The lpr gene causes an intrinsic T cell abnormality that is required for hyperproliferation.

The lpr gene induces marked lymphoproliferation characterized by the massive accumulation of T cells of an unusual phenotype and concomitant autoimmune disease. To clarify the mechanism of the lpr effect, bone marrow cells from B6-lpr/lpr (Ly-1.2) and B6-+/+ (Ly-1.1) mice were transferred into lethally irradiated B6-lpr/lpr mice. As has been previously reported, recipients of the B6-lpr/lpr bone marrow showed the typical lpr phenotype with marked lymphadenopathy, splenomegaly and increased levels of autoantibodies; while the recipients of B6-+/+ bone marrow had normal sized lymph nodes and spleen and no autoantibodies. A third group of mice received an equal mixture of bone marrow cells from the B6-lpr/lpr and B6-+/+ donors. These mice showed both lymphadenopathy and autoantibody production comparable to that of recipients of the B6-lpr/lpr marrow alone. Immunofluorocytometric analysis of the lymphoid populations in these mixed bone marrow recipients established that the T cells from the lpr/lpr and +/+ donors were equivalently represented in the peripheral blood and thymus. In striking contrast, the T cells that accumulated in abnormally large numbers in the lymph nodes were almost entirely from the lpr donor. Their surface phenotype was Thy-1+(dull), Ly-1.2+(dull), Lyt-2-, L3T4-, 9F3+, and 3A1+, which is consistent with that found in intact lpr mice. These results indicate that the lpr gene causes an intrinsic defect directly within the T cells that accumulate in large numbers in lpr mice. In addition, the presence of the +/+ T cells cannot prevent the expression of the lpr abnormalities.

Anti-idiotypic antibodies to the Coombs antibody in NZB F1 mice.

The F1 hybrids of NZB and several normal mouse strains are known to produce less anti-erythrocyte (Coombs) autoantibody and develop a milder hemolytic anemia than their NZB parents. We have found that serum from some (NZB x CBA)F1 mice agglutinated erythrocytes from certain Coombs-positive NZB mice, often in extremely high titer, whereas other (CBA x NZB)F1 sera agglutinated erythrocytes from different individual NZB mice. The agglutination was due to antibody, but was not due to rheumatoid factor activity. Because F(ab')2 fragments of the F1 sera agglutinated erythrocytes coated with F(ab')2 fragments of the appropriate NZB sera, the observed reactivity was probably caused by idiotype-anti-idiotype interactions. In addition, because F1 sera could not agglutinate mouse erythrocytes coated with monovalent NZB Fab' fragments, the recognized idiotype probably involved the antigen-binding site. Anti-idiotypic antibodies against anti-erythrocyte autoantibodies may play an important role in the regulation of autoantibody formation.

Subclass-restricted IgG polyclonal antibody production in mice injected with lipid A-rich lipopolysaccharides.

The effects of five distinct bacterial lipopolysaccharides (LPS) on the induction of polyclonal IgM and IgG antibodies, including polyclonal autoantibody formation, were investigated in several strains of mice. Injections of most LPS preparations that contained polysaccharide transiently induced only IgM polyclonal antibodies. However, LPS from Salmonella minnesota R595 (R595 LPS), which had a particularly high content of lipid A but lacked O-antigen polysaccharide, induced a markedly prolonged IgM and IgG polyclonal antibody response in mice, including athymic nude mice, but not in LPS-unresponsive C3H/HeJ mice. Polyclonal IgM and IgG production peaked in sera on day 8 and day 15, respectively, and remained higher than control values 2 mo after the injection. The IgG induced by R595 LPS was strictly restricted to IgG2b and Igg3 subclasses in normal mice. In contrast, in athymic nude mice which have normally lower levels of IgG1 and IgG2a than normal mice, R595 LPS stimulated the production of all the IgG subclasses and reconstituted serum levels of IgG1 and IgG2a up to, but not higher than, control values of normal mice. These findings suggest that different mechanisms regulate production of each IgG subclass after stimulation with LPS.

Presence of anti-Sm reactivity in autoimmune mouse strains.

The investigation of the fine specificities of antinuclear antibodies (ANAs) has been fruitful in terms of the nosology and immunopathogenesis of human autoimmune syndromes. Particular reactivities serve as "markers," in that patients with certain syndromes have a much higher incidence of such ANAs than do patients with other diseases. In this category is the almost exclusive against the nuclear acidic protein Sm. Reactivity to Sm can be detected by precipitation in agar, complement fixation, or passive hemagglutination (1,2). Autoimmune mouse strains have also provided a fertile field for the investigation of the basic phenomena of self-activity. In particular, the NZB strain and its hybrid NZB x NZW have been considered excellent models for human SLE and have therefore been studied in great detail (3,4). In addition, Murphy et al at The Jackson Laboratory, Bar Harbor, Maine, have developed several new inbred mouse strains that spontaneously develop SLE-like syndromes (5,6). These are the BXSB strain, which has a male dominant disease characterized by little antiative DNA antibody; the MRL/1, which develops massive, nonmalignant lymphadenopathy, associated with enormous increases in serum immunoglobulin levels and fulminant renal disease; and the MRL/n, which does not develop SLE-like disease until well into the 2nd yr of life, but like the MRL/1 develops high titers of ANA and fatal glomerulonephritis. The MRL/1 differs from MRL/n in only about 10 percent of its genome, including the gene responsible for the MRL/1's lymphoproliferation. In the current study, we have used the technique of double immunodiffusion (ID) in agarose with standard human reference sera (of known ANA specificity) to survey a large number of mice from the NZB x NZW, MRL/1, MRL/n, BXSB, and other strains. We report here the finding of the anti-Sm marker" antibody almost uniquely in MRL/1 and MRL/n animals. These two related strains may serve as experimental models to explore the mechanism stimulating the production of this unique autoantibody in SLE.

Hidden autoantibodies against common serum proteins in murine systemic lupus erythematosus. Detection by in vitro plaque-forming cell assay.

The autoantibodies found in human and murine systemic lupus erythematosus (SLE) are generally directed against cells or components of cells such as nuclear antigens. This predilection may be due to the unusual immunogenicity of certain autoantigens, or to unusual patterns of antibody crossreactivity. Alternatively, the observed spectrum of reactivities may reflect the in vivo absorption of those autoantibodies directed against soluble antigens. To test whether hitherto undetected autoantibodies against serum proteins might exist in murine SLE, we developed assays that were independent of the possibility of absorption of autoantibodies by serum autoantigens; large numbers of plaque-forming cells (PFC) directed against mouse albumin and mouse transferrin were easily detected in the spleens of MRL/Mp-lpr/lpr, BXSB, and NZB mice. The secreted antibodies were relatively specific for the mouse proteins, since only limited cross-reactivity was seen with albumin and transferrins of other species in inhibition experiments. The production of these hidden antibodies could not be the result of diffuse polyclonal B cell activation, since the PFC to mouse transferrins and albumin were not always accompanied by comparable numbers of PFC against related albumins and transferrins. The results indicate that autoantibody production in murine lupus is a generalized phenomenon, not limited to the production of autoantibodies to nuclear or other cell-bound antibodies. However, the relative specificity of the autoantibodies for self-antigens indicates that diffuse polyclonal B cell activation cannot be the mechanism responsible, and argues that a selective mechanism, probably driven by antigen, accounts for production of autoantibodies in SLE.

Conventional B cells, not B-1 cells, are responsible for producing autoantibodies in lpr mice.

Mice homozygous for the lpr gene develop autoantibodies and polyclonal B cell activation similar to what is seen in human systemic lupus erythematosus patients. We have previously shown that an lpr-specific intrinsic B cell defect was necessary for autoantibody production in this model. In the current study, we have further defined these autoantibody-producing B cells. Two major subsets of B cells have been described. B-1 cells (CD5+ B cells) can be distinguished from conventional B cells on the basis of phenotype, cytokine secretion, gene expression, anatomical location, and function. In addition, B-1 cells have been implicated in autoimmunity in several murine and human studies. To address the question of which B cell subset produces autoantibodies in lpr mice, we used immunoglobulin heavy chain (Igh) allotype-marked peritoneal (B-1 cell source) and bone marrow (conventional B cell source) cells from lpr mice to establish B cell chimeras. We used two general approaches. In one, we reconstituted sublethally irradiated mice with B-1 cells of one allotype and bone marrow cells of another allotype. In the second method, we suppressed endogenous B cells in neonatal mice with allotype-specific anti-IgM antibody, and injected peritoneal cells of another allotype. After antibody treatment was stopped, the mouse's conventional B cells recovered, but the B-1 subset was only reconstituted by the donor. In both types of chimeras, antichromatin, rheumatoid factor, and anti-single stranded DNA (ssDNA) autoantibodies were produced by the conventional B cell bone marrow source. In addition, an age-related decrease in peritoneal B-1 cells was seen, even in unmanipulated lpr mice. These data show that lpr B-1 cells are not important producers of autoantibodies. Conventional B cells are the source of autoantibodies directed at chromatin, ssDNA, and IgG.

Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I.

Mice homozygous for the lpr gene have a defect in fas (CD95), a cell surface receptor that belongs to the tumor necrosis factor receptor family and that mediates apoptosis. This genetic abnormality results in lymphoproliferation characterized by the accumulation of CD4-CD8- (double negative [DN]) T cells, autoantibody production, and background strain-dependent, end-organ disease. Our previous results suggested that major histocompatibility complex (MHC) class I may be involved in the development of DN cells. To test this hypothesis, we derived C57BL/6-lpr/lpr (B6/lpr) mice that were deficient for the beta 2-microglobulin gene (beta 2m lpr) and had no detectable class I expression. At 6 mo of age, compared with B6/lpr littermates with normal class I genes, these mice showed greatly reduced lymphadenopathy, mostly due to a dramatic decrease in the number of DN cells. Significant changes in the percentage of other T cell subsets were noted, but only gamma/delta+ T cells showed a marked increase in both percentage and absolute numbers. Analysis of T cell receptor V beta expression of the remaining DN T cells in beta 2m -lpr mice showed a shift to a CD4-like repertoire from a CD8-like repertoire in control B6/lpr mice, indicating that a small MHC class II selected DN population was unmasked in lpr mice lacking class I. We also found that the production of immunoglobulin G (IgG) autoantibodies (antichromatin and anti-single stranded DNA), total IgG and IgG2a, but not total IgM or IgM rheumatoid factor, was significantly reduced in the beta 2m -lpr mice. This work suggests that >90% of DN T cells in lpr mice are derived from the CD8 lineage and are selected on class I. However, a T cell subset selected on class II and T cells expressing gamma/delta are also affected by the lpr defect and become minor components of the aberrant DN population.

Splenic immunoglobulin-secreting cells and their regulation in autoimmune mice.

We have investigated in vitro the magnitude, nature, and regulation of spontaneous and mitogen-induced Ig secretion by splenic lymphocytes from several autoimmune murine strains (NZB, NZB X W, MRL/l BXSB) and appropriate, normal mice. All autoimmune strains had increased numbers of mature splenic B lymphocytes, which secreted and/or contained Ig, compared to age-matched normal strains. In NZB and NZB X W mice, the high frequency of mature B cells was apparent early in life, whereas in MRL/l and BXSB mice it was first noted shortly before the clinical onset of disease. Spleen cells from young autoimmune mice of all four strains secreted predominantly IgM, but with aging and the appearance of disease, the cells switched to IgG secretion predominantly. In contrast, spleen cells from normal mice were predominantly IgM, but with aging and the appearance of disease, the cells switched to IgG secretion predominantly. In contrast, spleen cells from normal mice were predominantly IgM secretors throughout the animals' lives. Approximately 15% of the total Ig-secreting cells in older NZB, NZB X W, and MRL mice were committed to secretion of anti-ssDNA antibodies. In both autoimmune and normal spleen cells, the B-cell population alone contained fewer secreting cells than the total lymphocyte population, indicating that T cells were required to achieve maximal levels of plaque-forming cells. Spleen cells of NZB and NZB X W mice had a greater response to lipopolysaccharide (LPS) than other autoimmune and normal strains. Responsiveness to LPS, as measured by the frequency of induced Ig-secreting cells, was considerably diminished with age and onset of disease in all autoimmune but not in normal strains. LPS-induced Ig secretion by B cells of autoimmune and normal mice was subject to regulation by splenic T cells. No significant differences were observed between concanavalin-A (Con A) stimulated spleen cells from young and older autoimmune mice and normal control strains in effectively suppressing spontaneous and LPS-induced Ig secretion. Moreover, B cells from autoimmune mice and from normal strains were equally receptive to Con A-induced suppressor signals. T cells from young and older NZB and BXSB mice added to a standard number of B cells from syngeneic young mice provided equal help in enhancing LPS-induced Ig secretion, and this help in turn was equivalent to that provided by T cells from normal mice of the same H-2 haplotype. The exception was the MRL/l strain; T cells from older animals provided considerably more help than T cells from young MRL/l or T cells from young and older H-2-compatible normal mice.

Distribution of lymphocytes identified by surface markers in murine strains with systemic lupus erythematosus-like syndromes.

The frequencies and absolute numbers of B and T cells in the lymphoid organs of five murine strains (NZB, (NZB X NZW)F1, BXSB, MRL/l, and MRL/n) with SLE-like syndromes were examined. We assessed the frequencies of cells bearing surface Ig, C3d and IgG Fc receptors, and theta-antigen. The sequential expression of Ig isotopes on developing B cells and the Ig isotypes expressed on adult B cells were ascertained. In addition, the Ly subsets and the expression of Ia antigens coded for by the I-J subregion of the mouse H-2 complex were examined. Compared to normal, older mice, New Zealand mice had low frequencies and absolute numbers of B cells, BXSB mice had a moderate B-cell proliferation, and MRL/l mice had normal absolute numbers of B cells but a reduced frequency concomitant with a massive T-cell proliferation. Old New Zealand mice and BXSB mice had reduced frequencies and absolute numbers of T cells compared to old controls. The developmental Ig-isotype diversity during the 1st wk of age was similar in normal mice and those with autoimmune manifestations. Mature B cells were present in lymphoid organs of New Zealand mice and BXSB mice as evidenced by the high frequency of C3d receptor-bearing cells and Ig-isotype expression (high ratio of IgM- to IgD-bearing cells) in adult spleen cells. Numbers of IgG Fc receptor-bearing cells were reduced in autoimmune mice with advanced age and disease. The proliferating T cells in MRL/l mice were found to be theta-antigen positive but Ly null. These theta+-, Ly null cells may have arisen from Ly123+ T cells. MRL/l and BXSB mice seemed normal in their content of T cells bearing Ia antigens coded for by the I-J subregion of H-2. Overall, mice with autoimmune manifestations appear to express perturbations in T and B cells with development of disease, and their patterns of change vary from one strain to another.

Treatment of experimental erosive arthritis in rats by injection of the muralytic enzyme mutanolysin.

A single intravenous injection into rats of 0.4 mg of the muralytic enzyme mutanolysin, given as long as 3 d after an arthropathic dose of peptidoglycan-polysaccharide polymers derived from group A streptococci (PG-APS), resulted in a complete resolution of acute arthritis and the prevention of chronic joint disease. When administration of mutanolysin was delayed until 14 d after the injection of PG-APS, a great reduction in the severity of chronic inflammation was still observed. Quantitation of the amount of PG-APS present in the limbs, spleen, and liver by a solid phase enzyme-linked immunoassay indicated that the tissues of mutanolysin-treated rats contained as much PG-APS as tissues of PBS-treated control rats. In addition, rats treated with mutanolysin immediately after receiving an intraperitoneal injection of PG-APS developed a transient limb edema similar to that seen in rats after the injection of PG-APS digested to a small fragment size in vitro with mutanolysin. We hypothesize that mutanolysin acts in vivo by degrading PG-APS to small fragments that persist but are no longer arthropathic.

Autoantibodies in chronic graft versus host result from cognate T-B interactions.

A chronic graft-versus-host reaction (GVH) induced in nonautoimmune mice causes a syndrome that closely resembles SLE. In this model, donor T cells react against incompatible host Ia structures and generate excessive help, which activates a subpopulation of self-reactive B cells. We have studied whether these self-reactive B cells are activated by direct interaction with alloreactive T cells or by nonspecific bystander effects. Two types of chimeras were made: double-parental chimeras, differing at both Ia and Igh allotype [B6.C20 + bm12----(B6.C20 x bm12)F1]; and control chimeras [(B6.C20 x bm12)F1----(B6.C20 x bm12)F1]. A chronic GVH syndrome was induced in the chimeras by infusion of B6 or bm12 spleen cells. Coombs and antichromatin autoantibodies were measured using Igh allotype-specific immunoassays. The double-parental chimeras that received bm12 cells made autoantibodies principally of the Igha allotype, indicating that the bm12 T cells interacted only with the Iab-bearing host B cells. Conversely, double-parental chimeras that received B6 cells made mostly Ighb autoantibodies, indicating direct cognate interaction with the Iabm12-bearing host B cells. The control chimeras made autoantibodies of both allotypes. These results indicate that autoantibodies in chronic GVH result from direct T-B interactions and not from nonspecific T cell-derived factors.

An intrinsic B cell defect is required for the production of autoantibodies in the lpr model of murine systemic autoimmunity.

Mice homozygous for the gene lpr develop marked lymphadenopathy and a spectrum of autoantibodies closely resembling that of human systemic lupus erythematosus. The unusual T cell phenotype of the expanded lymphocyte population and the T-dependence of several antibodies in this strain have suggested that primary T cell abnormalities underlie the autoimmune syndrome. Using double chimeras, we now show that expression of the lpr gene in B cells is absolutely necessary for autoantibody production. Combinations of anti-Thy 1.2 + C' treated bone marrow from congenic strains of C57BL/6 mice, differing only at the immunoglobulin heavy chain (Igh) and lpr loci, were transferred into lethally irradiated B6/lpr mice. Double chimerism was documented by allotype-specific surface IgD and IgM immunofluorescence assay of peripheral blood and by allotype-specific enzyme-linked immunosorbent assay for total IgM in serum. Despite the presence of both +/+ and lpr B cells, IgM and IgG2a anti-chromatin as well as IgM anti-IgG were entirely the products of lpr B cells. Total serum IgG2a and IgG1 were also dominated by the lpr phenotype but not to the same extent. A similar experiment using B6/lpr-Igha recipients confirmed these findings. Additional experiments in which B6/lpr recipients were infused with ratios of donor bone marrow favoring B6.C20 +/+ over B6/lpr showed that even though +/+ B cells were overrepresented, autoantibodies were only of the lpr allotype. In addition, in the presence of lpr B cells, normal B cells showed little response to an exogenous, T cell-dependent antigen. The data thus indicate that lpr B cells manifest an intrinsic abnormality which is essential for autoantibody production in the lpr model.

Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains.

MRL/1 and BXSB male mice have a systemic lupus erythematosus (SLE)-like disease similar to but more acute than that occurring in NZB X W mice. The common elements of lymphoid hyperplasia, B-cell hyperactivity, autoantibodies, circulating immune complex (IC), complement consumption, IC glomerulonephritis with gp70 deposition, and thymic atrophy were found in all three kinds of SLE mice. On the basis of these common elements, SLE seen in these mice can be considered a single disease in the same sense that human SLE is one disease. The differences in the SLE expressed in the different mice are no greater than those found in an unselected series of humans with SLE. However, the significant quantitative and qualitative variations in abnormal immunologic expression suggest that different constellations of factors, genetic and/or pathophysiologic, may operate in the three murine strains and that each constellation is capable of leading, via its particular abnormal immunologic consequences, to the activation of common immunopathologic effector mechanisms that cause quite similar SLE-like syndromes. From an experimental point of view, the availability of several inbred murine strains of commonplace histocompatibility types that express an SLE-like syndrome makes possible innumerable manipulations which should help to elucidate the nature and cause(s) of this disorder.

Isolation of circulating immune complexes using Raji cells. Separation of antigens from immune complexes and production of antiserum.

Raji cells were used for the isolation of complement-fixing antigen-antibody complexes from serum. Immune complexes bound to these cells were radiolabeled at the cell surface with lactoperoxidase. The complexes were then eluted from the cells with isotonic citrate buffer pH 3.2 or recovered by immunoprecipitation of cell lysates. The antigen and antibody moieties of the complexes were isolated by dissociating sucrose density gradient centrifugation or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of preformed immune complexes were successfully isolated from serum with this approach. In addition, these techniques were used to isolate and identify the antigens in immune complexes in the serum of rabbits with chronic serum sickness and rats with Moloney virus-induced sarcomas. Methods were also developed for the production of antisera against the antigenic moiety of immune complexes isolated from serum. Repeated challenge of rabbits with whole Raji cells with bound complexes or eluates from such cells resulted in antibody production against the antigens of the immune complexes, although reactivity against cellular and serum components was also elicited. Monospecific antisera against the antigens in immune complexes were produced by immunizing rabbits with the alum-precipitated antigen isolated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These techniques may be useful in isolating antigens in immune complex-associated diseases of unknown etiology.

Stochastic control of anti-Sm autoantibodies in MRL/Mp-lpr/lpr mice.

MRL/Mp-lpr/lpr autoimmune mice consistently show an approximately 25% incidence of the systemic lupus erythematosus marker autoantibody anti-Sm. In the present report, we show that the failure to find anti-Sm antibodies in three-quarters of 5-mo-old MRL/lpr mice was not an artifact of an insensitive assay, but rather that the mice fell into two populations as regards their anti-Sm positivity. Based on an extensive analysis of the incidence of anti-Sm positivity in 5-mo-old mice according to their cage of residence, we found no evidence for genetic, environmental, or parental influences on the propensity of an individual animal to become anti-Sm positive. Also, the gender of the mouse, its Sm antigen level, or its length of survival were not related to anti-Sm antibody, nor was the anti-Sm antibody status of either parent. Some animals became anti-Sm positive after 5 mo of age, but this was less likely than becoming positive before 5 mo of age. Finally, a survey of 205 autoimmune C57BL/6-lpr/lpr mice confirmed the uniqueness of the MRL background for this autoantibody response. These results together indicate that the possibility of making anti-Sm antibodies is under genetic control, but that the expression of this capability in an individual animal is governed by stochastic events. We hypothesize further that such random processes may involve the expression of particular immunoglobulin variable-region genes combined with mechanisms of extensive somatic mutation or positive feedback amplification, which would transmute an initial monoclonal response into an eventual polyclonal one.

Distinctive immune response patterns of human and murine autoimmune sera to U1 small nuclear ribonucleoprotein C protein.

The Ul small nuclear ribonucleoprotein (snRNP), a complex of nine proteins with Ul RNA, is a frequent target of autoantibodies in human and murine systemic lupus erythematosus (SLE). Anti-Sm antibodies recognizing the B'/B, D, E, F, and G proteins of Ul snRNPs are highly specific for SLE, and are nearly always accompanied by anti-nRNP antibodies recognizing the Ul snRNP-specific 70K, A, and/or C proteins. Previous studies suggest that human anti-nRNP antibodies recognize primarily the U1-70K and Ul-A proteins, whereas recognition of Ul-C is less frequent. We report here that autoantibodies to U1-C are more common in human autoimmune sera than believed previously. Using a novel immunoprecipitation technique to detect autoantibodies to native Ul-C, 75/78 human sera with anti-nRNP/ Sm antibodies were anti-Ul-C (+). In striking contrast, only 1/65 anti-nRNP/Sm (+) MRL mouse sera of various Igh allotypes was positive. Two of ten anti-nRNP/Sm (+) sera from BALB/c mice with a lupus-like syndrome induced by pristane recognized Ul-C. Thus, lupus in MRL mice was characterized by a markedly lower frequency of anti-U1-C antibodies than seen in human SLE or pristane-induced lupus. The results may indicate different pathways of intermolecular-intrastructural diversification of autoantibody responses to the components of Ul snRNPs in human and murine lupus, possibly mediated by alterations in antigen processing induced by the autoantibodies themselves.

Regulation of the anti-Sm autoantibody response in systemic lupus erythematosus mice by monoclonal anti-Sm antibodies.

The administration of certain monoclonal anti-Sm antibodies (2G7, 7.13) induced most MRL/lpr mice to become anti-Sm positive by 5 mo of age, although other anti-Sm monoclonals (Y2, Y12) suppressed the spontaneous response. Positive anti-Sm antibody enhancement occurred efficiently only in MRL/lpr mice and not in other systemic lupus erythematosus mice that have little spontaneous anti-Sm production. The enhancement by anti-Sm antibodies was specific for the anti-Sm response. The mechanism of the passive antibody enhancement was apparently not isotype- or idiotype-related. The fine specificity of the anti-Sm monoclonal antibody may be essential to its enhancing or suppressing effects, since both enhancing monoclonals recognized only the D Sm polypeptide, whereas both suppressing monoclonals saw the D and the B polypeptides. Furthermore, analysis of serial bleeds from unmanipulated MRL mice that developed anti-Sm positivity showed that the D specificity almost always appeared first. We hypothesize, therefore, that those animals in which an anti-Sm response is initiated by D-specific B-cell clones can become serologically positive with the aid of a positive feedback loop. In contrast, animals in which the initial specificity is for both B and D peptides would be prevented from developing a full anti-Sm response.

Subclass restriction and polyclonality of the systemic lupus erythematosus marker antibody anti-Sm.

Anti-Sm antibodies are highly specific markers for the diagnosis of systemic lupus erythematosus (SLE). This specificity suggests that the immunoregulation of these autoantibodies would reflect fundamental immune abnormalities in this disorder. As a clue to this immunoregulation, we have investigated the isotype distribution of anti-Sm antibodies by enzyme-linked immunosorbent assays. We have found that the anti-Sm response is markedly restricted to the IgG1 heavy chain isotype. On the other hand, the light chain distribution reflects that in normal serum, while isoelectric focusing analysis fails to show an oligoclonal pattern. The related specificity, anti-ribonucleoprotein, is also restricted to IgG1, while the SLE-specific antibody anti-double-stranded DNA is mostly IgG1 with a lesser contribution by IgG3. These results suggest that antinuclear antibodies that are strongly associated with SLE are produced by a T cell-dependent response, probably driven by antigen. The immunoregulation of the response to several autoantigens may be quite similar.

Antigen nonspecific effect of major histocompatibility complex haplotype on autoantibody levels in systemic lupus erythematosus-prone lpr mice.

MHC-linked genes strongly influence susceptibility to autoimmune diseases and also regulate responses to exogenous antigens. To begin to understand the mechanism of this MHC effect on disease, we have investigated MHC-congenic mouse strains that develop spontaneous autoimmunity because of the lpr gene. C57BL6/lpr (B6/lpr) mice (H-2b) are known to have substantial levels of autoantibodies to chromatin, single stranded DNA (ssDNA3), and IgG of different murine subclasses (rheumatoid factor). We have crossed the H-2d and the H-2bm12 (la mutant) haplotypes onto the B6/lpr background. Surprisingly, levels of all the autoantibodies were markedly lower in B6/lpr.H-2d, but levels in B6/lpr.H-2bm12 were no different from those in B6/lpr mice. The downregulating influence of the H-2d allele was dominant, and there was no effect on autoantibody fine specificities. The genetics of the H-2d effect and its diffuse influence on multiple autoantibody specificities, in addition to the lack of effect of the bm12 mutation, which modifies the peptide-binding groove of I-A, together raise the question of whether MHC-linked genes other than classical (IR) genes may be responsible for MHC disease associations in this model.

The polypeptide structure of the Sm and RNP nuclear antigens.

Sm and nuclear ribonucleoprotein are ubiquitous nuclear antigens towards which important autoantibodies are directed in systemic lupus erythematosus and other human autoimmune syndromes. Using physicochemical techniques and affinity adsorptions, we have purified the polypeptide components of these antigens. The Sm antigen contained polypeptide chains of 15,000 and 17,000 mol. wt. The RNP antigen, which is known by immunochemical techniques to contain the Sm antigen, had the same two polypeptides as well as a larger one of 85,000 mol. wt. This larger peptide was quite labile and apparently broke down into smaller components with manipulation. In addition, the process of affinity purification of the Sm polypeptides gave a product which had increased positive charge. Amino acid analysis of the Sm polypeptides confirmed the presence of relatively large numbers of basic residues. The purified Sm antigen provided an effective reagent for the investigation of autoreactivity to Sm. The differences in structure from our results and those published by others are probably accounted for by the lability of the constituent polypeptides.