Induction of aryl hydrocarbon hydroxylase in human pulmonary alveolar macrophages by cigarette smoking.

Pulmonary alveolar macrophages were obtained from healthy volunteers by saline pulmonary lavage, and aryl hydrocarbon hydroxylase was measured in the cells. Enzyme activity was low in cells from five nonsmokers with a mean of 0.008+/-0.004 U/10(6) cells. Cells obtained from nine cigarette smokers contained higher enzyme levels, with a mean of 0.095+/-0.024 U/10(6) cells. A former cigarette smoker was lavaged on five occasions. Enzyme activity during two lavages 4 mo apart were 0.010 and 0.009 U/10(6) cells, respectively. 1 wk after smoking was resumed, the enzyme activity rose slightly to 0.013, and reached 0.041 U/10(6) cells by 1 mo. Upon cessation of smoking, the enzyme activity returned to control levels by the next lavage, 2 mo later. These data indicate that aryl hydrocarbon hydroxylase may be induced in pulmonary alveolar macrophages of subjects chronically exposed to cigarette smoke.

Lipopolysaccharide (LPS) alters phosphatidylcholine metabolism in elicited peritoneal macrophages.

We investigated the effects of LPS on mouse peritoneal macrophage phospholipids using radiolabeled precursors. LPS (200 ng/ml) stimulated incorporation of [32P] into all classes of phospholipids within 0.5 hr, and after 2 hr the increase was 60% greater than controls. Separation of the phospholipid classes by thin-layer chromatography revealed a selective increase in incorporation of label into phosphatidylcholine (PC) (90% increase compared to approximately 50% in the other phospholipids). In macrophages labeled with [3H]-choline, LPS stimulated both the incorporation of label into PC and the release of incorporated label into the medium. The time dependencies of stimulated [3H] release and [32P] incorporation were similar. These data are consistent with the hypothesis that LPS activates macrophages via a PC-specific phospholipase-dependent mechanism.

Induction of aryl hydrocarbon hydroxylase in human pulmonary alveolar macrophages and peripheral lymphocytes by cigarette tars.

Cigarette smoking is associated with alterations in pulmonary alveolar macrophages (PAMs), including increased cytoplasmic inclusions and induction of the aryl hydrocarbon hydroxylase (AHH) system. Nonpigmented PAMs from nonsmokers were able to ingest and accumulate pigment from lysed PAMs of smokers, however, this pigment did not induce AHH activity in either PAMs or peripheral lymphocytes. In contrast, the cigarette tars significantly induced AHH levels in PAMs and in peripheral lymphocytes from either nonsmokers or smokers. This provides further evidence that components in cigarette smoke can explain the in vivo induction of AHH documented in cells from smokers.

Pulmonary inflammatory responses during viral pneumonia and secondary bacterial infection.

Pulmonary bactericidal mechanisms are reduced during viral pneumonia. It has been proposed that secondary bacterial pneumonia occurs because the host's ability to mount an inflammatory response is suppressed. These studies examine the pathobiology of parainfluenza 1 virus and viral-associated staphylococcal pneumonia in a murine model. The sequence of leukocyte and fluid protein changes were studied in the lung and blood. An influx of polymorphonuclear leukocytes into the lungs occurred early in the viral infection, and coincided with lung macrophages aggregation. Maximal increases in pulmonary leukocytes occurred during the period associated with maximum suppression of lung bactericidal mechanisms (days 7-9). During this period, the host was capable of mounting an additional inflammatory response to staphyloccal challenges. Finally, viral pneumonia resulted in a prolonged elevation in the numbers of pulmonary macrophages, lymphocytes, and granulocytes. Thus, changes in lung biology persisted well after resolution of the initiating infections.

BMS-182123, a fungal metabolite that inhibits the production of TNF-alpha by macrophages and monocytes.

A fungal metabolite, BMS-182123, which inhibited bacterial endotoxin-induced production of tumor necrosis factor (TNF-alpha) in murine macrophages and human peripheral blood monocytes (in vitro), was isolated from the culture broth of Penicillium chrysogenum strain V39673. The effective BMS-182123 concentration (IC50) resulting in 50% inhibition of lipopolysaccharide-induced TNF-alpha production in murine macrophages and human monocytes was 600 ng/ml and 4.0 microgram/ml, respectively. BMS-182123 suppressed the lipopolysaccharide-induced TNF-alpha promoter activity and did not affect the stability of posttranscriptional mRNA. Addition of hydrophobic resin, Amberlite XAD-8 (1%), to the fermentation enhanced the production of BMS-182123 by 5.5 fold. A total of 577 mg pure BMS-182123 was recovered from a 250-liter fermentation supplemented with 1% Amberlite XAD-8.

Alveolar macrophage dysfunction associated with viral pneumonitis.

Viral infections are known to predispose to bacterial infections of the lung. Studies on the virus-induced suppression of pulmonary bactericidal mechanisms have identified the defect with abnormalities in the alveolar macrophage phagocytic system. In the investigation presented herein, we have dissected some of the subcomponents of the phagocytic process and found virus-induced defects in phagocytic ingestion, phagosome-lysosome fusion, and intracellular killing.

Immune-enhanced phagocytic dysfunction in pulmonary macrophages infected with parainfluenza 1 (Sendai) virus.

Cultured alveolar macrophages infected with parainfluenza 1 (Sendai) virus were treated with specific antiviral immune serum and their phagocytic activity for opsonized erythrocytes (EA), Candida krusei, and Staphylococcus epidermidis quantitated. Membrane Fc receptor and candida binding activity were unaffected by the viral infection. In contrast, the virus infection decreased the phagocytic ingestion of EA. The addition of immune serum induced new phagocytic defects in that the treatment of virus-infected macrophages decreased the binding of EA and candida and reduced the ingestion of the yeast and the staphylococci. In addition, treatment with immune serum also enhanced the phagocytic defects induced by the virus infection alone, further reducing the binding and ingestion of EA. Neither virus infection nor treatment with immune serum affected the intracellular killing of S. epidermidis. These data demonstrated that virus infection of alveolar macrophages in vitro induce phagocytic defects that are accentuated by the treatment of the macrophages with antiviral antibody.

Lung defenses against viral and bacterial challenges during immunosuppression with cyclophosphamide in mice.

Pulmonary virus infections predispose to secondary bacterial pneumonias by suppressing the antibacterial defenses of the lung. Cyclophosphamide (CY) treatment interferes with antiviral defenses and also impairs pulmonary bactericidal activity. To determine whether CY aggravates secondary bacterial pneumonias, mice were infected by aerosol inhalation with para-influenza 1 (Sendai) virus and injected intraperitoneally with either 0.5, 1.0, 2.5, and 5.0 mg of CY on Days 1 and 5 of the infection. On Day 7, the lungs of control (CY-treated but not infected) and virus-infected mice were lavaged and the total and differential number of free pulmonary cells quantitated. At the same time, other groups of mice were challenged aerogenically with Staphylococcus aureus and the number of initially viable bacteria remaining in their lungs quantitated at 4 and 24 h thereafter. The CY treatment induced a dose-dependent neutropenia, which was paralleled by the number of free pulmonary cells recovered from the lungs. Pulmonary bactericidal activity was also suppressed by CY treatment, with the percentage of staphylococci remaining at 24 h in the lungs of control animals being 0.5 +/- 0.2% and 1.5 +/- 0.5%, 4.0 +/- 1.5%, 8.5 +/- 2%, and 36 +/- 5%, respectively, for the increasing doses of CY. In virus-infected animals, CY treatment suppressed the inflammatory response in a dose-dependent manner, with the total number of free lung cells recovered from the highest dose group being only 5% of that recovered from untreated animals. Virus infection depressed the antibacterial defenses of the lung so that in untreated animals, 80 +/- 9% of the staphylococcal remained at 24 h. Treatment with the 2 higher doses of CY caused the bacteria to proliferate extensively in the lungs to 280 +/- 53% and 792 +/- 112%, respectively, for the 2.5 mg and 5.0 mg CY doses. In contrast, treatment with 0.5 mg and 1.0 mg of CY significantly enhanced the intrapulmonary killing of S. aureus in virus-infected lungs so that the bactericidal values at 24 h were 28 +/- 3% and 21 +/- 2%, respectively. These data demonstrated that immunosuppression modulates virus-induced suppression of pulmonary antibacterial defenses with high doses of CY aggravating and low doses ameliorating the defect.

Alterations in lung macrophage antimicrobial activity associated with viral pneumonia.

Secondary bacterial infections are a common sequelae of viral pneumonias. To study 2 functions of the phagocytic defenses of the lung, macrophages were obtained by lung lavage from parainfluenza 1 virus-infected and noninfected mice. The phagocytic capacities (binding, ingestion, and killing) of these cells were assessed in vitro against viable Candida krusei. Viral pneumonia resulted in a progressive suppression (through day 7 of the infection) of the ability of macrophages to bind candida to their surfaces by nonimmunological or complement receptors; ingestion and intracellular killing of candida were also decreased. After day 7, all these functions returned and, in fact, cells with enhanced activities were present on day 17. After introduction of virus into the lungs, the lung macrophage population increased significantly between days 3 and 7 of infection. This resulted in an increase in the phagocytic potential of the lung, despite the virus-associated suppression of the phagocytic activity in a portion of the macrophages. However, the ability of the macrophages to kill ingested microorganisms was also reduced, resulting in an overall deficiency in the lung macrophage defenses. It was concluded that viral pneumonia was associated with at least two suppressive effects on the lung macrophage-decreased receptor activity and microbicidal activity-resulting in a deficiency in the lung phagocytic defenses represented by these cells. These effects were maximal 1 week after infection and could account for the increased susceptibility of these lungs to secondary bacterial pneumonias. In contrast, during the period of convalescence, the lung macrophage antimicrobial activities were increased and reflected in enhanced resistance of the lungs to infections.

In vitro migration of human alveolar macrophages: effects of cigarette smoking.

The responsiveness of alveolar macrophages lavaged from healthy volunteers to migration inhibitory factor (MIF) was tested by using the capillary-tube assay method. In every instance, macrophages from nonsmokers responded to MIF as demonstrated by a depression in migration of at least 30%, whereas MIF did not inhibit migration of macrophages from smokers. Cells from smokers migrated at a rate three times faster than cells from nonsmokers. Migration of macrophages from nonsmokers with delayed hypersensitivity skin reactions to Candida albicans antigen was inhibited when antigen was present in the tissue culture medium. Antigen did not inhibit macrophages from subjects who lacked delayed hypersensitivity to the antigen, or from subjects who were cigarette smokers. Since alveolar macrophages can respond to MIF in vitro, they may play a role in cell-mediated immune reactions in the lung. Cigarette smoking may interfere with this participation.

Chemotactic responsiveness of human alveolar macrophages: effects of cigarette smoking.

Pulmonary alveolar macrophages from six cigarette smokers demonstrated higher random migration and greater chemotactic responsiveness to casein than did macrophages from seven nonsmokers. These observations are consistent with the concept that pulmonary macrophages are metabolically activated by cigarette smoking.

Lung macrophage defense responses during suramin-induced lysosomal dysfunction.

Lysosomes form an integral part of the degradative mechanisms of the phagocytic cells. Mice were injected with suramin, a lysosomotrophic drug, to investigate the effects of lysosomal pathology on the cell biology and in situ bactericidal activity of the pulmonary macrophage. Treatment with suramin resulted in marked alterations in the cell biology of the macrophage: (i) increased vacuolization and protein content, (ii) suppressed intracellular phagosome-lysosome fusion, (iii) decreased activity of the lysosomal enzymes beta-glucuronidase and N-acetyl-glucosaminidase, and (iv) enhanced exocytosis of acid phosphatase during phagocytosis. Addition of suramin, in vitro, to cell lysates resulted in a reduction in the catalytic activities of acid phosphatase, beta-glucuronidase, and N-acetyl-glucosaminidase; thereby suggesting that selective interaction, in vivo, between suramin and lysosomes containing beta-glucuronidase and N-acetyl-glucosaminidase may have occurred. Plasma membrane 5'-nucleotide phosphodiesterase activity was increased in macrophages recovered from suramin-treated animals. Although the "resting-state" reduction of nitroblue tetrazolium (NBT) was lower in these macrophages, cells stimulated by a phagocytic challenge demonstrated normal increases in NBT reduction. Phagocytosis, in vitro, and pulmonary bactericidal activity were not altered. These data demonstrate that suramin altered numerous aspects of the phagocyte's lysosomal system. Despite these changes in the cell biology of the pulmonary macrophage, the cell's defense functions were not reduced.

The participation of antiviral immune mechanisms in alveolar macrophage dysfunction during viral pneumonia.

Virus infections transiently suppress pulmonary antibacterial defenses by causing dysfunctions in the alveolar macrophage phagocytic system. This impairment of pulmonary bactericidal activity is not associated in time with virus proliferation, but rather with the period of time of rapidly declining virus titers and the expression of the antiviral immune response in the lungs. This temporal relationship suggests that the impairment of pulmonary bactericidal activity might be secondary to the antiviral immune response rather than a direct effect of virus replication. Immune depletion of mice during the course of influenza virus pneumonia ameliorated the virus-induced bactericidal defect and prevented bacterial multiplication in the lungs. In contrast, immune reconstitution reestablished the alveolar macrophage phagocytic defect. These data indicate that virus-induced suppression of pulmonary antibacterial defenses may be, in part, immunologically mediated.

Alveolar macrophage ingestion and phagosome-lysosome fusion defect associated with virus pneumonia.

Virus-induced suppression of pulmonary phagocytic defenses is associated with defects in the intracellular processing of bacteria by alveolar macrophages. To determine whether the intracellular defect is related to a failure in phagosomelysosome fusion, mice were infected with a sublethal dose of Sendai virus, and the capacity of phagocytic cells, obtained by lung lavage, to exhibit phagosomelysosome fusion was quantitated during the course of the viral infection. Lysosomes of alveolar macrophages were prelabeled with acridine orange, the cells were challenged with Candida krusei, and fusion was determined with fluorescence microscopy by the discharge of the dye into the yeast-containing phagosome. Ultrastructural cytochemical studies verified the validity of the fluorescent fusion assay. Simultaneous experiments were performed to determine whether the viral infection also suppressed phagocytic ingestion by alveolar macrophages. Phagosome-lysosome fusion was progressively inhibited during the viral infection, reaching a low at day 7 when only 13 +/- 3% of the phagocytic cells fused as compared with 97 +/- 3% in cells from uninfected control animals; respectively, 55 +/- 5% as compared with 74 +/- 2% of the phagocytic cells contained yeasts. Thereafter, phagosome-lysosome fusion progressively increased reaching near normal levels (92 +/- 3%) on day 17 of the infection. At the same time period, phagocytic uptake was enhanced to a level where 97 +/- 3% of the cells contained yeasts. These data demonstrated that virus-induced suppression of intrapulmonary killing of bacteria involves functional lesions that retard the ingestion of inhaled organisms by alveolar macrophages and inhibit intracellular processing by degradative lysosomal enzymes by interfering with phagosome-lysosome fusion.

Alterations of pulmonary defense mechanisms by protein depletion diet.

Pulmonary defense mechanisms were quantitated in mice that were fed a protein-free diet (PFD) for periods of 2 and 3 weeks. Despite the severe weight loss and emaciation induced by the diet, the bactericidal mechanisms in their lungs were preserved against aerogenic challenges with staphylococcus aureus, Proteus mirabilis, and Listeria monocytogenes. Phagocytic assays of alveolar macrophages that were retrieved by pulmonary lavage from PFD-fed animals showed a decrease in Fc receptor-mediated binding activity but no alteration in the ingestion of sensitized erythrocytes. In contrast, the PFD induced defects in both the attachment phase and the engulfment phase of the phagocytic process when the challenge organism was Candida krusei. The PFD suppressed the pulmonary inflammatory response after mice were infected with influenza virus strain PR8; such mice also failed to eliminate infectious virus from their lungs. Virus infection in control mice suppressed pulmonary antibacterial defenses against challenges with S. aureus and P. mirabilis, and defect that was ameliorated in the lungs of PFD-fed mice with viral pneumonia. The data demonstrated that pulmonary defense mechanisms were modulated by a PFD but that the observed effect was dependent on the agent used to test host defenses.

Pulmonary and systemic defenses against challenge with Staphylococcus aureus in mice with pneumonia due to influenza A virus.

Pulmonary and systemic defenses against hematogenous challenge with 32P-labeled Staphylococcus aureus were measured 10 min, 8 hr, and 24 hr after intravenous injection of the bacteria in a mouse model of influenza virus pneumonia. Infection with influenza A virus did not alter bactericidal defenses in the liver and spleen, but pulmonary bactericidal activity measured 24 hr after infection was suppressed in virus-infected animals; 20% +/- 3% of the initially injected, viable bacteria were recovered from lungs of pneumonitic mice after 24 hr as compared with 9% +/- 1% from lungs of the uninfected mice. These data demonstrate that pulmonary infection with influenza virus does not alter antibacterial defenses of the liver and spleen but does suppress bactericidal activity in the lung.

Effect of in vivo administration of gold sodium thiomalate on rat macrophage function.

It has been shown that gold accumulates in macrophages. In vitro studies have also shown that long-term anti-inflammatory and immuno-regulatory effects on these cells may be responsible for the effectiveness of gold in the treatment of rheumatoid arthritis. However, the relevance of this information to the in vivo circumstance is largely untested. In this study, the effect of gold sodium thiomalate (AuTM) on rat alveolar macrophage (AM) lysosomal enzymes, bacterial killing, and metabolic activities associated with phagocytosis were assessed after in vivo administration. The activities of beta-glucuronidase, acid phosphatase, and lysozyme were inhibited 1 day following a single AuTM injection (50 mg/kg, subcutaneous). However, lysozyme returned to normal, while the activities of beta-glucuronidase and acid phosphatase were elevated from 4 to 12 days thereafter. When AuTM was administered weekly for 8 weeks, the activities of acid phosphatase and beta-glucuronidase were elevated throughout, while lysozyme was largely unaffected. The increased lysosomal enzyme activities were not due to contamination of polymorphonuclear leukocytes. These long-term effects of AuTm on enzyme activity were in marked contrast to its in vitro effect which inhibited the activities of beta-glucuronidase and acid phosphatase. No effect of AuTM administration on the release of beta-glucuronidase upon phagocytosis of opsonized zymosan was observed. At 1 day following a single AuTM injection or 3 days after a second weekly injection, in vivo bactericidal activity of AM toward S. aureus was diminished. This bacterial killing defect was not due to decrease phagocytosis; the in vivo binding and ingestion of bacteria were normal. The defect correlated with imparied metabolic activities associated with phagocytosis, namely a significant decrease in the reduction of nitroblue tetrazolium and the stimulation of the hexose monophosphate shunt. This may be an attractive anti-inflammatory effect in light of the destructive potential of the reactive oxygen species produced by macrophages in an arthritic circumstance.

Alveolitis induced by influenza virus.

Previous studies of influenza virus infections have focused on the acute pathologic manifestations associated with the virus pneumonia; however, there is evidence suggestive of persistent pathologic processes with possible long-term consequences. Herein we have examined the long-term outcome of virus pneumonia in mice infected by aerosol inhalation of a sublethal dose of influenza A/PR8/34 virus. At 3, 5, 7, 9, 15, 30, 60, 90, 120 days, and a year thereafter, the lavageable lung cell populations and differential counts were quantitated. Consistent with previous studies we demonstrated an inflammatory cellular response during the acute phase of the infection. However, this inflammatory response did not completely resolve, the pulmonary leukocytosis remaining stable from Day 30 through a year after virus infection. For example, on Day 30, virus-infected lungs yielded 12.4 +/- 0.9 X 10(5) cells per lavage of which 15 +/- 3% were polymorphonuclear leukocytes, 18 +/- 4% were lymphocytes, and 67 +/- 5% were alveolar macrophages. In contrast, 7.2 +/- 0.5 X 10(5) cells per lavage were obtained from uninfected lungs of which more than 98% were alveolar macrophages. Histopathologic examination of virus-infected lungs showed an ongoing inflammatory response resulting in patchy mononuclear interstitial pneumonia, deposition of collagen in the affected areas, and marked hyperplasia of bronchial-associated lymphoid tissue. Infectious virus could not be recovered after Day 9. However, in contrast to loss of infectivity, viral antigen persisted at high concentrations in the lung. We conclude that influenza virus infection induced a long-term alveolitis that is associated with persistence of viral antigen. These data open the possibility that influenza virus infections may play a role in interstitial lung disease.

Immunotoxicity of organophosphorus compounds. Modulation of cell-mediated immune responses by inhibition of monocyte accessory functions.

Several organophosphorus compounds (OP) used commercially as flame retardants and plasticizers and related chemicals were evaluated for their effects on human in vitro cell-mediated immune responses. At nontoxic concentrations ranging from 0.1 to 20 microM, two of the tested compounds, triphenylphosphine oxide (TPPO) and tetra-o-cresylpiperazinyl diphosphoamidate (TCPD) caused significant suppression of antigen-specific lymphocyte proliferation (P less than 0.01). Mitogenesis was less sensitive to OP treatment and was affected only by TCPD. When monocytes and lymphocytes were treated separately with OP, washed, and recombined, it appeared that these OP mediated their suppressive effects by interfering with a monocyte function rather than acting directly on lymphocytes. Further, triphenyl phosphate (TPP), triphenyl thiophosphate (TPTP) as well as TPPO and TCPD were tested for direct inhibition of monocyte antigen presentation, and all four compounds were found to cause significant inhibition at concentrations as low as 1 microM (P less than 0.001).