Undernutrition without malnutrition restricts the numbers and proportions of Ly-1 B lymphocytes in autoimmune (MRL/I and BXSB) mice

MRL/Mp-lpr/lpr (MRL/l) and BXSB mice represent inbred mouse strains in which lymphoproliferative disease and autoimmune disease that includes lethal renal disease routinely occurs by 6 months of age. Chronic energy intake restriction increases longevity and health span of MRL/l and BXSB mice as it does in mice of other short-lived as well as long-lived strains. Chronic energy intake restriction forestalls development of the lymphoproliferative process, prevents development of renal lesions, decreases levels of circulating immune complexes, and permits maintenance of vigorous immunologic function with age. We have reported that in autoimmune-prone mice, a population of Ly-1 B lymphocytes that is associated with autoimmune disease and is greatly expanded among cells of the spleen, peritoneal exudate, and peripheral blood can be reduced in proportion as a consequence of undernutrition without malnutrition. Herein, we demonstrate that in MRL/l and BXSB mice, chronic energy intake restriction imposed at weaning inhibited accumulation of Ly-1 B lymphocytes throughout the lymphoid system, i.e., among cells of the spleen, thymus, mesenteric lymph nodes, bone marrow, peritoneal exudate, and peripheral blood when these tissues or fluids were studied at age 3 or 5 months. These results extend our previous finding that autoimmune-prone mice possess unusually large numbers of Ly-1 B cells in their lymphoid tissues which can be reduced in frequency as a function of diet toward the levels present in long-lived autoimmune-resistant mice.     

Comparison of immunological effects of cholera toxin on autoimmune MRL/lpr and BXSB mice.

MRL/lpr and BXSB mice were treated weekly or biweekly with cholera toxin (CT) in intravenous dose of 2 micrograms/mouse. CT treatment notably alleviated proteinuria in MRL/lpr mice, but did not influence the course of lupus nephritis in BXSB male mice. Flow cytometric analysis showed that anomalous B220+ T cells in spleen and thymus were reduced in CT-treated MRL/lpr mice while no significant change in lymphocyte populations was induced in BXSB male mice by this treatment. The suppressive effect of CT treatment on Con A response and the augmentative action on LPS response were observed in MRL/lpr mice. The latter may reflect increased B cells in relative number in the peripheral lymphoid organs. Mitogenic responses in CT-treated BXSB male mice remained unchanged in comparison with those of untreated group. Increased production of IL-6 by spleen cells was demonstrated in MRL/lpr mice treated with CT while in BXSB mice the level of IL-6 was not changed by the treatment with CT. Production of IFN gamma was suppressed by CT treatment in both strains of mice. This may be attributed to the inhibitory effect of CT on IFN gamma-producing Th1 cells as reported previously (Munoz et al, J. Exp. Med. 172: 95-103, 1990). However, CT treatment did not inhibit anti-DNA antibody production in BXSB mice, whereas the autoantibodies were markedly decreased in MRL/lpr mice treated with CT.     

Proliferative activity of autoimmune MRL mouse serum IgG to interleukin 3-dependent cell line through autocrine mechanism

Serum of an autoimmune MRL/Mp-lpr/lpr (MRL/l) mouse supported the proliferation of interleukin 3 (IL-3)-dependent cell line, FDC-P2. This IL-3-like activity initially appeared at 1 month of age and increased with age. Females showed higher titers than did males. MRL/Mp-+/+ mouse sera also exhibited such activity, though somewhat later in life only in female. Other autoimmune mice, NZB, NZB/NZW F1, and BXSB, demonstrated no such activity in either males or females, young and old. The active component of MRL/l sera was shown to be IgG. F(ab')2 or Fc fragments of MRL/l-IgG lost such activity. Not all IL-3-dependent cell lines, however, responded to MRL/l-IgG. We subcloned MRL-IgG responding and nonresponding clones from FDC-P2 cells and both were still dependent to IL-3. Such nonresponding IL-3-dependent cell lines, however, could be stimulated by the culture supernatant of the responding cell line, FDC-P2/185-4, after being stimulated with MRL/l-IgG. In this culture supernatant, IL-3 was found, thus the existence of an autocrine system was suggested in the IL-3-dependent MRL/l-IgG responding cell line.     

Immunohistochemical examination of Peyer's patches in autoimmune mice

Summary The distribution of T-cells and B-cells in Peyer's patches was examined in three autoimmune model mice, MRL/Mp-lpr/lpr, BXSB, NZBWF1/J mice and normal BALB/c mice, between one and ten months old. A multiple layering technique was used for immunohistochemical detection of lymphocyte surface antigens of T-cells (Thy1.2, Lyt1, Lyt2) and B-cells (surface IgM) and peanut agglutinin receptor for germinal center cells. The T-cell population of female MRL/Mp-lpr/lpr mice increased markedly with age, and the B-cell population of the male BXSB mouse tended to increase. However, little change was observed with age in the NZBWF1/J mice. The immunohistochemical properties of the Peyer's patches in the three autoimmune model mice were different.     

Immunohistochemical examination of Peyer's patches in autoimmune mice

The distribution of T-cells and B-cells in Peyer's patches was examined in three autoimmune model mice, MRL/Mp-lpr/lpr, BXSB, NZBWF1/J mice and normal BALB/c mice, between one and ten months old. A multiple layering technique was used for immunohistochemical detection of lymphocyte surface antigens of T-cells (Thy1.2, Lyt1, Lyt2) and B-cells (surface IgM) and peanut agglutinin receptor for germinal center cells. The T-cell population of female MRL/Mp-lpr/lpr mice increased markedly with age, and the B-cell population of the male BXSB mouse tended to increase. However, little change was observed with age in the NZBWF1/J mice. The immunohistochemical properties of the Peyer's patches in the three autoimmune model mice were different.     

Defective B cell tolerance induction in New Zealand black mice. I. Macrophage independence and comparison with other autoimmune strains

B cell unresponsiveness was examined in vitro by using spleen cells from autoimmune NZB, BXSB/Mp male, MRL/Mp-Ipr/Ipr (MRL/l), and control mice, and the tolerogen trinitrophenyl human gamma-globulin (TNP-HGG). The B cell subset responsive to TNP-Brucella abortus in each autoimmune and control strain that was tested was highly susceptible to tolerance induction with the use of high epitope density conjugates (TNP30HGG and TNP32HGG). When a tolerogen with a lower epitope density was used (TNP7HGG), several control strains were all rendered tolerant in a thymic-independent and hapten-specific manner. NZB B cells were resistant to all concentrations of TNP7HGG tested, whereas B cells from BXSB/Mp male and MRL/1 mice were resistant to low concentrations of this tolerogen. NZB mice were resistant in addition to tolerance induction with TNP9HGG, TNP10HGG, and TNP12.7HGG. Experiments were performed to determine whether splenic macrophages played a role in resistance to tolerance in NZB mice. The mixing of NZB and control DBA/2J T cell-depleted splenocytes revealed no modulatory effects by the accessory cells in culture. Moreover, B cells rigorously depleted of macrophages by double Sephadex G-10 column passage exhibited characteristic patterns of resistance or susceptibility in NZB and control strains, respectively. These findings support the conclusion that resistance to tolerance in NZB mice is determined at the B cell level and are consistent with the hypothesis that diverse immunoregulatory disturbances contribute in varying degrees to the development of systemic lupus erythematosus in different inbred strains of mice.     

Natural thymocytotoxic autoantibodies in autoimmune and normal mice.

Natural thymocytotoxic autoantibodies (NTA) were found in all mouse strains. Among those strains that show autoimmune syndromes resembling human systemic lupus erythematosus (SLE), the NZB and NZBxNZW had high levels of NTA, the BXSB had moderate levels, and the MRL/1 and MRL/n had very low levels. In addition, some normal strains had high levels, sometimes even higher than the autoimmune strains. The NTA were mostly IgM and were present, but not concentrated, in the cryoprecipitates of teh autoimmune mouse strains. In most strains, they were directed toward an antigen shared by thymocytes and brain. The failure to find high levels of NTA in all autoimmune mouse strains, as well as the finding of very high levels in some normal strains, make it unlikely that such auto-antibodies are a fundametnal etiologic factor in all murine SLE.     

Activities of dipeptidyl peptidases in BXSB mice and MRL/lpr mice with lupus erythematosus-like syndrome

Activities of peptidases were examined in tissues of male BXSB and male MRL/Mp-lpr/lpr (MRL/lpr) mice which are animal models of human systemic lupus erythematosus. Female BXSB and male MRL/+ + mice without histopathological changes were used as controls. Activity of DPP II in the spleen, kidney, and liver showed an increase at 13 and 20 weeks of age, while that of DPP IV was decreased at 20 weeks of age, as compared to control mice. The ratio of DPP II/DPP IV activities in the tissues was significantly increased and these findings agree with our previous results in the tissues of NZB mice and in the serum of patients with lupus erythematosus, underscoring the importance of hydrolytic enzymes in the pathogenesis of autoimmune diseases.     

Responses of B cells from autoimmune mice to IL-5

Three strains of mice (NZB/W F1 X NZW (NZB/W), BXSB, and MRL/Mp-lpr/lpr (MRL/lpr] develop an autoimmune disease that is clinically and immunologically similar to human SLE. A characteristic of these mice is polyclonal B cell hyperactivity. To explore whether this may be related to hyper-responsiveness to B cell stimulatory factors, we investigated the proliferative and secretory responses of B cells from these mice to semi-purified natural and rIL-5, a major regulator of B cell development in the mouse. As this lymphokine stimulates growth and differentiation of activated B cells, attention was focused on in vivo-activated B cell populations, obtained from the interface of 50/65% Percoll density gradients, from normal or autoimmune mice. This cell population from NZB/W mice secreted IgM and incorporated [3H]TdR at significantly higher levels in response to IL-5, and was more sensitive to IL-5, than a comparable population from several normal murine strains. NZB/W female and male mice displayed heightened responses to IL-5, indicating that this is characteristic of the strain in general and is not associated with the accelerated severe disease of the females. Small resting B cells from NZB/W and normal mice were insensitive to IL-5 stimulation. In contrast to NZB/W mice, no difference was observed in the magnitude of either proliferative or Ig secretory responses between in vivo-activated B cell populations from autoimmune BXSB and MRL/lpr or normal mice. Thus, B cell hyper-responsiveness to IL-5 is a characteristic of NZB/W mice but not of two other lupus-prone murine strains. As one unique feature of NZB/W mouse B cells compared to normal and other autoimmune B cells is an elevated proportion of Ly-1+ B cells, the possibility of IL-5 hyper-responsiveness being associated with this B cell subpopulation was investigated. Fluorescence-activated cell sorter sorted Ly-1+ and Ly-1- B cells both responded to IL-5, however Ly-1+ B cells consistently showed a higher stimulation index in both proliferative and Ig secretory responses to this lymphokine.     

Characteristics of in vitro production of antibodies to DNA in normal and autoimmune mice.

The in vitro production of antibodies to dsDNA was studied with spleen cells from normal and autoimmune mice. After culture for 4 days, the binding of dsDNA in the culture supernatant was measured by a radioimmunoprecipitation assay. The production of antibodies to dsDNA by spleen cells appeared at 15 hr after culture and reached a plateau at 24 hr. No antibodies were produced by thymus cells or splenic T cells. The specificity for dsDNA was shown by competitive inhibition with nonradioactive nucleic acids. Autoimmune strains of mice (NZB/NZW, BXSB, MRL/1) produced more antibodies to dsDNA than did several control strains. Young B/W mice and control strain mice produced mainly IgM antibodies, whereas older B/W mice produced predominantly IgG antibodies to dsDNA. The in vitro production of antibodies to dsDNA by aged B/W spleen cells was macrophage and T cell dependent.     

The relationship between plasma fibronectin levels and autoimmune disease activity in MRL/l mice

Plasma fibronectin levels increased significantly over time in MRL/l mice with progressive autoimmune disease. At 100 and 120 days of age both male and female MRL/l mice exhibited significantly higher fibronectin (Fn) levels than the more resistant MRL/l controls. Male mice at early time points had Fn levels no greater than controls due perhaps to the later onset of disease in MRL/l males. In contrast, female MRL/l mice, when compared with MRL/n controls, had higher Fn levels from 40 days of age. The proteinuria in these animals was also above MRL/n controls from the first time point taken (Day 40). In a temporal study with female MRL/l mice, Fn levels peaked at age 120 days and reflected the pattern of the survival curve, indicating that plasma Fn levels have an association with disease activity.     

Cell cycle analysis of lymphocyte activation in normal and autoimmune strains of mice

The spontaneous spleen cell proliferation and the proliferation induced by in vivo or in vitro stimulation with such polyclonal B cell activators (PBA) as LPS, poly rI.rC, and anti-mu were studied in normal and autoimmune mice. The various murine models of autoimmunity differ in the level of naturally occurring splenic cellular hyperactivity as well as in the ability of their spleen cells to be further stimulated in vitro by polyclonal stimulators. Both the NZB strain and the MRL/Ipr strain had markedly increased numbers and percentages of spontaneously proliferating spleen cells, whereas the BXSB strain did not. Nonautoimmune strains were found to have very small numbers of activated cells in the spleen. However, such normal strains could be induced in vivo to mimic the natural splenic hyperactivity observed in older NZB and MRL/Ipr autoimmune strains by the injection of polyclonal B lymphocyte stimulators. In contrast, old hyperactive NZB mice were not further induced to undergo proliferation by in vivo administration of such stimulators. Density-separated, T depleted, spleen cells of normal and autoimmune mice were stimulated in vitro with PBA in 48-hr cultures. Cells from old MRL/Ipr and NZB mice were abnormal in both the anti-mu response and the LPS response; BXSB mice had normal anti-mu responses. These studies suggest that there is no prerequisite for spontaneous splenic hyperactivity in the development of autoimmunity. In addition, different PBA stimulate separate subsets of B cells that differ in their state of activation in the various autoimmune strains. Finally, different B cell subsets appear to be abnormal in different types of autoimmune mice.     

Functional defects of culture-grown bone marrow-derived macrophages from autoimmune MRL/MpJ-lpr/lpr mice

To investigate the primary defects and development of macrophages in MRL/MpJ-/pr/lpr (MRL/l) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L-cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow-derived macrophages (BMM phi) from MRL/l mice had lower antigen presenting activity as detected by the induction of antigen-specific T cell proliferation, than age- and sex-matched control mice (CBA/J). Cell surface antigens (Ia and Mac-1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMM phi from MRL/l contained a much smaller number of Ia antigen-positive macrophages than those from normal mice. Treatment of BMM phi with an Ia-inducing of factor (IFN-gamma) markedly increased the expression of Ia antigens. This increase was significantly greater in BMM phi from MRL/l mice than in BMM phi from control mice. Expression of Mac-1 antigen was not different in BMM phi from the two strains. The Fc-mediated phagocytosis of IgG-coated sheep red blood cells was decreased in BMM phi from MRL/l mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex-bead incorporation was also impaired in MRL/l mice. The functional defects of MRL/l BMM phi found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.     

Increased spontaneous polyclonal activation of B lymphocytes in mice with spontaneous autoimmune disease.

Early in life, mice of four kinds [NZB, (NZB X NZW)F1, MRL/1, and male BXSB] with autoimmune disease spontaneously produced far more (greater than 3 S.D.) anti-hapten antibody-forming cells in spleens and greater concentrations of anti-hapten antibodies in sera than immunologically normal strains of mice (AKR, BALB/c, C57BL/6, DBA/1-J, DBA/2J, LG/J, 129, NZW, and female BXSB). This increased nonspecific antibody production by the abnormal animals' B cells correlated well with the spontaneous development of anti-single-stranded DNA antibodies, but not with serum levels of the viral envelope glycoprotein, gp70. These results suggest that the spontaneous formation of autoantibodies in mice whose immunologic disorder is manifested by a lupus-like disease may result from polyclonal activation of B cells by endogenous or exogenous B cell activators.     

Dysfunction of Ia-positive antigen-presenting cells in autoimmune mice

Antigen-presenting activity of spleen macrophages for T-cell activation was studied in vitro using autoimmune prone MRL/Mp-lpr/lpr (MRL/l) mice, and their normal counterparts, MRL/Mp-+/+ (MRL/n) mice. In vitro induction of trinitrophenyl (TNP)-specific proliferative T-cell response in lymph node lymphocytes from picryl chloride-painted mice was markedly impaired in MRL/l mice. The presenting activity of TNP-hapten by spleen macrophages for proliferative T cells was also impaired in MRL/l mice. The impairment of both T-cell and antigen-presenting macrophage activities was marked in older MRL/l mice, but not in younger ones. The impaired antigen-presenting activity of macrophages was not due to the development of suppressor macrophages. In accordance with the impaired antigen-presenting activity of macrophages the population of Ia-positive cells in spleen macrophages and the production of interleukin 1 by spleen macrophages were also reduced in older MRL/l mice. These results suggest that Ia-positive macrophages are impaired in autoimmune prone mice and that the dysfunction of Ia-positive macrophages plays an important role in the pathogenesis of autoimmune diseases.     

Changes in the compositions of the null cell compartment with murine autoimmunity

The subpopulations that comprise the null cell compartment were examined sequentially in various strains of autoimmune-prone mice. Different patterns emerged that were consistent within strains but differed from strain to strain. Abnormalities appear earlier in life in short-lived mice, such as male BXSB and MRL/1 mice, than in relatively long-lived strains, such as female BXSB and NZB mice. The accumulation of T cells in MRL/1 mice was accompanied by null cell changes that contrasted with those that developed in AKR/J mice after their spleens were infiltrated with leukemic T cells. It would seem that lymphocyte perturbations with murine autoimmunity also involve their precursor cells and that these precursor cell changes vary in different strains, perhaps in relation to different genetic factors.     

Altered expression of self-reactive T cell receptor V beta regions in autoimmune mice.

Intrathymic tolerance results in elimination of T cells bearing self-reactive TCR V beta regions in mice expressing certain combinations of I-E and minor lymphocyte stimulatory (Mls) phenotypes. To determine if autoimmune strains of mice have a defect in intrathymic deletion of self-reactive TCR V beta regions, expression of V beta 3, V beta 6, V beta 8.1, and V beta 11 were examined in lpr/lpr and +/+ strains of mice; MRL/MpJ(H-2K, I-E+, Mlsb,), C57BL/6J(H-2b, I-E-, Mlsb,), C3H/HeJ(H-2k, I-E+, Mlsc), AKR/J(H-2k, I-E+, Mlsa); and in autoimmune NZB/N(H-2d, I-E+, Mlsa) and BXSB(H-2b, I-E-, Mlsb) mice. The results suggest that, during intrathymic development, self-reactive T cells are deleted in autoimmune strains of mice as found in normal control strains of mice. However, the TCR V beta repertoire is skewed in autoimmune strains compared to normal strains of mice. For example, MRL-lpr/lpr mice, but not other lpr/lpr strains, had increased expression of V beta 6 relative to expression in control MRL(-)+/+ mice, which is associated with collagen-induced arthritis. These data are consistent with a model of normal affinity for negative selection of self-reactive T cells in the thymus of autoimmune strains of mice followed by expansion of autoreactive T cell clones in the peripheral lymphoid organs. The peripheral lymphoid organs of lpr/lpr mice contain an expanded population of abnormal CD4-, CD8-, 6B2+ T cells. Elimination of self-reactive peripheral T cells suggests that these abnormal cells are derived from a CD4+ subpopulation in the thymus. Flow cytometry analysis of peripheral lymph node T cells from MRL-lpr/lpr mice reveal three populations of CD4+ T cells expressing low, intermediate and high intensity of B220 (6B2). This supports the hypothesis that in lpr/lpr mice, self-reactive CD4+ T cells are eliminated in the thymus, and that these cells lose expression of CD4 and acquire expression of 6B2 in the periphery.     

Increase in potential activities of protein phosphatases PP1 and PP2A in lymphoid tissues of autoimmune MRL/MpJ-lpr/lpr mice.

The differential assay conditions for protein phosphatases PP1, PP2A, and PP2C were extensively studied by using crude extracts from mouse lymphoid tissues as enzyme sources. Under these conditions, the protein phosphatase activities were measured in MRL/MpJ-lpr/lpr mice (MRL/lpr mice), autoimmune-prone mice, and MRL/MpJ(-)+/+ mice (MRL/+/+ mice) and C3H/HeJ mice as the controls. In MRL/lpr mice, significant alterations in protein phosphatase activities from those in the control mice were demonstrated. In spleen and liver from MRL/lpr mice, potential activities of PP1 and PP2A were distinctly elevated over those of the control mice. These elevations appeared to be due to accumulation of the abnormal lymphocytes that emerged in MRL/lpr mice. Although the PP1 activity in MRL/lpr lymph nodes was lower than those of normal spleen and thymus, it was greatly increased by Co(2+)-trypsin treatment so that the PP1 activity of MRL/lpr lymph nodes was the highest among those of all the tissues examined. In contrast, PP2C activity showed no remarkable alteration in the autoimmune disease model mice as compared with that in the control mice. These results demonstrated a specific elevation in potency of protein dephosphorylation in the tissues of MRL/lpr mice, suggesting a new explanation for the defect in signal transduction in this disease.     

Effects of oral dimethyl sulfoxide and dimethyl sulfone on murine autoimmune lymphoproliferative disease

The results from several studies examining the effects of DMSO on autoimmune phenomena have been inconclusive, possibly because of differences in experimental models, treatment regimens and doses employed. In the present investigation, autoimmune strain MRL/lpr, C3H/lpr, and male BXSB mice were placed on a continuous treatment regimen with 3% DMSO or 3% DMSO2 in the drinking water, ad libitum, commencing at 1 to 2 months of age, before spontaneous disease development could be detected. This represented doses of 8-10 g/kg/day of DMSO and 6-8 g/kg/day of DMSO2. Both compounds were observed to extend the mean life span of MRL/lpr mice from 5 1/2 months to over 10 months of age. All strains showed decreased antinuclear antibody responses and significant diminution of lymphadenopathy, splenomegaly, and anemia development. Serum IgG levels and spleen IgM antibody plaque formation, however, did not differ from control values. There was no indication of involvement of systemic immunosuppressive or antiproliferative effects, and treated animals were observed to remain healthy and vigorous with no signs of toxicity. These results demonstrate that high doses of both DMSO and its major in vivo metabolite, DMSO2, provide significant protection against the development of murine autoimmune lymphoproliferative disease. Possible mechanisms of protection are discussed.     

Enzymatic histamine catabolism in vertebrate ontogenesis. A comparative study

1. The synthesizing and degrading activities of histamine were determined in the liver and small intestine of developing guinea pig and chick embryos. 2. Though increasing with age, HDC values were always 2-3 orders of magnitude lower than those of degrading enzymes. 3. DAO activity on the other hand was 10-100 fold higher than HMT at all ages studied, suggesting a decisive role for oxidative deamination in control of tissue histamine levels. 4. Generally histamine levels were higher in tissues of developing guinea pig than chick embryo, however, in the laying hen intestine histamine concentration was approximately 5 times greater than in the adult guinea pig intestine.