Genetic aspects of the reactions of humoral immunity to collagen in man and animals. II. Modifications of the autoimmune status to collagen type I in NZB x NZW (F1) mice by sex hormones during ontogeny

The sex hormones, estradiol and testosterone, are able to modulate the status of spontaneous reactions of humoral immunity to type I collagen in ontogenesis of NZB x NZW (F1) females. Administration of estradiol to puber and unpuber females leads to a significant increase in the reactivity levels. The autoimmune status to type I collagen in NZB x NZW (F1) males is nonreactive to sex hormones influence. The results obtained corroborate the suggestion of the important role of sex hormones in formation of sex dimorphism and age variability to autoimmunity to type I collagen.     

Studies of tolerance to DNA in NZB/NZW F1 mice.

Administration of sDNA-poly-D-lysine (DNA-PDL) to newborn NZB/NZW F1 mice (B/W) was previously shown to prolonge survival and to decrease nephritis and DNA antibodies. In this study, B/W mice treated from birth with DNA-PDL were found to be tolerant to immunization with sDNA on PDL or mBSA carriers in adjuvants. Tolerance to sDNA was present and was hapten-specific carrier-dependent. IgG and IgM anti-nDNA circulating antibodies were suppressed. Continuous tolerization was necessary to maintain tolerance. Tolerance to sDNA could be transferred by spleen cells, by tolerized thymus cells, and by tolerized bone marrow cells, suggesting that both T and B cells participated in this phenomenon.     

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.     

Genetic aspects of humoral immunity reactions to collagen in man and animals. III. An analysis of the sexual dimorphism of the levels of autoantibodies to collagen type I in systemic lupus erythematosus patients and their modifications in ontogeny

The study of polymorphism levels of the autoantibodies to collagen type I in patients with systemic lupus erythematosus revealed the sex dimorphism of the character studied. Negative correlation of the levels of autoantibodies to collagen type I and those of sex hormones as well as modulation of the character under the action of xenobiotics were observed.     

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.     

Regulation of the immune response in autoimmune NZB/NZW F1 mice. I. The spontaneous generation of splenic suppressor cells.

The lymphoproliferative responses of rat peripheral blood lymphocytes to phytohemagglutinin (PHA) were studied following treatment with single or multiple doses of cyclophosphamide. A dose-dependent lymphocytopenia was observed with both regimes. The remaining lymphocytes had decreased responses to PHA. Serum collected 24 hr after a single injection of cyclophosphamide and used at a concentration of 5% enhanced the response of cells from normal or cyclophosphamide-treated rats. Serum collected after a course of treatment did not have this effect, but it lacked the marked suppressive activity, at a concentration of 20%, which was shown by normal rat serum. The enhancing activity was not dialysable. Doses of cyclophosphamide adequate to abolish primary antibody production to sheep erythrocytes did not totally abrogate responsiveness to PHA. Thus, the pattern of immunological defects in cyclophosphamide-treated rats consisted of decreased primary antibody production, lymphocytopenia with a decreased response of the remaining lymphocytes to PHA, and diminution of serum suppressive activity.     

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.     

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.     

Regulation of immune function by calorie restriction and cyclophosphamide treatment in lupus-prone NZB/NZW F1 mice

We compared the effects of calorie restriction (CR) and cyclophosphamide (CTX) on the progression of lupus nephritis and immunological changes in NZB/NZW F1 mice. Ad libitum (AL)/CTX and CR delayed onset of proteinuria and significantly decreased serum levels of anti-dsDNA, anti-histone, and circulating immune complex antibodies. CTX and CR prevented the increase in and activation of B cells, the decline in CD8(+) T cells, and maintained a higher proportion of na?ve CD4(+) and CD8(+) cells. MHC class I antigen and LFA-1 expression on CD8(+) T cells and MHC class II antigen on B cells were also decreased. AL/CTX and CR prevented the increase in production of IL-10 and up-regulated IL-2 production in T cells ex vivo. We concluded that both CR and CTX can delay the onset of autoimmune disease, in part by maintaining higher numbers of na?ve T cells and the immune responsiveness of T cells and decreasing the proportion of B cells.     

Dietary fat and immune function. II. Effects on immune complex nephritis in (NZB x NZW)F1 mice

(NZB x NZW)F1 mice initiated on fat restriction at weanling were significantly protected from the development of immune complex glomerulonephritis. Whereas the mice on high-fat intake demonstrated immune depositions both in capillary walls and mesangial areas in a diffuse granular pattern, those on a low-fat diet with caloric content similar to the high-fat diets exhibited mesangial confinement of the depositions of immunoglobulins, complement, and retroviral gp70. In association with these divergent patterns of immune deposition, the mice on high-fat diets had evidence of extensive diffuse cellular proliferation, wire loop lesion, and sclerosis in the glomeruli. In contrast, most of the mice on the low-fat diet showed only mesangial cell and matrix proliferations. In addition, the group of mice fed high saturated fat showed more severe glomerular pathology as compared to those fed high unsaturated fat. Paradoxically, levels of circulating immune complexes (as measured by the polyethylene glycol precipitation technique) in the high saturated fat group were low and did not correlate with the findings by light and immunofluorescence microscopy. These findings suggest that dietary fat restriction can serve as either a prophylactic or effective therapeutic approach to murine lupus nephritis.     

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.     

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.     

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.     

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.     

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.