Natural autoantibodies are involved in the haemolytic anaemia of NZB mice.

NZB is a mouse strain that spontaneously develops autoimmune haemolytic anaemia at 10-12 months of age. We analysed the autoantibodies present throughout their life and compared them to natural autoantibodies found in the normal mouse. Sera and Coombs' antibodies eluted from red blood cells (RBC) were tested for their activities against RBC and a panel of antigens: actin, myoglobin, myosin, tubulin, spectrin, DNA and trinitrophenyl bovine serum albumin (TNP-BSA), F(ab')2 and Fc fragments of IgG by using enzyme immunoassays (EIA) and Western blotting analysis of RBC membrane extracts. In NZB mouse sera, activities of IgM and IgG against the whole panel, compared to those of sera from age-matched BALB/c mice, increased progressively throughout life with oscillating values in parallel with the anti-RBC activity. Two periods of autoantibody production seem to exist: the first is characterized by a fluctuating high level of IgM and stable level of IgG natural autoantibodies, and the second by a rise of IgG natural autoantibodies in parallel with IgG anti-RBC antibodies. The presence of idiotype D23 (IdD23), which is characteristic of natural polyspecific autoantibodies, was high on serum IgM and low on IgG autoantibodies throughout life. To further analyse autoantibody level oscillations, we tested IgM and IgG fractions after their separation from whole serum and observed highly enhanced autoantibody activities of both IgM and IgG. These autoreactivities markedly diminished when the separated IgM and IgG fractions were recombined, suggesting humoral control of the autoreactivity as we had already noted for IgG in normal animals. During the first period of autoantibody production, IgM and IgG antibodies eluted from RBC (Combs' antibodies) and those eluted from serum using an RBC-immunoadsorbent (circulating antibodies) reacted with all RBC membrane components, with all antigens of the panel and with F(ab')2 and Fc. Some of these reactivities were comparable to those exhibited by a monoclonal antibody recognizing bromelain-treated RBC. In the second period, both IgM and IgG Coombs' antibodies reacted more strongly with spectrin, and exhibited new specificities, for example against the band 3 polypeptide. IdD23 was abundant on Combs' IgG antibodies in the second period. Taken together, these data suggest that IgM and IgG natural autoantibodies, able to recognize not only RBC antigens but also other antigens, particularly F(ab')2 and Fc fragment of IgG, predominate in Coomb's antibody population.(ABSTRACT TRUNCATED AT 400 WORDS)

NZB serum factor (NZB-SF)-B precursor cell maturation factor. II. In vivo effects of NZB-SF or mAb against NZB-SF on B lineage cell populations.

In vivo effects of NZB serum factor (NZB-SF), which enhances the maturation of B precursor cells in vitro, were examined. Immunoaffinity-purified NZB-SF from young NZB mice was injected into B6 mice intraperitoneally twice weekly, five times total (5 micrograms/dose/mouse). Control mice were given 0.01% albumin. Then the B lineage cell populations defined phenotypically (sIg+ cells, B220+ cells, and AA4.1+ cells) or the numbers of colony-forming B lineage cells were examined. NZB-SF-treated B6 mice exhibited a decrease in the percentage of B precursor cells in marrow, even though the percentage of sIg+ cells in marrow or spleen did not differ from controls. In contrast, the frequency of colony-forming B cells in marrow and spleen, especially sIg- colony-forming B cells in marrow, increased significantly in NZB-SF-treated mice as compared to controls. In addition, monoclonal antibody (mAb) against NZB-SF was injected weekly for 9 weeks into NZB mice beginning at 7 weeks of age. mAb vs NZB-SF at a dose of 5 micrograms per mouse per injection, as stated above, prevented the decline of sIg- colony-forming B lineage cells which usually occurred in the adult NZB mice (greater than 16 weeks). This treatment also prevented, in part, the decline of the B220+ cell population which normally occurs in the marrow with increasing age. Thus NZB-SF impressively influences the composition of B lineage cell populations in normal B6 mice and may account for abnormal changes of B lineage cell populations observed in NZB mice.

Up-regulation and down-regulation of cell surface and mRNA expression of CD5 antigen by various humoral factors on murine 70Z/3 pre-B cell leukemia cell line: IL-4 down-regulates CD5 antigen expression.

CD5, a pan-T cell antigen, is expressed on a minor subset of normal B lymphocytes and on cells of most B lineage tumors or transformed B cells in both man and animal models. In the present study, the effects of various humoral factors on CD5 expression by cells of a subcloned 70Z/3 murine pre-B leukemia cell line were investigated. Among the humoral factors studied, only LPS up-regulated CD5 expression on 70Z/3 cells (three- to fourfold) in a dose-dependent manner. However, this up-regulatory effect of LPS was not observed when cells were cultured in serum-free medium. NZB-serum factor (NZB-SF), a cytokine we have identified and shown to enhance the maturation and proliferation of immature B cells, synergistically enhanced CD5 expression in the presence of suboptimal doses of LPS. IL-4 down-regulated CD5 expression by 70Z/3 cells induced by LPS or LPS plus NZB-SF in a dose-dependent manner. IL-4 also suppressed spontaneous CD5 expression by 70Z/3 cells. No other cytokine tested showed an inhibitory effect. LPS, IFN-gamma, NZB-SF, and IL-1 enhanced sIg expression on 70Z/3 cells and their action on sIg expression was not inhibited by IL-4. Thus, the down-regulatory action of IL-4 on CD5 expression appeared specific for this antigen. IFN-gamma, which inhibits IL-4 induced CD23 and DR expression on B cells, does not abolish the down-regulatory action of IL-4 on CD5 expression by 70Z/3 cells. Changes in mRNA levels on coding CD5 were also examined following the incubation of 70Z/3 cells (24 hr) in the presence of humoral factors which can influence CD5 Ag expression. The levels of mRNA for CD5 Ag were moderately increased in the presence of LPS and NZB-SF. IL-4 appeared to suppress the actions of NZB-SF and LPS at least in part by reducing the levels of mRNA encoding CD5.

Elevated titers of cell-free interleukin 2 receptor in serum of lupus mice.

Activation/proliferation of mouse and human T and B cells is associated with expression and subsequent release of interleukin 2 receptors (IL 2R) into the milieu. The soluble form of IL 2R, at least in part, retains its ability to bind to IL 2 and to anti-receptor antibodies, but its exact structure remains unknown. Because systemic lupus erythematosus (SLE) is associated with T and B cell activation, we have used monoclonal anti-IL 2R antibodies in an ELISA to measure levels of IL 2R in sera of various lupus strains. High levels of the released receptor were found at an active clinical stage in sera of four autoimmune strains of mice homozygous for the lpr (lymphoproliferation) gene that causes T cell expansion, massive lymphoid organ enlargement, and promotes the autoimmune process. High levels were also found in lupus mice characterized primarily by B cell proliferation (BXSB males) and in (NZB X W)F1 mice characterized by T and B cell activation. Similarly high IL 2R serum levels could be induced experimentally in normal mice injected with immunostimulants such as bacterial lipopolysaccharide or Freund's complete adjuvant. The results indicate that IL 2R serum levels may provide a good marker of ongoing lymphoid cell activation/proliferation, and thus might be useful in the follow-up of patients with systemic autoimmune or other lymphoproliferative disorders. The biologic roles, if any, of the soluble form of IL 2R and its effects in normal and abnormal conditions remain to be determined.

Erythrocyte deformability changes in autoimmune hemolytic anemia during development of NZB mice and their (NZB/NZW)F1 hybrid.

NZB and B/W hybrid mice develop compensated hemolytic anemia during the first year of their life. By the age of 3-5 months, their erythrocytes show evidence of spherocytosis, increased osmotic fragility and decreased whole cell deformability, as measured by ektacytometry, a laser diffraction technique. The presence of spherocytes with decreased surface area/volume ratio was confirmed by scanning electron microscopy and osmotic gradient ektacytometry. Whereas these abnormalities persisted and worsened in the NZB mice with further growth, they gradually improved and reverted to normal by the age of 12 months in B/W mice. This spontaneous improvement seems to be due to the accumulation of red cell membrane lipids reflecting the lipemia of immune complex nephritis in B/W mice. The implications of these findings in the modulation of autoimmune hemolytic anemia are discussed.

Chromosome damaging agent of low molecular weight in the serum of New Zealand black mice.

Two substrains of NZB mice were developed by selective matings according to chromosome breakage frequencies in direct bone marrow preparations: HB, a line with increased chromosome breakage, and LB, a line with low or normal breakage rates. The serum of NZB mice from the HB strain contains a chromosome breaking factor that produces chromosome abnormalities in human lymphocytes. This clastogenic substance could not be detected in serum of NZB line. Nor does it exist in serum of Balb, C3H, C57 B1, Swiss, or AKR mice, in which no chromosomal breakage was observed in bone marrow. The breakage factor has a molecular weight between 1000 and 10,000 daltons.

[Diurnal variations in the cellular composition of peripheral blood in mice of different genotypes]. / Sutochnye kolebaniia kletochnogo sostava perifericheskoi krovi u myshei raznykh genotipov.

Changes of the circadian rhythm of the general numbers of nucleus-containing cells and leucograms of peripheral blood in NZB mice with autoimmune spontaneous hemolytic anemia and in C57BL mice without visible disturbances in the system of immunostructural homeostasis were the most obvious in the white blood in NZB mice: a) disappearance of the circadian rhythm of the general number of nucleus-containing cells in peripheral blood owing to the inversion of circadian rhythm of the absolute number of neutrophiles; b) changes of circadian fluctuations of lymphocytes-neutrophiles ratio. The observed differences in circadian rhythms of hematological indices in NZB and C57BL mice seem to stem from insufficiency of adrenal cortex function and/or changes in functional state of hemopoietic tissue which can occur during the development of autoimmune processes.