by which they cause disease, and the means by which data produced in the laboratory can be used to estimate risks to human health. Disorders of the respiratory tract continue to be a major health concern, and inhaled toxicants are thought to contribute substantially to respiratory morbidity. As the largest laboratory dedicated to the study of basic inhalation toxicology, ITRI provides a national resource of specialized facilities, personnel, and educational activities serving the needs of government, academia, and industry. The papers in this report are organized along topical lines, rather than by research program, so that research within specific disciplines is more readily identified. In contract to previous Annual Reports, the papers include summaries of research funded by both DOE and non-DOE sources, to more fully represent the scope of the Institute's activities. The source of funding is acknowledged for each paper. One section consists of summaries of research on the effects of injected actinides, conducted for DOE at the University of Utah.

Rabbit alveolar macrophages were exposed in culture medium to asbestos, beryllium sulfate, and beryllium oxide. The specific activities of the lysosomal hydrolases, acid phosphatase beta-N-acetylglucosaminidase and beta-glucuronidase plus the glycolytic enzyme, phosphohexose isomerase were determined in the medium, whole-cell homogenates, mitochondrial fractions, and supernatant. These hydrolases increased significantly in the medium but not in the mitochondrial fraction of cells exposed to dusts. Asbestos and beryllium sulfate were highly cytotoxic for alveolar macrophages in vitro and the data suggested that these agents were not associated with an increase in enzyme synthesis but rather a direct cytotoxic effect at the macrophage membrane level. For induction of enzyme release in vitro, a higher concentration of beryllium oxide was needed when compared with asbestos and beryllium sulfate. The cytotoxicity and enzyme release induced by these agents may represent an important nonspecific mechanism by which they induce inflammation and perhaps local proliferation of fibroblasts.

In a study designed to assess the potential sensitizing and granulomagenic capacities of selected metallic salts, rabbits were inoculated intradermally with zirconium aluminum glycinate (ZAG), sodium zirconium lactate (NZL), aluminum chlorhydrate (ACH), BeSO4, and ovalbumin (OVA) by single and multiple injections. Animals immunized with BeSO4 and with OVA developed delayed skin reactivity as well as antigen-specific alveolar macrophage migration inhibition. Neither single nor multiple injections of ZAG or ACH resulted in clear-cut positive skin reactivity, macrophage migration inhibitory factor (MIF) production, or lymphocyte stimulation. Rabbits inoculated with multiple injections of NZL (500 μg) showed some marginally positive macrophage migration inhibition and skin reactivity. Histologically, ZAG and ACH were found to induce well-organized foreign-body granulomas after intradermal injection in both normal and inoculated rabbits. NZL and BeSO4 also induced skin granulomas, but these were less organized and distinct. Cell viability and ultrastructural studies indicated that BeSO4 was highly toxic for isolated alveolar macrophages in vitro at concentrations above 10 μg/ml, but NZL and ZAG did not exert such an effect at these dose levels. BeSO4 also depressed lymphocyte stimulation in sensitized animals which demonstrated delayed skin reactivity and macrophage migration inhibition.

The blastogenic response of lymphocytes from patients with malignant neoplasms was evaluated by stimulation with three phytomitogens (PHA, PWM, and Con A). The response of patient lymphocytes to all three mitogens was significantly lower than that of control lymphocytes, and most patients with abnormal PHA responses also responded abnormally to PWM and Con A. However, a few patients with normal PHA responses were abnormal to Con A, suggesting the suppression of a Con A-sensitive population. The observation that PWM responses were abnormal in patients with lowered PHA lymphocyte stimulation indicates that both T and B lymphocyte mitogen responses were suppressed in these patients. Plasma from patients was capable of either inhibiting or enhancing lymphocyte mitogen stimulation. However, inhibitory plasmas were generally from patients with abnormal mitogen responses.

The response of the lower respiratory tract to bacterial and fungal antigens has received increasing attention because of relevance to the many forms of hypersensitivity pneumonitis and to host protective mechanisms. Previous animal studies in several species have demonstrated local cell-mediated immunity and antibody production in the lower respiratory tract after immunization with appropriate microbial protein antigens. 1 Reynolds HT Thompson RE Devlin HB Development of cellular and humoral immunity in the respiratory tract of rabbits of Pseudomonas lipopolysaccharide. J Clin Invest. 1974; 53: 1351 Crossref PubMed Scopus (19) Google Scholar , 2 Biberfield G Macrophage migration inhibition in response to experimental mycoplasma pneumonia infection in the hamster. J Immunol. 1973; 110: 1146 Google Scholar , 3 Fernold GW Clyde WA Bienenstock J Immunoglobulin containing cells in lungs of hamsters infected with mycoplasma pneumonia. J Immunol. 1972; 108: 1400 Google Scholar The lower respiratory tract response to thermophilic actinomycetes which are rich sources of antigen in production of human hypersensitivity pneumonitis has not been studied in detailed fashion. The purpose of this study was to further characterize the pulmonary humoral and cellular response of rabbits to particulate Micropolyspora faeni (M faeni) antigen administered intratracheally (IT) in saline solution.

Chronic mucocutaneous candidiasis in two siblings of consanguineous parents suggested an autosomal recessive transmission of the disease. We evaluated the two affected persons and 21 members of their kindred for an inherited immunological defect. Six members of the kindred, including both patients, had negative skin-delayed hypersensitivity to Candida. The lymphocytes of both patients and three asymptomatic relatives had diminished in vitro blastogenic response when cultured with Candida albicans. Because the defect occurred in clinically unaffected relatives, we concluded that the lack of blastogenic response to C. albicans was not the only determinant for or may be unrelated to the clinical manifestations of the disease.

The immunologic events involved in the pathogenesis of human hypersensitivity pneumonitis are poorly understood. To delineate better the cellular and humoral bronchopulmonary response to host challenge with an important agent for hypersensitivity pneumonitis, an animal model was developed. Rabbits intratracheally inoculated with Micropolyspora faeni antigen developed histologic lesions resembling hypersensitivity pneumonitis in man, characterized by a mononuclear cell interstitial reaction and a marked increase in the number of intra-alveolar cells. This reaction was intense 8 days after immunization and progressively decreased in intensity, with complete resolution occurring, in some instances, by 3 weeks. The increased intra-alveolar cells were predominantly macrophages, but soon after immunization, greater numbers of lymphocytes and granulocytes were present. Associated with the intra-alveolar macrophage response was the appearance of "activated" macrophages, based on increased glucose oxidation and ultrastructural appearance. The immunized rabbits demonstrated Arthus-type skin reactions, as well as delayed hypersensitivity skin reactions after intradermal injection of M. faeni antigen. Alveolar macrophage migration was significantly inhibited in immunized rabbits in the presence of M. faeni antigen, suggesting that specifically sensitized lymphocytes were present in the free bronchoalveolar cell population. Intratracheal inoculation with M. faeni resulted in the production of anti-M. faeni-precipitating antibodies in the sera of all animals and in bronchoalveolar secretions in 15 of 16 inoculated rabbits. The concentrations of IgG and IgA in lung-wash fluid were significantly greater in immunized animals. In the immunized rabbits, the mononuclear cell response, positive M. faeni-induced direct migration inhibition factor assays, and delayed skin reactivity strongly suggested the presence of a delayed hypersensitivity component in disease pathogenesis. The presence of precipitating antibody and the increased concentrations of immunoglobulins were consistent with stimulation of the humoral immune system; however, the role of humoral mechanisms in the pathogenesis of the pulmonary lesions is less certain than that of delayed hypersensitivity.

Rabbits were sensitized with either a soluble protein antigen (BSA) or a particulate thermophilic actinomycete antigen (Micropolyspora faeni) via the respiratory route, followed by monitoring of sequential morphologic changes and the humoral plus cellular immunologic response. Primary respiratory tract sensitization with BSA resulted in a humoral anti-BSA response, Arthus and delayed skin reactivity, and in some cases specific antigen-induced alveolar macrophage migration inhibition, all in the absence of pulmonary lesions. Lesions characterized by mild multifocal perivascular mononuclear cell infiltrates in the lungs developed only after secondary BSA aerosol challenge. In contrast to these findings, "primary" respiratory tract sensitization with M. faeni particulate antigen in saline solution resulted in the gradual development of extensive and progressive pulmonary interstitial and alveolar mononuclear cell infiltrates. These lesions were uniformly associated with specific serum precipitating antibody and delayed skin reactivity. Alveolar macrophage migration was significantly inhibited by Micropolyspora faeni in virtually of these animals. These results, while not excluding a primary irritant effect or Type II or III alergic tissue injury, suggest a role for delayed (cell-mediated) hypersensitivity in the pathogenesis of particulate actinomycete-induced pulmonary lesions. They also indicate that primary immunization with soluble purified protein antigens via the respiratory route can lead to systemic humoral and cell-mediated immunity without production of pulmonary lesions.

Serum protein concentrations and lymphocyte function, were determined in prisoner volunteers undergoing prolonged biweekly plasmapheresis and appropriate control subjects. Prolonged biweekly removal of 800 ml of plasma was well tolerated clinically. Neither circulating lymphocyte counts, PHA-induced in vitro lymphocyte 3H-TdR incorporation, or delayed hypersensitivity were affected. However, mean immunoglobulin, β1c-globulin and albumin levels decreased significantly during the initial year of plasmapheresis and individual values were below the accepted normal minimum range in over half the donors tested. Low levels of IgG, IgA, IgM, β1c-globulin and albumin were also detected in several ‘normal’ prisoner volunteers. These findings suggest that individual immunoglobulin and other serum protein concentrations should be quantitated initially to eliminate poor-risk donors, and monitored at intervals during the course of plasmapheresis.

Carrageenan, a high molecular weight, sulfated polygalactose known to be toxic for macrophages in vitro was utilized in in vivo experiments to further study the role of this cell type in the initiation and expression of delayed hypersensitivity. When administered intraperitoneally to sensitized guinea pigs around the time of skin testing, carrageenan suppressed the expression of existing delayed hypersensitivity reactions to BSA. In addition, when administered at the time of immunization, carrageenan suppressed the development of subsequent delayed hypersensitivity. Carrageenan, in vitro did not appear to be cytotoxic for lymphocytes either structurally or functionally. It did not possess mitogenic properties and did not interfere with phytohemagglutinin induced lymphocyte transformation.

Carrageenan, a high molecular weight sulfated polygalactan with known macrophage toxic properties was added to cultures of normal and sensitized lymphocytes from human peripheral blood and guinea pig lymph nodes to determine its effect on phytohemagglutinin, and antigen-induced 3H-TdR lymphocyte incorporation. Carrageenan was not toxic for guinea pig or human lymphocytes per se and induced no lymphocyte transformation in vitro. With high doses in the range of 1000 µg/ml and above, some decrease in 3H-TdR uptake was noted. Phytohemagglutinin-induced human and guinea pig lymphocyte transformation was not affected by carrageenan but a marked dose-dependent suppression of antigen-induced 3H-TdR lymphocyte uptake was noted in both species. This suppressive effect was not due to reagent or antigen dilution. It was observed when carrageenan was added to cell cultures prior to antigen or up to 6 hr later, but not when added after 17 hr. These findings suggest that carrageenan acts at an early stage of macrophage-antigen processing and are consistent with previous reports of inhibition of antigen-induced lymphocyte transformation using anti-macrophage serum. They provide further evidence that phytohemagglutinin-induced lymphocyte transformation is not mediated by macrophages and that macrophage-lymphocyte interaction occurs with cells from the peripheral blood as well as from lymph nodes.