Semiquantitative technique for estimating Pneumocystis carinii burden in the lung.

We developed a technique to estimate the amount of Pneumocystis carinii found in bronchoalveolar lavage fluid. P. carinii associated with 500 nucleated cells in the bronchoalveolar lavage fluid had little between-observer and within-observer variation. Varying the technique of the lavage did not change the amount of P. carinii recovered. This technique was used in patients treated for P. carinii pneumonia. Those patients who did not respond to treatment had more P. carinii in their bronchoalveolar lavage fluid than those who responded.

Release of tumor necrosis factor by alveolar macrophages of patients with sarcoidosis.

Tumor necrosis factor (TNF) is a monokine released by macrophages and is important in the inflammatory response. We compared the spontaneous release of TNF by alveolar macrophages (AMs) from patients with symptomatic pulmonary sarcoidosis, some of whom were receiving corticosteroid therapy, with AMs from control smokers. TNF was released from AMs of sarcoidosis patients at significantly higher levels than was released by control AM subjects (sarcoids, 15 U/ml [0 to 1140 U (median [range]]); controls, all less than 5 U/ml, p less than 0.01). By using a specific polyclonal antibody, the detected TNF was found to be TNF-alpha. Among the sarcoidosis patients, the amount of TNF released was significantly lower for those patients given treatment with corticosteroids (5 U/ml [0 to 15 U/ml]) compared with untreated persons (50 U/ml [0 to 1140 U/ml], p less than 0.05). In vitro studies demonstrated that incubation of peripheral blood monocytes or AMs with dexamethasone for 42 hours suppressed subsequent release of TNF. We conclude that AMs from sarcoidosis patients often spontaneously release TNF and this release is suppressed by prolonged corticosteroid therapy.

The use of an indirect fluorescent antibody test for detecting Pneumocystis carinii.

Pneumocystis carinii pneumonia is a major cause of morbidity and mortality in immunocompromised patients. An indirect fluorescent antibody (IFA) test has been developed using monoclonal antibodies specific for antigens on the surface of P carinii. We tested the sensitivity and specificity of this IFA test for detecting P carinii in respiratory specimens of immunocompromised patients with pulmonary symptoms undergoing bronchoscopy. Both the bronchial wash and bronchoalveolar lavage specimens of patients with and without P carinii pneumonia were studied. The bronchoalveolar lavage and bronchial wash specimens were examined using modified Wright-Giemsa and methenamine silver stains. In addition, aliquots of the specimen were fixed and stained with IFA and read with a fluorescent microscope. Fifty-nine patients were found to have P carinii organisms. The bronchial wash specimen has been shown to be less sensitive than the bronchoalveolar lavage specimen for detecting the presence of P carinii. In the bronchial wash specimen from these 59 patients, only 60% had positive modified Wright-Giemsa stains, and 70% had positive methenamine silver stains. The IFA stain was positive in 93% of the specimens tested (significantly higher than the other two stains). There was only one false-positive IFA test result among the 54 patients tested with negative results. We found the IFA stain to be superior to conventional stains when examining less-than-adequate specimens, such as those from bronchial washes.

Lung derived surface active material (SAM) inhibits natural killer cell tumor cytotoxicity.

Natural killer cells have been identified in lung tissue but have been found to have significantly less tumor cytotoxicity than natural killer cells found elsewhere in the body. The natural killer cells in the lung are still functional, since their killing can be enhanced by Interleukin-2. The surface active material (SAM) of lung lining fluid has been shown to have immunomodulating activity, including the suppression of lymphocyte blast transformation and enhancement of macrophage tumor cytotoxicity. We studied the effect of SAM purified from lung lining fluid on natural killer cell tumor cytotoxicity. SAM markedly inhibited tumor killing (Percent cytotoxicity of cells alone: 41 +/- 7.3% (mean +/- SEM); cells plus SAM: 10 +/- 9.7%, p less than 0.02). Further studies demonstrated that some phospholipids, including phosphatidylcholine, the major phospholipid of SAM, also significantly inhibited natural killer cell tumor killing. This inhibition of natural killer cell tumor cytotoxicity could be reversed by the addition of Interleukin-2 to the natural killer cells. These studies demonstrated that the surfactant found in lung lining fluid significantly inhibited natural killer cell tumor cytotoxicity and this effect could be reversed by Interleukin-2.

Spontaneous hydrogen peroxide release from alveolar macrophages of patients with active sarcoidosis: comparison with cigarette smokers.

Alveolar macrophages from patients with active pulmonary sarcoidosis have been shown to secrete several factors, such as interleukin-1 and alveolar macrophage-derived growth factor. We examined alveolar macrophages from nonsmoking patients with sarcoidosis undergoing bronchoalveolar lavage (BAL) for evaluation of disease activity. We compared these cells with macrophages from smoking and nonsmoking control patients. The amount of hydrogen peroxide released by the macrophages either spontaneously or after stimulation by phorbol myristate acetate was measured. The alveolar macrophages from smokers spontaneously released hydrogen peroxide, as we previously observed. The macrophages from the patients with sarcoidosis also released detectable amounts of hydrogen peroxide, but the macrophages from the non-smokers did not. Alveolar macrophages from all three groups released hydrogen peroxide when stimulated with phorbol myristate acetate. The macrophages from all three groups were examined for the presence on the surface membrane of beta-galactosidase, an enzyme that appears on the surface of older, activated macrophages. The macrophages in the BAL fluid of the patients with sarcoidosis had less beta-galactosidase staining than did those from the smokers, although they released comparable amounts of hydrogen peroxide. These findings suggest that alveolar macrophages in the BAL fluid of patients with sarcoidosis are younger, more monocyte-like, and activated by various factors, including gamma-interferon.

Bleomycin causes alveolar macrophages from cigarette smokers to release hydrogen peroxide.

Alveolar macrophages (AM) were retrieved by bronchoalveolar lavage from 20 patients. Following AM purification by glass adherence, the effect of bleomycin on hydrogen peroxide and lactic dehydrogenase release was assessed by colorimetric methods. All AM from nine nonsmokers had no detectable spontaneous release of hydrogen peroxide. The AM did not release hydrogen peroxide when stimulated with bleomycin (25 mU/mL). AM from all eleven smokers spontaneously released hydrogen peroxide (36 +/- 5.3 nm/10(6) AM, mean +/- SEM). AM from eight of the eleven smokers released more hydrogen peroxide when incubated with bleomycin (smokers 55 +/- 6.8 nm/10(6) AM, p less than 0.05). There was no difference in the amount of lactic dehydrogenase released by AM spontaneously or when incubated with bleomycin at 25 mU/mL, suggesting that this dose of bleomycin was not directly toxic to the AM. Results from this study further support the premise that bleomycin pulmonary toxicity may result from the generation of reactive oxygen metabolites and that cigarette smoking may predispose to bleomycin pulmonary toxicity.

Enhancement of macrophage and monocyte cytotoxicity by the surface active material of lung lining fluid.

The surface active material (SAM) of alveolar lining fluid has been shown to have immunologic activity. We studied the effect of SAM on monocyte-macrophage cytotoxicity against a tumor cell line. Alveolar macrophages were studied from 15 subjects without cancer. Tumor growth, as assessed by tritiated thymidine incorporation, was significantly inhibited by the macrophages alone (tumor alone median 39,401 cpm, macrophages plus tumor median 12,153 cpm, P less than 0.01). Tumor cytotoxicity was enhanced by preincubating the macrophages with lipopolysaccharide (median 37 cpm, P less than 0.01) or coincubating the tumor cells and macrophages with SAM (median 5474 cpm, P less than 0.01). Similar results were seen when using blood adherent mononuclear cells. There was increasing cytotoxicity for the adherent mononuclear cells with increasing amounts of SAM. When the various phospholipids of SAM were studied, it was found that phosphatidylcholine, sphingomyelin, and phosphatidyl glycerol all enhanced adherent mononuclear cell cytotoxicity, whereas phosphatidylinositol inhibited adherent mononuclear cell cytotoxicity. These studies suggest that SAM may have important immunoregulatory function for the alveolar macrophage.

Variation of differential cell counts of bronchoalveolar lavage fluid.

Bronchoalveolar lavage (BAL) is a common research and clinical tool to retrieve cells from the lower respiratory tract. Differential cell counts of the nucleated cells retrieved by BAL provide important diagnostic and prognostic information. There is much variation between laboratories in the reported normal percentages of lymphocytes and macrophages of cells retrieved by BAL. We compared three methods of identifying cells in the BAL fluid: a modified Wright-Giemsa (WG) stain, a nonspecific esterase stain, and a monoclonal marker for T lymphocytes. There was good agreement between the percentage of macrophages identified by WG and nonspecific esterase and the percentage of lymphocytes determined by WG stain and monoclonal marker. Intrasubject and intersubject agreement of the differential cell counts determined by the WG stain was good. We concluded that cells from BAL fluid can be analyzed using the WG stain alone.

Spontaneous hydrogen peroxide release from alveolar macrophages of some cigarette smokers.

Alveolar macrophages were retrieved by bronchoalveolar lavage (BAL) from 30 patients, 24 smokers and six nonsmokers. The macrophages were separated from other cells in the BAL fluid by glass adherence. The amount of hydrogen peroxide released into the media by these macrophages was then measured by a new method of determining hydrogen peroxide concentration. Two groups were found. Group 1, who did not spontaneously release hydrogen peroxide, were mostly nonsmokers (six of nine), and group 2, who spontaneously secreted hydrogen peroxide (87.5 +/- 17.08 nmol/10(6) macrophages [mean +/- SEM]), were all smokers (21 of 21). When the alveolar macrophages in group 1 were stimulated with phorbol myristate acetate, they secreted as much hydrogen peroxide as the stimulated macrophages of group 2 (group 1: 125.0 +/- 92.08 nmol/10(6) macrophages, group 2: 116.7 +/- 14.82 nmol/10(6) macrophages). We conclude that there is a subset of smokers whose alveolar macrophages spontaneously release hydrogen peroxide.

Decreased phosphatidylcholine in the lung fluid of patients with sarcoidosis.

Surfactant decreases the immune response of lymphocytes. Pulmonary sarcoidosis is a disease characterized by an increased number and activity of lymphocytes in the lung. We measured the lipids and lymphocytes retrieved from the lung by bronchoalveolar lavage. Thirteen patients with active pulmonary sarcoidosis had a significantly higher percentage of lymphocytes (18 +/- 21%, mean +/- standard error of the mean) than 10 control subjects (4 +/- 1.9%, p less than 0.01). Using an external marker, we found the absolute amount of disaturated phosphatidylcholine to be higher in the control group (174 +/- 17.4 micrograms/ml lung fluid) than in the sarcoid group (91 +/- 1.9 micrograms/ml of lung fluid, p less than 0.002).