Quantitative Genetic Variation in CD19 Expression Correlates with Autoimmunity¹

Humoral immune responses and the production of autoantibodies are regulated in part by signaling through B cell Ag receptors. Autoimmunity and immune responses are further regulated or “fine tuned” by signal transduction molecules that amplify or inhibit Ag receptor signaling during responses to self and foreign Ags (1, 2). These regulatory molecules include a subset of functionally interrelated cell surface receptors, such as CD19, CD21, and CD22, and their intracellular signaling components including Lyn, Btk, Vav, and the SHP1 protein tyrosine phosphatase (2, 3). Significant alterations in function or expression of these molecules can predispose for autoantibody production. For example, transgenic mice that overexpress CD19 by 3-fold exhibit loss of tolerance and generate spontaneous autoantibodies (4, 5). Lyn-deficient mice exhibit glomerulonephritis due to the presence of immune complexes containing autoantibodies (6, 7). Motheaten viable (me^v/me^v) mice with SHP1 mutations demonstrate elevated levels of spontaneous autoantibodies, hypergammaglobulinemia, and tissue deposition of immune complexes (8). Thus, these “response regulatory” molecules of B cell signal transduction may play critical roles in autoantibody production. In addition, unidentified polygenic variations present in a variety of mouse strains regulate Ag receptor signaling and the generation of autoreactive B cell clones (9). However, the extent that individual genetic alterations or subtle alterations in the functions of these molecules can quantitatively influence the development of autoimmunity remains relatively unknown. Therefore, in this study, we have assessed the extent to which small alterations in cell surface CD19 expression can bias mice toward autoantibody production.

CD19 is a B cell-specific member of the Ig superfamily expressed by early pre-B cells from the time of heavy chain rearrangement until plasma cell differentiation. The cell surface density of CD19 is tightly regulated during B cell differentiation, particularly in mice (10, 11). After B cell maturation, cellular activation induced by various stimuli, such as anti-IgM Abs, LPS, and IL-4, does not affect CD19 expression in either mice or humans (4, 12). Nonetheless, the B1 subset of mouse B cells expresses CD19 at levels 60% higher than conventional B cells (4). Mouse lines that overexpress CD19 have been generated by the B cell-specific expression of a human CD19 (hCD19)⁴ transgene (13). Since hCD19 and mouse CD19 (mCD19) are functionally equivalent in vivo when expressed at comparable site densities (11), these different mouse lines express overall CD19 at various cell surface densities. In these mice, CD19 expression levels correlate directly with altered B cell function, B cell hyperactivity, and autoantibody production (4, 11). Dose-dependent changes in B cell development and function resulting from CD19 overexpression in vivo presumably result from the fact that the cytoplasmic domain of CD19 is a central regulatory component of B cells upon which multiple signaling pathways converge (2). Perhaps most important, CD19 regulates a Src family protein tyrosine kinase activation loop in resting and Ag receptor-stimulated B cells that establishes basal signaling thresholds (14, 15).

Since previous “gene titration” studies in mice have shown that 2- or 3-fold increases in CD19 expression can predispose mice to autoantibody production, we assessed whether more subtle changes in CD19 expression could alter B cell homeostasis. Remarkably, a genetically determined quantitative increase in CD19 expression by 15–29% induced autoantibody production in mice that are otherwise genetically wild-type. CD19 expression was also 20% higher on B cells from autoimmune patients with systemic sclerosis (SSc) compared with healthy individuals. Therefore, it is possible that modest alterations in CD19 function or expression contribute to the development of autoimmunity. Moreover, similar subtle alterations in the expression or function of other important regulatory molecules may predispose to autoimmune susceptibility in other syndromes.

hCD19-transgenic (TG)-1 (C57BL/6 × B6/SJL) mice and hCD19TG-4 (C57BL/6 × B6/SJL) mice were described previously (13). hCD19TG-1 (C57BL/6 × B6/SJL) mice were backcrossed with C57BL/6 mice for either 7 or 12 generations before use in these studies. The hCD19TG-1 line backcrossed with C57BL/6 mice for seven generations overexpresses CD19 by 2.6-fold (4, 11). However, the hCD19TG-1 line backcrossed with C57BL/6 mice for 12 generations expressed hCD19 at levels similar to human blood B cells and thereby only overexpressed CD19 by 2-fold (data not shown). The molecular basis for the decrease in hCD19 expression in the hCD19TG-1 line is unknown. Reduced hCD19 expression could have resulted from a decrease in the number of hCD19 gene copies in this line of mice or subtle genetic changes. Nonetheless, hCD19TG-1 mice that were backcrossed with C57BL/6 mice for 12 generations and overexpress CD19 by 2-fold were used as positive controls for the current experiments unless noted otherwise. Wild-type littermates generated from breedings of hemizygous transgenic mice were used as negative controls. Results with wild-type littermates of hCD19TG-1 and hCD19TG-4 mice were similar and were therefore pooled. All mice were between 2 and 3 mo of age when used for this study. Mice were housed in a specific pathogen-free barrier facility. All mice were regularly checked for infections, pathogens, and parasites by clinical veterinarians. All tests have been negative for >3 years. All studies and procedures were approved by the Committee on Animal Experimentation of Kanazawa University School of Medicine and the Animal Care and Use Committee of Duke University.

Abs used in these studies included the anti-mCD19 mAbs, rat IgG2a clone 6D5 (Caltag, Burlingame, CA), and mouse IgA clone MB19-1 (4). Antihuman 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 IgG1 clone SJ25-C1; Caltag). For immunofluorescence staining, fresh heparinized whole blood samples were placed on ice immediately after collection. Blood samples (50 μl) were stained at 4°C using predetermined saturating concentrations of the test mAb for 20 min as previously described (11, 16). Blood erythrocytes were lysed after staining using the Coulter Whole Blood Immuno-Lyse kit as detailed by the manufacturer (Coulter). Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Five thousand cells with the forward and side light scatter properties of mononuclear cells were analyzed for each sample, with fluorescence intensity shown on a four-decade log scale. Fluorescence contours are shown as 50% log density plots. Positive and negative populations of cells were determined using unreactive isotype-matched mAbs (Coulter) as controls for background staining. Background levels of staining were delineated using gates positioned to include 98% of the control cells.

Cell surface densities of hCD19 and mCD19 were determined by staining blood lymphocytes using PE-conjugated anti-mCD19 (6D5) and anti-hCD19 (SJ25-C1) mAbs. The two Ab preparations had fluorochrome:Ab molar ratios of 1.0. After direct immunofluorescence staining and flow cytometry analysis, the number of PE molecules bound on the surface of CD19⁺ lymphocytes was determined using the QuantiBRITE PE Fluorescence Quantitation kit (Becton Dickinson Immunocytometry Systems, San Jose, CA) and software provided by the manufacturer. The PE Fluorescence Quantitation kit provides beads conjugated with four levels of PE that are used to generate a standard curve by flow cytometry analysis. Linear regression analysis is then used to determine the number of PE molecules bound to mAb-stained cells during flow cytometry analysis. Lymphocytes were incubated with various concentrations of the test mAb for 40 min at 4°C immediately before flow cytometry analysis. The percentage of CD19⁺ cells among lymphocytes in each sample was determined by flow cytometry analysis with total cell numbers determined using a hemocytometer. For Scatchard analysis (17), the number of cell-bound PE-mAb molecules was determined by calculating the total number of cell-bound PE molecules per sample for comparison with the total number of mAb molecules added to each sample. The maximal binding capacity of each mAb preparation was determined as described (18).

ELISAs were conducted as described previously using affinity-purified mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA Abs (Southern Biotechnology Associates, Birmingham, AL) to generate a standard curve (19, 20). The relative concentration of each Ig isotype in individual samples was calculated by comparing the mean OD value obtained for duplicate wells to a semilog standard curve of titrated standard Ab using linear regression analysis.

ANA was assayed by indirect immunofluorescence staining with sera diluted 1:50 using HEp-2 substrate cells (Medical & Biological Laboratories, Nagoya, Japan) as described elsewhere (21). Ig isotype-specific ANAs were performed using FITC-conjugated goat F(ab′)₂ fragment anti-mouse IgG (γ-chain-specific), anti-mouse IgM (μ-chain-specific), and anti-mouse IgG + IgM + IgA (Southern Biotechnology Associates) Abs. For two-color immunofluorescence staining of mouse and human serum samples, Ab binding was visualized using species-specific tetraethyl sulforhodamine-conjugated goat F(ab′)₂ anti-mouse Ig Abs (BioSource International, Camarillo, CA) and FITC-conjugated goat F(ab′)₂ anti-human Ig Abs (Medical & Biological Laboratories).

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

Nineteen patients (16 females and 3 males, 23–72 years old) who fulfilled the 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 proposed by LeRoy et al. (23): 13 patients (10 females and 3 males) had limited cutaneous SSc and 6 (all female) had diffuse cutaneous SSc. None of the SSc patients had received oral steroids, d-penicillamine, or immunosuppressive drugs.

All patients with SLE fulfilled the criteria proposed by the American College of Rheumatology (24) and had active SLE as determined by the SLE Disease Activity Index (25) that ranged between 8 and 20 for these patients. Three patients had not been medicated and two patients had received low-dose steroids (prednisolone, 10–20 mg/day), but had disease relapses at the time of blood collection. None of the SLE patients had received immunosuppressive drugs. Thirty-two age- and sex-matched healthy volunteers served as normal controls for the SSc and SLE patients. Laboratory data (serum levels of IgM, IgG, IgA, CH50, C3, and C4, erythrocyte sedimentation rates, C-reactive protein, and ANA titer) and blood samples were obtained at the same time. The protocol was approved by the Kanazawa University School of Medicine, and informed consent was obtained from all patients.

All data are shown as mean values ± SD unless indicated otherwise. ANOVA was used to analyze the data and Student’s t test was used to determine the level of significance of differences in sample means.

Transgenic mouse lines expressing various cell surface densities of hCD19 have been previously described (13). Mice from the hCD19TG-1 line produce spontaneous autoantibodies (4, 5). Moreover, autoantibody production correlates with the level of CD19 overexpression and CD19 expression correlates directly with gene dosage such that heterozygous mice express half as much hCD19 as their homozygous littermates (4). The hCD19TG-1 line (backcrossed with C57BL/6 mice for seven generations) overexpresses CD19 by 2.6-fold based on comparisons of hCD19 expression between mouse and human blood B cells (4, 11). Since there are no mAbs that react with both mCD19 and hCD19, these comparisons are based on the assumption that mouse and human B cells express similar cell surface densities of CD19.

To correlate CD19 expression with autoantibody production, the number of hCD19 and mCD19 molecules expressed on the surface of B cells in CD19TG mouse lines was quantified by immunofluorescence staining with saturating concentrations of IgG mAbs specific for hCD19 or mCD19. Two lines of hCD19TG mice were used for these studies: hCD19TG-1 mice that were backcrossed with C57BL/6 mice for seven generations (4, 11) and hCD19TG-4 mice that carry fewer copies of the hCD19 transgene. hCD19 was expressed by all B cells and only B cells among hematopoietic cells from hCD19TG mice (Fig. 1,A). Cell surface hCD19 expression by blood B cells of hCD19TG-4^+/− mice was 22 ± 3% of that expressed by hCD19TG-1^+/− mice (n = 5; Fig. 1,B). Hemizygous hCD19TG-1^+/− mice that were backcrossed with C57BL/6 mice for seven generations express hCD19 at levels comparable to human blood B cells (4, 11). Endogenous mCD19 expression in hCD19TG-4^+/− mice was 94 ± 2% (n = 5) of levels observed in wild-type littermates (Fig. 1, A and B). Thus, if circulating B cells from mice and humans express CD19 at comparable site densities, overall CD19 expression levels in hCD19TG-4^+/− mice were 116 ± 5% of wild-type mCD19 levels as determined by the intensity of immunofluorescence staining.

CD19 cell surface density was further assessed by Scatchard analysis using IgG mAbs specific for either hCD19 or mCD19. Ab binding was quantified using standardized PE-conjugated beads and quantitative flow cytometry analysis. hCD19TG-1^+/+ mice that were backcrossed with C57BL/6 mice for 12 generations were used for these studies since these mice had hCD19 expression levels equivalent with those of human B cells, as determined by quantitative flow cytometry analysis (data not shown). Using this approach, human blood B cells expressed 35,800 ± 1,750 (± SEM) anti-CD19 mAb binding sites (Fig. 1,C). B cells from hCD19TG1^+/+ mice expressed 36,800 ± 1,520 sites and hCD19TG-4^+/− mice expressed 6,740 ± 170 mAb binding sites (Fig. 1,C). Endogenous mCD19 expression was assessed similarly; wild-type mice expressed 20,800 ± 570 sites, hCD19TG1^+/+ mice 16,600 ± 730 sites (80% of wild-type), and hCD19TG-4^+/− mice 20,000 ± 410 (96% of wild-type) anti-mCD19 mAb binding sites (Fig. 1 C). With a strict interpretation of these results, hCD19TG-1^+/+ and hCD19TG-4^+/− B cells expressed overall CD19 at 257 and 29% higher levels than their wild-type littermates, respectively. Although these results suggest that human blood B cells express CD19 at ∼70% higher levels than mouse B cells, variability in ligand binding between the anti-hCD19 and anti-mCD19 mAb could also explain these apparent differences. Therefore, if human and mouse B cells express CD19 at comparable densities, then hCD19TG-1^+/+ and hCD19TG-4^+/− B cells express overall CD19 at 183 and 15% higher levels than their wild-type littermates, respectively. Thus, hCD19TG-4^+/− B cells overexpress CD19 by 15–29%.

The effect of the small increase in CD19 expression on B cell differentiation in hCD19TG-4^+/− mice was assessed by quantifying B cell numbers and measuring serum Ab levels. Overall, the numbers of B cells found in hCD19TG-4^+/− mice and their wild-type littermates were not significantly different, whereas peripheral B cell numbers were significantly reduced in hCD19TG-1^+/+ mice (Table I). Despite apparently normal B cell numbers in hCD19TG-4^+/− mice, serum IgG1 (p < 0.05), IgG2a (p < 0.05), and IgG2b (p < 0.001) levels were significantly increased in hCD19TG-4^+/− mice when compared with wild-type littermates, while serum IgM, IgG3, and IgA levels were normal (Fig. 2). Serum Ig levels in hCD19TG-1^+/+ mice were generally higher than those in hCD19TG-4^+/− mice, especially IgM, IgG2a, and IgG2b. Serum Ig levels did not differ between males and females (data not shown). Thus, small increases in CD19 expression resulted in significantly increased production of selected serum Ab isotypes in hCD19TG-4^+/− mice. However, humoral responses following immunizations with a T cell-dependent Ag were not significantly increased in hCD19TG-4^+/− mice (data not shown).

Autoantibody levels in hCD19TG-4^+/− mice were determined to assess the influence of elevated CD19 expression. ANAs were detected in 52% of hCD19TG-4^+/− mice and 95% of hCD19TG-1^+/+ mice, but were rarely detectable in wild-type littermates (Fig. 3; Table II). These autoantibodies were predominantly of the IgG isotype. A homogenous chromosomal staining pattern with more intense staining of mitotic cells was observed in 21% of the serum samples from hCD19TG-4^+/− mice (Fig. 3,c). The frequency (86%) and intensity of homogenous staining was higher for sera from hCD19TG-1^+/+ mice (Fig. 3,e). Sera from 28% of hCD19TG-4^+/− mice reacted with spindle poles (mitotic centers) of mitotic cells (Fig. 3,b), whereas this staining pattern was observed in only 10% of sera from hCD19TG-1^+/+ mice (Fig. 3,d). Since human autoantibodies recognizing centrioles, which are components of spindle poles, give a staining pattern similar to that observed for some hCD19TG-4^+/− sera (Fig. 3,f; and Refs. 26, 27, 28), two-color immunofluorescence staining was conducted with human anti-centriole Ab-positive serum and sera from hCD19TG-4^+/− mice. Both the mouse and human sera stained similar intracellular determinants, although the localized regions recognized by the mouse serum were larger than those of the human anticentriole Abs (Fig. 3, f and g). The antispindle pole autoantibodies stained mitotic cells, whereas the anticentriole Abs stained both mitotic and interphase cells. Anticentromere Abs were not detected in sera of hCD19TG-4^+/− or hCD19TG-1^+/+ mice. The frequency, specificity, and intensity of ANAs were similar between males and females (data not shown). Thus, the predominant ANA specificity in hCD19TG-4^+/− sera was for spindle poles, while homogenous staining was most commonly observed with hCD19TG-1^+/+ mouse sera.

Autoantibody production was further assessed by ELISA. Serum IgG autoantibody levels were significantly increased in hCD19TG-4^+/− 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% increase, p < 0.001) autoantibody titers were significantly higher in hCD19TG-4^+/− mice. Mean IgM antihistone Ab (142%, p < 0.05) and IgM rheumatoid factor (RF; 211%, p < 0.05) titers were also increased. Serum levels of all autoantibody specificities in hCD19TG-1^+/+ mice were generally higher than those in hCD19TG-4^+/− mice. Anti-DNA topoisomerase I Ab levels did not increase in either hCD19TG-4^+/− or hCD19TG-1^+/+ mice as quantified by ELISA (data not shown).

Autoantibody levels increased with age in hCD19TG-4^+/− mice. Mean titers of IgM anti-ssDNA Abs were significantly higher in 10-mo-old hCD19TG-4^+/− mice (n = 12) compared with 2-mo-old hCD19TG-4^+/− mice (n = 12; 207% increased, p < 0.01). Similarly, IgM RF levels were significantly higher in 10-mo-old hCD19TG-4^+/− mice (479% increased, p < 0.05). Mean IgG anti-ssDNA, IgM anti-dsDNA, IgG anti-dsDNA, IgM antihistone, and IgG antihistone Ab levels were similar in young and old mice.

Histopathological analysis of kidneys from 2- to 8-mo-old hCD19TG-4^+/− mice showed a normal architecture without detectable Ab deposits (data not shown). Swelling or deformity of joints, skin eruptions, or a thickened dermis were not observed. Mortality in transgenic mice was similar to that of wild-type littermates. In the hCD19TG-1^+/+ line of mice, there is a significant decrease in the reproductive capacity of female mice following birth of their first litter. However, none of the hCD19TG mice developed overt symptoms suggestive of lupus or scleroderma-like disease.

Since small genetic changes in cell surface CD19 expression induced autoantibody production in mice, B cells from autoimmune patients with SSc and SLE were assessed for abnormal CD19 expression. The cell surface density of CD19 on peripheral blood B cells from patients and healthy control individuals was examined quantitatively by immunofluorescence staining with flow cytometry analysis. SSc patients had significantly higher mean CD19 expression levels than normal controls (20%, p < 0.0001; Figs. 5 and 6 ). Similarly, mean CD21 expression was 23% higher in SSc patients than in normal controls (p < 0.001). However, there was not a significant correlation between CD19 and CD21 expression in SSc patients (n = 19, r = −0.07, Fig. 6,A), although CD21 expression was significantly correlated with CD19 expression in normal controls (n = 32, r = 0.48, p < 0.01, Fig. 6,A). CD19 and CD21 expression levels were similar in male and female patients (data not shown). Mean CD20, CD22, and CD40 expression levels were similar in SSc patients and normal controls. Higher CD19 expression in SSc patients did not result from increased cell size since the forward and side light scatter properties of CD20⁺ cells in SSc patients were not measurably different from those of normal controls (data not shown). Elevated CD19 expression did not result from B cell activation since HLA-DR, CD25, CD54, CD80, and CD86 expression levels were not increased on B cells from SSc patients (data not shown). In contrast to SSc patients, CD19 and CD21 expression levels were reduced on B cells from SLE patients (Fig. 6 B). Limited numbers of samples were available for SLE patients who had not undergone treatment or who had been in an active state of the disease for only a short time period. Therefore, the results obtained with these SLE patients may not represent a broader SLE population since the clinical manifestations of SLE are heterogeneous. Rather, the results with SLE patients are shown for comparison to demonstrate that increased CD19 expression correlated with SSc, but was not observed in other autoimmune diseases such as SLE. Thus, elevated CD19 and CD21 expression levels were the primary phenotypic abnormalities observed for B cells from SSc patients.

Correlations of cell surface molecule expression by blood B cells with various clinical immunologic parameters were assessed to determine whether high CD19 and CD21 expression correlated with the immunologic status of patients with SSc. Increased CD19 expression correlated positively with serum IgG and IgM levels in SSc patients (r = 0.610, p < 0.01 and r = 0.579, p < 0.02, respectively), whereas there was not a significant correlation between CD21 expression and serum Ig levels (data not shown). Although all SSc patients produced clinically significant levels of serum ANA, CD19 or CD21 expression did not correlate with ANA titers (data not shown). The numbers and percentages of circulating B-1a cells (CD5⁺ CD20⁺) and B cells (CD19⁺ CD20⁺) were similar for SSc patients and normal controls (data not shown).

The concept of a fine balance between humoral immunity and autoimmunity is reinforced by the finding that a 15–29% increase in cell surface CD19 expression (Fig. 1) significantly increases autoantibody production in mice (Figs. 3 and 4; Table II). Furthermore, these results suggest that modest changes in the expression or function of regulatory molecules in addition to CD19 may shift the balance between tolerance and immunity to autoimmunity. Since most SSc patients overexpressed CD19 to an extent comparable to the levels induced genetically in hCD19TG4^+/− mice, small changes in CD19 expression might also contribute to human autoimmunity (Figs. 5 and 6). In addition, genetic changes that result in graded alterations of B lymphocyte signaling thresholds may help explain why different autoimmune diseases produce characteristic patterns of autoantibodies. For example, CD19 expression levels in transgenic mice correlate directly with an increased capacity for autoantibody production (4), yet autoantibodies against spindle poles were detected more frequently in hCD19TG-4^+/− mice than in hCD19TG-1^+/+ mice (Table II). This suggests that differing autoantibody specificities may result from different “autoimmunity susceptibility gene” dosages or degrees of B cell dysregulation during systemic autoimmunity. Thereby incremental changes in the expression or function of important regulatory molecules may qualitatively modify the specificity of autoantibodies as well as amplify autoantibody production quantitatively.

The current gene titration studies suggest that genetically determined quantitative changes in CD19 expression alone can induce autoantibody production in a mouse genetic background not associated with autoimmunity. Although hCD19TG mice do not produce autoantibodies at levels equivalent to those found in other mouse models of spontaneous autoimmune disease, the production of autoantibodies is nonetheless remarkable given that most mouse models of autoimmunity result from radical genetic defects or the introduction of Ag receptor transgenes that dramatically alter the immune systems of the mice being studied (29). Rather, subtle genetic alterations, like CD19 overexpression in this study, may more accurately reflect the genetic basis for predisposition to autoimmunity. In addition, most mouse models of autoimmunity do not accurately reflect the varied and complex autoimmune syndromes found in humans. That increased expression of CD19 correlated with autoantibody production in SSc patients, but not in SLE patients (Fig. 6) also follows, since different mechanisms are likely to correlate with autoantibody production among the different autoimmune disorders. Genetic alterations similar to increased CD19 expression may also explain why autoantibody specificities and clinical manifestations are different among the autoimmune diseases. As with most transgenic mouse lines, hCD19TG-1^+/+ and hCD19TG-4^+/− mice originated in a (C57BL/6 × SJL)F₁ genetic background (13). It is therefore possible that background genes present in either of these mouse strains complement increased CD19 expression. However, wild-type offspring of hemizygous hCD19TG mice were similar to C57BL/6 mice and did not produce significant autoantibodies (Table II). Moreover, that incremental doses of CD19 expression induced autoantibody production in two independent lines of mice suggests that CD19 overexpression is the major contributor to autoantibody production in this mouse model. In addition, autoantibodies remain a feature of the hCD19TG-1^+/+ line even after being backcrossed with C57BL/6 mice for 12 generations. Therefore, it is likely that overexpression of CD19 in isolation can disrupt tolerance and induce autoantibody production (5).

That a 15–29% increase in CD19 expression affects B cell function affirms a significant regulatory role for CD19 ( Figs. 2–6). These results are also consistent with the notion that cell surface CD19 expression levels are tightly regulated. In support of this, a major regulatory function for CD22/SHP1 is to down-regulate CD19 tyrosine phosphorylation following B cell Ag receptor engagement (30). In addition, the CD19 cytoplasmic domain has dose-dependent functional activities that appear independent of cell surface engagement or signal transduction through other members of the CD19 complex (31). The CD19 cytoplasmic region of ∼240 amino acids contains 9 conserved tyrosine residues which mediate its interactions with Lyn, Lck, Fyn, phosphatidylinositol 3-kinase, and the adapter proteins Vav, Cbl, Shc, and Grb2. Importantly, the CD19 cytoplasmic domain regulates an endogenous and Ag receptor-induced Src family protein tyrosine kinase amplification loop that regulates Vav phosphorylation and B cell signal transduction thresholds (14, 15). Since the CD19 cytoplasmic domain can up-regulate Src family protein tyrosine kinase activity in isolation, it is likely that small alterations in CD19 expression levels will have parallel affects on Src family kinase activity within B cells. Consistent with this, endogenous and Ag receptor-induced Lyn kinase activities and Vav phosphorylation are up-regulated in B cells that overexpress CD19 by 3-fold and diminished in CD19-deficient B cells (15). We were unable to quantitate significant alterations in B cell development (Table I) or Lyn and Vav phosphorylation in B cells isolated from hCD19TG-4^+/− mice (data not shown). Similarly, humoral immune responses were not significantly increased in hCD19TG-4^+/− mice (data not shown). Thereby a 15–29% increase in CD19 expression may not have obvious effects on B cell function. Given this, autoantibody production in hCD19TG-4^+/− mice (Table II) is even more remarkable. Thus, the autonomous ability of CD19 to regulate endogenous kinase activity in B cells may contribute to the development of autoimmunity when CD19 expression is increased.

CD19 physically associates with CD21, CD81, and Leu-13 on the surface of B cells (2, 32). Associations between CD19 and CD21 may explain why both CD19 and CD21 expression levels were 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 selective and correlated with autoimmunity in these patients, it would be virtually impossible to prove a cause and effect relationship in this situation. Nonetheless, the finding that similar increases in cell surface CD19 expression by SSc patients (20% increase) and transgenic mice (15–29% increase) results in autoantibody production ( Figs. 3–6) suggests that CD19 regulation may be functionally linked with autoantibody production in this human autoimmune disease. In addition, hCD19TG-1^+/+ and hCD19TG-4^+/− mice produced high-titer ANA Abs (Figs. 3 and 4; Table II) and ANAs are detected in >90% of patients with SSc (33). Alternatively, increased CD19 expression and autoantibody production in SSc patients may serve as hallmarks for linked, yet unrelated genetic changes that predispose to sclerosis.

Both genetic and environmental factors have been implicated in the 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 of the fibrillin 1 gene is suggested to cause SSc susceptibility (36). However, skin sclerosis was not observed in hCD19TG-1^+/+ or hCD19TG-4^+/− mice over a 1-year period (data not shown). Furthermore, anti-DNA topoisomerase I and anticentromere Abs, which are highly specific for SSc (23, 33), were not detected in hCD19TG-4^+/− or hCD19TG-1^+/+ mice. This may be explained by 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 resulting from combinations of multiple disease susceptibility genes. This may explain why autoantibodies reacting with various other intracellular components, such as RNA polymerase (RNP), histones, ssDNA, centriole, U1RNP, heterogeneous nuclear RNP, U3RNP, ubiquitin, and pyruvate dehydrogenase complex, are also detected in sera from SSc patients (26, 39, 40, 41, 42, 43, 44). Nonetheless, hCD19TG-4^+/− 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 Ab production in hCD19TG-4^+/− mice may be significant since antihistone Abs are detected in 44% of diffuse cutaneous SSc patients, and the presence of antihistone Abs correlates with severe pulmonary fibrosis in patients with diffuse cutaneous SSc (40). Production of anti-spindle pole Abs in some hCD19TG-4^+/− mice may be also significant since 60% of patients with anticentriole Abs are diagnosed with SSc-related disorders (26, 27, 28). Therefore, high CD19 expression by B cells from patients with SSc may contribute to the development of autoantibodies in these patients while other disease characteristics may be caused by different genetic abnormalities.

The relationship between the induction of autoantibodies and the clinical manifestations of most autoimmune diseases is not clear. This is also true for CD19-overexpressing mice that produce autoantibodies, yet do not demonstrate readily discernible features of human autoimmune disease. Nonetheless, these studies demonstrate that subtle alterations in expression of a single cell surface receptor can lead to autoantibody production. Therefore, it is likely that many of the susceptibility genes that contribute to human autoimmunity represent similar subtle alterations in the expression or function of related regulatory molecules. Although investigators have traditionally regarded lymphocytes as either positive or negative for specific cell surface molecules, these studies reinforce the concept that it may be more important to quantify the amount of each receptor expressed on the surface of cells. Subtle differences in gene dosage or protein expression or function may be particularly important in instances when you are trying to understand the molecular basis for abnormal function of a cell population or surface molecule.

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