Genetic aspects of the reaction of humoral immunity to collagen in humans and animals. Report I. Analysis of polymorphism of autoantibody levels to collagen type I in mice NZB X NZW (F1)

The study of polymorphism of humoral immunoreactions to the type I (AC) collagen in CBA/Lac, C57B1/6-J inbred mice and NZB X NZW (F1) hybrids showed the presence of genetically determined variability of the above mentioned trait. The analysis of intralinear dispersions of AC levels in NZB X NZW (F1) mice revealed sex dimorphism and age variability of the trait. A suggestion was made that sex hormones are important factors in ontogenic formation and modulation of autoimmunity to the type I collagen in NZB X NZW (F1) 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.     

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.     

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.     

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.     

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.     

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.     

Studies of immune responses in mice prone to autoimmune disorders: I. Heterogeneity of the affinities of antihapten antibodies produced by NZB,NZW, and related strains of mice

Mice of the NZB and NZW strains and their F₁ hybrid produce antihapten plaque-forming cell (PFC) responses to T-dependent antigens (trinitrophenylated bovine gamma globulin and dansylated keyhole limpet hemocyanin) which are of unusually restricted heterogeneity of affinity, are relatively lacking in low-affinity PFC, and are of relatively high average affinity. Since some low-affinity PFC are present in NZB mice early after immunization, the results suggest a particularly marked down-regulation of low-affinity antibody production by these strains. The non-autoimmune-prone F₁ hybrid (NZB × CBA) produces a typical heterogeneous response containing a high proportion of low-affinity PFC. Thus, the tendency to down-regulate low-affinity PFC is not inherited as a simple Mendelian dominant trait. The response of NZB mice to T-independent antigens does not show the same restricted heterogeneity of affinity. In fact, late after injections of trinitrophenylated Ficoll, NZB mice tend to have more heterogeneous responses than nonautoimmune-prone BALB/c mice in which a marked down-regulation of high-affinity antibody-producing PFC is seen. The possible relationship between these unusual features of the immune response of NZB and some related strains and their tendency to develop autoimmune disease is discussed.     

Repetitive usage of immunoglobulin VH and D gene segments in CD5+ Ly-1 B clones of (NZB x NZW)F1 mice.

The usually small Ly-1 B cell population is markedly increased in older mice by expansion of certain clones. This results in a cellular picture very similar to human B chronic lymphocytic leukemia. Here we report a molecular analysis of the immunoglobulin gene rearrangements of the Ly-1 B cell populations in (NZB x NZW)F1 females. We find that (i) the number of clones found in the peritoneum (a major tissue source of Ly-1 B cells) decreases with age till mono- or biclonality is common by approximately 6 months, (ii) many clones from different mice show the same size rearrangements at both the Ig heavy and light chain loci and (iii) the IgH rearrangements found in a clone isolated from the spleen of one mouse are a subset of those found in the peritoneum of the same mouse, implying migration occurs from the peritoneum to the spleen. Molecular cloning and sequencing of the IgH rearrangements from the peritoneal clones of one B/W mouse revealed that all productive rearrangements used the identical unmutated VH and D elements joined to different JHS. Indeed, two VDJH4 rearrangements were recovered which were identical but for six junctional (N region) nucleotides. The conservation of VH and D segment usage in the rearrangements of these Ly-1 B cell clones could indicate some strong selective pressure for clonal expansion (for example antigen selection) operates via the immunoglobulin molecules of these cells. Southern analyses of other (NZB x NZW)F1 mice with this cloned VH and the usage of the same or similar VH genes among a number of Ly-1 B origin tumors in other mouse strains indicate the generality of this repetitive VH gene usage in individual mice.     

Lead and immunity: II. Suppression of humoral immune response to Hymenolepis nana in mice.

Serum protein changes in mice treated for varying durations with lead nitrate and subsequently infected with 1000 H. nana eggs were compared with their counterpart controls, only treated and only infected groups. Decreased values of beta and gamma globulins in all the experimental groups along with higher worm recoveries indicate suppression of humoral immune response by lead in association with higher worm recoveries indicate suppression of humoral immune response by lead in association with the cellular components since these globulins are known to contain antibodies. Lead treatment for a duration of 45 days proved to be most effective in suppressing the immune response.     

Genetic control of lymphocyte suppression. I. Lack of suppression in aged NZB mice is due to a B cell defect.

Con A-activated cells from old NZB mice were found capable of inhibiting the polyclonal response of cells from young NZB and BALB/c animals. Furthermore, Con A-preactivated spleen cells from young NZB and BALB/c mice did not significantly affect the response of spleen cells from old NZB mice. These results suggest that the defective suppressive activity in old NZB mice may be traced to a defect at the B cell level.     

Immunity to type XI collagen in mice. Evidence that the alpha 3(XI) chain of type XI collagen and the alpha 1(II) chain of type II collagen share arthritogenic determinants and induce arthritis in DBA/1 mice.

To determine whether native bovine type XI collagen (BXI) is arthritogenic, five strains of inbred mice were immunized with BXI/CFA. Arthritis was not observed in any of these strains, though it was prevalent in DBA/1 and B10.RIII controls immunized with bovine type II collagen (BII). Antisera from BXI-immunized mice reacted with mouse type XI collagen (MsXI), weakly with the alpha-chains of BXI, and minimally with mouse type II collagen (MsII). However, antisera to BII reacted with MsII and MsXI, indicating antibodies to conformation-independent epitopes shared by alpha 1(II) and alpha 3(XI). Mice immunized with BXI containing a small amount of BII developed arthritis much like those immunized with BII; sera from these mice reacted with MsXI and MsII. Delayed-type hypersensitivity responses differed from IgG responses, i.e., BXI elicited responses to alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II); BII, to alpha 3(XI) and alpha 1(II) exclusively. To determine whether alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II) are arthritogenic, DBA/1J mice were immunized with each alpha-chain. Arthritis was seen in mice injected with alpha 3(XI) or alpha 1(II). Sera to both alpha-chains reacted similarly with MsII and peptide fragment alpha 1(II)-CB11. Epitope mapping using polyclonal and mAb to type II collagen revealed that all polyclonal and 11 of 14 mAb reacted with alpha 3(XI) and alpha 1(II), whereas three mAb reacted only with alpha 1(II). In conclusion, BXI is immunogenic but not arthritogenic in five strains of mice, whereas alpha 3(XI) and alpha 1(II) are arthritogenic and immunogenic in DBA/1 mice and share greater than or equal to 11 epitopes recognized by autoantibody.     

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

Immunologic abnormality in NZB/NZW F1 mice. Thymus-independent occurrence of B cell abnormality and requirement for T cells in the development of autoimmune disease, as evidenced by an analysis of the athymic nude individuals

Both NZB nu/+ and NZW nu/+ mice were microbially clean by cesarean section. The (NZB x NZW)F1 hybrid (NZB/W) nu/nu mice and nu/+ littermates were then generated by mating of NZB nu/+ with NZW nu/+mice under specific pathogen-free conditions. The female NZB/W F1 nu/nu mice did not develop autoimmune kidney disease, whereas all of nu/+ female littermates mice exhibited proteinuria and died of renal failure with a 50% survival time of 35 wk. Namely, nude mice had no signs of proteinuria up to the time of their death caused by other diseases rather than glomerulonephritis, and their mean survival time was greater than 45 wk. Nude mice had also no anti-ssDNA antibody in their serum. However, splenic B cells of NZB/W nude mice exhibited hyper-responsiveness to both LPS and B151-TRF2, a T cell-derived polyclonal B cell-stimulation factor, and produced large numbers of Ig-secreting cells and anti-TNP plaque-forming cells as well as anti-ssDNA antibody comparable to the nu/+ littermate mice. Interestingly, thymus-engrafted NZB/W nude mice developed autoimmune disease exemplified by the induction of anti-ssDNA antibody and proteinuria at approximately the same time as their nu/+ littermates. These results indicate that the B cell hyper-responsiveness found in NZB/W mice is apparently determined by the T cell-independent process, and T cells are obligatorily required for the development of autoimmune disease in NZB/W mice.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.