Human alveolar macrophages release a factor that inhibits phagocyte function.

Human alveolar macrophages release in vitro a factor that inhibits both random migration and chemotaxis of human polymorphonuclear neutrophils (PMN). This factor is not cytotoxic and is recovered in culture supernatants of alveolar cells from most nonsmoking normal subjects. The inhibitor can be detected 30 min after cell cultures are established and is still produced after 24 h in culture. Its release was inhibited by cycloheximide. When supernatants are separated by molecular sieving (I-60 Waters HPLC column), most of the inhibitory activity is recovered in the low-molecular-weight fractions of the chromatogram (less than 1,000 D). The inhibitor has a broad spectrum of activity against known chemoattractants in that it reduces significantly the chemotaxis of PMN induced by the formyl peptide FMLP, by the complement fragment C5a, and by leukotriene B4; it also decreases the chemotactic activity associated with a monocyte-derived interleukin 1 preparation and the chemotactic activity derived from alveolar macrophage culture supernatants. The inhibitory factor is partially heat labile, is sensitive to aminopeptidase M, and is nonpolar. Both phorbol myristate acetate (PMA) and FMLP-induced superoxide release by PMN are diminished significantly in the presence of this inhibitory factor (p less than 0.01 for PMA and p less than 0.05 for FMLP). The inhibitor also reduces monocyte chemotaxis but has no effect on monocyte random migration. Finally, studies with [3H]FMLP indicate that this inhibitor does not act at the site of receptor binding on PMN. Thus, human alveolar macrophages can release in vitro both neutrophil chemotactic factors and an apparent neutrophil-inhibiting factor that may modulate positively and negatively the movement and the respiratory burst of neutrophils in the alveolar space.

Neutrophil chemotactic activity produced by normal and activated human bronchoalveolar lavage cells.

Activated macrophages can secrete a number of mediators that can attract inflammatory cells and enhance secretion of phlogistic substances from these cells. The ultimate effect of activated bronchoalveolar lavage (BAL) cells may be fibrotic lung injury. Inasmuch as pulmonary sarcoidosis is a disease associated with spontaneous activation of macrophages and lymphocytes among BAL cells, cells obtained from patients with sarcoidosis were compared with normal cells. We report that adherent BAL cells in culture from patients with sarcoidosis (n = 21) release during a resting period in vitro more chemotactic activity for neutrophils (PMNs) than do BAL cells from normal individuals (n = 14). After density fractionation of the respiratory cells by albumin gradient, cells from high-density fractions in the group with sarcoidosis secrete more chemotactic activity for neutrophils than cells from less dense fractions. The PMN chemotactic activity spontaneously released in vitro by BAL cells from patients with sarcoidosis correlates with the percentage of PMNs recovered by BAL. Immunochemical bioassay and high-performance liquid chromatographic (HPLC) analysis of BAL cell supernatants revealed a complex pattern of chemotactic factors to be present. Generally, three peaks of chemotactic activity were noted on HPLC 1-60 separations at greater than 20 kd, 8 to 10 kd, and less than 1 kd apparent molecular weights. Significantly, interleukin-1 was present in these supernatants, whereas complement components and leukotriene B4 were absent. Sarcoid BAL cells, principally alveolar macrophages, are activated in vivo as manifested by spontaneous secretion of chemotactic factors for PMNs in vitro. Interleukin-1 and other less well characterized molecules were detected. The presence of PMNs among the lavage cells of some patients with sarcoidosis appears to be an in vivo biologic correlate of this activation. These data provide additional criteria of BAL cell activation in patients with pulmonary sarcoidosis and provide further evidence concerning factors that attract inflammatory cells into the lung.

Chronic beryllium disease in a precious metal refinery. Clinical epidemiologic and immunologic evidence for continuing risk from exposure to low level beryllium fume.

Five workers at a precious metal refinery developed granulomatous lung disease between 1972 and 1985. The original diagnosis was sarcoidosis, but 4 of the workers were subsequently proved to have hypersensitivity to beryllium by in vitro proliferative responses of lymphocytes obtained by bronchoalveolar lavage. Review of medical records of coworkers and extensive industrial hygiene surveillance of the plant demonstrated that 4 cases occurred in the furnace area where air concentrations of beryllium fume were consistently below the permissible exposure limit of 2 micrograms/M3. A single case has been recognized from parts of the refinery where exposures to cold beryllium dust often exceeded the standard by as much as 20-fold. These data demonstrate that chronic beryllium disease still occurs and confirm the importance of specific immunologic testing in patients suspected of having sarcoidosis but with potential exposure to beryllium. The data raise concern about the adequacy of modern industrial controls, especially in the setting of exposure to highly respirable beryllium fume.

Use of a central laboratory for analysis of bronchoalveolar lavage fluid.

The logistics and related costs were assessed for providing analysis of bronchoalveolar lavage (BAL) fluid in a central laboratory for practicing pulmonologists in the area around New Haven, Connecticut. During a 4-yr period (1981-1985) we arranged for a courier service to transport 175 BAL specimens, obtained by 22 participating physicians at the time of fiberoptic bronchoscopy, directly to our laboratory. A large battery of information was generated on each specimen, and the data were returned to the responsible physician within 24 h. Any remaining cells and the cell-free supernatants were used for additional research purposes. The primary benefits of this program included: rapid and standardized analysis of lavage fluid cell counts for the clinician to use along with historical and other more routine laboratory tests in guiding patient diagnosis and an augmented number of lavage specimens available to the academician for research purposes. The costs were judged to be reasonable but need to be factored into the overall costs of patient diagnostic evaluations.

Bronchoalveolar lavage cells and proteins in patients with the acquired immunodeficiency syndrome. An immunologic analysis.

Several components of cellular and humoral immunity were examined in bronchoalveolar lavage fluid and blood of 15 patients with the acquired immunodeficiency syndrome, and the results were compared to data from 25 healthy controls (including 5 asymptomatic homosexual men). Compared with that of controls, bronchoalveolar lavage fluid from patients tended to have more lymphocytes and significantly more neutrophils; a lower OKT4/OKT8 ratio, due to an increase in total OKT8 cells; and normal total OKT4 cell counts, despite a significant decrease in numbers of OKT4 cells in peripheral blood. Patients also had significantly more IgG-releasing cells and higher IgG levels than controls in lavage fluid. These data show that, in the lung lining fluid of patients with the acquired immunodeficiency syndrome, significant alterations in cellular and humoral immunity exist that differ in several important respects from immunity in controls and from corresponding changes in patients' peripheral blood.

Immunoglobulin G subclass proteins in serum and lavage fluid of normal subjects. Quantitation and comparison with immunoglobulins A and E.

Although total concentration of immunoglobulin G has been quantitated in the lower respiratory tract of humans, the contribution of the 4 subclass species of IgG to total recoverable IgG protein has not been assessed. We have developed sensitive, micro-ELISA assays specific for the individual subclasses and employed them to measure serum and local intrapulmonary levels of these proteins. We have compared lung lavage and serum concentrations of these proteins (relative to albumin) and also compared these immunoglobulins with IgA and IgE. The results of serum level measurements of subclass proteins are similar to results reported by others; IgG1 and IgG2 are present in lung lavage in concentrations similar to their serum concentration, serum and lavage levels are directly related, and IgG4 is increased in lavage compared with that in serum, suggesting increased local synthesis or accumulation of this protein within the lower respiratory tract. Local intrapulmonary concentrations of both IgA and IgE also are increased compared with those in their serum concentrations. Local IgG3 is variable, with some subjects having increased amounts compared with that in serum, whereas others have concentrations similar to those in serum. These data suggest a preferential accumulation of IgG4 in the lower respiratory tract. It is possible that IgG4, like IgA and IgE, plays a special role in the immune defense of the lung.

Proteins of the cystic fibrosis respiratory tract. Fragmented immunoglobulin G opsonic antibody causing defective opsonophagocytosis.

In the disease cystic fibrosis (CF), pulmonary infection with Pseudomonas aeruginosa is a common clinical complication that determines most morbidity and almost all excess mortality. We postulated that in this disease a defect in Pseudomonas-reactive IgG antibodies may contribute to chronic Pseudomonas infections. Bronchoalveolar lavages were performed upon 13 patients with CF, 7 patients with chronic bronchitis characterized by recurrent Pseudomonas infections, and 4 normal volunteers. The levels of various proteins important to host defenses and proteases were determined; enzyme inhibition studies were performed. CF respiratory immunoglobulin levels were significantly elevated when compared with both normals and patients with chronic bronchitis (P less than 0.05). Albumin and transferrin levels were decreased in the CF lung fluids. CF elastolytic activity was strikingly elevated (means = 6.02 micrograms/mg total protein) and the inhibitory profile suggested such activity resembled a serine-proteinase. Alpha-1-antitrypsin antigenic levels were not altered in CF respiratory fluids. There was a tendency for the lavage IgG to fall as elastase levels rose (r = -0.29). IgG opsonins for two Pseudomonas immunotypes were isolated with affinity chromatography for functional and immunochemical studies. Bacterial phagocytic rates in the presence of these Pseudomonas-reactive IgG opsonins derived from CF lavage fluid were depressed (0.3% uptake/unit time) when compared with similarly titered positive controls (uptake = 1.3%/unit time, P less than 0.001). Additionally, normal pulmonary macrophage intracellular killing of Pseudomonas was severely altered in the presence of opsonins derived from CF respiratory fluids. At some time points, less than 30% of the bacteria were killed. CF IgG opsonins contain a cleavage fragment (100,000 D, 5S sedimentation coefficient) with antigenic determinants similar to the Fab portion of IgG. The presence of such a fragment was inversely correlated with phagocytic functional activity. Intact IgG comprised as little as 18% of the CF lavage fluid specimens. Aliquots of intact human IgG, when mixed with the CF opsonins, augmented Pseudomonas uptake and improved intracellular killing. Conversely, peptide fragments of IgG opsonins, which are proteolytically derived in vitro, duplicated in our system the defect observed with opsonins derived from CF lung fluids; bacterial uptake was inversely related to the concentration of F(ab')2 and to a greater degree, to Fc present in the opsonic mixture. We concluded that IgG respiratory opsonins are fragmented, inhibiting phagocytosis and serving a permissive role in the chronic Pseudomonas pulmonary infection in the disease CF.

Receptors for human IgG subclasses on human alveolar macrophages.

The biology of individual heavy chain subclasses of human IgG (IgG1-4) in lung host defenses has become important now that specific deficiencies of certain subclasses (IgG2 and IgG4) can be associated with chronic sinopulmonary infections and that IgG4 can be increased in forms of hypersensitivity lung disease. Because IgG is an important opsonic antibody that promotes attachment of bacteria or particles to phagocytes, the relative binding of IgG subclasses to membrane receptors on human alveolar macrophages might predict the efficacy of specific opsonin-mediated phagocytosis. With in vitro cultured normal alveolar macrophages, various IgG complexes were assessed for receptor binding with a rosetting assay. For respiratory cells in culture for 24 h, about 25% of the macrophages bound IgG3 and about 10% bound IgG1; binding with IgG2 and IgG4 complexes was minimal. In macrophage cultures maintained for as long as 6 days, this pattern of binding persisted. However, in very short-term cultures, 30 min and 105 min after cell adherence had occurred, binding was much greater for IgG3 complexes (about 60%); likewise IgG1 and IgG4 bound to about 20% of the cells. The IgM erythrocyte complexes, usually showing no binding at later time points in culture, bound to 20% of the cells, acutely. Therefore, our studies found that IgG3 consistently bound to more alveolar macrophages than the other subclasses, including IgG1. Also, the duration in culture of adherent cells may significantly affect the pattern of binding.

Air-space immunoglobulin production and levels in bronchoalveolar lavage fluid of normal subjects and patients with sarcoidosis.

The purpose of this study was to examine the relationship between immunoglobulin production and immunoglobulin levels in bronchoalveolar lavage (BAL) fluid and serum of normal subjects and patients with sarcoidosis. Eleven normal volunteers and 17 patients were studied. In normal subjects, no important relationship existed between the number of immunoglobulin-secreting cells and immunoglobulin levels in BAL or serum. By contrast, in patients with sarcoidosis, a highly significant correlation existed between the number of IgG secreting cells and IgG/alb% in BAL (p = 0.008) and between the number of IgG secreting cells in BAL and serum IgG mg/ml (p = 0.002). Similar associations did not exist for IgA and IgM. These data demonstrate for the first time the relationship between immunoglobulin production and immunoglobulin levels in normal persons, and convincingly show that immunoglobulin production at sites of disease activity is responsible for hypergammaglobulinemia in BAL and serum of patients with sarcoidosis.

Cystic fibrosis pseudomonas opsonins. Inhibitory nature in an in vitro phagocytic assay.

Pseudomonas aeruginosa infection plays a primary pathogenetic role in the chronic respiratory tract disease of cystic fibrosis (CF) patients. Despite pronounced humoral immune responses, reflected by high levels of antibodies against Pseudomonas in serum and in sputum, the antibodies do not eliminate this bacterium. In the present study we have used affinity chromatography with a lipopolysaccharide substituted immunoadsorbent gel to isolate high titers (meanCF = 1:256) of immunotype specific Pseudomonas IgG antibodies from the sera of nine CF subjects, and have evaluated the functional ability of these antibodies to promote phagocytosis and intracellular killing of P. aeruginosa in an in vitro human alveolar macrophage culture system. The phagocytic and intracellular bactericidal kinetics revealed that CF IgG antibodies function in an inhibitory fashion. Both the rate of phagocytosis (rateCF = 204 cpm/unit time) and absolute bacterial uptakes maximal at 120 min (uptakeCF = 18 x 10(3) 14C cpm) were inhibited compared with appropriate positive controls (hyperimmune serum, HIS; [rateHIS = 399; uptakeHIS = 29 x 10(3), P less than 0.005]). The ability of such CF-derived opsonins to potentiate macrophage intracellular bactericidal processes was mildly impaired (bacterial survivalCF = 15 x 10(3) colony forming units (CFU)/min, survivalHIS = 9 x 10(3)). Further characterization of this defect, assessed with functional studies of the Fab and Fc portions of the immunoglobulin molecule, revealed an impairment in the attachment of these specific antibodies to the alveolar macrophage membrane Fc gamma receptors. Preliminary studies of the physical-chemical properties of these immunoglobulins were normal. The expression of this inhibitory activity in vivo may facilitate Pseudomonas colonization and the subsequent established infections in the respiratory tracts of CF subjects.

Quantitation of carcinoembryonic antigen in the lung lining fluid of normal smokers and nonsmokers.

Bronchoalveolar lavage was performed in 47 volunteers: 19 nonsmokers and 28 smokers. Total protein, albumin, immunoglobulins G and A, and carcinoembryonic antigen (CEA) were measured in the concentrated lavage effluent. Although a significant increase (p < 0.001) in the ratio of CEA to total protein recovered from the group of smokers was found, this increase primarily reflected the greater increase that occurred in a subgroup of 7 smokers. However, the increases in lavage CEA correlated weakly (p = 0.096) with smoking history in pack-years, and not at all with plasma CEA concentrations. Results regarding the number of cells recovered and immunoglobulin-to-albumin concentration ratios in these subjects were similar to those reported by others. Thus, CEA was increased in the lavage fluid of a subgroup of otherwise normal young smokers. It is possible that CEA might serve as a useful indicator of future airway disease in certain young smokers.

Alveolar macrophage-derived chemotactic factor: kinetics of in vitro production and partial characterization.

Alveolar macrophages are the initial phagocytic cells that encounter foreign material and particulates deposited in the terminal airways. We have examined a mechanism by which these cells, after phagocytic challenge, may control or amplify the inflammatory response in lung parenchyma. Normal human alveolar macrophages (AM) were studied from eight subjects. With in vitro culture, AM produced and released two substances into culture media which have potent chemoattractant activity for blood polymorphonuclear granulocytes (PMN) and negligible activity for mononuclear cells. Release of these factors is maximally stimulated by aggregated human immunoglobulin (Ig)G or zymosan particles; however, simple adhesion of the macrophages to plastic surfaces is also sufficient to stimulate release of these chemotactic substances. The larger substance (10,000 daltons) is immunologically distinct from C5a and interacts with a different PMN membrane receptor than that known to exist for formyl-methionyl-leucyl-phenylalanine. Its chemotactic activity is sensitive to the enzymatic effect of trypsin. Although producing a single elution peak on gelfiltration chromatography, electrofocusing in polyacrylamide gels yielded five peaks of radioactivity. Chemotactic activity was localized to a fraction with a pI = 5.0. The smaller molecular weight substance has been less well characterized. Thus, the human AM can produce at least two factors which attract PMN and this capability may augment the local inflammatory response in the lung.

Use of Pseudomonas aeruginosa lipopolysaccharide immunoadsorbents to prepare high potency, mono-specific antibodies.

Potent, mono-specific anti-Pseudomonas immunoglobulins were isolated from serum and lung lavage fluid from patients with cystic fibrosis using immunotype specific Pseudomonas aeruginosa lipopolysaccharide (LPS) substituted immunoadsorbent gel. Iodinated monovalent Pseudomonas LPS somatic antigens, Fisher immunotypes, were used as ligands for two different insoluble gel matrices. LPS iodination, using the water insoluble chloroglycoluril reagent, permitted quantitation of the percent LPS bound as a ligand. The coupling efficiencies of epoxy-activated and cyanogen bromide-activated Sepharose matrices for various Pseudomonas immunotype specific LPS preparations were compared. Although each of the 7 LPS somatic antigens produced an equivalent amount of coupling, higher percentages of coupling were found using the cyanogen bromide-activated gel when compared to the epoxy-activated gel. IgG fractions prepared from cystic fibrosis sera and lung lavage fluid were passed through the LPS affinity gels, and Pseudomonas LPS immunotype specific antibodies were eluted. The presence of specific antibody activity against individual Pseudomonas immunotypes was determined with a passive micro-hemagglutination assay. Bronchial lavage fluid seemed to be as effective as serum as a source of Pseudomonas specific antibody. Use of such a LPS substituted gel permits direct recovery of Pseudomonas monospecific antibodies suitable for physical-chemical analyses and for biologic functional assays.