key: cord-0007373-abottt5s
authors: Rose, Richard M.; Wasserman, Andrew S.; Weiser, Weishui Y.; Remold, Heinz G.
title: Deficient Responses of Pulmonary Macrophages from Healthy Smokers to Antiviral Lymphokines in Vitro
date: 1986-10-03
journal: J Infect Dis
DOI: 10.1093/infdis/154.4.611
sha: 4d3a13ff21c4dc4e1e7ebba5d79c5c22985a1f7f
doc_id: 7373
cord_uid: abottt5s

The antiviral function of pulmonary macrophages obtained by broncholavage of healthy smokers and nonsmokers was studied. Compared with nonsmokers' cells, smokers' macrophages produced significantly more virus during in vitro infection with herpes simplex virus type 1 (HSV-1). Exposure of macrophages to either antiviral macrophage-activating factor or interferon-y for 20 hr before infection resulted in diminished production of HSV-1 by both types of macrophages. However, in contrast to smokers' cells, exposure of nonsmokers' macrophages to these antiviral lymphokines totally prevented viral replication. This difference could not be attributed to diminished adsorption of virus to smokers' macrophages or to an increased proportion of extracellular to intracellular virus in smokers' cell cultures. The effect of smoking on viral infectivity did not appear to be mediated by secretion of a soluble factor by the macrophage because incubation of nonsmokers' cells with supernatant from smokers' cell cultures did not affect the growth of HSV-1.

The antiviral function of pulmonary macrophages obtained by broncholavage of healthy smokers and nonsmokers was studied. Compared with nonsmokers' cells, smokers' macrophages produced significantly more virus during in vitro infection with herpes simplex virus type 1 (HSV-l). Exposure of macrophages to either antiviral macrophage-activating factor or interferon-y for 20 hr before infection resulted in diminished production of HSV-l by both types of macrophages. However, in contrast to smokers' cells, exposure of nonsmokers' macrophages to these antivirallymphokines totally prevented viral replication. This difference could not be attributed to diminished adsorption of virus to smokers' macrophages or to an increased proportion of extracellular to intracellular virus in smokers' cell cultures. The effect of smoking on viral infectivity did not appear to be mediated by secretion of a soluble factor by the macrophage because incubation of nonsmokers' cells with supernatant from smokers' cell cultures did not affect the growth of HSV-l.

Cigarette smoking increases both the risk and severity of viral infection of the respiratory tract. Healthy young adult smokers have more frequent acute infections of the respiratory tract than do nonsmokers of the same age [1, 2] . During episodes of acute nonspecific illness in the respiratory tract, smokers report more symptoms [3] and develop more signs [4] in the lower respiratory tract than do nonsmokers. Although the etiology of acute illness in the respiratory tract was not characterized in some of these studies, a substantial proportion of acute nonspecific illness in the respiratory tract is caused by viral infection [5] .

Smoking has been found to influence the impact of respiratory tract infection on the host in studies in which definite viral etiology has been specified. During an epidemic of influenza (HINI) in young soldiers, smoking was associated with increased risk of development of clinically apparent infection as well as increased likelihood of a severe influenza syn-drome [6] . In chronic bronchitis in which smoking is the major causative factor [7] , rates of viral infections in the respiratory tract are greater in smoking than in nonsmoking controls for rhinoviruses [8] , respiratory syncytial virus [9] , influenza A and B viruses, parainfluenza virus types 1, 2, and 3, and coronavirus OC 43 [9, 10] . Viral infection of the respiratory tract is also responsible for substantial morbidity in chronic bronchitis; viruses have been implicated in up to one-third of all exacerbations of bronchitis [11, 12] .

In spite of the impact of cigarette smoking on viral infection of the respiratory tract, little is known about how smoking alters respiratory defenses against viruses. The pulmonary macrophage is a principal cellular defense in the lung against infectious agents, including viruses [13, 14] . Because various functional, metabolic, and structural alterations have been demonstrated in this cell as a result of exposure to tobacco smoke, it is reasonable to speculate that certain critical antiviral functions of the pulmonary macrophage may also be affected by smoking. For this reason we assessed the infectivity of herpes simplex virus type 1 (HSV-l) for pulmonary macrophages from healthy smokers and nonsmokers. Because macrophage mechanisms for restricting viral growth may be activated in vivo by lymphokines, soluble mediators of the immune response [15, 16] , we also investigated the possibility that smoking affects lung defenses by altering macrophage responsiveness to antiviral lymphokines.

Source of m acroph ages. Pulmonary macrophages were harvested from healthy smokers and nonsmokers as described previously [17] . All volunteers for this study were screened by standardized questionnaire [18] , chest auscultation, and spirometry [19] . Individuals were excluded from this study if they had a history or evidence of chronic pulmonary disease, abnormal lung sounds, or abnormal spirometry. To ensure that macrophages were free of respiratory viruses and had not been recently exposed to endogenous antiviral mediators such as interferon, we only accepted volunteers for broncholavage if they were free of all respiratory tract symptoms for the prior two weeks. All smokers identified in this fashion smoked at least one pack of cigarettes per day (range, one to 2.5 packs per day) for two years (range, two to 12 years).

Because preliminary studies confirmed the work of Daniels et al. [20] , which demonstrated that monocytes obtained from HSV-l-seropositive and -seronegative donors were equally permissive for viral replication, the serological status of the donor was not taken into account in these studies.

Macrophage infection. Pulmonary macrophages were isolated from broncholavage fluid by centrifugation at 100 g for 10 min at 20 C. They were maintained in RPMI-1640 medium supplemented with 25 mM HEPES buffer, 50 ug of gentamicin/ml, 2 mM L-glutamine, and 10070 heated-inactivated fetal calf serum. Cells (5 x 10 5 ) were seeded in 35-mm plastic dishes with 2-mm grids (Miles Laboratories, Naperville, Ill) to facilitate counting. Cells remaining adherent after incubation for 3 hr were >97070 viable, as assessed by ability to exclude trypan blue, and >94070 phagocytic of latex beads 1.1 urn in diameter. Cells from smokers and nonsmokers were similar with respect to viability and phagocytic ability. However, smokers' cells exhibited morphological differences from nonsmokers' cells by light microscopy: smokers' cells appeared larger, spread more rapidly on plastic, and contained numerous refractile intracytoplasmic inclusions.

Within 24 hr of isolation, macrophages were infected with KOS strain HSV-l at an moi of 20:1 by exposure of cells to virus (0.1 ml) for 60 min at 37 C in an atmosphere of 95070 room air and 5070 CO2 • Unadsorbed virus was removed by three washes with media.

At intervals after infection, virus present in the Rose et of. supernatant of macrophage cultures was quantitated by determination of the number of viral pfu in replicate cultures of Vero cells [21] . In this assay 1% pooled human serum was used as a source of HSV-I-specific antibody to neutralize virus released into the culture medium after lysis of infected cells and to prevent spread of released virus to noninfected cells.

Adsorption of HSV-l to macrophages was determined after exposure of cells to viral inoculum for 1 hr, followed by extensive washing of cells with serum-free medium to remove unadsorbed virus. Adsorbed virus was then quantitated by freeze-thawing cells at -70 C and measurement of the number of viral pfu in the lysate. Freeze-thawing cells in this manner resulted in complete disruption of macrophage cultures (as seen by light microscopy) from both smokers and nonsmokers. Cell-associated virus was quantitated at intervals after infection in a fashion analogous to the method used for determination of initial adsorption of HSV-l to cells.

Cells were quantitated by counting pulmonary macrophages remaining on one of the four central grids of the 35-mm dish. This technique was feasible because all nonadherent cells were nonviable. Virus-specific cytolysis was determined as described previously [22] by comparison of the number of remaining cells in infected cultures at intervals after infection to those present in infected wells at 1 hr after exposure to virus. The latter number was multiplied by the percentage of cytolysis in uninfected cells at the time under analysis to account for nonspecific cell death in macrophage cultures.

Modification of infection in nonsmokers' cells by exposure to supernatant from cultures of smokers' macrophages was attempted by incubation of nonsmokers' cells with supernatants (50070 by volume) from one-to three-day-old cultures of smokers' cells for 24 hr before viral infection.

Preparation oflymphokines and macrophage activation. Human interferon-gamma (IFN-y) was obtained from Meloy Laboratories (Springfield, Va). Antiviral macrophage-activating factor (AV-MAF) was prepared from concanavalin A (10 ug/rnljstimulated human leukocyte concentrates obtained as a by-product of plateletpheresis as described previously [23] . This procedure involves partial purification of leukocyte supernatants by gel filtration on a column (2.5 x 100em) of Sephadex" G-IOO(Pharmacia Fine Chemicals, Piscataway, NJ), followed by isoelectric focusing of the effluent on ampholine

Replication of HSV-I in untreated pulmonary macrophages from smokers and nonsmokers is shown in figure 1 . Peak viral growth occurred 24 hr after initiation of infection in cells from both sources; viral growth in smokers' macrophages was 10 times greater than growth in nonsmokers' cells (P < .01).

This result could not be explained by differences in cell number between smokers' and nonsmokers' cell cultures because the initial number of adherent cells in these cultures before infection was similar and because >800/0 cell lysis was observed in both smokers' and nonsmokers' cell cultures by 48 hr after infection. At the time of greatest viral replication (24 hr), infectivity of HSV-I was greater for smokers' cells when corrected for cell count (pfu per cell, 3.39 ± 0.51 and 0.35 ± 0.10 for smokers' and nonsmokers' cell cultures, respectively).

(50 ul/ml) for 20 hr before infection resulted in diminished viral replication in both smokers' and nonsmokers' cells compared with cells exposed to mediator control (figure 2). In cells from nonsmokers, inhibition of viral replication was observed at both 24 and 48 hr after infection. In contrast, smokers' cells exposed to AV-MAF were not capable of totally suppressing viral growth, as evidenced by an increase in the amount of virus recovered during the first 24 hr after infection. Exposure to AV-MAF protected cells from smokers and nonsmokers to a simi-Control macrophage cultures were exposed to the mediator control fraction (50 ul/rnl) in a similar fashion.

Quantitation of cellular protein. The cellular protein concentration was determined in uninfected macrophage cultures grown in serum-free medium after incubation for 3 hr. Nonadherent cells were first removed by gentle washing, and intracellular protein in the remaining adherent cells was released into the culture supernatant by freeze-thawing once at -70 C. The concentration of protein was quantitated by a modification [25] of the Lowry assay, based on the differential A S 9 S of Coomassie'" brilliant blue 0-250 (Bio-Rad, Richmond, Calif) when protein binding occurs.

Statistical analysis. Results were analyzed by a noninpaired Student's t test [26] . 24 48 Hours after Infection Previous studies in human pulmonary macrophages from nonsmoking donors and a transformed human macrophage-like cell line, V937, demonstrated that the fraction containing antiviral activity against HSV-I in macrophage effector cells was the one with an isoelectric point between 1.4 and 2.2 [22] . This fraction contained no detectable IFN-y activity when tested in a classical interferon assay with human fibroblasts [24] and was not neutralized by monoclonal antibody to IFN-y [22] . This fraction is therefore referred to as AV-MAF.

A mediator control fraction was prepared in an analogous manner from the supernatant of unstimulated leukocytes. Concanavalin A (10 ug/ml) was added to mediator control supernatant after removal of cells.

Macrophages were activated by exposure to either IFN-y (500 Vlml) or AV-MAF (50 ul/rnl) for 20 hr at 37 C in an atmosphere of 950/0 room air and 50/0 CO 2 • Before infection, mediator was removed by three washes of the cells in serum-free medium. In previous studies these conditions were the optimal concentration and incubation period to achieve activation of antiviral mechanisms of nonsmoker macrophages in this in vitro system [22] . The concentration of AV-MAF used in these experiments was expressed as J.lI of the lymphokine-containing fraction concentrated to I/50th the volume of the original supernatant by vacuum concentration. was observed in the number of virions per cell at 24 hr of infection compared with cells exposed to a lower viral inoculum (20 virions per cell): 0.39 ± 0.18 pfu per cell for the larger inoculum versus 0.35 ± 0.1 as shown earlier for the smaller inoculum. Maximal viral replication and lymphokine-mediated viral inhibition were similarly unaltered (data not shown). Exposure of cells to an inoculum of >100 virions per cell was associated with cytotoxicity within 8 hr after exposure and was not tested further. lar degree from virus-specific cytolysis. At 48 hr after infection, mean ± SD cytolysis in smokers' cell cultures was 77.70/0 ± 40/0 with addition of mediator control and 48.60/0 ± 10/0 with addition of AV-MAF, compared with 80.680/0 ± 1.60/0 and 48.90/0 ± 1.80/0 for nonsmokers' cell cultures, respectively.

Exposure of macrophages to IFN-y (500 U/ml) diminished viral growth in both smokers' and nonsmokers' cells but only totally inhibited viral replication in nonsmokers' cells ( figure 3) .

To examine the possibility that the different response of smokers' cells to AV-MAF could be attributable to the use of suboptimal concentrations of this lymphokine to activate cells, we pretreated smokers' macrophages with different amounts of AV-MAF before initiation of infection (table I) . In these studies the maximal antiviral effect was achieved with 50 J..LVml, the amount used in the experiment shown in figure 2. Incubation of cells with 200 ul of AV-MAF/ml resulted in 100/0-150/0 cell death and was not tested further. Exposure of smokers' cells for periods as long as 48 hr or as short as 8 hr to AV-MAF before infection did not result in diminished viral growth compared with cells exposed to this lymphokine for 20 hr (data not shown).

In three experiments nonsmokers' cells were exposed to an increased moi ( NOTE. The ratio of cell-associated HSV-I to the total amount of virus recovered was determined by separate quantitation of viral pfu in celllysates (cell-associated virus) and culture supernatants and addition of these values to obtain the total amount of virus for each interval studied. Data are mean values from three experiments.

Because exposure to tobacco smoke alters the ultrastructure of the pulmonary macrophage plasma membrane [27] , it is possible that the abnormalities in viral infectivity and lymphokine responses observed in smokers' cells are related to alterations in initial adsorption of virus to the cell surface. Smokers' macrophages exposed or not exposed to lymphokine adsorbed more virus on a per cell basis than did nonsmokers' cells after exposure for 1 hr to HSV-l (table 2). For determination of whether this finding is the result of altered binding of HSV-l to the macrophage plasma membrane or is attributable to the larger volume of the smokers' macrophage [28] , virus initially adsorbed to cells was quantitated with respect to the amount of protein present in cell lysates of macrophage cultures to account for differences in cell size. There were no differences between smokers' and nonsmokers' cells or lymphokineexposed and control cells in terms of amount of cellassociated virus per ug of macrophage protein (table 2). Thus, smokers' macrophages adsorbed more HSV-l at 1 hr after infection on a per cell basis than did nonsmokers' cells, but this difference appears attributable to the larger surface area of smokers' cells.

To examine the possibility that differences between smokers' and nonsmokers' cells are related to an altered capacity of smokers' cells to excrete virus, we measured the ratio of cell-associated virus to the total amount of virus recovered in celllysates and supernatants. This value was comparable between smokers' and nonsmokers' cells (table 3) . It does not appear therefore that increased release of virus into culture medium from smokers' cells accounts for en-hanced infectivity of HSV-l for smokers' cells or for the diminished response of smokers' cells to antiviral lymphokines.

To investigate the possibility of whether smokers' macrophages secrete a substance capable of altering viral infectivity in nonsmokers' cells, we incubated macrophages from nonsmokers with supernatants from unstimulated smokers' macrophages for one day before infection.

In three experiments viral replication in nonsmokers' cells was unaffected by prior exposure to supernatant from cultures of smokers' cells. Maximal viral replication was 2.8 ± 1.7 x 10 5 pfu/ml for cells exposed to medium alone and 1.76 ± 1.1 x 10 5 pfu/ml for cells exposed to medium containing smokers' supernatant (50070 by volume).

These studies demonstrate that cigarette smoking can alter lung cellular defenses against HSV-l in two distinct ways: (l) Smoking enhances infectivity of HSV-l for macrophages, and (2) smoking leads to suboptimal activation of antiviral activity in macrophages by two lymphokines (AV-MAF and IFN-y). These data describe the interaction of macrophages with a single virus; the relevance of these findings to other respiratory viruses is unclear. However, these observations raise the possibility that abnormal antiviral responses of macrophages may be one pathogenic mechanism underlying the increased incidence and severity of viral infection in the respiratory tract in smokers [1] [2] [3] [4] [5] [6] [8] [9] [10] . Although exposure to tobacco smoke can affect other aspects of lung defense, such as mucociliary transport [29] , noncellular defenses may be less important than phagocytic cells in eradication of infectious agents from the respiratory tract [30] . For this reason alteration of critical antiviral functions in pulmonary macrophages by cigarette smoke could have a substantial impact on the fate of viruses that reach the lower respiratory tract. Inability of smokers' macrophages to restrict viral replication could lead to increased production of virus within pulmonary airways and parenchyma at an early phase of infection, when the spread of virus cannot be limited by immune factors. Diminished activation of antiviral mechanisms in smokers' macrophages by lymphokines may also interfere with the effectiveness of macrophages at a later stage of infection, after cellular immunity has developed.

In the in vitro system used in these studies to evaluate macrophage antiviral responses, a single viral agent, HSV-l, was tested. This virus is an uncommon cause of respiratory tract infection in immunocompetent humans: In one series, HSV-l was recovered in 5070 of all episodes of communityacquired infections of the lower respiratory tract, compared with an isolation rate of 1.4070 in healthy controls [31] . This virus may also be a cause of serious pneumonitis in immunosuppressed individuals [32, 33] . Nevertheless, HSV-l has been an exceptionally useful agent to probe antiviral responses of macrophages in vitro because it grows rapidly in macrophages, produces cell lysis, and can be inhibited by lymphokine-activated macrophages [22, 34, 35] . One of the lymphokines used in these studies, IFN-y, has been associated with augmentation of mononuclear phagocyte activity against neoplastic and infectious processes [36] [37] [38] . On the other hand, AV-MAF is an unique lymphokine that, like IFN-y, is capable of activating antiviral activity of macrophages [22] but can be separated from IFN-y in the supernatant of human peripheral blood leukocytes by preparative isoelectric focusing [22] [23] [24] . The suboptimal responses of smokers' pulmonary macrophages to both these lymphokines may have important implications for the effect of smoking on other lymphokinemediated activities of macrophages, such as killing tumor cells and other intracellular parasites (e.g., Legionella pneumophila), because both lung cancer and legionellosis are more common in smokers than nonsmokers [7, 39] .

These studies extended the spectrum of functional abnormalities in macrophages that have been at-tributed to cigarette smoke. Smoking has been shown to depress antibacterial activity of pulmonary macrophages in vitro [40] [41] [42] and inhibit phagolysosome fusion [43] , a crucial mechanism for eradication of certain intracellular microbes. Only one previous study assessed the impact of smoking on macrophage responses to lymphokines. Human pulmonary macrophages from smokers had diminished responses to macrophage migration-inhibitory factor [44] . These functional alterations may be based on metabolic or structural changes in macrophages produced by exposure to tobacco smoke. For instance, macrophage levels of the energy-generating enzymes adenosine triphosphatase [45] and glucose-3phosphate dehydrogenase [46] are diminished in smokers' cells and may be responsible for lowered rates of phagocytosis observed in smokers' macrophages. Similarly, smoking-related ultrastuctural changes, consisting of increased blebs and lamellipodia, in the macrophage plasma membrane have been implicated in altered pinocytosis and endocytosis [27] .

Although in the present studies we did not elucidate the mechanism(s) responsible for increased infectivity and diminished lymphokine response in smokers' macrophages, these alterations could not be explained by increased release of intracellular virus into culture medium during infection (table 3) or by differences between the optimal concentration of lymphokine needed to produce an antiviral state in smokers' and nonsmokers' cells (table 1) . Moreover, macrophage abnormalities associated with smoking do not appear to be the result of exposure of cells to a soluble factor secreted by smokers' macrophages because incubation of nonsmokers' cells with supernatant from smokers' cell cultures did not alter the infectivity of HSY-l for cells treated in this manner.

Altered antiviral responses of smokers' macrophages may be due in part to enhanced initial adsorption of HSY-l (table 2). Because the amount of virus adsorbed to smokers' and nonsmokers' cells is similar when corrected for intracellular protein concentration (table 2), increased uptake of HSY-l by smoker's cells appears to be related to a larger surface area available for attachment of virus. This conclusion is in contrast to a previous study that found only a small size difference in these cell populations on the basis of direct measurement of cell diameter [28] . This method of assessing cell size could underestimate true cell volume compared with measurements of cellular protein concentration. By the latter method smokers' cells appeared nearly twice as large as nonsmokers' cells. On a per cell basis, smokers' macrophages are exposed to more than twice the amount of adsorbed virus as are nonsmokers' cells (table 2) . It may be, therefore, that increased infectivity of HSV-I for smokers' cells results from greater initial adsorption of virus, diminished ability of the extra protein in smokers' cells to limit viral replication, or both.

Thus, smoking appears to depress the intrinsic antiviral activity of macrophages against HSV-I and diminish the augmentation of this activity by lymphokines. Further studies are needed to clarify the relevance of these observations for other respiratory viruses and other lymphokine-directed macrophage functions. The importance of smoking in acquired antiviral and immune deficiencies of macrophages remains speculative.

Cigarette smoking and respiratory infections in a class of student nurses

Smoking habit and incidence of respiratory tract infections in a group of adolescent males

Smoking, pulmonary function, and respiratory symptoms in a college-age group

Association between cigarette smoking and acute respiratory tract illness in young adults

The Tecumseh study of respiratory illness. II. Patterns of occurrence of infection with respiratory pathogens, 1965-1969

Cigarette smoking as a risk factor for epidemic A(H.N.) influenza in young men

Smoking and health: a report of the Surgeon General. U.S. Department of Health, Education, and Welfare publication (PHS) 79-50066

Susceptibility to rhinovirus infection in chronic bronchitis

The Tecumseh study of respiratory illness. X. Relation of acute infections to smoking, lung function and chronic symptoms

The Tecumseh study of respiratory illness. VIII. Acute infection in chronic respiratory disease and comparison groups

Stouch WHo Role of infection in chronic bronchitis

Williams OR Viral and Mycoplasma pneumoniae infections in exacerbations of chronic lung disease

Pulmonary alveolar macrophage: defender against bacterial infection of the lung

Role of macrophages in natural resistance to virus infections

In vitro approaches to the mechanism of cellmediated immune reactions

Cellular hypersensitivity and immunity

Analyses of sequential bronchoalveolar lavage specimens from healthy human volunteers

Adult questionnaire. Epidemiology standardization project

The maximal expiratory flow-volume curve. Normal standards, variability, and effects of age

Abortive and productive infections of human mononuclear phagocytes by type I herpes simplex virus

Restriction of herpes simplex virus by macrophages. An analysis of the cell-virus interaction

An acidic lymphokine distinct from interferon-y inhibits the replication of herpes simplex virus in human pulmonary macrophages

Studies on human migration inhibitory factor: characterization of three molecular species

Dissociation of human interferon-gamma-like activity from migrationinhibition factor

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Surface morphology of the human alveolar macrophage

Human alveolar macrophages: comparison of phagocytic ability, glucose utilization, and ultrastructure in smokers and nonsmokers

Relationship of smoking history and pulmonary function tests to tracheal mucous velocity in nonsmokers, young smokers, ex-smokers, and patients with chronic bronchitis

Defense mechanisms of the respiratory membrane

A study of the combined role of viruses, mycoplasmas and bacteria in adult pneumonia

Herpes simplex virus infection of the adult lower respiratory tract

Herpes simplex virus pneumonia. Clinical, virologic, and pathologic features in 20 patients

Comparison of antiviral and antitumor activity of activated macrophages

Lysis of herpes virus-infected cells by macrophages activated with free or liposome-encapsulated lymphokine produced by a murine T cell hybridoma

Similarities of murine gamma interferon and lymphokine that renders macrophages cytotoxic

Macrophage activation for tumor cytotoxicity: induction of macrophage tumoricidal activity by lymphokines from EL-4, an continuous T cell line

Identification of interferon-y as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity

Sporadic community-acquired Legionnaires' disease in the United States. A case-control study

Tobacco smoke: effects on pulmonary host defense

The depressant effect of cigarette smoke on the in vitro antibacterial activity of alveolar macrophages

An animal model of cigarette smoking in beagle dogs: correlative evaluation of effects on pulmonary function, defense, and morphology

Abnormal phagolysosome fusion in pulmonary alveolar macrophages of rats exposed chronically to cigarette smoke

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

Correlated effects of cigarette smoke components on alveolar macrophage adenosine triphosphatase activity and phagocytosis

Cigarette smoke-a proposed metabolic lesion in alveolar macrophages