Modulation of autoimmunity in NZB mice by cyclophosphamide-induced, nonspecific suppressor cells

A group of NZB mice received six biweekly injections of cyclophosphamide-induced nonspecific suppressor cells, with treatment commencing at 2 mo of age. Mice were evaluated for Coombs and natural thymocytotoxic antibody at 6-wk intervals thereafter, and for anti-DNA autoantibodies, total IgM and IgG levels, and renal histology at selected time points. The administration of suppressor cells resulted in marked and prolonged suppression of both Coombs and natural thymocytotoxic antibody reactivity in the majority of animals while not measurably affecting the levels of anti-DNA autoantibodies, the total IgM and IgG levels, or the life span of the mice.     

Distribution of anti-histone-antibody-secreting cells in NZB/NZW mice

Using a histone-specific plaque assay, we examined anti-histone-antibody (AHA) production at the organ level in the autoimmune NZB/NZW strain. The spleen had the highest absolute numbers of AHA-secreting cells. High percentages of immunoglobulin-secreting cells producing AHA were characteristic of spleen and bone marrow but not lymph node. AHA-secreting cells were detected in NZB/NZW mice with elevated serum activity but not in mice with normal serum levels. Serum AHA activity correlated with the number of AHA-secreting cells in the spleen but not with the total number of immunoglobulin-secreting cells in the spleen nor with the total serum immunoglobulin level. These findings concerning the organ distribution of AHA-secreting cells contrast with results of other investigators studying autoantibodies of other specificities. Furthermore, our results suggest that AHA production does not solely result from a generalized increase in total immunoglobulin synthesis present in NZB/NZW mice.     

Demonstration of active suppressor cells in spleens of young NZB mice

NZB mice, a strain prone to the development of autoimmune disease, have during the first 2 weeks of life suppressor cells in their spleens which can in coculture with adult spleen cells suppress the antibody response to sheep red blood cells (SRBC) generated in culture by the adult cells. The suppressive activity of spleen cells from NZB mice in the first week after birth is similar to that of spleen cells from 4-day-old C57BL/6 mice, a strain which does not spontaneously develop autoimmune disease. As in “normal” strains of mice, suppressor cell activity in NZB mice is diminished at 2 weeks and undetectable at 3 weeks of age. The data indicate that there is no defect inherent in the suppressor cells detected in the spleens of newborn and young NZB mice and suggest that the development of autoimmune responses does not result from a lack of suppressor cells in the young animals.     

Defective isotype-specific regulation of IgA anti-erythrocyte autoantibody-forming cells in NZB mice

B cell hyperactivity characterizes many autoimmune diseases. In NZB mice this is manifested by a variety of immunologic aberrations, including increased B cell proliferation and hyper IgM and IgA secretion in vitro. Recent studies have shown that IgA secretion can be suppressed or enhanced in an isotype-specific manner by a soluble factor(s), called IgA-binding factor (IgABF), produced by IgA FcR-bearing T cells. We now show that T cells from young NZB mice, cultured with high concentrations of IgA, produce an IgABF that has aberrant biologic activity when compared to IgABF produced from IgA FcR+ T cells of BALB/c mice. Although BALB/c IgABF normally suppresses proliferation and secretion by IgA-producing B cells, neither proliferation nor IgA secretion from normal murine IgA-B cells is suppressed by NZB IgABF. In fact, IgA secretion is significantly enhanced by NZB IgABF. We also present the first evidence of IgA anti-mouse erythrocyte (anti-MRBC) autoantibody-forming cells present in the spleens of NZB mice. Whereas BALB/c IgABF suppresses the in vitro generation of IgA anti-MRBC autoantibody-forming cells by NZB spleen cells, NZB IgABF enhances this response. Of particular interest is the development of IgA anti-MRBC autoantibody-forming cells in cultures of spleen cells from nonautoimmune BALB/c mice in the presence of NZB IgABF. These studies suggest that isotype-specific T cells factors might play an important role in the development of autoantibody-forming cells.     

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.     

Natural killer-type lysis of thymocytes from young mice by normal spleen cells in vitro

Normal spleen cells from 6- to 10-week-old mice, enriched for natural killer (NK) cells on a discontinuous polyvinylpyrrolidone-silica (Percoll) gradient, lyse thymocytes of young mice (less than or equal to 19 days old) in a short-term 51Cr release assay. The highest NK-type activity was found in band 3 (density less than or equal to 1.077 g/ml) of a four-step gradient. In some experiments band 2 (density less than or equal to 1.070 g/ml) also showed NK activity. Activity was not unequivocably detectable in cells before separation or in bands 1 and 4. These results also show that the thymocyte sensitivity is dependent on the age of the target cell. Sensitivity of very young thymocytes (less than or equal to 7 days old) was higher than that of thymus cells from 8- to 19-day-old donors. Moreover, it seemed that syngeneic target thymocytes were lysed more effectively than allogeneic. Thus, an NK-type cell population may have the ability to lyse immature thymic target cells at an early stage of their differentiation. This could be of importance as a physiological mechanism for controlling the T cell repertoire and its reactivity.     

Properties of fractionated spleen cells from NZB/W mice.

The unit gravity sedimentation technique was used to separate spleen cells from sevveral strains of mice. Settling patterns (plot of cell number against settling rate) were similar for BALB/c, DBA/2, C3H/He, and NZB/W mice of different ages. In particular, no subpopulation was found by this technique to be missing from the spleens of old NZB/W mice.A number of functional studies performed with the separated cells proved more informative than the settling patterns themselves. Fractions of cells which sedimented at a rate of between about 6 mm/hr and 10 mm/hr were enriched in responsiveness to PHA, Con A, and allogeneic cells. These fractions obtained from old NZB/W mice lacked such activities. However, the active fractions from young NZB/W spleens, which were enriched in θ-bearing cells, could restore the responsiveness of old NZB/W mice to primary immunization with sheep erythrocytes. These studies indicate that functional separation of spleen cells from NZB/W mice is possible and that activities lacking in whole spleens from old NZB/W mice are also lacking in the separate fractions. The ability to restore helper T cell function in old NZB/W mice with active fractions from young NZB/W mice has implications for further study and treatment of their autoimmune disease.     

Similar in vivo expansion of B cells from normal DBA/2 and autoimmune NZB mice in xid recipients

B cells from normal DBA/2 and autoimmune NZB mice were transferred into H-2-compatible xid recipients where they engrafted without irradiation or other manipulation of the host. The properties of these cells and their interaction with the host environment were analyzed at the single cell level with a splenic focus assay. When similar numbers of NZB and DBA/2 anti-DNA-producing B cell precursors were transferred, they expanded at similar rates in xid recipients. The rate of expansion varied with the strain of the recipient: it was fastest in autoimmune-prone NZB . xid and slowest in DBA/2 . xid hosts. Cells producing antibodies reactive with the autoantigen DNA proliferated substantially faster than those reactive with the non-autoantigen trinitrophenylated keyhole limpet hemocyanin. These results suggest that 1) B cells from NZB mice do not behave differently from DBA/2 B cells, 2) the internal milieu of the recipient into which the cells are transferred has an important effect on B cell proliferation, and 3) B cells capable of autoantibody production may have a selective growth and/or differentiation advantage relative to other B cells.     

Genetic studies in NZB mice. II. Hyperdiploidy in the spleen of NZB mice and their hybrids.

The presence of hyperdiploidy was studied in New Zealand black (NZB) mice and the progeny of NZB X DBA/2 crosses and backcrosses. Hyperdiploidy was observed in the spleens of a majority of NZB mice but not in DBA/2 mice at 1 year of age. In crosses of NZB with the DBA/2 strain, hyperploidy was observed only in backcrosses to NZB. Hyperdiploidy appeared to be determined by a recessivley inherited trait and was not related to the presence of other immunological abnormalities, including splenomegaly, hypergammaglobulinemia, and spontaneous antibodies cytotoxic for T cells and reactive with single-stranded DNA. Abnormal cells were not present in Concanavalin A-stimulated 48-h spleen cultures. There was no difference in the in vitro sister chromatid exchange rate between the autoimmune NZB strain and the non-autoimmune DBA/2 strain. Identification of NZB chromosomes by banding analysis showed that chromosomes 15 and 17 were frequently present in more than two copies in hyperdiploid spleen cells. NZB chromsomes also had reduced C-banding in an autosomal pair. These studies indicate that chromosomal abnormalities which occur in NZB mice may be useful as genetic and cytogenetic markers.     

Cytokine production by NZB, C58, and NZB X C58 recombinant inbred mice

Humans with autoimmune disease and autoimmune mouse strains such as NZB have been shown to produce reduced levels of the cytokines interleukin 1 and interleukin 2. The NZB X C58 recombinant inbred (N X 8 RI) strains exhibit certain of the autoimmune characteristics of the NZB strain. Their abilities to produce interleukin 1 and interleukin 2 have been tested. Deficiencies in the production of one or both of these cytokines were observed in the N X 8 RI strains. Decreased interleukin 2 production was not due to inability to respond to concanavalin A or allogeneic stimulation, nor to altered kinetics of the response. No correlation was observed between cytokine deficiencies and the inherited autoimmune characteristics previously studied in these strains. One especially interesting strain was N X 8 RI 16, which made high amounts of interleukin 2 but no detectable interleukin 1.     

Autoantibodies against human tropomyosin isoform 5 in ulcerative colitis destroys colonic epithelial cells through antibody and complement-mediated lysis

INTRODUCTION: Patients with ulcerative colitis (UC) have IgG1 antibodies in serum and colon against human tropomyosin isoform 5 (hTM5), a cytoskeletal microfilament protein found intracellularly and on the surface of colonic epithelial cells (EC). These antibodies may be pathogenic in UC. METHODS: Sera from patients with UC (n=110) or Crohn's disease (CD) (n=50) and from healthy individuals (Hl) (n=30) were preincubated with recombinant hTM5 or bovine serum albumin (BSA), then cultured for 4h with (51)Cr-labelled colonic adenocarcinoma cells (LS180). Cytotoxicity was determined by (51)Cr release assay. RESULTS: All serum samples lysed up to 36% of LS180 cells regardless of the source of the serum. However, adding hTM5 to UC, but not to CD or HI, sera reduced cytotoxicity by up to 75%. This hTM5-induced inhibition of cytotoxicity was found especially with sera from UC patients with active disease, and was found even after total colectomy. The hTM5-induced inhibition was mediated by purified IgG from UC sera. Complement was involved since hTM5-induced inhibition of cytotoxicity declined with either heat inactivation of the sera or premixing sera with Fc fragments. CONCLUSIONS: This study shows that hTM5-specific IgG autoantibodies present in UC sera destroy LS180 cells by antibody and complement-mediated lysis. Such a phenomenon was not seen in CD or HI. This suggests an autoantigenic role of hTM5 and anti-hTM5 antibodies in the pathogenesis of UC. This observation may lead to novel diagnostic and therapeutic possibilities.