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.     

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.     

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.     

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.     

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.     

Alterations of stress related proteins in genetically altered mice revealed by two-dimensional differential in-gel electrophoresis analysis

Transgenic, knockout and knockin mice are useful tools for linking specific genes with behaviour and other complex biological processes. However, complications arising due to compensatory changes, genetic background differences and other factors could lead to difficulty in interpreting the resulting changes in phenotype. We have used fluorescence two-dimensional differential in-gel electrophoresis in combination with matrix-assisted laser desorption/ionization-time of flight mass fingerprinting to investigate the possibility that distinct genetic alterations can lead to common protein expression changes in genetically modified mice. Brain proteomes were compared from two transgenic mouse strains (Tg2576 x TgPS1 and Tg2576), two knockout mouse strains (5-HT(7)R -/- and GABA(A)Ralpha5 -/-) and one knockin mouse strain (GABA(A)Ralpha1-H101R). Both of the transgenic models showed an isoform change in the heat shock 70 related protein, mortalin. The knockout and knockin models showed similar changes in mortalin expression along with an alteration of the anti-oxidant protein 2. The observed proteomic alterations indicate that stress-responsive protein pathways may be altered artefactually in all of the mouse models used in this study and highlights an area where caution is needed in interpreting proteomic changes in genetically modified mice.     

Autoantibodies to the thymus and skin epithelium in NZB/N mice and (NZBxNZW)F1 hybrids

Antibodies reacting with thymus and skin epithelial cells were revealed by indirect immunofluorescence in sera of NZB/N mice and (NZB X NZW)F1 hybrids (B/W) 1-2 and 4-5 months of age. Similar antibodies were not found in sera of BALB/c mice. The inhibition experiments with DNA have shown that antibodies reacting with the thymus and skin epithelium differ from those reacting with the cellular nucleus. Positive reactions with the epithelium were obtained in all thymus and skin tissue samples of humans, guinea-pigs and NZB/N, B/W and BALB/c mice, including autologous tissues of NZB/N and B/W mice. Thus, antibodies reacting with thymus and skin epithelial tissues belong to autoantibodies. These autoantibodies are revealed during the first month of life before the onset of autoimmune processes. The role of these autoantibodies in the damage of thymus epithelium and the development of immunoregulatory disturbances, typical of autoimmune processes, needs further study.     

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.     

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.     

Maintenance of NF-kappaB activation in T-lymphocytes and a naive T-cell population in autoimmune-prone (NZB/NZW)F(1) mice by feeding a food-restricted diet enriched with n-3 fatty acids

We have previously shown that feeding a fish oil (FO) supplemented diet in combination with 40% food restriction (FO/FR) has a greater impact on extending life span in lupus-prone (NZB x NZW)F1 mice than either FO ad libitum (FO/AL) or corn oil food restricted (CO/FR) alone. Lupus disease is associated with increased Th-2 (i.e., IL-6 and IL-10) cytokine production and reduced IL-2 production and NF-kappaB activation. We hypothesized that the mechanism of action by which FO/FR increases life span may involve alterations in T-lymphocyte signaling and subsequent cytokine production. To test this hypothesis, we isolated and then stimulated splenic T-lymphocytes ex vivo with anti-CD3 and -CD28 monoclonal antibodies. We report here that CO/FR and FO/FR and to a lesser extent FO/AL offset disease-associated losses in Th-1 cytokine production, CD69 expression, and NF-kappaB activation in splenic T-lymphocytes activated ex vivo. Similarly, CO/FR and FO/FR prevented the disease-dependent rise in Th-2 cytokine production ex vivo and CD69 expression in vivo. In essence, the T-lymphocyte phenotype in the old CO/FR and FO/FR groups was identical to that in the young disease-free mice. Taken together, the data suggest that both CO/FR and FO/FR increase life span, in part, by maintaining a youthful immune phenotype in autoimmune-prone mice. However, FO/FR appears to represent a more potent dietary strategy in delaying disease-associated immune dysregulation than CO/FR.     

Alterations in the expression of Ia antigens in F1 hybrid mice

The Ia.8 and 9 specificities detected either by conventional or monoclonal antisera (Ia.m3, 4) are present in strains bearing the b H-2 haplotype, but absent from those with the k haplotype. It would be expected that the (b x k)F₁ hybrids would have approximately half the amount of these specificities found on the b parent, but the Ia.8 and 9 specificities are absent or reduced in this F₁ hybrid, though not on F₁ LPS blasts. Examination of appropriate H-2 congenic strains demonstrated that only the k haplotype confers the absence of these specificities on H-2 ^b — it was not observed with b, d, q, r or s haplotypes. In the k haplotype the gene(s) responsible for this effect is mapped to the I-A ^k subregion. The reason for this low expression effect is not clear but the observation has important implications for the relationship of Ia specificities and Ir genes and may serve to explain the low responder status of certain F₁ hybrids, e. g., to TNP-mouse serum albumin, as observed elsewhere.     

Failure of (SJL/J x PL/J)F1 hybrid mice to develop immunologic tolerance to V beta 17a+ T cells results in F1 anti-SJL mixed lymphocyte reaction.

It has been reported previously that spleen cells from (SJL x PL) F1 hybrid mice are not tolerant to SJL parental cells as assessed by a one-way MLR. The possibility that the F1 anti-SJL reaction was due to the effect of lymphokines produced by the irradiated SJL T cells in response to I-Eu expressed on the F1 hybrid cells was eliminated since inclusion of anti-I-E mAb was without effect. Cell separations showed the responder cells to be plastic and nylon wool nonadherent Ia- T cells. Separation of the SJL spleen cells showed that the stimulator cells were nonadherent, passed through a nylon wool column, and were Ia-. the F1-anti-SJL MLR was blocked 70 to 90% by inclusion of mAb KJ23a in the culture medium that indicated that the stimulatory cell population was V beta 17a+ T cells. This was confirmed by the use of V beta 17a+ and V beta 17a-T cell clones as stimulators. To determine whether failure to develop tolerance to this T cell subset in F1 hybrid mice might be responsible for the F1-anti-parent MLR, (SJL x PL)F1 mice were treated at birth and 48 h thereafter with anti-I-E mAb for 7 wk. Spleen cells from antibody-treated F1 mice were nonreactive with irradiated SJL parental cells in contrast to spleen cells from control mice which indicated that V beta 17a+ T cells were eliminated by negative selection before the development of tolerance.     

Beneficial effects of treatment with transglutaminase inhibitor cystamine on macrophage response in NZB/W F1 mice

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disorder of unknown etiology. However, the definitive mechanisms remain obscure. Recently, transglutaminase 2 (TG2) was implicated in the pathogenesis of SLE. Cystamine, which inactivates TG2 activity by forming a mixed disulfide, may interfere with and inhibit other thiol-dependent enzymes such as caspases. To investigate the effects of cystamine in SLE pathogenesis, this in vivo study assessed the serum and macrophage response after administration of cystamine to NZB/W F(1) mice. The experimental results demonstrated for the first time a significant reduction in TG2 and matrix metalloproteinase (MMP)-9 activity; tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, TG2, tumor necrosis factor alpha, and tumor growth factor beta mRNA expression; and anticardiolipin autoantibodies (aCL) in NZB/W F(1) mice following cystamine administration. It strongly suggests the therapeutic potential of cystamine in SLE.     

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.     

Lupus prone (SWR x NZB)F1 mice produce potentially nephritogenic autoantibodies inherited from the normal SWR parent

The incidence of nephritis in autoimmune NZB mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed 65 monoclonal anti-DNA autoantibodies derived from SNF1 mice and compared them with those obtained from the NZB parent. The majority of the SNF1-derived anti-DNA antibodies were IgG and cationic in charge. By contrast, 77% of the NZB-derived antibodies were IgM. Moreover, all three NZB-derived IgG anti-DNA antibodies were anionic. The cationic property of the SNF1-derived IgG autoantibodies was not restricted to any particular antigenic specificity pattern or IgG subclass, nor was there a preference for the allotype of either parent. However, we identified a set of highly cationic (pI at 8.2 to 8.8 pH) IgG2b anti-DNA antibodies from SNF1 hybrids that had the SWR allotype. Isoelectric focusing of intact antibodies and isolated heavy and light chains showed that the highly cationic charge of these antibodies was determined by the variable regions of their heavy chains. Because IgG anti-DNA antibodies with cationic charge are especially pathogenic, those antibodies bearing the allotype of the normal SWR parent may account for the high incidence of severe nephritis in the F1 hybrids. The results indicate that pathogenic autoantibodies, which are encoded by genes of the nonautoimmune SWR parent, are expressed in the SNF1 mice due to some cellular and genetic regulatory influence of the NZB parent.     

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.     

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.