An ELISA for anti-thymocyte autoantibody in NZB mice. Comparison with the cytotoxic assay.

An ELISA is described for the detection of anti-thymocyte antibody found in the serum of NZB autoimmune mice. This assay avoids the disadvantages of previous assay techniques which require carefully selected complement sources and/or radioactive labelled target cells. Thymocytes from a variety of strains of mice can be used as the antigenic targets, and the plates stored as long as 1 month. The lymphoma line EL4 can be used as the antigenic target in this assay, but it not as sensitive to most serum pools as thymocytes.

Whole blood culture for measuring mitogen induced T cell proliferation provides superior correlations with disease state and T cell phenotype in asymptomatic HIV-infected subjects.

Proliferative responses to a panel of mitogens were compared in parallel for two sources of cells, whole blood (WB) and conventionally prepared peripheral blood mononuclear cells (PBMC), obtained from asymptomatic HIV seropositive and control subjects. Weak but statistically significant correlations of the proliferative responses were observed. Use of either lymphocyte source produced significant differences in the proliferative responses between the HIV seropositive and control subjects, but the use of WB was more powerful, with a smaller sample size being required to discriminate between the proliferative responses of the two study groups. Furthermore, proliferative responses using WB gave strong and highly significant correlations with a number of important changes in the surface marker phenotype of the lymphocyte populations in the HIV seropositive subjects including CD4, CD8, CD4:CD8 ratio and certain CD8 subsets, whereas strong correlations were not observed with the PBMC. The response of WB lymphocytes to staphylococcal enterotoxin B (SEB) was highly reproducible and provided the best discrimination between HIV-infected and control subjects. We conclude that the use of WB for measuring lymphoproliferation is easy, rapid, accurate, and discriminative for assessing and following the changes in immune function which occur in HIV seropositive subjects, applicable in the clinical as well as in the research setting.

Mechanism of the immunoregulatory effect of cloned T cells on proliferation in mixed lymphocyte responses.

The mechanism of the suppression of the mixed lymphocyte response (MLR) by T cell clones derived from the murine AMLR was studied in a system employing .45 microns membrane chambers to isolate the cellular reactants. The clones effectively suppressed MLR regardless of the MHC haplotype of the reactants in the absence of direct cellular contact, via a soluble factor able to pass through this membrane. Utilizing specific neutralizing antibodies, this factor was shown to be principally TNF alpha, with a contributing effect of gamma interferon. Production of TNF alpha by the cloned T cells was confirmed by direct assay of clone culture supernatants.

Animal models of fibrosis.

This review of the animal models for scleroderma described in the literature demonstrates that there are available a number of induced and spontaneous systems in which to study various aspects of this complex disorder. Each model has its strengths in mimicking certain aspects of the disease--inflammatory, immunologic, or fibrotic--as well as important differences or unstudied aspects, as emphasized in the foregoing discussion. It is apparent that each of these models can contribute to our knowledge of the mechanisms underlying this presently incurable disorder. The most extensively studied model from the histologic, immunologic and biochemical viewpoint is the TSK/+ mutant mouse. Its principal deficiencies are the absence of inflammatory, immunologic, vascular, gastrointestinal, and articular involvement. Our new findings reported in this review demonstrate that certain previously undefined immunologic alterations commonly observed in autoimmune diseases are present in this mutant mouse. Table 3 summarizes the available data on the two most studied models, the TSK/+ mouse and the UCD-L200 chicken, and compares them with SSc. It can be readily seen that there is the need for further studies of many aspects of these models and of the human disease to extend our present knowledge and hopefully achieve a better understanding of the underlying causes and pathologic mechanisms. The existence of genetic mutant animals holds the promise of applying the techniques of molecular biology to address these questions.

Immunoregulatory effects of cloned T cells on B-cell responses: comparison of autoimmune and non-autoimmune derived clones.

T-cell clones were derived from autoimmune prone (NZB) and non-autoimmune (C58) strains of mice and tested for their effects in several assays of B-cell responsiveness. Clones from the C58 strain suppressed lipopolysaccharide LPS-stimulated B-cell proliferation, activation and immunoglobulin synthesis. In contrast, an NZB-derived clone enhanced these measures of B-cell response. The effects of the NZB clone were more notable on splenic target populations taken from mice 6 months of age or older. MHC-compatible DBA/2 spleen cells also showed enhancement of B-cell activation, but not of immunoglobulin synthesis by the NZB clone. It has been shown previously that all of the clones suppress T-cell proliferative responses. A potentially important skewing of the immune system toward humoral rather than cellular responses is therefore mediated by this clone derived from an autoimmune strain.

Immunological function of kidney-infiltrating lymphocytes from aged NZB mice.

A striking aspect of autoimmune kidney disease in the NZB mouse strain is the perivascular infiltration of lymphoid cells. Upon release by enzymatic digestion of kidney tissue from animals 6 months of age or older, these cells have been found to exhibit a high level of immunologic activity not seen in younger mice or in nonautoimmune strains. Kidney-derived cells were found to respond to T and B cell mitogens at levels ranging up to those observed for peripheral blood, and in some cases splenic lymphocytes, from the same animals. An enhanced proliferative response to autologous and allogeneic stimulation was observed compared to these other lymphoid sources. Both spontaneous and LPS-stimulated immunoglobulin synthesis were noted with all three populations, which could be totally or partially blocked by cycloheximide. Selective localization of autoantibody-producing cell populations was observed, with anti-erythrocyte antibody restricted to splenocytes and PBL, and the anti-dsDNA implicated in immune complex formation found only in kidney-derived cell culture supernatants.

T-suppressor clones derived from murine AMLR.

Panels of cloned T-cell lines were derived from the autologous mixed lymphocyte reactions of NZB and C58 mice. These clones were all Thy 1+. In addition, various clones expressed appropriate Ia, Lyt 1 and/or Lyt 2 antigenic specificities. None of these clones produced the lymphokines IL-2, CSF or AMLR-helper factor. The clones suppressed fresh syngeneic AMLR and MLR responses when added at low cell numbers at the initiation of culture. This suppression was not abrogated by treatment with mitomycin c or reversed by the addition of a source of T-cell growth factor. The mechanism of suppression was not cytotoxicity, as the clones were non-cytotoxic for either syngeneic or allogeneic cells. Many of the clones appeared to require the presence of Lyt 2+ cells in the MLR responding population to suppress, and therefore can be classified as T-suppressor inducers. Two clones did not require the Lyt 2+ subset to suppress the MLR, and are therefore T-suppressor effectors.

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.

Autologous mixed lymphocyte reactions in NZB mice: analysis in recombinant inbred lines shows a T cell defect unrelated to autoantibody production.

The autologous mixed lymphocyte reaction (AMLR) has been examined in the AMLR-deficient strain, NZB, in C58 mice, which have normal AMLR responses, and in the (NZB x C58) recombinant inbred (NX8 RI) lines. Half of the NX8 RI lines had deficient AMLR and half were comparable in reactivity to the C58 parent strain. AMLR unresponsiveness did not correlate in the NX8 RI lines with H-2 or any other marker known to affect immune recognition. Non-T cells from H-2k NX8 RI lines which had deficient AMLR were able to stimulate an MLR when cultured with T cells of C58 origin. AMLR-positive H-2d lines responded to NZB stimulators, whereas NZB T cells did not respond to any of the H-2d NX8 RI lines but did exhibit strong alloreactivity. Thus, the AMLR defect in NZB mice is due to a T cell lesion. Furthermore, since AMLR deficiency in the NX8 RI lines did not correlate with a positive Coombs test or the production of naturally occurring thymocytotoxic antibody, we conclude that autoimmunity in NZB mice is a complex disease resulting from the inheritance of multiple independently segregating genes.

Immunological characterization of (tight skin/NZB)F1 hybrid mice with connective tissue and autoimmune features resembling human systemic sclerosis.

A new murine model of Systemic Sclerosis (SSc) has been developed by breeding the Tsk+/+pa tight skin mouse (TSK) and the autoimmune disease-prone NZB strain to produce an F1 hybrid displaying the connective tissue abnormalities of the TSK parent and the autoimmune abnormalities of the NZB parent. The interscapular skin thickness in the (TSK/NZB)F1 was significantly greater than in the (+pa/NZB)F1 litter mates. Protein biosynthesis in skin punch biopsies was over 3.5 times higher in the (TSK/NZB)F1 mice than in controls. Hydroxyproline analyses confirmed that the increase in protein synthesis was primarily in collagen. These (TSK/NZB)F1 mice were tested for a number of cellular and humoral autoimmune manifestations. Autoantibodies, including antinuclear antibodies (ANA) and anti-DNA, were present in their sera, and the proliferative response to autologous lymphocyte stimulation (AMLR) was decreased as is commonly observed in murine and human autoimmune disorders. These results indicate that the (TSK/NZB)F1 mice display connective tissue and immunologic abnormalities resembling those present in human SSc and, therefore, these mice may be a valuable model for the study of the disease.

Age-related decline in cell-mediated immunity in the C58 leukemic mouse strain.

The leukemia-prone C58 strain of mouse was examined for age-related changes in cellular immune function. Proliferative responses of lymphocytes to autologous and allogeneic stimulator cells [autologous mixed lymphocyte response (AMLR) and mixed lymphocyte response (MLR), respectively] and to mitogens were tested both prior to and around the usual age of disease onset which occurs at 7-8 months. Leukemia in these animals was defined by elevated peripheral blood and splenic white blood cell counts. The AMLR declined greater than 30% by 6-7 months of age and was virtually absent by 8 months of age even in animals that were not overtly leukemic. The MLR declined precipitously (greater than 95%) at 9 months of age. Both declines occurred at a younger age in C58 mice than in nonleukemic strains. Mixing experiments with cells from young and old animals indicate a defect in the Ly 1+23-, L3T4+ responding T cells. No evidence indicating a role for suppressor cell activity in this decline of cell-mediated immunity could be found. Deficiencies in cytokine (IL-2 and IL-1) production were not observed except in the oldest mice tested. Around the usual time of disease onset, splenic natural killer (NK) cell activity declines sharply even in nonleukemic mice. Cell-mixing experiments showed no evidence of suppressor cell activity by spleen cells from older mice, leukemic or nonleukemic, on the NK cell activity of young adult animals. Interferon alpha, beta treatment enhanced the NK activity of cells from old mice but did not restore the level of activity seen in young mice. Evidence has therefore been found for a premature decline in cellular immune function in two responses with proposed immunoregulatory roles, the AMLR and NK cell activity. It is possible that their decline could play a predisposing role in the onset of this retroviral leukemia or that these cell populations may be the target of the retrovirus.

Independent segregation of NZB immune abnormalities in NZB x C58 recombinant inbred mice.

The study of NZB x C58 recombinant inbred mouse strains has revealed independent segregation of naturally occurring thymocytotoxic antibody and Coombs' anti-erythrocyte autoantibody. The lack of concordance of either of these autoantibodies with known heavy and light chain markers suggests that the autoantibodies are produced as a result of regulatory gene defects rather than alterations of antibody structural genes. Further, lack of concordance of the various autoimmune traits with each other or with H-2 or virus expression suggests that the autoimmune phenotype is not the result of a single "autoimmunity' gene but rather the outcome of faulty regulation of a number of independently segregating genes.

Occurrence of interleukin-1 in human synovial fluid: detection by RIA, bioassay and presence of bioassay-inhibiting factors.

Synovial fluid (SF) from patients with osteoarthritis (OA), rheumatoid arthritis (RA), and various other arthridites was analyzed to assess the prevalence of interleukin-1 (IL-1) using both radioimmunoassay competitive inhibition specific for the beta form of IL-1 and the D10.G4.1 cell line bioassay which measures both alpha and beta forms of IL-1. Using radioimmunoassay competitive inhibition, IL-1 beta was found in 45% and 60% of individual samples from patients with OA and RA respectively. When RA and OA SF were examined in sequentially obtained samples, IL-1 beta occurred in one or more samples from 8 of 10 patients studied, suggesting the probability that it can be produced at some time in SF by all patients with these conditions. No correlation between SF leukocyte counts and the occurrence of IL-1 beta was noted and no effect of antiinflammatory drug treatment on the prevalence of IL-1 beta was found. When tested for the presence of IL-1 by the D10.G4-1 cell line, 66% and 50% of RA and OA patients respectively were found to contain IL-1. These were not in total concordance with results obtained by RIA. Of all SF tested, seven were negative by RIA but positive by D10.G4.1 and these are considered to contain IL-1 alpha. Seven samples which were RIA positive and D10.G4.1 negative were tested for their ability to inhibit IL-1 responses in the bioassay. Five of these contained inhibitor and one markedly enhanced the proliferative response of D10.G4.1 to a known amount of IL-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for autoimmunity in the tight skin mouse model of systemic sclerosis.

The tight skin mouse strain has been proposed for use as an animal model of systemic sclerosis because this animal exhibits a condition that has biochemical and pathologic similarities to the human disease. To date, however, evidence of inflammatory and immunologic changes in the tight skin mouse has been scarce. We demonstrated the presence of antinuclear antibodies in approximately half of these mice ages 8 months and older. This suggests that there is an autoimmune component in their disease process. The antibodies were identified as anti-topoisomerase I by a characteristic staining pattern on HEp-2 cells and by Western blot analysis. Except for a low incidence of anti-DNA antibodies, none of the other parameters tested, including mitogen responses, lymphokine production, and anti-erythrocyte antibodies, was indicative of immune system dysregulation.

Ontogeny of interleukin 2 responsive cells in the fetal thymus.

The ontogeny of IL 2-responsive cells in the thymus of CBA/J mice was examined in neonatal animals and in fetuses at 14, 16, and 18 to 20 days gestation. The thymocytes were tested for responsiveness to 2 micrograms/ml Con A, TCGF, IL 2, and co-stimulation by Con A plus TCGF or IL 2. These responses were compared with those of thymocytes of 6- to 8-wk-old CBA/J. Thymocytes (1 X 10(5)) were cultured, and the reaction was measured at maximum response (96 hr). Neonatal animals gave an unusually high response to TCGF or partially purified IL 2 alone, approximately five times greater than the adult. A low but significantly enhanced proliferation, stimulated by partially purified IL 2 alone, was observed with 14-day fetal thymocytes, even though cultures of these cells in medium alone had higher background proliferation than any other age tested. In the co-stimulator reaction, proliferation significantly above background was measured at 16 days of gestation with Con A plus TCGF. The magnitude of the co-stimulator reaction increased with age, especially between the 16th and 18th day of gestation and immediately after birth.

Colony stimulating factor occurs in both inflammatory and noninflammatory synovial fluids.

Synovial fluids (SF) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) and various other arthritides were examined for the presence of colony stimulating factors (CSF). CSF was found in 7 of 13 (54%) SF from OA patients and in 8 of 12 (67%) SF from RA patients. It was also found in SF from patients with other arthropathies including 5 of 5 samples from patients with septic arthritis. Inhibition studies employing monospecific antisera indicated that in both RA and OA, CSF was of the macrophage type (M-CSF). While CSF was found in both inflammatory and noninflammatory effusions, significantly greater numbers of colonies were stimulated by RA SF than by OA SF and in general greater numbers of colonies correlated with higher SF leukocyte counts. Our data suggest that CSF as well as other cytokines may be involved in the perpetuation of joint destruction that occurs in various rheumatological conditions.

Functional consequences of perceived interleukin deficiencies? Analysis employing NZB x C58 recombinant inbred mice.

NZB mice have previously been shown to be deficient in the production of interleukins 1 and 2 (IL-1, IL-2) during the development of autoimmune disease. One or both of these defects have been inherited in certain of the NZB X C58 recombinant inbred strains (N X 8 RI). Certain of these strains have been selected to examine further the effect of decreased production of IL-1 and/or IL-2. The interleukin deficiencies found in vitro were not due to the presence of an inhibitor/suppressor nor was any activity found intracellularly upon water lysis of stimulated cells. Despite profound IL-1 and/or IL-2 deficiencies measured in vitro, all of the N X 8 RI lines examined were found to be capable of producing IL-1 in vivo as shown by their serum amyloid A response to endotoxin injection. We conclude from these studies that defects in IL-1 production measured in vitro do not reflect inability to produce this lymphokine in vivo. Young, IL-1 deficient NZB mice generated CTL to TNP-self but old, IL-2 deficient NZB mice did not. Since all other strains were found to generate cytotoxic T cells to TNP-self regardless of interleukin defects, we also conclude that the cytotoxic T cell defect in NZB mice is due to some presently unknown factor in addition to IL-2 deficiency. The relationship of decreased production of interleukins to the development of autoimmunity remains undefined.