Quantitation kit (Becton Dickinson Immunocytometry Systems, San

Jose, CA) and software provided by the manufacturer. The PE Fluores-cence Quantitation kit provides beads conjugated with four levels of PEthat are used to generate a standard curve by flow cytometry analysis.Linear regression analysis is then used to determine the number of PEmolecules bound to mAb-stained cells during flow cytometry analysis.Lymphocytes were incubated with various concentrations of the test mAbfor 40 min at 4°C immediately before flow cytometry analysis. The per-centage of CD191 cells among lymphocytes in each sample was deter-mined by flow cytometry analysis with total cell numbers determined usinga hemocytometer. For Scatchard analysis (17), the number of cell-boundPE-mAb molecules was determined by calculating the total number ofcell-bound PE molecules per sample for comparison with the total numberof mAb molecules added to each sample. The maximal binding capacity ofeach mAb preparation was determined as described (18).

Mouse Ig isotype-specific ELISAs

ELISAs were conducted as described previously using affinity-purifiedmouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA Abs (Southern Biotech-nology Associates, Birmingham, AL) to generate a standard curve (19, 20).The relative concentration of each Ig isotype in individual samples wascalculated by comparing the mean OD value obtained for duplicate wellsto a semilog standard curve of titrated standard Ab using linear regressionanalysis.

Antinuclear Ab (ANA) analysis

ANA was assayed by indirect immunofluorescence staining with sera di-luted 1:50 using HEp-2 substrate cells (Medical & Biological Laboratories,Nagoya, Japan) as described elsewhere (21). Ig isotype-specific ANAswere performed using FITC-conjugated goat F(ab9)2 fragment anti-mouseIgG (g-chain-specific), anti-mouse IgM (m-chain-specific), and anti-mouseIgG 1 IgM 1 IgA (Southern Biotechnology Associates) Abs. For two-color immunofluorescence staining of mouse and human serum samples,Ab binding was visualized using species-specific tetraethyl sulforhodam-ine-conjugated goat F(ab9)2 anti-mouse Ig Abs (BioSource International,Camarillo, CA) and FITC-conjugated goat F(ab9)2 anti-human Ig Abs(Medical & Biological Laboratories).

ELISAs for autoantibodies

Serum autoantibody levels were determined by ELISA as described pre-viously (4). Briefly, 96-well microtiter plates (Costar, Cambridge, MA)were coated overnight at 4°C with 5mg/ml ssDNA (Sigma, St. Louis, MO),dsDNA (MBL), histone (Sigma), or rabbit IgG (Sigma). Plates were incu-bated for 1.5 h with serum samples diluted 1:100 in TBS containing 1%BSA (Sigma). After washing three times, the plates were incubated withperoxidase-conjugated goat anti-mouse IgG (g-chain-specific) or goat anti-mouse IgM (m-chain-specific) Abs (Southern Biotechnology Associates)for 1 h. Substrate solution containing 0.0125%o-phenylenediamine (Sig-ma) and 0.015% H2O2 in 0.1 M sodium citrate buffer (pH 4.5) was addedand the OD of the wells was subsequently determined. Relative levels ofautoantibodies were determined for each group of mice using pooled serumsamples. Sera were diluted at log intervals (1:10–1:105) and assessed forrelative autoantibody levels as above, except the results were plotted as ODvs dilution (log scale). The dilutions of sera giving half-maximal OD val-ues were determined by linear regression analysis, thus generating arbitraryunit per milliliter values for comparison between sets of sera.

SSc and systemic lupus erythematosus (SLE) patients

Nineteen patients (16 females and 3 males, 23–72 years old) who fulfilledthe criteria for SSc proposed by the American College of Rheumatology(formerly the American Rheumatism Association) (22) were examined.Patients with SSc were grouped according to the classification system pro-posed by LeRoy et al. (23): 13 patients (10 females and 3 males) hadlimited cutaneous SSc and 6 (all female) had diffuse cutaneous SSc. Noneof the SSc patients had received oral steroids,D-penicillamine, or immu-nosuppressive drugs.

All patients with SLE fulfilled the criteria proposed by the AmericanCollege of Rheumatology (24) and had active SLE as determined by theSLE Disease Activity Index (25) that ranged between 8 and 20 for thesepatients. Three patients had not been medicated and two patients had re-ceived low-dose steroids (prednisolone, 10–20 mg/day), but had diseaserelapses at the time of blood collection. None of the SLE patients hadreceived immunosuppressive drugs. Thirty-two age- and sex-matchedhealthy volunteers served as normal controls for the SSc and SLE patients.Laboratory data (serum levels of IgM, IgG, IgA, CH50, C3, and C4, eryth-rocyte sedimentation rates, C-reactive protein, and ANA titer) and bloodsamples were obtained at the same time. The protocol was approved by the

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Kanazawa University School of Medicine, and informed consent was ob-tained from all patients.

Statistical analysis

All data are shown as mean values6 SD unless indicated otherwise.ANOVA was used to analyze the data and Student’st test was used todetermine the level of significance of differences in sample means.

ResultsCD19 expression in transgenic mice

Transgenic mouse lines expressing various cell surface densities ofhCD19 have been previously described (13). Mice from thehCD19TG-1 line produce spontaneous autoantibodies (4, 5).Moreover, autoantibody production correlates with the level ofCD19 overexpression and CD19 expression correlates directlywith gene dosage such that heterozygous mice express half asmuch hCD19 as their homozygous littermates (4). ThehCD19TG-1 line (backcrossed with C57BL/6 mice for seven gen-erations) overexpresses CD19 by 2.6-fold based on comparisons ofhCD19 expression between mouse and human blood B cells (4,11). Since there are no mAbs that react with both mCD19 andhCD19, these comparisons are based on the assumption that mouseand human B cells express similar cell surface densities of CD19.

To correlate CD19 expression with autoantibody production, thenumber of hCD19 and mCD19 molecules expressed on the surfaceof B cells in CD19TG mouse lines was quantified by immunoflu-orescence staining with saturating concentrations of IgG mAbsspecific for hCD19 or mCD19. Two lines of hCD19TG mice wereused for these studies: hCD19TG-1 mice that were backcrossedwith C57BL/6 mice for seven generations (4, 11) and hCD19TG-4mice that carry fewer copies of the hCD19 transgene. hCD19 wasexpressed by all B cells and only B cells among hematopoieticcells from hCD19TG mice (Fig. 1A). Cell surface hCD19 expres-sion by blood B cells of hCD19TG-41/2 mice was 226 3% ofthat expressed by hCD19TG-11/2 mice (n5 5; Fig. 1B). Hemi-zygous hCD19TG-11/2 mice that were backcrossed withC57BL/6 mice for seven generations express hCD19 at levelscomparable to human blood B cells (4, 11). Endogenous mCD19expression in hCD19TG-41/2 mice was 946 2% (n5 5) of levelsobserved in wild-type littermates (Fig. 1,A and B). Thus, if cir-culating B cells from mice and humans express CD19 at compa-rable site densities, overall CD19 expression levels in hCD19TG-41/2 mice were 1166 5% of wild-type mCD19 levels asdetermined by the intensity of immunofluorescence staining.

CD19 cell surface density was further assessed by Scatchardanalysis using IgG mAbs specific for either hCD19 or mCD19. Abbinding was quantified using standardized PE-conjugated beadsand quantitative flow cytometry analysis. hCD19TG-11/1 micethat were backcrossed with C57BL/6 mice for 12 generations wereused for these studies since these mice had hCD19 expressionlevels equivalent with those of human B cells, as determined byquantitative flow cytometry analysis (data not shown). Using thisapproach, human blood B cells expressed 35,8006 1,750 (6SEM) anti-CD19 mAb binding sites (Fig. 1C). B cells fromhCD19TG11/1 mice expressed 36,8006 1,520 sites andhCD19TG-41/2 mice expressed 6,7406 170 mAb binding sites(Fig. 1C). Endogenous mCD19 expression was assessed similarly;wild-type mice expressed 20,8006 570 sites, hCD19TG11/1

mice 16,6006 730 sites (80% of wild-type), and hCD19TG-41/2

mice 20,0006 410 (96% of wild-type) anti-mCD19 mAb bindingsites (Fig. 1C). With a strict interpretation of these results,hCD19TG-11/1 and hCD19TG-41/2 B cells expressed overallCD19 at 257 and 29% higher levels than their wild-type litter-mates, respectively. Although these results suggest that human

blood B cells express CD19 at;70% higher levels than mouse Bcells, variability in ligand binding between the anti-hCD19 andanti-mCD19 mAb could also explain these apparent differences.Therefore, if human and mouse B cells express CD19 at compa-rable densities, then hCD19TG-11/1 and hCD19TG-41/2 B cellsexpress overall CD19 at 183 and 15% higher levels than theirwild-type littermates, respectively. Thus, hCD19TG-41/2 B cellsoverexpress CD19 by 15–29%.

Effect of CD19 overexpression on B cell development

The effect of the small increase in CD19 expression on B celldifferentiation in hCD19TG-41/2 mice was assessed by quantify-ing B cell numbers and measuring serum Ab levels. Overall, the

FIGURE 1. Quantitation of CD19 expression by hCD19TG mouselines. A, hCD19 expression is B cell specific. Blood lymphocytes fromwild-type and hCD19TG-41/2 mice were examined using two-color im-munofluorescence staining for mCD19 (MB19-1-biotin plus PE-avidin)and hCD19 (B4-FITC) expression with flow cytometry analysis. Quadrantgates indicate negative and positive populations of cells as determinedusing isotype-matched unreactive control mAbs. Horizontal dashed linesare provided for reference.B, Quantitative analysis of mCD19 and hCD19expression. Blood lymphocytes from wild-type, hCD19TG-11/2 (back-crossed for seven generations) and hCD19TG-41/2 mice were examinedusing single-color indirect immunofluorescence staining for mCD19(MB19-1-biotin plus PE-avidin) and hCD19 (B4-PE) expression with flowcytometry analysis. Dashed histograms represent background staining asdetermined using isotype-matched unreactive control mAbs.C, CD19 ex-pression density. Blood lymphocytes from humans (PBL) and from wild-type (WT), hCD19TG-11/1 (TG11/1, backcrossed for 12 generations),and hCD19TG-41/2 (TG41/2) mice were examined using single-color im-munofluorescence staining for mCD19 (6D5-PE) and hCD19 (SJ25-C1-PE) with flow cytometry analysis. The number of Abs bound per cell wasdetermined as described inMaterials and Methods. A typical Scatchardplot for human blood B cells is shown in theleft paneland results for threehumans or mice are shown in theright panel. All results are representativeof those obtained with at least three 2-mo-old mice of each genotype.

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numbers of B cells found in hCD19TG-41/2 mice and their wild-type littermates were not significantly different, whereas peripheralB cell numbers were significantly reduced in hCD19TG-11/1 mice(Table I). Despite apparently normal B cell numbers in hCD19TG-41/2 mice, serum IgG1 (p , 0.05), IgG2a (p , 0.05), and IgG2b( p , 0.001) levels were significantly increased in hCD19TG-41/2

mice when compared with wild-type littermates, while serum IgM,IgG3, and IgA levels were normal (Fig. 2). Serum Ig levels inhCD19TG-11/1 mice were generally higher than those inhCD19TG-41/2 mice, especially IgM, IgG2a, and IgG2b. SerumIg levels did not differ between males and females (data notshown). Thus, small increases in CD19 expression resulted in sig-nificantly increased production of selected serum Ab isotypes inhCD19TG-41/2 mice. However, humoral responses following im-munizations with a T cell-dependent Ag were not significantlyincreased in hCD19TG-41/2 mice (data not shown).

Effect of CD19 overexpression on autoantibody production

Autoantibody levels in hCD19TG-41/2 mice were determined toassess the influence of elevated CD19 expression. ANAs were de-tected in 52% of hCD19TG-41/2 mice and 95% of hCD19TG-11/1 mice, but were rarely detectable in wild-type littermates (Fig.3; Table II). These autoantibodies were predominantly of the IgGisotype. A homogenous chromosomal staining pattern with moreintense staining of mitotic cells was observed in 21% of the serumsamples from hCD19TG-41/2 mice (Fig. 3c). The frequency(86%) and intensity of homogenous staining was higher for serafrom hCD19TG-11/1 mice (Fig. 3e). Sera from 28% ofhCD19TG-41/2 mice reacted with spindle poles (mitotic centers)of mitotic cells (Fig. 3b), whereas this staining pattern was ob-served in only 10% of sera from hCD19TG-11/1 mice (Fig. 3d).Since human autoantibodies recognizing centrioles, which arecomponents of spindle poles, give a staining pattern similar to thatobserved for some hCD19TG-41/2 sera (Fig. 3f; and Refs. 26–28), two-color immunofluorescence staining was conducted withhuman anti-centriole Ab-positive serum and sera from hCD19TG-41/2 mice. Both the mouse and human sera stained similar intra-cellular determinants, although the localized regions recognized bythe mouse serum were larger than those of the human anticentrioleAbs (Fig. 3,f andg). The antispindle pole autoantibodies stainedmitotic cells, whereas the anticentriole Abs stained both mitoticand interphase cells. Anticentromere Abs were not detected in sera

of hCD19TG-41/2 or hCD19TG-11/1 mice. The frequency, spec-ificity, and intensity of ANAs were similar between males andfemales (data not shown). Thus, the predominant ANA specificityin hCD19TG-41/2 sera was for spindle poles, while homogenousstaining was most commonly observed with hCD19TG-11/1

mouse sera.Autoantibody production was further assessed by ELISA. Se-

rum IgG autoantibody levels were significantly increased inhCD19TG-41/2 mice compared with their wild-type littermates(Fig. 4). Mean IgG anti-ssDNA (346% higher,p , 0.001), anti-dsDNA (194% increase,p , 0.005), and anti-histone (755% in-crease,p , 0.001) autoantibody titers were significantly higher inhCD19TG-41/2 mice. Mean IgM antihistone Ab (142%,p ,0.05) and IgM rheumatoid factor (RF; 211%,p , 0.05) titers werealso increased. Serum levels of all autoantibody specificities inhCD19TG-11/1 mice were generally higher than those inhCD19TG-41/2 mice. Anti-DNA topoisomerase I Ab levels didnot increase in either hCD19TG-41/2 or hCD19TG-11/1 mice asquantified by ELISA (data not shown).

Autoantibody levels increased with age in hCD19TG-41/2

mice. Mean titers of IgM anti-ssDNA Abs were significantlyhigher in 10-mo-old hCD19TG-41/2 mice (n 5 12) comparedwith 2-mo-old hCD19TG-41/2 mice (n 5 12; 207% increased,p , 0.01). Similarly, IgM RF levels were significantly higher in10-mo-old hCD19TG-41/2 mice (479% increased,p , 0.05).Mean IgG anti-ssDNA, IgM anti-dsDNA, IgG anti-dsDNA, IgMantihistone, and IgG antihistone Ab levels were similar in youngand old mice.

Table I. B cell development in mice that overexpress CD19a

Tissue

Frequency (%) and No. (31026) of B Cells

Wild type hCD19TG-41/2 hCD19TG-11/1

Bone marrow% IgM2 B220low 54.36 2.8 51.96 3.5 49.26 6.4% IgM1 B220low 22.36 1.2 25.16 1.0 24.06 1.4% IgM1 B220high 11.96 1.5 9.96 0.6 6.46 0.7*

Blood% B2201 63.06 3.2 54.46 0.9 14.16 2.4**No. B2201 7.06 0.4 5.56 0.6 0.86 0.1**

Spleen% B2201 46.36 1.2 43.66 1.2 26.96 1.7**No. B2201 43.76 7.6 36.76 1.0 15.96 2.9*

Peritoneal cavity% CD51 B2201 23.96 2.6 31.16 4.8 48.86 3.3**No. CD51 B2201 0.96 0.1 1.06 0.3 2.06 0.1**

a Values represent the number or percentage (6SD,n 5 3) of lymphocytes (basedon side and forward slight scatter properties) expressing the indicated cell surfacemarkers. The background percentage of cells that were positive (,1%) was subtractedfrom the values shown. The number or percentage of cells was significantly differentfrom that for wild-type littermates,p, p , 0.05; pp, p , 0.01.

FIGURE 2. The effect of increased CD19 expression on serum Ig levelsin transgenic mice. Serum Ig levels of 2-mo-old wild-type, hCD19TG-41/2

(TG-41/2), and hCD19TG-11/1 (TG-11/1) mice were determined by iso-type-specific ELISA, with results from each mouse represented as a dot.Horizontal bars represent mean Ig levels.

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Histopathological analysis of kidneys from 2- to 8-mo-oldhCD19TG-41/2 mice showed a normal architecture without de-tectable Ab deposits (data not shown). Swelling or deformity ofjoints, skin eruptions, or a thickened dermis were not observed.Mortality in transgenic mice was similar to that of wild-type lit-termates. In the hCD19TG-11/1 line of mice, there is a significantdecrease in the reproductive capacity of female mice followingbirth of their first litter. However, none of the hCD19TG micedeveloped overt symptoms suggestive of lupus or scleroderma-likedisease.

CD19 expression by B cells from SSc and SLE patients

Since small genetic changes in cell surface CD19 expression in-duced autoantibody production in mice, B cells from autoimmunepatients with SSc and SLE were assessed for abnormal CD19 ex-pression. The cell surface density of CD19 on peripheral blood Bcells from patients and healthy control individuals was examinedquantitatively by immunofluorescence staining with flow cytom-etry analysis. SSc patients had significantly higher mean CD19expression levels than normal controls (20%,p , 0.0001; Figs. 5and 6 ). Similarly, mean CD21 expression was 23% higher in SScpatients than in normal controls (p , 0.001). However, there wasnot a significant correlation between CD19 and CD21 expressionin SSc patients (n5 19, r 5 20.07, Fig. 6A), although CD21expression was significantly correlated with CD19 expression innormal controls (n5 32, r 5 0.48,p , 0.01, Fig. 6A). CD19 andCD21 expression levels were similar in male and female patients(data not shown). Mean CD20, CD22, and CD40 expression levelswere similar in SSc patients and normal controls. Higher CD19expression in SSc patients did not result from increased cell sizesince the forward and side light scatter properties of CD201 cellsin SSc patients were not measurably different from those of normalcontrols (data not shown). Elevated CD19 expression did not resultfrom B cell activation since HLA-DR, CD25, CD54, CD80, andCD86 expression levels were not increased on B cells from SScpatients (data not shown). In contrast to SSc patients, CD19 andCD21 expression levels were reduced on B cells from SLE patients

(Fig. 6B). Limited numbers of samples were available for SLEpatients who had not undergone treatment or who had been in anactive state of the disease for only a short time period. Therefore,the results obtained with these SLE patients may not represent abroader SLE population since the clinical manifestations of SLEare heterogeneous. Rather, the results with SLE patients are shownfor comparison to demonstrate that increased CD19 expressioncorrelated with SSc, but was not observed in other autoimmunediseases such as SLE. Thus, elevated CD19 and CD21 expression

Table II. Frequency of ANAs in mice that overexpress CD19a

Staining Patternb

Mice

Wild-typelittermates(n 5 23)

hCD19TG-41/2

(n 5 29)hCD19TG-11/1

(n 5 21)

HomogenousIgM 1 (4) 3 (10) 17 (81)IgG 1 (4) 5 (17) 18 (86)Ig 1 (4) 6 (21) 18 (86)

Spindle polesIgM 0 0 0IgG 0 4 (14) 0Ig 0 4 (14) 0

Homogenous plus spindlepole staining

IgM 0 0 2 (10)IgG 0 4 (14) 2 (10)Ig 0 4 (14) 2 (10)

TotalIgM 1 (4) 3 (10) 17 (81)IgG 1 (4) 14 (48)c 20 (95)Ig 1 (4) 15 (52)c 20 (95)

a Values represent the number and percentage of positive sera from 2- to3-mo-old mice. ANA were detected by indirect immunofluorescence stainingusing HEp-2 cells as the substrate.

b ANA isotypes were determined using isotype-specific antimouse Ig Abs.c Serum from one mouse that produced a nucleolar staining pattern was

included.

FIGURE 3. ANA staining by sera from wild-type (a), hCD19TG-41/2 (b andc), and hCD19TG-11/1 (d ande) mice. IgG-specific ANAs were assayedby indirect immunofluorescence staining using HEp-2 substrate cells. Two-color immunofluorescence staining with a human serum sample containinganticentriole Abs (f) and a serum sample from one hCD19TG-41/2 mouse (g). Ab binding was visualized using tetraethyl sulforhodamine-conjugated goatF(ab9)2 anti-mouse Ig and FITC-conjugated goat F(ab9)2 anti-human Ig. Both sera stained the same location as dots (arrows). Anticentriole Abs recognizedcentriole pairs during interphase (f, arrowhead).

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levels were the primary phenotypic abnormalities observed for Bcells from SSc patients.

Correlations of cell surface molecule expression by blood Bcells with various clinical immunologic parameters were assessedto determine whether high CD19 and CD21 expression correlatedwith the immunologic status of patients with SSc. Increased CD19expression correlated positively with serum IgG and IgM levels inSSc patients (r5 0.610, p , 0.01 andr 5 0.579, p , 0.02,respectively), whereas there was not a significant correlation be-tween CD21 expression and serum Ig levels (data not shown).

Although all SSc patients produced clinically significant levels ofserum ANA, CD19 or CD21 expression did not correlate withANA titers (data not shown). The numbers and percentages ofcirculating B-1a cells (CD51 CD201) and B cells (CD191

CD201) were similar for SSc patients and normal controls (datanot shown).

DiscussionThe concept of a fine balance between humoral immunity andautoimmunity is reinforced by the finding that a 15–29% increasein cell surface CD19 expression (Fig. 1) significantly increasesautoantibody production in mice (Figs. 3 and 4; Table II). Further-more, these results suggest that modest changes in the expressionor function of regulatory molecules in addition to CD19 may shiftthe balance between tolerance and immunity to autoimmunity.Since most SSc patients overexpressed CD19 to an extent compa-rable to the levels induced genetically in hCD19TG41/2 mice,small changes in CD19 expression might also contribute to humanautoimmunity (Figs. 5 and 6). In addition, genetic changes thatresult in graded alterations of B lymphocyte signaling thresholdsmay help explain why different autoimmune diseases producecharacteristic patterns of autoantibodies. For example, CD19 ex-pression levels in transgenic mice correlate directly with an in-creased capacity for autoantibody production (4), yet autoantibod-ies against spindle poles were detected more frequently inhCD19TG-41/2 mice than in hCD19TG-11/1 mice (Table II).

FIGURE 4. Serum levels of anti-ssDNA, anti-dsDNA, antihistone, andRF Abs in wild-type, hCD19TG-41/2, and hCD19TG-11/1 mice. All se-rum samples were from 2-mo-old mice with relative Ab levels determinedusing Ig subclass-specific ELISAs. Results from each mouse are repre-sented as a single dot. Values in parentheses represent relative autoanti-body levels of pooled sera. The dilutions of pooled sera giving half-max-imal OD values in autoantigen-specific ELISAs was determined by linearregression analysis to generate arbitrary units per milliliter that could bedirectly compared between each group of mice.

FIGURE 5. Representative mean linear fluorescence intensity levels forcell surface molecule on B cells from SSc patients, SLE patients, andnormal controls. All samples were stained in parallel by two-color immu-nofluorescence staining and analyzed sequentially by flow cytometry withidentical instrument settings. Horizontal and vertical dashed lines in eachhistogram are provided for reference. Relative fluorescence intensity isshown on a four-decade log scale.

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This suggests that differing autoantibody specificities may resultfrom different “autoimmunity susceptibility gene” dosages or de-grees of B cell dysregulation during systemic autoimmunity.Thereby incremental changes in the expression or function of im-portant regulatory molecules may qualitatively modify the speci-ficity of autoantibodies as well as amplify autoantibody productionquantitatively.

The current gene titration studies suggest that genetically deter-mined quantitative changes in CD19 expression alone can induceautoantibody production in a mouse genetic background not asso-ciated with autoimmunity. Although hCD19TG mice do not pro-duce autoantibodies at levels equivalent to those found in othermouse models of spontaneous autoimmune disease, the productionof autoantibodies is nonetheless remarkable given that most mousemodels of autoimmunity result from radical genetic defects or theintroduction of Ag receptor transgenes that dramatically alter theimmune systems of the mice being studied (29). Rather, subtlegenetic alterations, like CD19 overexpression in this study, maymore accurately reflect the genetic basis for predisposition to au-toimmunity. In addition, most mouse models of autoimmunity donot accurately reflect the varied and complex autoimmune syn-dromes found in humans. That increased expression of CD19 cor-related with autoantibody production in SSc patients, but not in

SLE patients (Fig. 6) also follows, since different mechanisms arelikely to correlate with autoantibody production among the differ-ent autoimmune disorders. Genetic alterations similar to increasedCD19 expression may also explain why autoantibody specificitiesand clinical manifestations are different among the autoimmunediseases. As with most transgenic mouse lines, hCD19TG-11/1

and hCD19TG-41/2 mice originated in a (C57BL/63 SJL)F1

genetic background (13). It is therefore possible that backgroundgenes present in either of these mouse strains complement in-creased CD19 expression. However, wild-type offspring of hemi-zygous hCD19TG mice were similar to C57BL/6 mice and did notproduce significant autoantibodies (Table II). Moreover, that in-cremental doses of CD19 expression induced autoantibody pro-duction in two independent lines of mice suggests that CD19 over-expression is the major contributor to autoantibody production inthis mouse model. In addition, autoantibodies remain a feature ofthe hCD19TG-11/1 line even after being backcrossed withC57BL/6 mice for 12 generations. Therefore, it is likely that over-expression of CD19 in isolation can disrupt tolerance and induceautoantibody production (5).

That a 15–29% increase in CD19 expression affects B cell func-tion affirms a significant regulatory role for CD19 (Figs. 2–6).These results are also consistent with the notion that cell surface

FIGURE 6. Expression of cell surface mole-cules by blood B cells from SSc and SLE patients,and normal controls (CTL).A, Relative cell sur-face molecule densities were determined by com-paring mean CD19 and CD21 expression levelson blood B cells from individual patients.B,Mean cell surface molecule expression. Expres-sion of cell surface molecules was determined bytwo-color immunofluorescence staining with flowcytometric analysis as described in Fig. 5. Eachdot represents an individual patient. The horizon-tal bars represent mean values with statisticallysignificant differences between samples indicated.Values represent the mean linear fluorescencechannel numbers of each B cell population stainedfor each cell surface molecule.

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CD19 expression levels are tightly regulated. In support of this, amajor regulatory function for CD22/SHP1 is to down-regulateCD19 tyrosine phosphorylation following B cell Ag receptor en-gagement (30). In addition, the CD19 cytoplasmic domain hasdose-dependent functional activities that appear independent ofcell surface engagement or signal transduction through other mem-bers of the CD19 complex (31). The CD19 cytoplasmic region of;240 amino acids contains 9 conserved tyrosine residues whichmediate its interactions with Lyn, Lck, Fyn, phosphatidylinositol3-kinase, and the adapter proteins Vav, Cbl, Shc, and Grb2. Im-portantly, the CD19 cytoplasmic domain regulates an endogenousand Ag receptor-induced Src family protein tyrosine kinase am-plification loop that regulates Vav phosphorylation and B cell sig-nal transduction thresholds (14, 15). Since the CD19 cytoplasmicdomain can up-regulate Src family protein tyrosine kinase activityin isolation, it is likely that small alterations in CD19 expressionlevels will have parallel affects on Src family kinase activity withinB cells. Consistent with this, endogenous and Ag receptor-inducedLyn kinase activities and Vav phosphorylation are up-regulated inB cells that overexpress CD19 by 3-fold and diminished in CD19-deficient B cells (15). We were unable to quantitate significantalterations in B cell development (Table I) or Lyn and Vav phos-phorylation in B cells isolated from hCD19TG-41/2 mice (datanot shown). Similarly, humoral immune responses were not sig-nificantly increased in hCD19TG-41/2 mice (data not shown).Thereby a 15–29% increase in CD19 expression may not haveobvious effects on B cell function. Given this, autoantibody pro-duction in hCD19TG-41/2 mice (Table II) is even more remark-able. Thus, the autonomous ability of CD19 to regulate endoge-nous kinase activity in B cells may contribute to the developmentof autoimmunity when CD19 expression is increased.

CD19 physically associates with CD21, CD81, and Leu-13 onthe surface of B cells (2, 32). Associations between CD19 andCD21 may explain why both CD19 and CD21 expression levelswere higher on B cells from patients with SSc, while CD20, CD22,and CD40 expression levels were normal (Figs. 5 and 6). However,increased CD19 expression correlated most significantly with SSc.Although the changes in CD19 and CD21 expression appear se-lective and correlated with autoimmunity in these patients, itwould be virtually impossible to prove a cause and effect relation-ship in this situation. Nonetheless, the finding that similar in-creases in cell surface CD19 expression by SSc patients (20%increase) and transgenic mice (15–29% increase) results in auto-antibody production (Figs. 3–6) suggests that CD19 regulationmay be functionally linked with autoantibody production in thishuman autoimmune disease. In addition, hCD19TG-11/1 andhCD19TG-41/2 mice produced high-titer ANA Abs (Figs. 3 and4; Table II) and ANAs are detected in.90% of patients with SSc(33). Alternatively, increased CD19 expression and autoantibodyproduction in SSc patients may serve as hallmarks for linked, yetunrelated genetic changes that predispose to sclerosis.

Both genetic and environmental factors have been implicated inthe origins of SSc and the autoantibodies present in this disease.SSc is associated with certain MHC class I, II, or III genes (34),although non-MHC loci have also been implicated (35). In the“tight-skin” mouse model of human SSc, a genomic duplication ofthe fibrillin 1 gene is suggested to cause SSc susceptibility (36).However, skin sclerosis was not observed in hCD19TG-11/1 orhCD19TG-41/2 mice over a 1-year period (data not shown). Fur-thermore, anti-DNA topoisomerase I and anticentromere Abs,which are highly specific for SSc (23, 33), were not detected inhCD19TG-41/2 or hCD19TG-11/1 mice. This may be explainedby qualitative MHC differences between mice and humans as anti-DNA topoisomerase I and anticentromere autoantibody production

is closely associated with certain HLA-DR genes (33, 37, 38).Moreover, it is likely that SSc is a polygenic condition resultingfrom combinations of multiple disease susceptibility genes. Thismay explain why autoantibodies reacting with various other intra-cellular components, such as RNA polymerase (RNP), histones,ssDNA, centriole, U1RNP, heterogeneous nuclear RNP, U3RNP,ubiquitin, and pyruvate dehydrogenase complex, are also detectedin sera from SSc patients (26, 39–44). Nonetheless, hCD19TG-41/2 mice produced ANA, anti-ssDNA, antihistone, and RF Abs(Fig. 6), which are present in 30–50% of SSc patients (40, 44, 45)and patients with other autoimmune disorders (46). Antihistone Abproduction in hCD19TG-41/2 mice may be significant since an-tihistone Abs are detected in 44% of diffuse cutaneous SSc pa-tients, and the presence of antihistone Abs correlates with severepulmonary fibrosis in patients with diffuse cutaneous SSc (40).Production of anti-spindle pole Abs in some hCD19TG-41/2 micemay be also significant since 60% of patients with anticentrioleAbs are diagnosed with SSc-related disorders (26–28). Therefore,high CD19 expression by B cells from patients with SSc maycontribute to the development of autoantibodies in these patientswhile other disease characteristics may be caused by different ge-netic abnormalities.

The relationship between the induction of autoantibodies andthe clinical manifestations of most autoimmune diseases is notclear. This is also true for CD19-overexpressing mice that produceautoantibodies, yet do not demonstrate readily discernible featuresof human autoimmune disease. Nonetheless, these studies demon-strate that subtle alterations in expression of a single cell surfacereceptor can lead to autoantibody production. Therefore, it is likelythat many of the susceptibility genes that contribute to human au-toimmunity represent similar subtle alterations in the expression orfunction of related regulatory molecules. Although investigatorshave traditionally regarded lymphocytes as either positive or neg-ative for specific cell surface molecules, these studies reinforce theconcept that it may be more important to quantify the amount ofeach receptor expressed on the surface of cells. Subtle differencesin gene dosage or protein expression or function may be particu-larly important in instances when you are trying to understand themolecular basis for abnormal function of a cell population or sur-face molecule.

AcknowledgmentsWe thank M. Matsubara and Y. Yamada for technical assistance and Dr.David Pisetsky for helpful discussions.

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