Chronological assessment of asbestos exposure on cell composition in murine lung.

To elucidate immune pathogenic mechanisms in asbestosis, lung and spleen lymphoid cell populations were analyzed at defined time intervals (1, 2, 3, 6, and 12 weeks during exposure and 4, 24, and 48 weeks post-exposure) in asbestos-exposed and unexposed (control) mice. Polymorphonuclear leukocytes and macrophages were increased in the lung tissue histologic sections of asbestos-exposed mice compared to controls. No consistent changes were observed in percentages of lung or spleen helper, suppressor, or total lymphocyte populations after asbestos exposure. The numbers of B cells (identified by anti-IgG) in minced lung preparations of asbestos-exposed animals were increased after 12 weeks of exposure. There also was an increase in IgG production in asbestos-exposed mice after 12 weeks exposure and at 4 weeks post-exposure with a return to near baseline levels 24 and 48 weeks after initial exposure. Collectively, these studies demonstrate stimulatory effects of inhaled asbestos fibers on B cells and IgG production after 12 weeks of continuous inhalation of asbestos fibers in a dust generation chamber.

Phase-dependent response of the lung to NO2 irritant insult.

The biochemical and histopathological response of the lung following acute and repeated (subacute) exposure to nitrogen oxide (NO2) was examined. Activities of lactate dehydrogenase, beta-glucuronidase, choline kinase, and protease inhibitor were measured in murine pulmonary tissue immediately and two days following exposure. Nonenzymatic parameters, pulmonary protein content, and wet lung weight were also monitored. Immediately following acute exposure to NO2, only the nonenzymatic parameters were elevated. By two days following acute exposure, following subacute exposure; however, the nonenzymatic parameters were attenuated with respect to the enzymatic activities. The lung exhibits a dynamic response following damage by oxidants such as NO2. This response is divided into three distinct phases (exudative, proliferative, and tolerant), which can be characterized both biochemically and histopathologically.

Soy- and shrimp-specific IgE responses in orally and intraperitoneally immunized mice.

Female C3H/HeJ mice were immunized parenterally and/or intragastrically with two important food allergens, soy and shrimp. Although both preparations elicited specific IgE production when administered intraperitoneally, anti-shrimp titers were consistently higher. Soy or shrimp administration, i.g. with Pertussis adjuvant (intraperitoneally or intragastrically) did not induce detectable reaginic antibody. Animals treated with soy intragastrically were unable to produce a soy-specific IgE response upon intraperitoneal immunization. The combination of soy and Pertussis (intragastric) did, however, abrogate this tolerizing effect on IgE synthesis. In contrast to soy, the shrimp antigens (intragastric) did not act as tolerogens and were found to enhance subsequent reaginic responses produced by intraperitoneal immunization. These results are considered with respect to mechanisms operative in food allergy.

Analytical and functional assessment of lymphoid cells within murine lung.

In this study we sought to characterize the tissue leukocytes in murine lung which (when derived from peripheral lymphoid organs) have been associated with host defense against tumors and certain microbial agents. Numerical and functional properties of lung lymphoid populations obtained by collagenase digestion were evaluated in inbred mice. Flow cytometric analysis of monoclonal and polyclonal antibody labelled preparations revealed that lung cells so derived were 47% T-lymphocytes (helper-suppressor ratio 4.25), 23% B-cells (surface immunoglobulin+) and 26% asialo GM1 (natural killer) cells. Lung T-cells responded well to mitogen stimulation after partial depletion of adherent macrophages, but the magnitude of this response was less than that of autologous spleen cells. Natural killer cell (NK) activity against chromium labelled tumor target cells was also manifested by lung cells and was similar to that of peripheral spleen cells. This lung NK activity could be boosted in vitro by incubation with interferon and in vivo by treatment with poly I:C. Together, these data indicate that normal murine lung contains a large number of functional T-cell and NK cell mononuclear populations. The characterization of the cell populations presented here should facilitate future studies of their role(s) in host defense against infection and neoplasia.

Epstein-Barr virus-related herpesvirus from a rhesus monkey (Macaca mulatta) with malignant lymphoma.

A herpesvirus (RhEBV) was isolated from a lymphoblastoid cell line (LCL) that became established from a malignant lymphoma in a rhesus monkey. The predominant cell marker in the LCL was that of B lymphocytes. RhEBV-induced viral capsid (VCA) and nuclear antigens (NA) in the LCL were serologically related to similar antigens known to be induced by human Epstein-Barr virus (EBV). RhEBV was of nonhuman primate origin and was clearly differentiated from EBV in the anti-complement immunofluorescence reaction using human and non-human primate sera with antibodies to the NA induced by the respective viruses. While human sera reacted with NA induced by both EBV and RhEBV, monkey sera failed to recognize the NA induced by EBV. RhEBV-induced NA was present in nearly all the cells of a suspension prepared from the tumor tissue mass, but not in the monolayer fibroblasts derived from the tumor tissue or in the blood and lymph-node lymphocytes of clinically healthy animals. RhEBV induced in vitro transformation and establishment of LCLs from peripheral blood lymphocytes of normal rhesus and cynomolgus monkeys but not from those of 6 other non-human primate species tested. The LCLs, with predominant B-lymphocyte markers, established after treatment with RhEBV, all had evidence of the virus infection since nearly all cells in the culture expressed the virus-induced NA.

Effects of asbestos fibers on alveolar macrophage-mediated lymphocyte cytostasis.

Prolonged asbestos inhalation results in pulmonary inflammation and fibrosis. Since alveolar macrophages are active in regulation of immune responses in lung and appear to be involved in the pathogenesis of asbestosis, we evaluated the effects of asbestos exposure on the ability of these cells to regulate lymphocyte function. Alveolar macrophages obtained by lung lavage from BALB/C mice were treated in vitro with either UICC amosite or chrysotile asbestos and the effects on lymphocyte cytostasis compared with those of macrophages incubated with latex beads or zymosan. Macrophages (10%) incubated either alone or with latex beads for 48 hr effectively inhibited lymphocyte mitogenesis. However, alveolar macrophages incubated with either amosite or chrysotile asbestos did not demonstrate intact cytostatic activity. Decreased viability of chrysotile asbestos-treated macrophages correlated with loss of cytostatic effects, but alveolar macrophages exposed to amosite remained viable. We conclude, therefore, that exposure of alveolar macrophages to asbestos can result in loss of their ability to down-regulate lymphocyte proliferation, a finding which may be important in the pathogenesis of asbestos-related disease.

A longitudinal immunologic evaluation of hemophiliac patients.

Over an average span of one year, we performed a prospective clinical and immunologic evaluation of 30 patients with hemophilia. No patient developed life-threatening opportunistic infection or malignancy; however, the immunologic abnormalities and lymphadenopathy initially present in nine patients (lymphadenopathy group) persisted. In addition, five patients, representing 24% of the initial group without lymphadenopathy, developed generalized lymphadenopathy (converter group). One episode of idiopathic thrombocytopenia (ITP) and one episode of staphylococcal sepsis occurred in this "converter" group; one episode of ITP also occurred in the lymphadenopathy group. Sixteen patients remained asymptomatic. At the time of the follow-up evaluation, those differences in mononuclear cell (MNC) percentages and numbers noted initially among the three hemophiliac groups were no longer present. Natural killer cell function alone or in the presence of biologic response modifiers was not different among hemophiliac and control groups. Before developing lymphadenopathy, the converter group of patients had significantly better lymphocyte mitogenic function than did the other two groups of patients with hemophilia. However, lymphocyte mitogenic responses of all groups of patients with hemophilia significantly deteriorated over the course of the study. The abnormal mitogenic responses noted in these patients was explained in part by higher levels of spontaneous suppressor cell activity in mononuclear cell preparations from patients with hemophilia. We conclude that long-term immunologic studies of this patient population requires both quantitative and qualitative evaluations. Our data show that patients with hemophilia have progressive dysfunction of cell-mediated immunity.

Effects of anti-epsilon on total and specific IgE levels in adult mice.

Adult (2- to 3-month-old) female CBA/J mice were injected intraperitoneally with heavy chain specific rabbit anti-IgE (anti-epsilon) to determine its effects on total and specific serum IgE. Animals receiving 10 X 250 micrograms injections over a 50-day period displayed significantly increased (10 X levels of serum IgE compared to rabbit gamma-globulin or untreated controls. If animals were immunized with castor allergen (CA) prior to anti-epsilon treatments their IgE anti-CA titers were significantly suppressed, although their total IgE levels were not significantly different compared to controls. In a second series of experiments, mice which received increased quantities (3 X more) of anti-epsilon had no detectable serum IgE (within 30 days post anti-epsilon treatment), significantly decreased titers of IgE antibody, and reduced numbers of IgE bearing spleen and mesenteric lymph node cells. These studies indicate that anti-epsilon injections in adult mice can significantly alter total and specific IgE levels.

Immunologic techniques utilized in the diagnosis of occupational lung disease.

Humoral and/or cell-mediated immune responses may contribute to the tissue injury in patients with certain types of occupational asthma, hypersensitivity pneumonitis, silicosis, and asbestosis. Numerous diagnostic modalities are available to the clinician investigating the etiology of these disorders. Among the current immunologic techniques discussed in this article are immunoassays for specific anti-IgE antibody, gel diffusion reactions, immunoelectrophoresis, ANA assays, complement studies, and immune complex assays.

Humoral immunologic abnormalities in workers exposed to asbestos cement dust.

Serum specimens from 144 workers exposed to asbestos cement dust were examined for the presence of ANA, RF, immunoglobulins, and IC. These immunologic findings were compared with chest radiographic changes. A high percentage of workers had polyclonal hypergammaglobulinemia, and there was a statistically significant association between elevated levels of IgG and IgM and radiographic classification. Although a significant number of workers had an increased prevalence of ANA and elevated levels of IC, there was no correlation between these parameters and chest radiographs. These findings support B cell hyperactivity in workers exposed to asbestos and suggest that autoantibody production and IC are not directly involved in disease pathogenesis.

A murine model of asbestosis.

Balb/c mice were exposed to aerosolized chrysotile fibers and evaluated as a host for the study of asbestos-induced pulmonary disease. Histologically, an initial macrophage reaction was found to progress to "asbestos body" formation and diffuse focal interstitial fibrosis within 1 year of a chronic exposure period. This reaction was most intense in areas adjacent to respiratory bronchioles and alveolar ducts. Two morphologically distinct tumors at the pulmonary visceral surface were also discovered among a high percentage of asbestos-exposed mice. Bronchoalveolar lavage demonstrated an eventual alteration of the retrievable cell populations among the dusted animals. Evaluation of systemic B-lymphocyte activity suggested a stimulation of this cell subset following chrysotile inhalation. These results demonstrate that subsequent to chronic asbestos exposure, the mouse develops pulmonary and immunologic changes very similar to those noted in human asbestosis.

Functional analysis of monkey lymphocyte subsets defined by OKT4 and OKT8 Monoclonal Antibodies.

The percentages of rhesus monkey blood lymphocytes (PBL) reactive with OKT4 and OKT8 antibodies and the OKT4/OKT8 ratio showed significant correlations with the log of the immunoglobulin plaque-forming cell (PFC) response after stimulation with pokeweed mitogen (PWM). These correlations suggested that monkey OKT4+ cells function as "helper" cells and OKT8+ cells function as "suppressor" cells for the PFC response. This was confirmed by separation and study of enriched T- and B-cell subpopulations. OKT8-depleted (OKT4+) and OKT4-depleted (OKT8+) cells were obtained by treatment of purified T cells with antibody and complement. OKT4+ cells augmented the PWM-induced B-cell differentiation into PFC but OKT8+ cells did not. OKT8+ cells suppressed the PFC response by mixtures of B cells and OKT4+ cells. OKT8 antibodies also detected a suppressive cell subset in African green monkeys since the percentage of OKT8+ cells showed a negative correlation with the log PFC response. OKT4 antibodies failed to bind to African green monkey PBL.

Asbestos-induced alterations of human lymphoid cell mitogenic responses.

Using mitogenic assays, we have investigated the short term effects of two asbestos (amosite and chrysotile) fibers on lymphocyte functions in vitro. These oppositely charged fibers produced different alterations in mitogenesis. The blastogenic responses of concanavalin-A (Con-A) and pokeweed mitogen stimulated human peripheral blood mononuclear cells (PBMN) were significantly increased by the inclusion of 6 micrograms of chrysotile to the culture media. Amosite fibers proved to be inhibitory in all tests. When PBMN were depleted of monocytes, asbestos-related alterations of Con-A responsiveness were unchanged among the remaining cells. However, the addition of chrysotile to phytohemagglutinin (PHA) cultures resulted in a significant increase of the mitogenic response. When PBMN were enriched for T lymphocytes, and again cultured with the mitogens and fibers, the Con-A response still displayed impressive enhancement with chrysotile. In contrast to an intact PBMN population, PHA-induced blastogenesis among these T-enriched lymphocytes was significantly elevated. These experiments demonstrate that asbestos can induce significant changes in the functional integrity of PBMN following a relatively short exposure time in culture.

Immunologic techniques utilized in the diagnosis of occupational lung disease.

Numerous immunologic diagnostic modalities are available to the clinician investigating the etiology of occupational lung disorders. Certain tests--immunoassays for specific anti-IgE antibody, gel diffusion reactions, and immunoelectrophoretic techniques--may aid in the identification of specific antigens or analysis of antigens involved in the pathogenesis of environmental lung diseases. Other techniques--ANA assays, complement studies, and immune complex assays--should be regarded as complementary. For example, a specific non-histone nucleoprotein "marker," ANA has not been identified in any of the environmental fibrotic lung diseases, and ANA positivity does not always correlate with severity or progression of disease. Additionally complement assays and detection of circulating immune complexes do not identify specific antigens. Clearly, there are other assays useful in determining immunologic mechanisms that may be important in occupational lung diseases but were not discussed in this article. Bronchoalveolar lavage with cellular identification and therapeutically important when considering the activity of various lung disorders. Also, lymphokine and leukotriene identification, lymphocyte transformation studies, and assessment of macrophage function are contributing greatly to our understanding of the possible immunologic mechanisms involved in different environmental pulmonary diseases. Thus, the investigation of immunologic mechanisms offers an exciting and rewarding future in the evaluation of the pathologic mechanisms of occupational and environmental pulmonary disease.

IgE isotype suppression in anti-epsilon-treated mice.

Two groups of CBA/J mice received a total of eight intraperitoneal (i.p.) injections of heavy-chain-specific rabbit anti-IgE or rabbit gammaglobulin within 48 hr of birth through day 38. A third group of animals was untreated. All mice were subsequently immunized with four i.p. injections of castor allergen plus aluminum hydroxide. Results indicate that anti-treatments severely suppressed murine serum IgE levels as compared with control mice. In addition anti-epsilon-treated mice were initially unable to produce detectable reaginic antibody upon immunization with castor bean allergens (CA). Upon further CA immunization, these animals did produce an IgE antibody response, but this was still lower than that detected in control immunized mice. Other immunoglobulin levels in the anti-epsilon-treated mice were not suppressed as compared with those in the control mice. These results suggest that neonatally administered anti-epsilon antisera selectively diminished total IgE levels as well as antigen-induced IgE antibodies in mice.