Patrick J. Haley

THE ACUTE TOXICITY OF INHALED BERYLLIUM METAL IN RATS

We exposed rats once by nose only for 50 min to a mean concentration of 800 micrograms/m3 of beryllium metal (initial lung burden, 625 micrograms) to characterize the acute toxic effects within the lung. Histological changes within the lung and enzyme changes within bronchoalveolar lavage (BAL) fluid were evaluated at 3, 7, 10, 14, 31, 59, 115, and 171 days postexposure (dpe). Beryllium metal-exposed rats developed acute, necrotizing, hemorrhagic, exudative pneumonitis and intraalveolar fibrosis that peaked at 14 dpe. By 31 dpe, inflammatory lesions were replaced by minimal interstitial and intraalveolar fibrosis. Necrotizing inflammation was observed again at 59 dpe which progressed to chronic-active inflammation by 115 dpe. This inflammation worsened progressively, as did alveolar macrophage and epithelial hyperplasia, becoming severe at 171 dpe. Low numbers of diffusely distributed lymphocytes were also present but they were not associated with granulomas as is observed in beryllium-induced disease in man. Throughout the experiment, total numbers of cells were elevated within the BAL samples due primarily to increased numbers of neutrophils. Lymphocytes were not elevated in BAL samples collected from beryllium-exposed rats at any time after exposure. Lactate dehydrogenase (LDH), beta-glucuronidase, and protein levels were elevated in BAL fluid from 3 through 14 dpe but returned to near normal levels by 31 dpe. LDH increased once again at 59 dpe and remained elevated at 171 dpe. beta-Glucuronidase and protein levels were slightly, but not significantly, elevated from 31 through 171 dpe. Results indicate that inhalation of beryllium metal by rats results in severe, acute chemical pneumonitis that is followed by a quiescent period of minimal inflammation and mild fibrosis. Progressive, chronic-active, fibrosing pneumonitis is observed later. Chronic beryllium lung disease of man is an immunologically mediated granulomatous lung disease, whereas beryllium-induced lung lesions in rats appear to be due to direct chemical toxicity and foreign-body-type reactions.

The Immunotoxicity of Three Nickel Compounds following 13-Week Inhalation Exposure in the Mouse

Groups of B6C3F1 mice were exposed to aerosols of nickel subsulfide (Ni3S2), nickel oxide (NiO), or nickel sulfate hexahydrate (NiSO4.6H2O) 6 hr/day, 5 days per week for 65 days to determine the immunotoxicity of these compounds. Exposure concentrations were 0.11, 0.45, and 1.8 mg Ni/m3 for Ni3S2, 0.47, 2.0, and 7.9 mg Ni/m3 for NiO; and 0.027, 0.11, and 0.45 mg Ni/m3 for NiSO4. Thymic weights were decreased only in mice exposed to 1.8 mg Ni/m3 Ni3S2. Increased numbers of lung-associated lymph nodes (LALN), but not spleen nucleated cells, were seen with all compounds. Nucleated cells in lavage samples were increased in mice exposed to the highest concentrations of NiSO4 and NiO and to 0.45 and 1.8 mg Ni/m3 Ni3S2. Increased antibody-forming cells (AFC) were seen in LALN of mice exposed to 2.0 and 7.9 mg Ni/m3 NiO and 1.8 mg Ni/m3 Ni3S2. Decreased AFC/10(6) spleen cells were observed in mice exposed to NiO, and decreased AFC/spleen were seen for mice exposed to 1.8 mg Ni/m3 Ni3S2. Only mice exposed to 1.8 mg Ni/m3 Ni3S2 had a decrease in mixed lymphocyte response. All concentrations of NiO resulted in decreases in alveolar macrophage phagocytic activity, as did 0.45 and 1.8 mg Ni/m3 Ni3S2. None of the nickel compounds affected the phagocytic activity of peritoneal macrophages. Only 1.8 mg Ni/m3 Ni3S2 caused a decrease in spleen natural killer cell activity. Results indicate that inhalation exposure of mice to nickel can result in varying effects on the immune system, depending on dose and physicochemical form of the nickel compound. These nickel-induced changes may contribute to significant immunodysfunction.

Immunopathologic effects of nickel subsulfide on the primate pulmonary immune system

The effects of nickel subsulfide (Ni3S2) on the pulmonary immune system were evaluated in cynomolgus monkeys that had been previously immunized and repeatedly challenged with sheep red blood cells in specific lung lobes. After instillation of Ni3S2, at a final dose of 0.06 mumol/g lung, in one immunized and one control lobe of each monkey, bronchoalveolar lavage cells were evaluated for alterations in cell number and function. Neither numbers of antibody-forming cells nor natural killer (NK) cell conjugate formation were altered in immunized lobes of nickel-exposed animals regardless of individual lung lobe nickel exposure. Macrophage phagocytic activity was significantly decreased, and target cell killing by conjugate-forming NK cells was increased in all lobes independent of antigen or nickel exposure in all nickel-exposed animals. Histologic sections of nickel-exposed lobes were characterized by mild focal accumulations of macrophages and lymphocytes within the interstitium and alveoli and by perivenular lymphoid infiltration and follicle formation. We conclude that alveolar macrophages are sensitive to the toxic effects of nickel and that local instillation of Ni3S2 in the subhuman primate lung results in suppression of pulmonary alveolar macrophage function accompanied by a secondary increase in NK cell-mediated killing of target cells.

Clearance, Translocation, and Excretion of Beryllium following Acute Inhalation of Beryllium Oxide by Beagle Dogs

Beagle dogs inhaled radiolabeled beryllium oxide (7BeO) particles that were calcined at either 500 or 1000 degrees C, resulting in either high (mean of 50 micrograms/kg body wt) or low (mean of 17 micrograms/kg body wt) initial lung burdens (ILBs) of both preparations of BeO. Levels of beryllium in whole body, tissue, and excreta were measured by external gamma-ray counting. Dogs were euthanized in pairs at 8, 32, 64, and 180 days after exposure to determine beryllium distribution in tissues. Beryllium oxide calcined at 1000 degrees C was retained more tenaciously in the lungs (62% of the ILB retained at 180 days after exposure) than BeO calcined at 500 degrees C (14% of the ILB retained at 180 days after exposure). Most of the beryllium that was cleared from the lungs and not excreted was translocated to the tracheobronchial lymph nodes, skeleton, liver, and blood. More beryllium was translocated to the skeleton and liver at 180 days after inhalation of BeO prepared at 500 degrees C than at 1000 degrees C. The predominant mode of excretion at early times after exposure was through the feces, with urinary excretion assuming predominance at later times. These data are important for interpreting the toxic effects of beryllium in the exposed dogs. Furthermore, because little is known concerning the retention and clearance of inhaled beryllium in man, these results provide information that may be used to understand the disposition of beryllium in accidentally exposed humans.

Long-term Consequences of 239PuO2 Exposure in Dogs: Persistent T Lymphocyte Dysfunction

Young Beagle dogs were exposed by inhalation to aerosols of 239PuO2 and observed for their lifespans as part of a large, ongoing study of the biological effects of inhaled radionuclides. The purpose of our study was to compare certain immune responses of the 239PuO2-exposed dogs at middle age (7-10 years old) and old age (12-14 years old), with those of unexposed, age-matched or young (3-4 years old) animals. Some of the aged, exposed dogs had developed lung tumours. Lymphocyte proliferative responses to phytohaemagglutinin (PHA) were lower in aged control dogs than in either young or middle-aged control dogs. Both aged and middle-aged, radiation-exposed dogs had decreased responses to PHA when compared to age-matched controls. Responses to concanavalin A (Con A) were not affected by age in control dogs, but tended to decrease in the oldest group of radiation-exposed dogs. Responses to both PHA and Con A were severely depressed in tumour-bearing dogs. The cytolytic activity of natural killer cells was not affected by age, radiation exposure, or tumour presence. We concluded that inhalation of 239PuO2 by young Beagle dogs resulted in an earlier-than-normal decrease in the ability of T cells to respond to mitogenic stimulation. In other words the depressed responses to PHA that were observed might represent radiation-induced, accelerated ageing of the T cell response.

The effects of age on immune responses in the antigen-instilled dog lung. Antibody responses in the lung and lymphoid tissues following primary and secondary antigen instillation.

To evaluate the effects of age on immunity induced by lung immunization, 11 aged (12-17 years; median age = 14) and 12 young (2-5 years) male Beagle dogs were instilled with 10 mg of keyhole limpet hemocyanin (KLH) in the right cardiac lung lobe and 10(10) sheep red blood cells (SRBC) in the left cardiac lung lobe. Five aged and six young dogs were sacrificed at day 9 after primary antigen instillation. The remainder were given challenge antigen instillations of KLH and SRBC at day 21 and sacrificed 7 days later. Serum, bronchoalveolar lavage fluid and lung tissue from immunized and control lobes, tracheobronchial, mesenteric and popliteal lymph nodes, spleen, and blood were taken at sacrifice. Anti-KLH IgA, IgG and IgM antibody production by cells in lung tissue and lavage fluid from the KLH-exposed lobe was lower at primary immunization and challenge in aged than young dogs. Lavage fluid IgA and IgG levels from the KLH exposed lobe at primary immunization and challenge were lower in aged versus young dogs, while IgM levels were lower only after primary immunization. Localized lung immune memory responses were also markedly lower in aged dogs when compared with young dogs. Anti-SRBC responses were similar to the anti-KLH responses. Our data show that systemic immune responses are significantly lower in aged dogs following primary antigen instillation, but not after antigen challenge in the lung. This was not the case for localized lung immune responses, which were significantly lower in aged dogs even following antigen challenge. The data also show that antibody production by lavage cells is a good index of interstitial lung cell antibody production.

Beryllium-induced lung disease in the dog following two exposures to BeO

We have shown previously that dogs exposed once to aerosols of beryllium oxide (BeO) calcined at 500 or 1000 degrees C developed granulomatous lung lesions as well as Be-specific immune responses in the blood and lung. In this report, we investigate the immunopathologic consequences of exposing dogs twice to aerosols of BeO. Dogs previously exposed to aerosols of 500 or 1000 degrees C calcined BeO to achieve an initial lung burden (ILB) of either 50 or 17 micrograms/kg body wt were exposed a second time to BeO calcined at 500 degrees C, 2.5 years after the first exposure, to achieve an ILB of about 50 micrograms/kg body wt. Immune responses of peripheral blood and lung lymphocytes were measured at 0, 14, 30, 60, 90, 120, 150, 165, 180, and 210 days postexposure (dpe), and dogs were euthanized at 210 dpe. Be-specific immune responses occurred in blood at 30 dpe and again at 150 to 210 dpe. Only sporadic positive responses were seen among lung lymphocytes when cells were cultured in 10% fetal bovine serum. In contrast, samples collected at 165, 180, and 210 dpe and incubated with 10% dog serum showed a large number of positive responses in both blood and lung. Histologic lesions were characterized by perivascular and interstitial infiltrates of lymphocytes and macrophages with progression to patchy granulomatous pneumonia accompanied by focal septal fibrosis. We conclude that Be-induced granulomatous and fibrotic lung lesions are accompanied by Be-specific immune responses within the lung but these changes do not appear to be cumulative if enough time has elapsed between exposures.

Pulmonary Immune Response of Dogs after Exposure to 239PuO2

This study evaluated the cell-mediated (CMI) and humoral immune responses in four Beagle dogs five to six years after single inhalation exposures to different monodisperse 239PuO2 aerosols (0.72-1.4 microns activity median aerodynamic diameter). These exposures resulted in initial lung burdens ranging from 19 to 35 kBq. Four nonexposed dogs were used as age-matched controls. Anesthetized dogs were immunized by instillation of sheep red blood cells (SRBC) into selected lung lobes. Cells and fluids were obtained serially from blood samples and by bronchoalveolar lavage of the saline- and SRBC-treated lung lobes at 5-20 days after immunization. The CMI response evaluated by the leukocyte procoagulant activity test was similar in the saline- and SRBC-treated lobes of both groups of dogs. The humoral immune response was measured by the enzyme-linked immunosorbent assay. No differences were shown between the amount of antibody measured in the sera or lung lavages from control or Pu-exposed dogs. Histopathology of the tracheobronchial lymph nodes from the Pu-exposed dogs showed them to be fibrotic with no lymphoid cells, suggesting that these tissues could not respond to the antigen deposited in the lungs. However, both mediastinal and sternal lymph nodes did contain lymphoid tissue, and were likely to be the lymphoid tissues that produced the immunity to the antigen deposited in the lungs of the exposed dogs. Although both exposed and control dogs produced immune responses to the antigen instilled into their lungs, differences were observed in the number of neutrophils in lung lavages from the control and exposed animals. There was a dramatic influx of neutrophils into both the saline- and SRBC-treated lung lobes of the Pu-exposed dogs that was not seen in the age-matched controls. This suggests that the inhaled 239PuO2 produced chronically-active inflammation in the lung which may contribute to recruitment of lymphocytes to the lung following intrapulmonary deposition of antigen. In conclusion, the immune responses induced by lung immunization of dogs that had inhaled 239PuO2 were not suppressed by large doses of chronic alpha irradiation of the lungs and tracheobronchial lymph nodes, indicating that local pulmonary immune responses are preserved despite severe radiation-induced alteration of these tissues.