Self-reactive B cells in nonautoimmune and autoimmune mice

The defining feature of autoimmune disease is the presence of specific autoreactive lymphocytes. Systemic lupus erythematosus (SLE), for example, is characterized by a discrete set of antibodies directed to nuclear antigens; these include autoantibodies to DNA and snRNPs that are diagnostic for SLE. The murine model of SLE, the MRL-lpr/lpr mouse, likewise, has a similar autoantibody profile. To understand how SLE-associated autoantibodies are regulated in healthy individuals and to identify mechanisms underlying their expression in autoimmunity, we have developed a transgenic (tg) model system using multiple sets of tgs. The development of B cells bearing these tgs has been studied in BALB/c and MRL-lpr/lpr autoimmune backgrounds, and the relative fates of anti-ssDNA and anti-dsDNA tg B cells when they are a part of a diverse as well as monoclonal B cell repertoire have been evaluated.     

Preferential induction of autoantibody secretion in polyclonal activation by peptidoglycan and lipopolysaccharide. I. In vitro studies

Peptidoglycan (PG) and lipopolysaccharide (LPS) are bacterial cell wall components that are B cell mitogens and activators of polyclonal antibodies. Using the hemolytic plaque assay and its modifications with protein A-, IgG-, or DNA-coated erythrocytes, we determined the proportions of cells that secrete antibodies specific to various autologous and heterologous antigens in PG- and LPS-stimulated cultures of mouse splenic lymphocytes. Of all PG- and LPS-activated IgM-secreting cells, 47 to 75% produced antibodies specific to mouse IgG; 2.7 to 21% produced IgM antibodies that reacted with bovine IgG; 4.4 to 17%, with single-stranded (ss) DNA; 0.18 to 5.9% with double-stranded (ds) DNA; 0.16 top 3.3%, with bromelin-treated mouse red cells; 0.006 to 0.02%, with intact mouse red cells; and 0.35 to 1.9%, with sheep red cells. A similar distribution of cells secreting these antibodies was observed in unstimulated cultures. However, the absolute numbers of cells secreting these antibodies were substantially higher in the PG- and LPS-stimulated cultures. Similar results were obtained in BALB/c, CBA/H, and C57BL/6 mice. Since these strains of mice have different H-2 types, there was no association of a single H-2 type with the ability to form hgh numbers of autoantibody-secreting cells in vitro. The production of autoantibodies closely resembled polyclonal activation of all immunoglobulin-secreting cells in terms of the kinetics and dose-response. It did not involve any substantial specific anti-PG or anti-LPS response. Further studies on the specificities of polyclonally induced antibodies confirmed the specificity of anti-mouse IgG and anti-ssDNA antibodies. However, the formation of plaques with bovine IgG- or dsDNA-sensitized red cells was due to the cross-reacting anti-mouse IgG or anti-ssDNA antibodies, respectively. These results do not support the popular hypothesis of an equal polyclonal activation of lymphocytes secreting antibodies of all different specificities. Also, if preferential activation of cells secreting rheumatoid factor and anti-ssDNA antibodies occurs in vivo, it may have important pathologic consequences.     

Lateral cephalometric analysis of asymptomatic volunteers and symptomatic patients with and without bilateral temporomandibular joint disk displacement

Anecdotal evidence links silicone gel breast implants with the development of autoimmune connective tissue disease in women. To investigate whether silicone gel is capable of directly inducing and/or enhancing the development of autoimmune disease, female BALB/cAnPt (BALB/c) and New Zealand Black (NZB) mice were injected subcutaneously with silicone gel, pristane, a nonmetabolizable substance that can cause plasmacytomas in BALB/c and NZB mice, or saline and monitored for the development of glomerulonephritis and autoantibody production. NZB, but not BALB/c, mice spontaneously develop autoantibodies and an autoimmune hemolytic anemia by 12 months of age. Over a period of 10 months, biweekly screening for proteinuria revealed increases in urinary protein in NZB mice that received multiple injections of either silicone gel or pristane. In contrast, urinary protein was unaffected in identically treated BALB/c mice. Although, silicone gel had no effect on serum titers of antierythrocyte antibodies in NZB mice, the hematocrits were significantly decreased. Moreover, silicone gel both increased the concentration of IgM anti-type I collagen antibodies and skewed the immunofluorescent staining pattern of serum autoantibodies on HEp-2 cells. In contrast, silicone gel failed to induce the production of anti-erythrocyte or antinuclear antibodies in BALB/c mice and induced only slight increases in IgG anti-type I collagen antibodies. These results suggest that silicone gel can exacerbate the development of autoimmune disease in autoimmune NZB mice, but fails to induce disease in normal BALB/c mice. This is consistent with several epidemiological studies failing to demonstrate an increase in the incidence of autoimmune disease in women with breast implants. However, because silicone gel was able to exacerbate autoimmune disease in NZB mice, it may play a similar role in the development of autoimmune disease in a small percentage of women who are genetically susceptible to such diseases.     

IL-5 receptor positive B cells, but not eosinophils, are functionally and numerically influenced in mice carrying the X-linked immune defect

Mouse IL-5 (mIL-5) acts on B cells and eosinophils to induce growth and differentiation through the mIL-5 specific receptor (mIL-5R). The functional high-affinity mIL-5R is a heterodimer composed of alpha and beta chains. We investigated the expression of mIL-5R and the responsiveness of B cells and eosinophils to mIL-5 in X-linked immunodeficient (xid) mice. mIL-5R expression analyzed by using mAbs specific for alpha and beta chains revealed that xid B cells had fewer mIL-5R alpha +mIL-5R beta + than BALB/c B cells. In particular, a decrease in the number of peritoneal mIL-5R+ B cells among Ly-1 B cells (known as B-1 cells) was remarkable. Furthermore, the frequency of precursors of mIL-5 responsive B cells in xid mice was approximately 100-fold lower than that of BALB/c mice. Interestingly, sorted mIL-5R+ peritoneal B cells from xid mice displayed a low response to mIL-5. Intraperitoneal injection of mIL-5 into BALB/c mice induced polyclonal IgM production and an increase in the number of eosinophils. The same regimen failed to induce an increase in the same parameters in xid mice. However, xid mice showed mIL-5-induced eosinophilia in peripheral blood to a similar extent as BALB/c mice. Eosinophils from mIL-5-injected xid mice expressed both alpha and beta chains of mIL-5, and responded to mIL-5 with prolonged in vitro survival.     

Induction of lupus-associated autoantibodies in BALB/c mice by intraperitoneal injection of pristane

Intraperitoneal injection of pristane (2,6,10,14 tetramethylpentadecane) is a standard technique for obtaining monoclonal antibody-enriched ascitic fluid. However, pristane also induces plasmacytomas and an erosive arthritis resembling rheumatoid arthritis in BALB/c mice, probably as a consequence of enhanced interleukin 6 production. We report here that the production of autoantibodies characteristic of systemic lupus erythematosus (SLE) is a further consequence of injecting pristane in BALB/c mice. Anti-Su antibodies appeared as early as 1-2 mo after a single injection of 0.5 ml pristane, followed by anti-U1RNP and anti-Sm antibodies after 2-4 mo. Within 6 mo of pristane injection, 9 of 11 BALB/c mice had developed anti-Su, anti-U1RNP, anti-U2RNP, anti-Sm, and possibly anti-U5RNP antibodies. Autoantibodies were not produced by 20 BALB/c mice of the same age and sex that were not injected with pristane. Thus, autoantibodies characteristic of lupus were induced in mice that are not usually considered to be genetically susceptible to the disease. The induction of autoantibodies associated with SLE by pristane may be relevant to understanding the role of abnormal cytokine production in autoantibody production and the pathogenesis of autoimmune disease. Furthermore, the induction of high titer autoantibodies by pristane dictates caution in the use of ascitic fluid as a source of monoclonal antibodies, since the polyclonal antibodies induced by pristane may copurify with the monoclonal antibody secreted by an injected hybridoma.     

IFN-gamma prevents Th2 cell-mediated pathology after neonatal injection of semiallogenic spleen cells in mice

BALB/c mice injected at birth with 10(8) (A/J X BALB/c)F1 hybrid spleen cells develop an autoimmune host-vs-graft (HVG) disease as a result of activation of donor B cells by host CD4+ cells. The antidonor CD4+ cells seem to be Th2-like cells, inasmuch as they are profoundly deficient in IL-2 and IFN-gamma production, but secrete high levels of IL-4 and IL-10. As IFN-gamma is known to inhibit the development of TH2 cells, we attempted to modulate HVG disease by injecting rIFN-gamma. First, we found that 10 micrograms of rIFN-gamma given on days 1 and 3 after birth reduced the serum hyper-IgE of HVG mice by 90% and the serum hyper-IgG1, by 70%. In addition, rIFN-gamma administration significantly decreased the anti-DNA IgG1 titers and prevented the occurrence of anti-glomerular basement membrane and anti-laminin IgG1 Abs as well as the formation of immune deposits in renal glomeruli. These effects were not caused by the abrogation of chimerism, as indicated by the persistence of donor-type B cells in lymph nodes and of Igs bearing donor allotype in serum. MLC experiments indicated that the major effect of early rIFN-gamma administration was to restore the production of IL-2 and IFN-gamma by donor-specific T cells while these cells still secreted significant amounts of IL-4 and IL-10. Unresponsiveness of antidonor cytolytic T cells was not influenced by rIFN-gamma. We conclude that rIFN-gamma prevents the TH2-type response induced by the neonatal injection of semiallogeneic spleen cells and the associated pathology.     

Isolation and functional characterization of IL-2 responsive T cell clones from NZB x NZW F1 mice

Autoantigen-reactive T cells might play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Autoantigen-reactive T cell clones were generated from spleens of NZB x NZW F1 (BWF1) and normal control BALB/c mice with interleukin-2 (IL-2), a procedure that selects for in vivo activated antigen-reactive T cells. The antigen-specificity of the T cell clones was tested by using a panel of candidate autoantigens. The T cell clones from BWF1 mice but not those from BALB/c mice proliferated against heparan sulfate, the major glycosaminoglycan of glomerular basement membrane. None of the clones proliferated against dsDNA or cardiolipin. All the heparan sulfate-reactive T cell clones had the ability to selectively augment the production of IgG anti-dsDNA autoantibodies. When cultured with either heparan sulfate or Concanavalin A, the T cell clones produced high levels of IL-4 and IL-5 with no detectable IL-2 or IFN-gamma. In contrast, T cell clones derived from BALB/c mice augmented the production of total polyclonal IgG but not the production of anti-dsDNA antibodies. These studies indicate the existence of heparan sulfate-reactive T cells in BWF1 mice. Characterization of heparan sulfate-reactive T cells that could selectively augment anti-dsDNA production will permit the design of targeted and antigen-specific therapy.     

Immunological liver diseases in children

Pristane induces a lupus-like syndrome characterized by autoantibody production and glomerulonephritis in nonautoimmune strains of mice. Although it has been suggested that this syndrome results from nonspecific immune activation, there is little evidence so far that B cells are activated nonspecifically by pristane or that this promotes autoimmunity. In this study, we examined whether polyclonal hypergammaglobulinemia occurs in pristane-induced lupus, and its relationship to the production of anti-DNA, nRNP/Sm, and Su autoantibodies. In conventionally housed mice, there was a marked increase in total IgM and IgG3 2 weeks after i.p. pristane injection, followed by increased IgG1, IgG2a, and IgG2b levels. IgM levels were higher in pristane-treated specific pathogen-free (SPF) mice than in conventionally housed mice, whereas IgG and IgA levels were reduced. Pristane induced anti-nRNP/Sm and Su autoantibodies in SPF mice, but their onset was delayed and levels were lower than those in conventionally housed mice. There was no consistent relationship between total IgG1, 2a, and 2b hypergammaglobulinemia and production of anti-nRNP/Sm and Su autoantibodies. Moreover, the total Ig levels were similar in the anti-nRNP/Sm-positive and -negative groups. In contrast, production of IgM anti-ssDNA antibodies paralleled IgM hypergammaglobulinemia in some, but not all, mice. These studies indicate that pristane-induced lupus is associated with marked hypergammaglobulinemia, the magnitude of which is influenced by the microbial environment. However, anti-nRNP/Sm and Su autoantibody production is at least partly independent of polyclonal B cell activation. The data strongly suggest that pristane-induced lupus is not exclusively the consequence of nonspecific immune stimulation. They also point to the importance of microbial stimulation in the development of hypergammaglobulinemia in this inducible lupus model.     

Effects of bacterial DNA on cytokine production by (NZB/NZW)F1 mice

Microbial DNA has multiple immune effects including the capacity to induce polyclonal B cell activation and cytokine production in normal mice. We recently described the accelerated induction of anti-DNA Abs in NZB/NZW mice immunized with Escherichia coli (EC) dsDNA; paradoxically these mice developed less renal disease than unimmunized mice or mice immunized with calf thymus DNA. We postulated that alterations in cytokine production induced by bacterial DNA may play a key role in renal protection. To determine the effect of bacterial DNA on cytokine production in NZB/NZW mice, we measured the serum cytokine levels, cell culture supernatant cytokine levels, and number of cytokine-producing splenocytes in NZB/NZW mice injected with EC DNA, calf thymus DNA, or an immune active oligonucleotide. There was a 10- to 25-fold increase in the number of cells secreting IFN-gamma compared with IL-4 in mice immunized with EC DNA. IL-12-secreting cells were also increased by bacterial DNA immunization. In parallel with the increase in IFN-gamma secreting cells, there was a significant rise in serum IFN-gamma levels in mice receiving EC DNA. These results indicate that EC DNA modulates systemic cytokine levels in NZB/NZW mice, selectively increasing IL-12 and IFN-gamma while decreasing IL-4 production. The cytokine response of NZB/NZW mice to bacterial DNA may be of significance in disease pathogenesis and relevant to the treatment of lupus-like disease.     

The role of host (endogenous) T cells in chronic graft-versus-host autoimmune disease

Chronic graft-vs-host (cGVH) disease induced by the transfer of Ia-incompatible spleen cells from one normal mouse strain (such as B6.C-H2(bm12)/KhEg (bm12)) to another (such as C57BL/6) causes an autoimmune syndrome resembling systemic lupus erythematosus (SLE). The role of host-derived T cells in this response is not obvious. Previous reports suggested that host T cells might serve to down-regulate the autoimmune syndrome. To address this issue more definitively, we used CD4 knockout (KO) or CD8KO C57BL/6 (B6) mice as recipients in the bm12-->C57B6 cGVH model. CD4KO B6 mice injected with allogeneic bm12 spleen cells (bm12-->CD4KO group) showed no evidence of cGVH disease. They made no detectable autoantibodies, including anti-chromatin, anti-dsDNA, anti-ssDNA, and rheumatoid factor. They survived at least 20 wks after induction of cGVH disease; and they did not develop nephritis, based on the absence of detectable levels of proteinuria and normal renal histology at the time of sacrifice. By contrast, CD8KO B6 mice (bm12-->CD8KO group) and normal B6 mice (bm12-->B6 group) injected with bm12 spleen cells generally showed similar levels of mortality, nephritis, and autoantibodies, although the autoantibody titers declined somewhat after week 8 in the bm12-->CD8KO group. Control groups of recipients injected with B6 spleen cells showed no induction of autoantibodies. A surprising finding, however, was that the B6-->CD8KO group developed severe histologic glomerulonephritis in the absence of autoantibodies and with decreased immune deposits. These results indicate that endogenous (host) CD4+ T cells play an essential role in the cGVH autoimmune syndrome.     

No evidence of linkage between schizophrenia and D3 dopamine receptor gene locus in Icelandic pedigrees

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.     

T cell responses to myelin basic protein in experimental autoimmune encephalomyelitis-resistant BALB/c mice

In strains of mice that are susceptible to experimental autoimmune encephalomyelitis (EAE), cloned CD4+ T cells reactive with autologous myelin basic protein (MBP) have been shown to cause disease when transferred to naive syngeneic recipients. Recent reports indicate that under particular experimental conditions, 'resistant' strains of mice can also develop EAE, although cloned cells have not been isolated and characterized. An analysis of the characteristics of a panel of MBP-specific T cells and the antigen presenting capability of CNS-derived cells obtained from the resistant strain BALB/c is presented here. The data demonstrate that immunization of EAE-resistant BALB/c mice results in the activation of a heterogeneous group of T cells reactive with autologous MBP. Both peripheral antigen presenting cells, as well as microglia isolated from brains of BALB/c mice, are capable of stimulating these cloned MBP-specific T cells to proliferate. When optimally activated in vitro and then injected in vivo into syngeneic BALB/c recipients, three clones studied induced severe cachexia, resulting in loss of up to 35% of body weight before death. Two of the clones also induced clinical and histological EAE, while the third induced only occasional histological evidence of disease. Differences in epitope recognition, T cell receptor usage, cytokine profiles or regulatory mechanisms of self tolerance, may play important roles in preventing potentially destructive autoimmune reactions by these T cells capable of recognizing autologous myelin in the central nervous system.     

Immune response to the filaria Litomosoides sigmodontis in susceptible and resistant mice

Comparisons were made between the immune responses evoked during the course of chronic and patient infections of Litomosoides sigmodontis in susceptible BALB/c mice and non-patent infections in resistant B10.D2 mice. Early antigen specific responses of spleen cells were weak in both mouse strains. However, by day 58 post infection a strong Th2 response, as determined by production of IL-4, IL-5 and IL-10, was observed in BALB/c mice but not in B10.D2 mice. Antibody responses seemed to appear sooner in B10.D2 than in BALB/c mice, and these differentially recognised two antigens of 15 kD and 80 kD.     

The inflammatory response to nonfatal Sindbis virus infection of the nervous system is more severe in SJL than in BALB/c mice and is associated with low levels of IL-4 mRNA and high levels of IL-10-producing CD4+ T cells

SJL mice are susceptible to inflammatory autoimmune diseases of the central nervous system (CNS), while BALB/c mice are relatively resistant. To understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of SJL and BALB/c mice to infection with Sindbis virus, a virus that causes acute nonfatal encephalomyelitis in both strains of mice. Clearance of virus was similar, but SJL mice developed a more intense inflammatory response in the brain and spinal cord and inflammation persisted for several weeks. Analysis of lymphocytes isolated from brains early after infection showed an absence of NK cells in SJL mice, while both strains of mice showed CD4+ and CD8+ T cells. During the second week after infection, CD4+ T cells increased in SJL mice and the proportion of CD8+ T cells decreased, while the opposite pattern was seen in BALB/c mice. Expression of IL-10 mRNA was higher and IL-4 mRNA was lower in the brains of infected SJL than in BALB/c mice, while expression of the mRNAs of IL-6, IL-1beta, TNFalpha, and the Th1 cytokines IL-2, IL-12, and IFN-gamma was similar. Lymphocytes isolated from the CNS of SJL mice produced large amounts of IL-10. CNS lymphocytes from both strains of mice produced IFN-gamma in response to stimulation with Sindbis virus, but not in response to myelin basic protein. These data suggest that IL-10-producing CD4+ T cells are differentially recruited to or regulated within the CNS of SJL mice compared with BALB/c mice infected with Sindbis virus, a characteristic that may be related to low levels of IL-4, and is likely to be involved in susceptibility of SJL mice to CNS inflammatory diseases.     

Analysis of B cell autoepitopes and mechanisms for autoantibody production in autoimmune diseases

One of the representative immunological disorders in systemic autoimmune diseases is production of autoantibodies. Recent studies were concentrated on B cell epitopes of intranuclear autoantigens using recombinant proteins. They revealed autoepitopes homologous to those on exogenous agents and multiple epitopes, which respectively indicated molecular mimicry and antigen-driven immune responses as a mechanism for autoantibody production. Further, some autoantigens are thought to be catalyzed in a disease-specific manner. These antigen-specific factors would contribute to the autoantibody production, cooperating with antigen-nonspecific factors such as polyclonal B cell activation, overexpression of cytokines and dysfunction of T cells and antigen presenting cells.     

Resting B cells from autoimmune lupus-prone New Zealand Black and (New Zealand Black x New Zealand White)F1 mice are hyper-responsive to T cell-derived stimuli

To determine whether B cells from New Zealand Black (NZB) and (New Zealand Black x New Zealand White)F1 (NZB/W) mice possess intrinsic defects that lead to altered immune responsiveness, we purified resting B cells from these mice and compared their surface phenotype and function with those of resting B cells isolated from BALB/c and DBA/2 nonautoimmune mouse strains. Flow cytometric analysis of freshly isolated resting B cells revealed that NZB and NZB/W resting B cells are conventional B2-type cells similar to their nonautoimmune counterparts. Despite this, resting B cells from young NZB and NZB/W mice express lower levels of CD23 on their surface and aberrant levels of intracellular IgM. Upon stimulation, resting B cells from young NZB and NZB/W mice demonstrate increased proliferation, IgM secretion, or enhanced expression of costimulatory molecules in response to a variety of different T cell-derived stimuli, including cytokines and signals generated through CD40. Therefore, B cell hyper-responsiveness to T cell stimuli is immunodominant or codominant in NZB/W mice. Taken together, our results suggest that intrinsic B cell hyper-responsiveness may play a role in the pathogenesis of autoimmune disease in NZB and NZB/W mice. The increased clonal expansion of these B cells together with increased Ig production and enhanced costimulatory capacity serve to amplify the immune response. In the context of normal but incomplete T cell tolerance, B cell hyperresponsiveness to the limited signals provided by partially tolerant T cells may be sufficient to yield an autoantibody response.     

Spontaneous development of plasmacytoid tumors in mice with defective Fas-Fas ligand interactions

B cell malignancies arise with increased frequency in aging individuals and in patients with genetic or acquired immunodeficiency (e.g., AIDS) or autoimmune diseases. The mechanisms of lymphomagenesis in these individuals are poorly understood. In this report we investigated the possibility that mutations at the Fas (lpr) and Fasl (gld) loci, which prevent Fas-mediated apoptosis and cause an early onset benign lymphoid hyperplasia and autoimmunity, also predispose mice to malignant lymphomas later in life. Up to 6 mo of age, hyperplasia in lpr and gld mice results from the predominant accumulation of polyclonal T cell subsets and smaller numbers of polyclonal B cells and plasma cells. Here, we examined C3H-lpr, C3H-gld, and BALB-gld mice 6-15 mo of age for the emergence of clonal T and B cell populations and found that a significant proportion of aging mice exclusively developed B cell malignancies with many of the hallmarks of immunodeficiency-associated B lymphomas. By 1 yr of age, approximately 60% of BALB-gld and 30% of C3H-gld mice had monoclonal B cell populations that grew and metastasized in scid recipients but in most cases were rejected by immunocompetent mice. The tumors developed in a milieu greatly enriched for plasma cells, CD23- B cells and immunodeficient memory T cells and variably depleted of B220+ DN T cells. Growth factor-independent cell lines were established from five of the tumors. The majority of the tumors were CD23- and IgH isotype switched and a high proportion was CD5+ and dull Mac-1+. Considering their Ig secretion and morphology in vivo, most tumors were classified as malignant plasmacytoid lymphomas. The delayed development of the gld tumors indicated that genetic defects in addition to the Fas/Fasl mutations were necessary for malignant transformation. Interestingly, none of the tumors showed changes in the genomic organization of c-Myc but many had one or more somatically-acquired MuLV proviral integrations that were transmitted in scid passages and cell lines. Therefore, insertional mutagenesis may be a mechanism for transformation in gld B cells. Our panel of in vivo passaged and in vitro adapted gld lymphomas will be a valuable tool for the future identification of genetic abnormalities associated with B cell transformation in aging and autoimmune mice.     

Restoration of an early, progressive defect in responsiveness to T-cell activation in lupus mice by exogenous IL-2

Splenic T-cells from lupus strain (NZB/W F1, Mrl/lpr) mice lack the ability to respond to concanavalin A (Con A) by secretion of IL-2 and hence expression of IL-2 receptor and proliferation. These defects were found not only in an aged group (> 5 months) of mice in which obvious clinical 'SLE like' symptoms and elevated levels of serum autoantibodies were observed, but also in mice as young as 4-wk. We demonstrate here that the defective mitogenic activation of T-cells from lupus mice is due to the inability of T-helper cells to produce IL-2 and this defect can be restored by exogenous IL-2 in vitro. Con A-induced cell proliferation and IL-2 receptor expression on CD3+ cells from lupus mice occur only in the presence of exogenous IL-2, whereas normal T-cells from BALB/c and CBA control mice are activated by the mitogen and undergo complete cell cycling in the absence of exogenous IL-2, as they are able to secrete sufficient endogenous IL-2. The detection of impaired T-helper function in young lupus mice, before development of overt disease, and the reversible nature of the defect indicate that defective IL-2 activity may be fundamental to the mechanism of development of pathology in SLE.     

Functional CD4+ T cell subsets defined by expression of CD45RC and NTA260 antigens and age-associated polarization in murine lupus

Using two mAb, one specific to the alternative exon 6-dependent epitope of CD45 molecules (JH6.2) and one a natural thymocytotoxic autoantibody (NTA) with an unknown reactive epitope (NTA260), we subdivided splenic CD4+ T cells from 2-month-old BALB/c mice into five phenotypically distinct subsets. CD45RC+NTA260- (S I) cells were phenotypically analogous to CD4+ T cells predominating in newborn mice and produced a significant amount of IL-2, but not so IL-4, IL-10 or IFN-gamma when stimulated with immobilized anti-CD3 mAb in vitro. They appeared to consist mainly of naive ThP cells. The CD45RC+NTA260+ (S II) subset also produced IL-2, but not other cytokines; however, the IL-2 levels produced were much higher than seen with the S I subset, thereby suggesting the predominance of further maturated ThP cells. The CD45RC-NTA260+ (S III) subset mainly produced IL-4, IL-10, IFN-gamma and less IL-2, and contained memory cells that helped the secondary antibody response to a recall antigen, and hence contained Th2 and probably a mixture of Th0 and Th1 cells. The CD45RC-NTA260- (S IV) subset was a poor responder to the immobilized anti-CD3 mAb. The CD45RCbrightNTA260dull (S V) subset consisted of a small number of cells that were phenotypically analogous to activated CD4+ T cells. While an age-associated decrease in the proportion of S I and less markedly in S II and in turn increase in S III subsets of CD4+ T cells occurred in normal BALB/c mice, autoimmune disease-prone (NZB x NZW)F1 mice showed a marked age-associated decrease in the proportion of not only S I, II but also III subsets. As aged (NZB x NZW)F1 mice carry CD4+ T helper cells for IgG anti-DNA antibody production, such age-associated polarization to the S IV subset appears to be critical in the pathogenesis of autoimmune disease in these mice.     

Autoimmunity develops in lupus-prone NZB mice despite normal T cell tolerance

NZB mice spontaneously develop an autoimmune disease characterized by production of anti-RBC, -lymphocyte, and -ssDNA Abs. Evidence suggests that the NZB mouse strain has all of the immunologic defects required to produce lupus nephritis but lacks an MHC locus that allows pathogenic anti-dsDNA Ab production. The capacity to produce diverse autoantibodies in these mice raises the possibility that they possess a generalized defect in self-tolerance. To determine whether this defect is found within the T cell subset, we backcrossed a transgene encoding bovine insulin (BI) onto the NZB background. In nonautoimmune BALB/c mice, the BI transgene induces a profound but incomplete state of T cell tolerance mediated predominantly by clonal anergy. Comparison of tolerance in NZB and BALB/c BI-transgenic mice clearly demonstrated that NZB T cells were at least as tolerant to BI as BALB/c T cells. NZB BI-transgenic mice did not spontaneously produce anti-BI Abs, and following antigenic challenge, BI-specific Ab production was comparably reduced in both BI-transgenic NZB and BALB/c mice. Further, in vitro BI-specific T cell proliferation and cytokine secretion were appropriately decreased for primed lymph node and splenic T cells derived from NZB BI-transgenic relative to their nontransgenic counterparts. These data indicate that a generalized T cell tolerance defect does not underlie the autoimmune disease in NZB mice. Instead, we propose that the T cell-dependent production of pathogenic IgG autoantibodies in these mice arises from abnormal activation of T cells in the setting of normal but incomplete tolerance.