Class-specific regulation of anti-DNA antibody synthesis and the age-associated changes in (NZB x NZW)F1 hybrid mice

Investigations of regulatory helper and suppressor T cells in the in vitro anti-DNA antibody synthesis in NZB x NZW (B/W) F1 hybrid mice were initiated by the development of an in vitro system in which G10-passed B cells from B/W F1 mice were cocultured with mitomycin C-treated T cells in the presence of Con A and either in the presence or in the absence of LPS. It was revealed that each IgG and IgM anti-DNA antibody synthesis was under the regulation of separate L3T4+ helper and Ly-2+ suppressor T cells. The function of these class-specific regulatory T cells was age-dependent. Although the helper effect of L3T4+ T cells on IgG antibody synthesis increased, the effect of L3T4+ T cells on IgM antibody production decreased in B/W F1 mice with aging. The IgG anti-DNA antibody production in the cocultures of L3T4+ T cells and B cells was suppressed by addition of Ly-2+ T cells from young but not aged B/W F1 mice, whereas the production of IgM anti-DNA antibodies was suppressed by Ly-2+ T cells from aged but not young B/W F1 mice. We also found that although IgM anti-DNA antibody-producing B cells were already present in 2-mo-old mice, B cells producing IgG antibodies under the influence of L3T4+ T cells appeared in mice at 7 mo of age. These data clearly indicate that separate class-specific regulatory T cells are involved in the production of IgM and IgG anti-DNA antibodies and that the total serum level of the antibodies is reflected by both their age-associated changes and the generation of antibody-forming B cells in B/W F1 mice.     

Mechanisms of T and B cell collaboration in the in vitro production of anti-DNA antibodies in the NZB/NZW F1 murine SLE model

B/W mice spontaneously develop IgG antibodies to DNA that cause lethal immune nephritis. T and B cell interactions in the in vitro anti-DNA antibody response of B/W mice were investigated, and two distinct families of helper T cells that drive these responses were defined. First, the anti-DNA antibody-forming cell (AFC) response was found to be increased in B/W mice with nephritis and was inhibited with the monoclonal antibody anti-L3T4, suggesting a major role for helper T cells. Purified splenic T cells from mice with nephritis were able to augment both the IgG and the IgM anti-DNA AFC response of young B/W B cells. T helper cells were cloned from spleens of NZB/W F female mice with high titer anti-DNA antibodies and nephritis. The cloned T cells augmented both IgG and IgM anti-DNA AFC responses of young B/W B cells. Four clones--27.9, 30.7, 30.8, and 30.10--were selected for further study. These cells proliferated, in the context of syngeneic (H2d/z) antigen-presenting cells (APC) but not to allogeneic APC. Analysis of the mechanism of T helper cell clone-mediated augmentation of anti-DNA AFC revealed two populations: "cognate" T helper cells, which specifically augment anti-DNA AFC (30.7 and 30.10), and non-antigen-specific T helper cells (27.9 and 30.8), which augment the response of B cells of differing specificity by a bystander mechanism, probably through increased release of B cell growth and differentiation factors.     

Interclonal and intraclonal diversity among anti-DNA antibodies from an (NZB x NZW)F1 mouse

The immunologic basis for the generation of autoantibodies that are characteristic of systemic autoimmunity in mice and humans remains obscure. Experiments directed toward the analysis of serum antibody and the cell populations that combine to generate antibody in autoimmune mice have led to the proposition that autoantibody production, including anti-DNA, results from the nonselective, polyclonal activation of B cells. The present results from the molecular analyses of anti-DNA autoantibodies from an individual (NZB x NZW)F1 autoimmune mouse, however, are inconsistent with a clonally nonselective model for autoantibody production and are most consistent with a clonally selective, Ag-driven model for anti-DNA autoantibody production. These results demonstrate that Ig V region structures contributed by germ-line V region genes; recombinational diversity, including unusual DH gene usage and DH-DH recombination; and somatic mutation during B cell clonal expansion are all important for generating antibody and presumably B cell Ig receptor specificity for nucleic acids including native, duplex DNA.     

In vitro immune response of cells of various lymphoid tissues in (NZB X NZW)F1 mice: evidence for abnormality of the mesenteric lymph node cells

Autoimmune-prone (NZB X NZW)F1 (B/W) mice have been shown to have a variety of immunologic perturbations. However, most studies have been performed with spleen cells. By using the Mishell-Dutton culture system, we examined the in vitro immune response of the various lymphoid tissue to determine whether an imbalance at a selective lymphoid site may exist in B/W mice. It was shown that the ability of mesenteric lymph node (MLN) cells of B/W mice to generate plaque-forming cells (PFC) in response to sheep red blood cells was consistently less than that of the spleen cells. This relationship held true in the aged mice. In contrast, the ability of the MLN cells of other strains not prone to develop autoimmunity to generate PFC was higher than that of the spleen cells. No significant difference in the mitogenic response of the lymphoid cells from various lymphoid tissue in the young B/W mice was seen, as compared with normal lymphoid cells from control mice. However, it was demonstrated that a relative decrease of B cells and immunoregulatory Lyt-123+ cells in the MLN in the B/W mice occurred early in life, and it was concluded that this abnormality may account for the low PFC response observed.     

Development of the B cell anti-DNA repertoire in (NZB x NZW)F1 mice. Relationship with the natural autoimmune repertoire.

The relationship between pathologic anti-DNA and natural autoantibodies (Auto Ab) remains unclear. In particular, it has not yet been elucidated whether pathologic anti-DNA antibodies originate from and are regulated by the pool of natural Auto Ab. To address this question, a large number of Ig-secreting hybridomas were derived from the unstimulated splenocytes of B/W mice, newborn to 12 mo of age, and their binding activities against a panel of self-Ag (DNA, actin, tubulin, myosin, and myoglobin), isotype, idiotypic determinants, and VH gene utilization were analyzed. A progressive increase in the number of Ig-secreting clones was observed and associated with a constant proportion (approximately 6%) of autoreactive B cell clones. However, dramatic changes in the pool of autoreactive B cell hybridomas were observed as the disease evolved, including the selective maintenance of IgM anti-DNA polyspecific antibodies, reduction in percentage of polyspecific IgM mAb with no DNA-binding activity, and the production of IgG anti-DNA antibodies of the IgG2 class. The kinetics, immunochemical properties, and idiotypic analysis of polyspecific IgM mAb with DNA-binding activity strongly suggest that they belong to natural Auto Ab and constitute the precursors of pathologic IgG anti-DNA antibodies. In addition, and IgM polyspecific antibody was demonstrated to bind IgG anti-DNA mAb through F(ab')2 interactions suggesting a regulatory role of natural antibodies and their participation in the control of pathologic Auto Ab production.     

Establishment of a nucleoside-specific suppressor T cell line from SLE prone (NZB/NZW)F1 mice

A long-term cultured suppressor T cell line (GTS-124) was established from an autoimmune mouse strain, (NZB X NZW)F1, by a two-part procedure: a) B/W F1 mice were made tolerant to guanosine (G) by administration of a tolerogen, the G-modified copolymer of D-glutamic acid and D-lysine (G-D-GL); and b) the spleen cells obtained from tolerant mice were repeatedly stimulated with mitomycin C-treated G-modified syngeneic spleen cells. The GTS-124 cells suppressed the secondary in vitro response to G-keyhole limpet hemocyanin (G-KLH) but did not suppress the response to unrelated antigens, sheep erythrocytes (SRBC), or trinitrophenyl-KLH (TNP-KLH). The expression of Thy-1 antigen on the cell surface of GTS-124 was demonstrated by flow cytometry. Growth of GTS-124 cells was dependent on IL 2. To determine whether GTS-124 cells could suppress the response to nucleosides other than G, KLH coupled with four nucleosides (adenosine [A], G, cytidine [C], and thymine riboside [T]) collectively (AGCT-KLH) was first used as the antigen in the assay system. The PFC response to the individual nucleosides (anti-A, -G, -C, and -T PFC) were effectively inhibited by GTS-124 cells, suggesting that the GTS-124 cells mediated cross-suppression toward all four nucleosides. A more stringent cross-suppression test was conducted by using only the T moiety bound to KLH (T-KLH) as antigen. The results showed that GTS-124 cells were capable of suppressing the T-specific response. The cross-suppression could be seen after repeated selection on a G-BSA-coated dish. These results provide direct evidence that the suppressor T cells induced by in vitro stimulation with G-modified self can indeed suppress the response to nucleosides other than G.     

The regulatory role of NZB T lymphocytes in the production of anti-DNA antibodies in vitro

Murine lupus is characterized by the production of numerous autoantibodies and immune complex glomerulonephritis. Anti-DNA antibodies are the hallmark of this disorder and may be associated pathogenetically with the glomerulonephritis. The cellular mechanisms underlying the regulation of the production of anti-DNA antibodies may prove to be the fundamental abnormalities responsible for the lupus syndrome seen in these mice. By using a system of spontaneous anti-DNA antibody production in vitro, we have determined that such production is characteristic of autoimmune NZB and MRL-lpr/lpr mice but not of the nonautoimmune control strains. Additional examination of the cellular mechanisms involved in the regulation of this response in NZB mice revealed: 1) this response is markedly T cell dependent, 2) NZB T cells are essential for maximal production of this autoantibody, and 3) NZB T cells actively interfere with normal immune regulatory mechanisms that lead to the production of anti-DNA antibodies spontaneously in vitro by nonautoimmune syngeneic B lymphocytes. Although these studies of anti-DNA antibody production in vitro disagree with previous work by others they successfully reproduce the results obtained earlier in experiments performed in vivo.     

Anti-tumor effects of allogeneic bone marrow transplantation in (NZB X NZW)F1 hybrids with spontaneous lymphosarcoma

Untreated female (NZB X NZW)F1 hybrid mice (B/W F1) were found to develop lymphosarcoma spontaneously as they aged. Tumor incidence was evaluated in B/W F1 mice immunosuppressed with total lymphoid irradiation (TLI) and in TLI-conditioned B/W F1 mice reconstituted with 3 X 10(7) BALB/c bone marrow (BM) cells. BALB/C leads to B/W F1 chimerism (79 to 89% BALB/c-type cells) was confirmed by typing peripheral blood lymphocytes with specific alloantisera and complement by using a microcytotoxicity assay. Chimeras showed no clinical signs of graft-vs-host disease (GVHD). TLI-treated mice seemed to show a slightly accelerated onset of lymphosarcoma as compared with untreated controls, but the difference was not significant (p = 0.08). BALB/c leads to B/W F1 chimeras reconstituted at 1 to 3 mo of age (25 mice) developed no tumors for an observation period of 18 mo after transplantation. In contrast, tumors developed in 24/130 of age-matched controls, and in 13/57 of TLI-treated nonreconstituted age-matched B/W F1 mice. Tumor incidence in BALB/c leads to B/W F1 chimeras transplanted at an older age (9 to 11 mo) was similar to that observed in age-matched TLI-treated B/W F1 mice and age-matched untreated controls. The data suggests that the high naturally occurring incidence of lymphosarcoma could be reversed by reconstituting TLI-treated mice with BM cells (p = 0.027). Thus, allogeneic BM transplantation may exert potent graft-vs-tumor effects (GVT) when tumor susceptible hosts are reconstituted at an early age, whereas GVT is relatively ineffective at an advanced age, which probably correlates with an advanced stage of tumor development. Allogeneic BM transplantation should be additionally explored as a potential clinical tool for eradication of certain solid tumors in adjunct to high-dose radiochemotherapy, inasmuch as GVT seems to be independent of GVHD.     

Total lymphoid irradiation reduces IgG autoantibody production and enhances specific antibody responses in NZB/NZW F1 mice

Thymus-independent primary antibody responses were studied in young and old (9 months) untreated and TLI-treated NZB/NZW and BALB/c mice. Untreated old NZB/NZW mice had a low primary response to Brucella abortus (BA) as compared to that of young NZB/NZW and BALB/c mice. However, TLI treatment resulted in a 130-fold increase in the IgG anti-BA primary antibody response at day 21 postimmunization, achieving similar levels to those of young NZB/NZW or nonautoimmune BALB/c mice. Anti-TNP responses to trinitrophenylated BA or Ficoll were masked by high background levels of anti-TNP antibodies. Despite the increase in the anti-BA response, spontaneous immunoglobulin secretion and autoantibody levels were markedly decreased after TLI in old NZB/NZW mice.     

Splenic phagocytes promote responses to nucleosomes in (NZB x NZW) F1 mice

Autoantigen presentation to T cells is crucial for the development of autoimmune disease. However, the mechanisms of autoantigen presentation are poorly understood. In this study, we show that splenic phagocytes play an important role in autoantigen presentation in murine lupus. Nucleosomes are major autoantigens in systemic lupus erythematosus. We found that nucleosome-specific T cells were stimulated dominantly in the spleen, compared with lymph nodes, lung, and thymus. Among splenic APCs, F4/80(+) macrophages and CD11b(+)CD11c(+) dendritic cells were strong stimulators for nucleosome-specific T cells. When splenic phagocytes were depleted in (NZB x NZW) F(1) (NZB/W F(1)) mice, nucleosome presentation in the spleen was dramatically suppressed. Moreover, depletion of splenic phagocytes significantly suppressed anti-nucleosome Ab and anti-dsDNA Ab production. Proteinuria progression was delayed and survival was prolonged in phagocyte-depleted mice. The numbers of autoantibody- secreting cells were decreased in the spleen from phagocyte-depleted mice. Multiple injections of splenic F4/80(+) macrophages, not those of splenic CD11c(+) dendritic cells, induced autoantibody production and proteinuria progression in NZB/W F(1) mice. These results indicate that autoantigen presentation by splenic phagocytes including macrophages significantly contributes to autoantibody production and disease progression in lupus-prone mice.     

Morphogenetic characteristics of the mammary glands of autoimmune F1 mice (NZB x NZW)

The morphogenesis of mammary glands was studied in the normal and autoimmune F1(NZW X NZB) mice. In the lactation cycle of the autoimmune mice the normal course of structural-functional rearrangements of parenchyma and stroma in the developing and involuting mammary glands was disturbed. A conclusion has been reached that the modification of stromal elements, first of all involved in the autoimmune disease, is the leading link in the abnormal development of mammary glands.     

Functional role of self IA molecules in polyclonal B cell activation using an autoreactive B cell clone derived from (NZB X NZW) F1 mice.

The mechanism of polyclonal B cell activation in autoimmune diseases was investigated by using an autoreactive B cell clone established by somatic hybridization with B cells derived from NZB X NZW (B/W) F1 mice. Briefly, splenic B cells from B/W F1 mice were fused with M12.4.1, a mutant of a B cell line, in the presence of polyethylene glycol and DMSO. NW47.7, a subclone of a resulting hybridoma, expresses B cell surface antigens on the cell membrane, namely IAd, IgM, B220, the receptors for the C3 fragment of complement (C3R), and the Fc portion of IgG (Fc gamma R). It also possesses a receptor molecule for mouse red blood cells treated with bromelain (Br-MRBC) on its surface, by rosette-forming assay with Br-MRBC. In contrast, parental M12.4.1 does not express IAd and IgM on the cell membrane, and does not bind to Br-MRBC under the same conditions. Thus, it is likely that NW47.7 may be an autoreactive B cell clone specific for Br-MRBC. Interestingly, NW47.7 was induced to generate a significant number of IgM-secreting cells when treated with Br-MRBC and rIL-5. Furthermore, mAb against IAd molecules, but not IAk and KdDd, markedly inhibited the differentiative effect of polyclonal activators such as LPS and rIL-5. Also, when MHC identical irradiated B cells were added to the culture of NW47.7 as a stimulator, the induction of IgM-producing cells was greatly augmented, but this augmenting effect was lost by interfering with direct contact of NW47.7 cells with stimulator B cells using a semipermeable membrane, as well as by the addition of mAb against IAd molecules. In addition, irradiated NW47.7, but not M12.4.1, by itself could enhance the secretion of IgM by NW47.7 as a stimulator, but this enhancing effect markedly decreased in the presence of anti-IAd mAb. The present results suggest that surface IA molecules on B cells are involved during the differentiative response to polyclonal activators, and may directly provide a differentiative signal for maturation of B cells into IgM-secreting cells.     

Reversal of advanced murine lupus in NZB/NZW F1 mice by treatment with monoclonal antibody to L3T4

Murine lupus in NZB/NZW F1 (B/W) mice can be prevented by weekly treatment with monoclonal antibodies (MAb) to L3T4 (on "helper/inducer" T cells) if treatment is begun prior to the onset of clinical illness. To determine whether anti-L3T4 could reverse as well as prevent murine lupus, we monitored a cohort of 30 B/W females until age 7 mo, when severe autoimmune disease was established, and then we examined the effects of weekly treatment with MAb to L3T4. The rate of target cell clearance by MAb was considerably slower in old B/W mice than it was in young B/W mice or in normal (BALB/c and C57BL/6) mice. Nonetheless, treatment with anti-L3T4 depleted 90% of circulating L3T4+ cells over 3 mo. In treated mice, the concentration of anti-DNA antibodies fell by 80%, renal insufficiency was reversed, and 1 yr survival was 75% compared to 17% in controls. These findings indicate that L3T4+ cells play an important role in perpetuating murine lupus in B/W mice even after severe disease is present. Because the L3T4 antigen in mice is homologous to the Leu-3/T4 (CD4) antigen in humans, these findings suggest that treatment with CD4 MAb may be effective in people with systemic lupus erythematosus.     

Regulation of an in vitro anti-DNA antibody response by thymocytes and T cells from NZB/W and normal mice

The spontaneous in vitro anti-DNA antibody response generated by preautoimmune and many normal mouse spleen cells was suppressed by the addition of syngeneic thymocytes or splenic T cells. Suppressive activity was found in normal mice (DBA/2J) and to an equivalent degree in the autoimmune (New Zealand Black X New Zealand White)F1 (B/W) strain. The suppressor cells were cortisone-resistant, radiosensitive and carried Lyt 1 and Lyt 2 markers. Nonspecific suppression was not involved since the primary and primed in vitro anti-sheep erythrocyte (anti-SRBC) responses were unaffected. Both spontaneous and lipopolysaccharide-stimulated anti-DNA antibody responses could be suppressed. There was no difference in the suppressive activity of cells from young or old, normal or autoimmune mice. These T cells may therefore play a role in preventing the anti-DNA antibody response in normal and young B/W mice, but evidently fail to influence the development of in vivo anti-DNA autoimmune responses in the old B/W mice.     

Intravenous infusion of PAF affects ovulation, fertilization and preimplantation embryonic development in NZB x NZW F1 hybrid mice

Platelet Activating Factor (PAF) is a bioactive phospholipid, which exhibits a variety of biological activities and plays a significant role in all aspects of reproduction. In this work, a single intravenous injection of various concentrations of PAF shortly after Human Chorionic Gonadotropin (HCG) administration as well as 24 and 48 h before HCG administration was studied in NZB x NZW F1 hybrid mice. Optimum results were observed when PAF was injected just after the administration of HCG. In this protocol, the concentrations of PAF exhibited bell-shaped response to every stage of development. Any concentration of PAF between 5.5 x 10(-11) and 5.5 x 10(-15)g/g b.w., caused an improved ovulation rate, an increased fertilization rate, an increased rate of cell cycle and an enhanced hatching blastocyst rate (P<0.05 for all stages). Injection of lyso-PAF had no effect in any stage. Our data show that the effect of PAF on early stages of embryo development in vitro is dependent on its way of administration, on the concentrations used as well as on the time PAF is injected.     

CD4+ T lymphocytes with constitutive CD40 ligand in preautoimmune (NZB x NZW)F1 lupus-prone mice: phenotype and possible role in autoreactivity

Lupus disease is marked by B lymphocyte hyperactivity and the production of Abs to dsDNA. The production of these anti-dsDNA Abs is T lymphocyte dependent. However, it is not clear how CD4+ T lymphocytes provide help for B lymphocytes to produce IgG anti-dsDNA Abs. One possible mechanism is suggested by studies showing that human patients with systemic lupus erythematosus and lupus mice have increased numbers of CD40 ligand (CD40L)+ T and B lymphocytes. The results described in this study reveal that young, clinically healthy lupus-prone New Zealand Black x New Zealand White F1 (BWF1) mice have naive CD4+ T cells with preformed CD40L. These cells contribute to a brisk response to immunization and to the production of anti-dsDNA Abs. In vitro experiments revealed that CD4+ T cells with preformed CD40L could, upon stimulation, provide antiapoptotic signals for B cells but could not induce proliferation or reduce activation threshold. These results suggest that the direct target cells for the effect of T cells with preformed CD40L in lupus may not be B lymphocytes.     

Tolerance to DNA in (NZB x NZW)F1 mice that inherit an anti-DNA V(H) as a conventional micro H chain transgene but not as a V(H) knock-in transgene

Lupus-prone (NZB x NZW)F(1) (BWF(1)) mice were made transgenic (Tg) for an anti-DNA Ab inherited either as a conventional V(H)3H9- micro H chain Tg (3H9- micro ) with or without a conventional V(kappa)8-kappa Tg, or a V(H)3H9 V(H) knock-in Tg allele (3H9R) with or without a V(kappa)4 V(kappa) knock-in Tg allele (V(kappa)4R). V(H)3H9 yields an anti-DNA Ab with most L chains including an anti-ssDNA with the V(kappa)8 Tg and an anti-dsDNA with the V(kappa)4 Tg. BWF(1) mice that inherited the conventional 3H9- micro had normal serum IgM, little to none of which was encoded by 3H9- micro, and only a small percentage of those mice had serum anti-DNA, none of which was transgene encoded. B cells expressing the conventional 3H9- micro Tg were anergic. BWF(1) mice that inherited the knock-in 3H9R Tg allele also had normal serum IgM, one-half of which was encoded by 3H9R, and produced anti-DNA encoded by the Tg allele. Most B cells expressing the knock-in 3H9R Tg also had an anergic phenotype. The results indicate that autoimmune-prone BWF(1) mice initially develop effective B cell tolerance to DNA through anergy, and anergy was sustained in 3H9- micro Tg peripheral B cells but not in 3H9R Tg B cells. B cells expressing the 3H9R knock-in Tg allele were able to achieve an activation threshold that B cells expressing the 3H9- micro conventional Tg could not. The maintenance of B cell tolerance to DNA in autoimmune-prone BWF(1) mice appears to differ from both normal mice and autoimmune-prone MRL(lpr/lpr) mice.     

Dietary fat and immune function. I. Antibody responses, lymphocyte and accessory cell function in (NZB x NZW)F1 mice

The influence of dietary fat on autoimmunity in lupus-prone (NZB x NZW)F1 mice has been demonstrated. In defining further the effects of dietary lipid on the immune system of this strain, female weanling mice were placed on four diets differing in quantity and type of fat. Their immunologic response was then studied by a variety of tests at 4 and 7 mo of age. Few differences were seen among the four groups at 4 mo of age. At 7 mo of age, however, the mice receiving diets high in saturated and unsaturated fats had a reduced mitogenic response to T cell mitogens and an enhanced response to the B cell mitogen LPS. Immunoglobulin levels and delayed hypersensitivity responses did not show any consistent differences among the diet groups. At 7 mo, however, mice receiving diets high in unsaturated fat demonstrated hyperresponsiveness to injected sheep red blood cells as measured by the hemolytic plaque technique. In addition, peritoneal leukocytes from the same diet group exhibited an increased response to bromelain-treated autologous erythrocytes which was decreased after treatment with anti-Thy-1 antiserum and complement. Phagocytosis by peritoneal macrophages was significantly decreased in the animals fed high-fat diets, particular high saturated fat. Similarly, natural killer cell activity was markedly reduced in the mice with a high intake of saturated lipid, a finding which correlated with the in vitro production of interferon. These results indicate that diets high in fat influence immune responses and thus can affect the onset and severity of autoimmune disease. A low-fat diet can reduce the development of disease by maintaining normal immune responses. The data also suggest that unsaturated fat may influence T helper cell activity and therefore antibody production, whereas saturated fats may affect cellular immune responses which are dependent on membrane contact.