Oestrogen induced suppression of collagen arthritis. IV: Progesterone alone does not affect the course of arthritis but enhances the oestrogen-mediated therapeutic effect.

The effects of 17 beta-oestradiol and progesterone on the development of type II collagen-induced arthritis (CIA) and the anti-type II collagen (CII) autoantibody response were investigated. Treatment with physiological doses of 17 beta-oestradiol, inducing serum levels below the estradiol peak at the end of pregnancy, abrogated the development of arthritis and suppressed the anti-CII autoantibody response. Treatment with progesterone alone did not have significant effects on the development of arthritis or on the anti-CII autoantibody response. However, a combined treatment with both progesterone and oestrogen in physiological doses induced a more pronounced suppression of CIA than the suppression induced with oestrogen treatment alone. These findings suggest that oestrogen, but not progesterone, may be the critical factor to explain the pregnancy-related down-regulation of CIA.

Inoculation candidiasis in a murine model of severe combined immunodeficiency syndrome.

To further elucidate the importance of T- and B-lymphocyte-mediated responses in host defense against systemic infection with Candida albicans, we studied this infection in a murine model of severe combined immunodeficiency (SCID). The course of inoculation candidiasis in these mice, which lack functional T and B lymphocytes, was compared with that in immunologically normal BALB/c mice. Mice were inoculated intravenously with 10(5) yeast cells. Quantitative cultures of liver, spleen, and kidneys were performed with necropsy specimens obtained 1, 3, 7, 10, 14, and 21 days after this intravenous inoculation. The differences in the time courses of recovery of organisms from liver and spleen specimens were not significantly different in the SCID mice compared with the BALB/c mice. The recovery of C. albicans from the kidneys was significantly lower in the SCID mice, indicating less persistence of the organism in the kidneys of the SCID mice than in those of the BALB/c mice. These data indicate that defense mechanisms other than T- and B-lymphocyte-mediated mechanisms are primarily responsible for host defense against inoculation candidiasis.

T cell hyperproliferation in autoimmunity prone obese strain (OS) chickens is independent of abnormal mitogen binding in vitro and can be demonstrated in vivo.

In contrast to systemic autoimmunity, spontaneous autoimmune thyroiditis of Obese strain (OS) chickens is associated with a marked T cell hyperreactivity in vitro, i.e. an increased proliferation and interleukin 2 (IL 2) secretion in response to Concanavalin A (ConA). In the present study we report an enhanced capacity of OS peripheral lymphoid cells (splenocytes and peripheral blood lymphocytes, PBL) to adsorb fluorescein isothiocyante (FITC) labelled ConA, but not phytohemagglutinin (PHA). However, the elevated ConA binding cannot be a prerequisite for in vitro ConA hyperreactivity as OS thymocytes are normal with respect to ConA binding but nonetheless exhibit elevated responses to this mitogen. Moreover, ConA binding does not correlate with the frequency of cells able to express IL 2 receptors upon short term ConA stimulation. The percentage of ConA activatable cells was found to be increased in OS- PBL as compared to normal control PBL, but was unaltered in OS splenocytes. This finding points to a further mechanism of T cell hyperreactivity in OS chicks in addition to the previously reported defects in nonspecific immunosuppression. Finally, enumeration of cells in the S phase revealed that enhanced proliferation of OS T lymphocytes was not restricted to the in vitro response to ConA and phytohemagglutinin (PHA) but also occurs in vivo.

A nasal allergy model developed in the guinea pig by intranasal application of 2,4-toluene diisocyanate.

An experimental model of nasal allergy has been developed in guinea pigs by intranasal application of 2,4-toluene diisocyanate (TDI). A 10% TDI solution in ethyl acetate was painted onto the nasal vestibuli of the animals once a day for 5-10 days. During the course of repeated application of TDI, the number of animals which secreted rhinorrhea containing eosinophils increased. Morphological survey of the nasal mucosa showed infiltration of eosinophils and some other changes indicative of acute inflammation. Moreover, mast cells were found not only in the subepithelial connective tissue but also in the epithelial layer. Nasal mucus obtained from the mucosa has been found to be an effective test material for studies of nasal allergy. A striking decrease of specific granules was found in some mast cells contained in the mucus. In parallel with the symptomatology, biochemical and serological studies suggested the involvement of type I allergy in the experimental system; TDI-specific histamine release from the nasal mucosa and positive passive cutaneous anaphylaxis were found 3 weeks after the application of TDI.

Increased helper cell activity of NZB mice against H-2-identical allogeneic cells.

The T cells of NZB mice become hyperreactive after stimulation with minor histocompatibility (MIH) antigens. This hyperreactivity has previously been demonstrated only for cytotoxic T cells of NZB, although there was some evidence for an increase of their T-helper cell activity facilitating the response. Here we report a quantitative analysis of T-cell help and help of T-cell subpopulations against autologous, MIH, and H-2 antigens in a limiting dilution assay. After stimulation of NZB T cells with autologous and H-2 antigens, the T-helper cell frequencies did not differ from that of normal mice. After stimulation with MIH antigens however, Lyt 1+2+ T-cells of NZB showed a higher response than those of BALB/c origin. The same difference was seen after prestimulation with ConA or the specific antigen. This demonstrates that NZB-helper cells are more easily activated by weak antigenic differences, and it is possible that this contributes to the prevalence of autoimmune disease in this strain.

Studies on immunological air pouch inflammation in the rat.

The development of a novel immune-based model of inflammation in the subcutaneous air pouch of the rat is described. Rats were sensitized by injection with methylated bovine serum albumin emulsified with Freund's complete adjuvant and challenged 14 days later by injection of heat-aggregated antigen in saline into a 6-day subcutaneous air pouch. Time course studies revealed a peak polymorph cell infiltration at 24 h with no exudate formation, followed by a separate mononuclear cell response, rapid exudate formation, and granuloma formation over the 2- to 7-day period. Separate peaks of increased vascular permeability were observed in the acute and chronic phases of the response. This reaction was considered to be an example of delayed-type hypersensitivity based on its extended time course, the characteristic time course of oedema formation in foot-pad-challenged rats, and the failure of serum from sensitized rats to elicit a reversed passive Arthus-type reaction. The sensitization schedule was also shown to enhance the acute inflammatory response to carrageenan. In addition, the 'flare-up' reaction to secondary antigenic challenge was investigated in 7-day postchallenge lesions.

Hyperthyroxinemic mice have reduced natural killer cell activity. Evidence for a defective trigger mechanism.

Natural killer (NK) activity of peripheral blood lymphocytes from hyperthyroxinemic patients (Graves' disease or thyroxine (T4)-treated) is severely depressed. In order to study the relationship of thyroid hormone to NK activity, a model for hyperthyroxinemia was induced in mice by addition of T4 to the drinking water. Control mice were hypothyroid (fed propylthiouracil) or normal. Serum T4 levels were elevated (within 2 wk) in mice fed thyroid hormone. Six weeks after initiation of the diets, in vitro NK activity was undetectable in the peripheral blood, spleen, or lung mononuclear cell populations harvested from hyperthyroxinemic mice. Control mice had NK activity within the normal range. Spleen cells from mice fed thyroid hormone and control mice were tested for their ability to release lytic factors (natural killer cytotoxic factors). Lymphoid cells were incubated for 20 hr with unlabeled Yac-1 cells. Supernatants were tested for their capacity to lyse 51Cr-labeled Yac-1 cells in a 20-hr chromium release assay. Unlike controls, supernatants from hyperthyroxinemic spleen cells incubated with Yac-1 targets were unable to lyse 51Cr-Yac-1 cells. The NK cells from the mice fed T4 synthesized lytic factors because nonspecific stimuli, such as 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore A23187, induced release of lytic factors capable of lysing Yac-1 targets into the media. These data support the hypothesis that excess thyroid hormone interferes with the triggering mechanism used by NK targets to cause release of lytic molecules from NK cells.

BioBreeding/Worcester (BB/Wor) rats are deficient in the generation of functional cytotoxic T cells.

The BioBreeding/Worcester (BB/Wor) rat provides a good model of spontaneous autoimmune diabetes. There are several sublines of the BB/Wor rat. The diabetes prone (DP) sublines develop diabetes at a frequency of 50 to 80% from 60 to 120 days of age. The DP rats are lymphopenic, have a severe deficit in phenotypic OX 19+ OX 8+ cytotoxic T cells (Tc), and lack RT 6.1 T cells. These rats have a relative increase in OX 19- OX 8+ natural killer (NK) cells and in NK activity as compared with the diabetes resistant (DR) sublines. The DR sublines have a normal complement of phenotypic Tc and RT 6.1 T cells, fewer NK cells, and lower NK activity than the DP rat. The ability to elicit functional Tc in the BB/Wor rat has not been well studied. In these experiments, by using a model of lymphocytic choriomeningitis virus (LCMV) infection in DP and DR rats, we have studied the functional activity of Tc in these lines. Seven days after infection with LCMV, DR rats develop lymphocytes which are cytotoxic for LCMV-infected syngeneic fibroblasts. These cytotoxic lymphocytes are phenotypic Tc (OX 19+ OX 8+), and do not kill Pichinde virus-infected syngeneic fibroblasts or LCMV-infected allogeneic fibroblasts. This cytotoxic activity is accompanied by an increase in phenotypic Tc from 17 to 33%. DP rats produced neither functional nor phenotypic Tc. These studies confirm that NK cells are the predominant cytotoxic lymphocyte in the BB/Wor rat and suggest that these rats may not utilize a Tc mechanism in islet destruction or another immunologic process such as graft rejection.

Levels and specificity of antibody in bronchoalveolar lavage (BAL) and serum in an animal model of trimellitic anhydride-induced lung injury.

A study was undertaken to characterize the antibody response in rats exposed to trimellitic anhydride (TMA) by inhalation. Total antibody levels directed to trimellitic rat serum albumin (TM-RSA) from TMA-exposed rats were assayed by an ammonium sulfate technique. Total antibody levels in bronchoalveolar lavage (BAL) and the matched serum were compared by correction for the albumin content of each. An ELISA was developed to detect IgG, IgA, and IgM directed toward TM-RSA in BAL and serum and to compare class-specific antibody levels in BAL and serum by normalizing for albumin content. The specificity of the rat IgG response was determined by ELISA inhibition with TM-RSA and TM-human serum albumin (TM-HSA) and compared with reciprocal inhibition studies with serum from TMA-exposed workers. The levels of total antibody in BAL were three to 15 times greater than the levels found in the matched serum pair. IgG, IgA, and IgM antibodies were detected in the BAL and the serum of TMA-exposed rats but not in control rats. In each of the four rats tested, all antibody classes were present in equal or greater amounts in the BAL than in the serum. Complete inhibition of the rat IgG binding in ELISA was observed when TM-RSA or TM-HSA were added as inhibitors. Human IgG was inhibited in ELISA only by TM-HSA. In an animal model of human lung disease, the levels of total antibody as well as class-specific antibodies directed against TM-RSA were greater in BAL than in serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Isotype progression and clonality of anti-Sm autoantibodies in MRL/Mp-lpr/lpr mice.

Antibodies to the nuclear ribonucleoprotein Sm are found in 25% of MRL/Mp-lpr/lpr mice, which develop a syndrome similar to human systemic lupus erythematosus. We have previously described that these autoantibodies are relatively restricted to the IgG2a isotype. In the current study, we analyze the isotype distribution of anti-Sm antibodies in these mice over time. The most common pattern observed was an initial response of the IgG2a isotype, which progressed such that this isotype was the major one at the time of peak response. No IgM to IgG class switch was observed. Additional studies directed at the clonality of the anti-Sm response indicated that both kappa- and lambda-light chains could be involved, and that the isoelectric focusing pattern of the anti-Sm antibodies was in general characteristic of multiple spectrotypes. These results also support a special role for the IgG2a isotype in the anti-Sm response in MRL/Mp-lpr/lpr mice. Despite this heavy chain isotype restriction, the response usually evidences substantial diversity, which suggests either multiple B cell clones or somatic mutation of antibody variable region genes.

Monoclonal autoantibodies to nuclear antigens from murine graft-versus-host disease.

Systemic lupus erythematosus-like graft-versus-host (GVH) disease was induced in 10-week-old male (C57BL/10 X DBA/2) F1 mice by the intravenous injection of spleen and thymus cells (2:1) from 10-week-old male DBA/2 mice. GVH mice were bled at regular intervals 1 month after injection. Antibody to nuclear antigens (ANA) were detected by immunofluorescence using HEp-2 cells as substrate, and antibody to histones and DNA were detected by enzyme-linked immunosorbent assay (ELISA). The titer and frequency of ANA were found to relate directly to the number of donor cells injected. In order to determine the spectrum of ANA in GVH disease, mice were reinjected with optimum cell numbers (120 X 10(6], and splenocytes from two mice with high titer ANA were fused to mouse myeloma cell line P3/X63Ag8.653. Hybridomas were analyzed for ANA by immunofluorescence and ELISA. Sixty-eight clones were found which secreted ANA. Of these, 59% produced antibody to double-stranded DNA, single-stranded DNA, and/or histones and the remainder gave a variety of nuclear immunofluorescence patterns including speckled, homogeneous, nuclear matrix, and nucleolar. This study indicates that GVH disease provides an excellent source of splenocytes for the production of ANA-producing hybridomas as well as a model for the study of autoimmunity.

Removal of glomerular deposits induced by either preformed immune complexes or by a chronic immune complex model in NZB/W mice.

The solubilization and removal of defined glomerular immune complex deposits by excess antigen was examined in NZB/W female mice. Glomerular deposits were induced by administering preformed immune complexes to young (2 to 4 mo) mice before they naturally acquired deposits from endogenous disease and to old (7 mo) mice with deposits from naturally acquired disease. The administration of excess antigen specifically removed deposits of preformed immune complexes in both groups. This was associated with a reduction in circulating large latticed complexes containing more than two antigen and two antibody molecules (greater than Ag2Ab2). Established deposits in old mice therefore did not interfere with removal of newly induced deposits of preformed immune complexes. Glomerular deposits were also induced in young mice by a chronic human serum albumin (HSA) immune complex model. The antigen in immune deposits induced by 2 wk of chronic antigen administration was solubilized and was removed within 48 hr of administering excess antigen. Circulating antibodies to the antigen were also reduced by excess antigen. Glomerular deposits of mouse immunoglobulin and complement were not significantly reduced by excess antigen but remained more intense than in mice of comparable age given preformed complexes. Thus deposits of other antigen antibody systems and possibly endogenous disease were induced by the chronic HSA immune complex model in NZB/W mice. However, defined antigen deposits within deposits containing multiple antigen antibody systems can clearly be removed by administering excess antigen.

Antibacterial immunity and its modeling in experimental tumor tetanus and wound tetanus of the mouse.

The experimental and theoretical analysis of the tumor-tetanus phenomenon has provided us with new insights into the pathogenesis of tetanus infection. Our theoretical model of clostridial propagation in the proliferating tissue is based upon the principle of mitosis-controlled rod division (hit and cloning model). It has lent itself to the description of early growth stages of the clostridial rod population in our experiments of tumor tetanus and of wound tetanus of the mouse. However, the later course of the tetanus lethality curves under antitoxin protection, about a week following injection of the tumor cell-spore or CaCl2-spore suspensions, reveals a pronounced delay in clostridial propagation. Based on our model we can explain this process by a humoral immune reaction directed against the clostridial rods taking into account the variability of elimination of the heterologous tetanus antitoxin applied. The experimental results are in good agreement with those obtained by computer simulation. The theoretical knowledge resulting from these studies can be used for the interpretation of the serodiagnostic tumor test with apathogenic clostridia as well as for the quantitative assessment of the malignancy of neoplastic growth.

Mechanisms of T and B cell collaboration in the in vitro production of anti-DNA antibodies in the NZB/NZW F1 murine SLE model.

B/W mice spontaneously develop IgG antibodies to DNA that cause lethal immune nephritis. T and B cell interactions in the in vitro anti-DNA antibody response of B/W mice were investigated, and two distinct families of helper T cells that drive these responses were defined. First, the anti-DNA antibody-forming cell (AFC) response was found to be increased in B/W mice with nephritis and was inhibited with the monoclonal antibody anti-L3T4, suggesting a major role for helper T cells. Purified splenic T cells from mice with nephritis were able to augment both the IgG and the IgM anti-DNA AFC response of young B/W B cells. T helper cells were cloned from spleens of NZB/W F female mice with high titer anti-DNA antibodies and nephritis. The cloned T cells augmented both IgG and IgM anti-DNA AFC responses of young B/W B cells. Four clones--27.9, 30.7, 30.8, and 30.10--were selected for further study. These cells proliferated, in the context of syngeneic (H2d/z) antigen-presenting cells (APC) but not to allogeneic APC. Analysis of the mechanism of T helper cell clone-mediated augmentation of anti-DNA AFC revealed two populations: "cognate" T helper cells, which specifically augment anti-DNA AFC (30.7 and 30.10), and non-antigen-specific T helper cells (27.9 and 30.8), which augment the response of B cells of differing specificity by a bystander mechanism, probably through increased release of B cell growth and differentiation factors.

Thymocyte-dependent immunity to toxoplasmosis in the normal and immunocompromised guinea-pig host.

Guinea-pigs made T-cell deficient by thymectomy and irradiation, and protected with syngeneic bone marrow cells (TXB) have a greatly reduced capacity to express normal cell-mediated immune functions, based on their poor responses to T-cell mitogens, prolonged acceptance of skin allografts, and susceptibility to the lethal effects of graft-versus-host disease. Further evidence for impaired T-cell activity in TXB guinea pigs was based on their inability to be fully sensitized to mycobacterial antigens, and increased susceptibility to an intradermally induced infection with the intracellular protozoan parasite, Toxoplasma gondii (RH strain). After challenge at multiple sites with 10(6) or 10(5) parasites, toxoplasmosis in thymus-intact, fully immunocompetent guinea pigs is a self-limiting and survivable infection, whereas the disease takes an acutely lethal course in the majority of TXB guinea-pigs. The latter also had more parasites disseminating to various tissues sites than their euthymic counterparts. The reduced capacity of TXB guinea-pigs to respond to mycobacterial products, and to generate anti-Toxoplasma immunity can be restored by an intravenous infusion of normal syngeneic thymocytes. These findings provide substantial direct evidence strengthening the concept that protection against toxoplasmosis is heavily dependent upon an intact T-cell component of the host's immune response.

The inflammatory reaction in the airways in an animal model of the late asthmatic response.

The late asthmatic response is defined as airway obstruction that occurs hours after antigen exposure in some atopic asthmatics. The importance of this reaction is that the airway obstruction may be severe, prolonged, and difficult to control unless corticosteroids are employed. In addition, this response may lead to an increase in airway reactivity. To investigate the immunopathogenesis of this disorder, an animal model in rabbits was developed. In this model, antigen-specific IgE was associated with the late asthmatic response and antigen-specific IgG was associated with blunting of the reaction. Antigen challenge of immune rabbits led to edema within the large airways shortly after antigen exposure, with infiltration of inflammatory cells (neutrophils and eosinophils) into the large and small airways during the late response. The infiltrates became more mononuclear with time and resolved over 10 days. As in humans, the late response was associated with an increase in airway reactivity and correlated temporally with infiltration of the airways with neutrophils and eosinophils. The contribution of granulocytic cells to the airway responses to antigen was studied by granulocyte depletion, which prevented both the late response and the heightened airway reactivity. In addition, transfusion of a neutrophil-rich population of white cells into granulocytopenic immune rabbits restored both responses. Thus, in this animal model, the antigen-induced late asthmatic response and subsequent increase in airway reactivity were dependent on the presence of granulocytes at the time of exposure to antigen.