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Immunotoxicology of Cigarette Smoke Condensates: Suppression of Macrophage Responsiveness to Interferon γ
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
149, 136 –143 (1998)
TO978346
Immunotoxicology of Cigarette Smoke Condensates: Suppression of Macrophage Responsiveness to Interferon g Kristen M. Braun,*,1 Toby Cornish,*,2 Alex Valm,* Jason Cundiff,* John L. Pauly,† and Samuel Fan*,3 *Department of Biology, Bradley University, Peoria, Illinois 61625; and †Department of Molecular Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263-0001 Received June 12, 1997; accepted November 14, 1997
Immunotoxicology of Cigarette Smoke Condensates: Suppression of Macrophage Responsiveness to Interferon g. Braun, K. M., Cornish, T., Valm, A., Cundiff, J., Pauly, J. L., and Fan, S. (1998). Toxicol. Appl. Pharmacol. 149, 136 –143. We have investigated systematically the effects of short-term exposure to main stream cigarette smoke condensates (CSC-MS) on basal and inducible functional capacities of murine peritoneal exudate macrophages. Macrophages treated with CSC-MS form granules that fluoresce orange under blue excitation, consistent with the speculation that they are polycyclic aromatic hydrocarbons (PAH). CSC-MS selectively suppressed interferon gamma (IFNg) induction of four macrophage functional capacities: enhanced phagocytosis of immunoglobulin-opsonized sheep red blood cells, TPA-induced H2O2 production, class II major histocompatibility complex expression, and nitric oxide synthesis. In contrast, two macrophage functions that are not induced by IFNg, basal electron transport and LPS-induced TNFa production, were enhanced by treatment with CSC-MS. These results suggest that the suppressive effects of CSC-MS on macrophage responsiveness were selective and were not due to nonspecific inhibition of general functions such as RNA or protein synthesis. Since macrophage responsiveness to IFNg can result in induction of functional capacities that are fundamental to immunity, the data suggest that CSC-MS maybe deleterious to the general health of the smoker. © 1998 Academic Press
Transient effects of short-term exposure to environmental chemicals on human health are difficult to assess. Yet such exposures may cause immediate or delayed deleterious effects to the subject. A case in point is cigarette smoke. The chemical constituents of cigarette smoke, including some polycyclic aromatic hydrocarbons (PAH), clearly exert potent long-term effects on the smoker as initiators and promoters of cancer (IARC, 1976; Hoffmann and Wynder, 1971). Less well defined are the short-term effects of acute and transient exposure to 1
Present address: Department of Biochemistry, University of Wisconsin, Madison, WI 53706. 2 Present address: Neuroscience Program, University of Illinois, Urbana, IL 61801. 3 To whom correspondences should be addressed at Department of Biology, Bradley University, Peoria, IL 61625. E-mail: [email protected]. 0041-008X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
tobacco smoke. Of particular concern are the immediate effects on the host’s immune defenses against infectious diseases and cancer. A potential target for functional modulation by tobacco smoke is the macrophage. Smokers who inhale have higher incidences of infection and cancer of the respiratory tract as well as a higher predisposition to cardiovascular diseases (IARC, 1976). Recently, environmental tobacco smoke (ETS) also became a subject of concern, as data emerged from studies indicating higher health risks for those who do not smoke but live in the same household as smokers compared with those who do neither (U.S. Surgeon General, 1986). The products of burning cigarettes are airborne and thus are apt to enter the human airways and eventually deposit in the alveoli of the lungs. Since alveoli are lined with macrophages, the lung is a potential site where macrophages encounter cigarette smoke. Lung macrophages would encounter constituents of cigarette smoke more frequently, and in larger quantities, than those resident in the rest of the body. In fact, macrophages recovered from smokers but not nonsmokers contain numerous granules that fluoresce upon illumination with blue light (Skold et al., 1989; Streck et al., 1994). The granules are thought to be ingested polycyclic aromatic hydrocarbons (PAH), a major component of the condensate fraction of cigarette smoke (CSC), primarily because PAH fluorescence has similar spectral characteristics as the granules (Skold et al., 1989). Macrophages can help prevent infectious diseases and cancer. Directly, macrophages can ingest and destroy infectious agents (Lepay et al., 1985), and they can kill neoplastic cells by a variety of mechanisms (Evans, 1986; Adams and Hamilton, 1988); indirectly, macrophages can initiate and regulate immune responses against infectious agents (Unanue and Askonas, 1968). Many of these functions are not constitutively expressed but are induced in response to stimuli such as interferon gamma (IFNg), bacterial lipopolysaccharide (LPS), or combinations of various stimuli (Adams and Hamilton, 1988). Thus, macrophages must maintain their responsiveness to a full range of stimuli and their capacity to perform their full range of functions for the host organism to thrive. By extension, environmental factors that modulate macrophages’ capacity to
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respond to stimuli or to perform functions would impair the host organism’s well being. Using cultured murine peritoneal macrophages as a model, we have investigated systematically the effects of short-term exposure to CSC prepared from mainstream smoke (CSC-MS) on basal and inducible functional capacities of macrophages. We report here that macrophages ingest CSC-MS. We also report that CSC-MS selectively suppresses macrophage functional capacities that are inducible by IFNg, while functional capacities that are basal to the macrophages or are inducible by LPS are either not affected or are enhanced.
METHODS Cigarette smoke condensates. Since CSC-MS are complex mixtures (Grimmer et al., 1987), obtaining a standardized reagent is improbable. Instead, we prepared large lots of CSC-MS, so that many experiments can be performed with a single CSC-MS preparation to make the results directly comparable within a trial. CSC-MS were collected from lit cigarettes (regular size, national brand) as crude smoke condensates by simulating periodic 2-s puffs. Condensates were the components of smoke that condense on glass Erlenmeyer flasks that had been baked at 280°C for 4 h to destroy bacterial LPS. Condensates were subsequently eluted-off with methanol, evaporated to dryness by roto-evaporation under reduced pressure, and then redissolved in methanol at five cigarette-equivalents per milliliter. This constitutes the 100% stock solution, which was diluted to 25% with methanol and stored at 4°C. Further dilutions for experimental use were in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech, Herndorn, VA) supplemented with 5% newborn bovine serum (NBS, Hyclone, Logan, UT). All data presented in this report refer to a single lot (the most recent) of CSC-MS (from mainstream smoke). However, similar results were obtained with two other preparations of CSC-MS for many of the functional capacities described here. The lot used in the reported experiments was intermediate in potency (i.e., modulatory activity) of the three lots that we have worked with. Macrophages. The experimental system is well characterized (Marino and Adams, 1980; Fan et al., 1991). Female C57BL/6 mice (Trudeau Institute, Saranac Lake, NY) were kept in a PHS-certified mouse housing unit at Bradley University and were fed tap water and Teklad LM-485 M/R Diet (Harlan Teklad, Madison, WI) ad libitum. They were used at between 7 to 15 weeks of age. Peritoneal macrophages were obtained by peritoneal lavage with Hank’s balanced salt solution (HBSS, GIBCO, Grand Island, NY) following established procedures (Marino and Adams, 1980; Fan et al., 1991). To increase yield, peritoneal macrophages were obtained from mice injected peritoneally with 1 ml 1.2% aqueous casein (nach Hammerstein) 3 days prior to harvest. Typically, each mouse yielded about 1 3 107 peritoneal exudate cells, of which about 95% were morphologically similar to macrophages. Harvested macrophages were suspended in DMEM with 5% NBS and dispensed into 96 flat-well microtiter plates (Falcon) at 2 3 105/well in 100 ml. After 2– 4-h incubation at 37°C in a 95% air, 5% CO2 environment, nonadherent cells were removed by washing with DMEM with 5% NBS, yielding macrophage monolayers. Fluorescence microscopy. Macrophage ingestion of CSC-MS was monitored by observing macrophages that were exposed to 0.05% CSC-MS, fixed in 0.1% formaldehyde in Dulbecco’s phosphate-buffered saline (DPBS), and mounted in 90% glycerol/10% 103 DPBS under blue (490 nm) illumination. Photomicroscopy was carried-out at 10003 magnification using an Olympus BX 40 microscope. Spectrophotometry. All spectrophotometry was performed in polystyrene microwell plates in a BioTek 311S microplate reader equipped with 405-, 450-, 570-, and 600-nm filters.
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Electron transport activity. Macrophage metabolic activity was assessed by the cells’ ability to produce intermediates of the electron transport chain, reflected by reduction of an orange dye (MTT, Molecular Probes, Eugene, OR) to yield a purple precipitate. The soluble dye was removed along with detached macrophages, and remaining precipitate was then dissolved in acidified isopropanol and quantified by spectrophotometry at 600 nm (Slater et al., 1963; Mosmann, 1983). This assay is commonly used to evaluate cellular viability, based on the assumption that mammalian cells that are unable to effect electron transport are nonviable. Class II major histocompatibility complex (class II MHC). The responsiveness of experimentally treated macrophages to stimulation by IFNg was assessed in part by the cells’ ability to express class II MHC molecules after treatment with IFNg. Macrophage monolayers were either treated with IFNg or left untreated. After a 36- to 40-h incubation, media in the wells were removed, and 50 ml 0.1% formaldehyde was added. The wells were washed twice with blocking buffer (DPBS containing 2% nonfat dry milk). Sixty microliters of diluted monoclonal antibody from the hybridoma 28-16-8S (Ozato and Sachs, 1981; IgM anti-Iab,d, culture fluids diluted 1/5 in blocking buffer) was added to each well and incubated for 1 h. The wells were washed several times with blocking buffer, and 60 ml peroxidase-conjugated goat anti-mouse immunoglobulin antibodies (Southern Biotechnology, Birmingham, AL) diluted 1/2000 in blocking buffer was added to each well. The plates were incubated for 45 min. The plates were washed four times in DPBS, and 100 ml of substrate solution (1 mg/ml 2,29-azino bis[3-ethylbenzthiazolinesulfonic acid] (ABTS), 0.012% H2O2 in 25 mM citrate/50 mM Na2HPO4, pH 9.0) was added to each well. Positive reactions were indicated by color changes in the substrate solution from colorless to green and were quantified by spectrophotometry at 405 nm. Respiratory burst. Another indication of macrophage responsiveness to IFNg is an increase in the macrophage’s capacity to mount respiratory bursts in response to stimulation by tetradodeconyl phorbol acetate (TPA). Respiratory bursts result in H2O2 production, which can be monitored by oxidation of phenol red (Pick and Mizel, 1981). Media were removed from IFNg-treated macrophage monolayers and replaced by 100 ml of assay mixture (0.01% phenol red, 100 U/ml horseradish peroxidase [Sigma, St. Louis, MO], 200 ng/ml TPA in DMEM 1 5% NBS). The cells were incubated for 1 h at 37°C before 20 ml of 1N NaOH was added, and the color product was quantified by spectrophotometry at 600 nm and compared to a H2O2 standard curve. Fc-mediated phagocytosis. Yet another indication of macrophage responsiveness to IFNg is an increase in the macrophages’ capacity to phagocytize antibody-coated sheep red blood cells (EA). Sheep red blood cells (E) in Alsever’s solution were washed in DMEM 1 5% NBS and diluted in DMEM 1 5% NBS to 8 3 107/ml. Anti-E (Cappel, Durham, NC) antibodies were then added to a final concentration of 1/2000 (forming EA). The media were removed from the macrophage monolayers, and 50 ml of diluted EA was added to each well to yield a EA/macrophage ratio of 20. Macrophages and EA were incubated together at 37°C for 30 min and then washed twice with DMEM with 5% NBS to remove unbound EA. Bound but uningested EA were then lysed by a brief treatment with 0.82% NH4Cl in 10 mM Tris, pH 8.0. The monolayers were then washed once with DMEM 1 5% NBS and lysed in 100 ml 1% Triton X-100 to solubilize intracellular hemoglobin, which is an indirect indication of the number of E present in the sample. Released hemoglobin was assessed by spectrophotometry at 405 nm and was expressed as percentage phagocytosis, which is the estimated amount of hemoglobin ingested normalized to the amount of hemoglobin in the EA inoculum. Tumor necrosis factor alpha (TNFa). The macrophages’ responsiveness to bacterial LPS is reflected in their ability to produce TNFa. TNFa produced over 18 h was assayed by ELISA (Perceptive Bioresearch Products, Cambridge, MA). Nitric oxide. Macrophages’ responsiveness to synergistic stimulation by IFNg 1 LPS was assessed by their production of NO in response to low concentrations of both agents. Nitrite, the stable degradatory product of NO, which accumulated over 16 –18 h in culture, was assessed by the Griess
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FIG. 1. Macrophage monolayers incubated with 0.05% CSC-MS for 2 h. Fluorescent micrograph under blue (490 nm) excitation, showing orange fluorescent granules.
reaction (Drapier and Hibbs, 1988). The final pink chromophore was quantified by spectrophotometry at 570 nm and was compared to a standard curve of NaNO2. Statistical analysis. All data presented were from single experiments representative of three to 10 separate experiments performed in quadruplicate, except for the experiments measuring TNFa production, which were performed in triplicate. Data points indicate means and standard deviation of the measurements. Data presented were analyzed for significant differences among points at the p , 0.05 level by Duncan’s New Multiple Range Test (Dowdy and Wearden, 1983). While the test indicates all significantly different points, we mark with asterisks only those points that differed significantly from controls that were not exposed to CSC-MS.
RESULTS
Macrophage Ingestion of CSC-MS As early as 2 h after CSC-MS was added to macrophage monolayers, granules that fluoresced orange under blue (490 nm) illumination began to appear within many, but not all, macrophages in the monolayer (Fig. 1). Control macrophages, characteristic of casein-elicited macrophages and in contrast to those elicited with Brewer’s thioglycollate broth (another common macrophage-eliciting agent, not used in this study), contained few granular inclusions that fluoresced green, not orange, under our observational conditions. In both control and CSC-MS-treated macrophages, the usual faint green background fluorescence was present.
Macrophage Basal Metabolism We first determined whether treatment with CSC-MS at the concentrations used, and in the duration of the intended assays, affects macrophage viability. As assessed by MTT reduction over 2 h, macrophages treated with CSC-MS for 18 h prior to assay exhibited increased mitochondrial electron transport activities compared to untreated control macrophages (Fig. 2). Macrophage TNFa Production in Response to Stimulation by LPS Macrophages produce cytokines in response to stimulation by LPS. One of these is TNFa. CSC-MS enhanced TNFa production in response to LPS over 18 h at both concentrations used (Fig. 3). Macrophage Fc-Dependent Phagocytic Capacity Induced by IFNg Macrophages are phagocytic, and their phagocytic activity can be further enhanced by treatment with IFNg. CSC-MS inhibited increase in phagocytosis in response to IFNg over 18 h at the highest concentration used, 0.1% (Fig. 4). Macrophage Respiratory Burst Capacity Induced by IFNg and Elicited by TPA Casein-elicited macrophages produce H2O2, a product of respiratory burst, in response to stimulation by TPA without
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ergistic induction of iNOS at physiological concentrations of IFNg. CSC-MS markedly inhibited NO production induced by low concentrations of IFNg and LPS over 18 h in a dosedependent manner, with a reduction of over 80% at 0.1% CSC-MS (Fig. 7). DISCUSSION
FIG. 2. Macrophage basal mitochondrial electron transport activity is modulated by CSC-MS. Macrophage monolayers were treated with varying concentrations of CSC-MS for 18 h, then MTT reduction was assessed over 2 h. CSC-MS-treated and control untreated macrophages both remained active in mitochondrial electron transport with the CSC-MS-treated macrophages increasing in activity. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
We demonstrate here that macrophages exposed to CSC-MS in vitro form granules that were not present in unexposed controls, and that were fluorescent under blue illumination (Fig. 1). This is consistent with prior suggestions that macrophages ingest components of CSC-MS to form these granules (Skold et al., 1989; Streck et al., 1994). This observation is also consistent with the observation that alveolar macrophages from smokers, to a much greater extent than those from nonsmokers, contain numerous granules that were fluorescent under blue illumination (490 nm, Skold et al., 1989; Streck et al., 1994). The authors of those studies also speculated, as we do, that the granules contain PAH, substantial constituents of CSC-MS. More importantly, our data also suggest that exposure of macrophages to CSC-MS has functional consequences. The most striking of these functional consequences was the macrophages’ failure to acquire functional capacities in response to IFNg, while their basal metabolic activities and response to
additional induction. TPA-stimulated H2O2 production can be further enhanced by treatment with IFNg. CSC-MS inhibited increase in TPA-stimulated H2O2 production in response to IFNg in a dose-dependent manner. Maximal inhibition was at 0.1% CSC-MS and was about 75% of maximum (Fig. 5). Macrophage Surface Class II MHC Expression in Response to Stimulation by IFNg Unlike human monocytes, murine macrophages express very little Class II MHC on their surfaces until stimulated by such cytokines as IFNg. Forty hours after macrophages were stimulated by IFNg, a substantial increase in surface Class II MHC was detected. CSC-MS markedly inhibited this increase in surface Class II MHC in a dose-dependent manner. Maximal inhibition was at 0.1% CSC-MS and was about 90% of maximum (Fig. 6). Macrophage Nitric Oxide Production Induced Synergistically by IFNg and LPS While high levels of IFNg will elicit the inducible NO synthase (iNOS) and result in NO production, small amounts of a second signal, such as bacterial LPS, is needed for syn-
FIG. 3. Macrophage LPS-induced TNFa production is modulated by CSC-MS. Macrophages were treated with 200 ng/ml LPS with varying concentrations of CSC-MS, and culture fluids were assayed by ELISA for TNFa. TNFa production increased with the amount of CSC-MS used. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
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FIG. 4. Macrophage IFNg-induced Fc-dependent phagocytic capacity is inhibited by CSC-MS. Macrophages were treated for 18 h with 5 U/ml IFNg with varying concentrations of CSC-MS and then incubated with antibodycoated SRBC for 30 min to assess phagocytic capacities. Ingestion of antibodycoated SRBC decreased to uninduced levels (indicated by the dotted line) at the highest concentration of CSC-MS used, 0.1%. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisk indicates the point that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
LPS not only remained intact but were enhanced. Indicators of macrophage responsiveness to IFNg that were suppressed by CSC-MS were as follows: (1) phagocytosis of antibody-coated SRBC (Fig. 4), (2) respiratory burst stimulated by TPA (Fig. 5), (3) surface expression of class II MHC (Fig. 6), and (4) production of NO with costimulation by low doses of LPS (Fig. 7). While these are important functions, our interest in them is first and foremost as indicators of macrophage responsiveness, and only secondarily as mediators and effectors of immunity. Thus, among the indicators, we included NO, although its induction in human macrophages and the possible pathophysiological consequences are not completely resolved (Granger et al., 1996). The observed suppression of macrophage responsiveness to IFNg was not due to general toxicity of CSC-MS to macrophages. Basal electron transport activity and LPS-induced TNFa production were not suppressed in CSC-MS-treated macrophages but were enhanced. The observed enhancement of TNFa production was not due to LPS contamination—all glassware used to collect CSC-MS was baked to remove LPS; also, NO production was not enhanced by CSC-MS, as would be expected if CSC-MS were contaminated by LPS. In putting these results within the context of the smoker, two considerations are of foremost concern: whether the murine
peritoneal macrophage is a suitable model system for human lung macrophages and whether the doses of CSC-MS used in this study are reasonable representations of that encountered in the lung and elsewhere in the body. While these experiments could be carried out with human or murine alveolar macrophages rather than murine peritoneal macrophages, they would require either eight to 10 times the mice used in this study or the acquisition of human tissues. At the time we initiated this study we did not have adequate rationale to undertake either of these approaches. Further, those models still would not obviate the arguments that macrophages, whether from the peritoneum or the lung (Brain, 1986; Lehnert, 1992), are heterogeneous in characteristics, or that culturing macrophages alters their responsiveness to stimuli. Our results on CSC-MS modulation of macrophage responsiveness to LPS are consistent with results from studies comparing alveolar macrophages from human smokers and nonsmokers, suggesting that murine peritoneal macrophages and human alveolar macrophages share some characteristics. Alveolar macrophages from smokers and nonsmokers did not differ significantly in either basal TNFa production or their capacity to produce TNFa in response to LPS (Saulty et al., 1994). Both Soliman and Twigg (1992) and Brown et al. (1989) showed that while LPS-induced IL-1 secretion in mac-
FIG. 5. Macrophage IFNg-induced and TPA-elicited respiratory burst capacity is modulated by CSC-MS. Macrophages were treated for 18 h with 5 U/ml IFNg and with graded concentrations of CSC-MS, and then assayed for TPA-induced H2O2 production over 1 h. Hydrogen peroxide production was inhibited by CSC-MS in a dose-dependent manner. Maximal inhibition was at 0.1% CSC-MS and was about 75% of maximum. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
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FIG. 6. Macrophage IFNg-induced surface Class II MHC expression is inhibited by CSC-MS. Macrophage monolayers were treated for 40 h with 5 U/ml IFNg with varying concentrations of CSC-MS and then fixed with 0.1% formaldehyde for ELISA determination of surface class II MHC expression. Surface class II MHC expression was inhibited by CSC-MS in a dosedependent manner, with maximal inhibition of about 90% of maximum at 0.1% CSC. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
rophages from smokers were lower than that from nonsmokers, intracellular IL-1 levels in macrophages from smokers were higher than that in macrophages from nonsmokers. This evidence suggests that smoking may inhibit macrophage IL-1 secretion but not IL-1 synthesis. Since IL-1 and TNFa production are similarly regulated (Collart et al., 1986; Koide and Steinman, 1987), these data corroborate our observation that CSC-MS enhanced LPS-induced TNFa production by murine peritoneal macrophages. Thus, the responses of murine peritoneal macrophages and human alveolar macrophages to CSC-MS seem to differ at a late step in the response cascade, secretion of peptides, rather than at the early steps where we propose modulation by CSC-MS takes place. The difference may be attributable to chronic smoke exposure by human smokers and the relatively short-term exposure suffered by our macrophages. This possible difference between human alveolar macrophage and murine peritoneal macrophage may not be relevant in our main observations here, since none of the four parameters of macrophage responsiveness to IFNg depended on protein secretion. Higashimoto et al. (1992), using murine lung macrophages, and Dubar et al. (1993), using guinea pig lung macrophages, showed respectively, that cigarette smoke trapped in aqueous
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solution and cigarette smoke gas phase decreased LPS-induced TNFa production. Both studies differed from ours in the sources of macrophages and toxicant used and thus are not directly comparable. In addition, the LPS concentration used by Higashimoto et al. was substantially higher than ours. The literature does not enable us to perform similar analysis on the effects of CSC-MS on macrophage responsiveness to IFNg. Sherman et al. (1991) have shown that macrophages from smokers, compared to those from nonsmokers, exhibit increased capacity for TPA-stimulated production of reactive oxygen intermediates but decreased capacity for phagocytosis and killing of Candida albicans. Ortega et al. (1992) also showed that murine lung macrophages exposed to whole cigarette smoke phagocytized latex beads less well than controls. Davis et al. (1988) showed that oxidation-mediated cytotoxicity of alveolar macrophages were higher in smokers than nonsmokers. All these studies exposed hosts to unfractionated cigarette smoke and then assessed basal, uninduced functions and are not directly comparable to the effects of CSC-MS on IFNg-induced capacity for respiratory burst and phagocytosis that we report here. Further, both Sherman et al. (1991) and Davis et al. (1988) used human alveolar macrophages that were chronically exposed to mainstream smoke, while our exposure protocol might be considered short term. However,
FIG. 7. Macrophage IFNg and LPS-induced NO production is inhibited by CSC-MS. Macrophages monolayers were treated for 18 h with 5 U/ml IFNg and 100 ng/ml LPS with varying concentrations of CSC-MS, and culture fluids were removed for measurement of nitrite by the Griess reaction. Nitrite accumulation was inhibited by CSC-MS in a dose-dependent manner, with maximal inhibition of about 80% of maximum at 0.1% CSC. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
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the observation that TPA increased oxidation-mediated cytotoxicity in alveolar macrophages from nonsmokers but not smokers (Davis et al. 1988) may bear on our observation that exposure to CSC-MS reduced the capacity of murine peritoneal macrophage, in response to IFNg to mount TPA-stimulated H2O2 production. Murine peritoneal macrophages elicited by a mild inflammatory stimulus such as casein could be a reasonable model for human lung macrophages. Humans, unlike experimental rodents, do not live under controlled animal care conditions. Humans, particularly those resident outside rural areas, are constantly exposed to air containing inflammatory material, and their lung macrophages would therefore consist of mixed populations of resident lung macrophages and macrophages that migrate from the circulation in response to inflammatory stimuli. Our model system harvested peritoneal macrophages from mice after an inflammatory stimulus and should consist of a mixture of resident and inflammatory macrophages as well. Because mainstream smoke is inhaled by the smoker and then some components are absorbed through the respiratory tract, our results must be interpreted first within the context of the respiratory tract where primary exposure and absorption occur, and then the rest of the body where absorbed toxicants are distributed. The progression of CSC-MS from the cigarette to the alveoli is a complex process, making determination of exact doses delivered to alveolar macrophages a difficult proposition. We estimate that if a single cigarette dose of CSC-MS were to deposit completely into alveolar spaces of one human lung, then the macrophages would have been exposed to a CSC-MS dose equivalent to 0.1% of our preparation. We expect fractional loss of CSC-MS due to trapping and dilution as the smoke progress down the respiratory tract, so that a 0.1% CSC-MS dose would require exposure to several cigarettes. Note, however, that substantial suppression of macrophage responsiveness to IFNg occurred at CSC-MS doses less than 0.1% (Figs. 4 – 6). CSC-MS levels would clearly diminish still further as the absorbed CSC-MS distribute throughout the body, even though elevated concentration of some CSC components in selected parts of the body remains a possibility. Thus our data do not substantiate a role for CSC-MS in modulating macrophage responsiveness to IFNg outside of the lung, although selective absorption of large quantities of some immunomodulatory components of CSC-MS remains a possibility. While these studies ideally would be performed by exposing healthy human individuals to cigarette smoke and then examining their lung macrophages, our model system does offer some conveniences not available with the ideal system. We were able to isolate defined populations of macrophages from healthy and syngeneic mice in sufficient quantities to determine their responses to individual stimuli. We were also able to determine the effects of toxicants on macrophage responses to stimuli and to provide information on dose responses.
CSC-MS may exert their effects on macrophage responses to IFNg at many points over the stimulus–response process: signal transduction, transcription initiation, mRNA synthesis and processing, translation and posttranslational peptide processing, transport, and secretion. Our results showed that macrophage basal metabolism and production of TNFa in response to LPS was not disrupted by CSC-MS, but enhanced, suggesting that general processes such as RNA or protein synthesis remained intact and highly functional after exposure to CSCMS. Therefore, CSC-MS likely affect processes that permit some level of selectivity, such as signal transcription or transcription initiation, both early events. Results from this study suggest that low levels of CSC-MS can suppress macrophage responsiveness to interferon g. While alterations in responsiveness to cytokines may have both beneficial and deleterious consequences in general, we believe that in this particular case, the consequences are not beneficial. We have not yet identified all functional capacities that macrophages acquire in response to interferon g. However, decreased capacities to phagocytose opsonized antigens, to produce reactive oxygen and nitrogen intermediates to kill phagocytosed organisms, and to present processed antigen to T-helper lymphocytes via surface class II MHC molecules cannot be conducive to effective host defense against infection. ACKNOWLEDGMENTS This work was supported by grants from the American Heart Association, Illinois Affiliate (GB/CGB-01) and the National Science Foundation (DUE9650507).
REFERENCES Adams, D. O., and Hamilton, T.A. (1988). Phagocytic cells: Cytotoxic activities of macrophages. In Inflammation: Basic Principles and Clinical Correlates (J. I. Gallin, I. M. Goldstein, and R. Snyderman, Eds.), pp. 471– 492. Raven Press, New York. Brain, J. D. (1986). Toxicological aspects of alterations of pulmonary macrophage function. Annu. Rev. Pharmacol. Toxicol. 26, 547–565. Brown, G. P., Iwamoto, G. K., Monick, M. M., and Hunninghake, G. W. (1989). Cigarette smoking decreases interleukin 1 release by human alveolar macrophages. Am. J. Physiol. 256, C260-C264. Collart, M. A., Belin, D., deCossodo, S., and Vassalli, P. (1986). g-Interferon enhances macrophage transcription of tumor necrosis factor/cachectin, interleukin-1, and urokinase genes which are controlled by short-lived repressors. J. Exp. Med. 164, 2113–2118. Davis, W. B., Pacht, E. R., Spatafora, M., and Martin, W. J., II. (1988). Enhanced cytotoxic potential of alveolar macrophages from cigarette smokers. J. Lab. Clin. Med. 111, 293–298. Drapier, J. C., and Hibbs, J. B. (1988). Interferon-g and tumor necrosis factor induce the l-arginine-dependent cytotoxic effector mechanism in murine macrophages. Eur. J. Immunol. 18, 1587–1592. Dowdy, S., and Wearden, S. (1983). Statistics for Research. Wiley, New York. Dubar, V., Gosset, P., Aerts, C., Voisin, C., Wallaert, B., and Tonnel, A. B. (1993). In vitro acute effects of tobacco smoke on tumor necrosis factor-
SMOKE CONDENSATES SUPPRESS MACROPHAGE RESPONSES alpha and interleukin 6 production by alveolar macrophages. Exp. Lung Res. 19, 345–359. Evans, R. (1986). The immunological network at the site of tumor rejection. Biochim. Biophys. Acta 865, 1–11. Fan, S., Fehr, H.-G., and Adams, D. O. (1991). Activation of macrophages for ADCC in vitro: Effects of IL-4, TNF, interferons-ab, interferon-g, and GM-CSF. Cell. Immunol. 135, 78 – 87. Grimmer, G., Naujack, K. W., Dettbarn, G. (1987). Gas chromatographic determination of polycyclic aromatic hydrocarbons, aza-arenes, aromatic amines in the particle and vapor phase of mainstream and sidestream smoke of cigarettes. Toxicol. Lett. 35, 117–124. Higashimoto, Y., Shimada, Y., Fukuchi, Y., Ishida, K., Shu, C., Teramoto, S., Sudo, E., Matsuse, T., and Orimo, H. (1992). Inhibition of mouse alveolar macrophage production of tumor necrosis factor alpha by acute in vivo and in vitro exposure to tobacco smoke. Respiration 59, 77-80. Hoffmann, D., and Wynder, E. L. (1971). A study of tobacco carcinogenesis. XI. Tumor initiators, tumor accelerators, and tumor promoting activities of condensate fractions. Cancer 27, 848 – 864. International Agency for Research on Cancer. (1976). Coumarin. IARC Monogr. 10, 113–119. Koide, S., and Steinman, R. M. (1987). Induction of murine interleukin-1. Stimuli and responsive primary cells. Proc. Natl. Acad. Sci. USA 84, 3802–3806. Lehnert, B. E. (1992). Pulmonary and thoracic macrophage subpopulations and clearance of particles from the lung. Environ. Health Perspect. 97, 17– 46. Lepay, D. A., Steinman, R. M., Nathan, C. F., Murray, H. W., and Cohn, Z. A. (1985). Liver macrophages in murine listeriosis: Cell-mediated immunity is correlated with an influx of macrophages capable of generating reactive oxygen intermediates. J. Exp. Med. 161, 1503–1512. Marino, P. A., and Adams, D. O. (1980). Interaction of bacillus CalmetteGuerin-activated macrophages and neoplastic cells in vitro. I. Conditions of binding and its selectivity. Cell. Immunol. 54, 11–25. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63. Ortega, E., Hueso, F., Collazos, M. E., Pedrera, M. I., Barriga, C., and
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Rodriguez, A. B. (1992). Phagocytosis of latex beads by alveolar macrophages from mice exposed to cigarette smoke. Comp. Immunol. Microbiol. Infect. Dis. 15, 137-142. Ozato, K., and Sachs, D. H. (1981). Monoclonal antibodies to mouse major histocompatibility complex antigens: 3. Hybridoma antibodies reacting to antigens of the H-2b haplotype reveal genetic control of isotype expression. J. Immunol. 126, 317-321. Pick, E., and Mizel, D. (1981). Rapid microassays for measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J. Immunol. Methods 46, 211–226. Sauty, A., Mauel, J., Philippeaux, M. M., and Leuenberger, P. (1994). Cytostatic activity of alveolar macrophages from smokers and nonsmokers: role of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha. Am. J. Respir. Cell Mol. Biol. 11, 631– 637. Sherman, M. P., Campbell, L. A., Gong, H., Roth, M. D., Tashkin, D. P. (1991). Antimicrobial and respiratory burst characteristics of pulmonary alveolar macrophages recovered from smokers of marijuana alone, smokers of tobacco alone, smokers of marijuana and tobacco, and nonsmokers. Am. Rev. Resp. Dis. 144, 1351–1356. Sko¨ld, C. M., Eklund, A., Hallde´n, G., and Hed, J. (1989). Autofluorescence in human alveolar macrophages from smokers: Relation to cell surface markers and phagocytosis. Exp. Lung Res. 15, 823– 835. Slater, T. F., Sawyer, B., and Strauli, U. (1963). Studies on succinate-tetrazolium reductase systems. III. Points of coupling of four different tetrazolium salts. Biochim. Biophys. Acta 77, 383–393. Soliman, D. M., and Twigg, H. L., III. (1992). Cigarette smoking decreases bioactive interleukin-6 secretion by alveolar macrophages. Am. J. Physiol. 263, L471–L478. Streck, R. J., Jezewski, H. M., Rodriguez, M. I., Hurley, E. L., Rich, G. A., Braun, K. M., and Pauly, J. L. (1994). A method for isolating human lung macrophages and observations of fluorescent phagocytes from the lungs of habitual cigarette smokers. J. Immunol. Methods 174, 67– 82. Unanue, E., and Askonas, B. (1968) The immune response of mice to antigen in macrophages. Immunology 15, 287–296. U.S. Surgeon General. (1986). The health consequences of involuntary smoking. DHHS (CDC) 87-8398. Washington, DC.
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Immunotoxicology of Cigarette Smoke Condensates: Suppression of Macrophage Responsiveness to Interferon g Kristen M. Braun,*,1 Toby Cornish,*,2 Alex Valm,* Jason Cundiff,* John L. Pauly,† and Samuel Fan*,3 *Department of Biology, Bradley University, Peoria, Illinois 61625; and †Department of Molecular Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263-0001 Received June 12, 1997; accepted November 14, 1997
Immunotoxicology of Cigarette Smoke Condensates: Suppression of Macrophage Responsiveness to Interferon g. Braun, K. M., Cornish, T., Valm, A., Cundiff, J., Pauly, J. L., and Fan, S. (1998). Toxicol. Appl. Pharmacol. 149, 136 –143. We have investigated systematically the effects of short-term exposure to main stream cigarette smoke condensates (CSC-MS) on basal and inducible functional capacities of murine peritoneal exudate macrophages. Macrophages treated with CSC-MS form granules that fluoresce orange under blue excitation, consistent with the speculation that they are polycyclic aromatic hydrocarbons (PAH). CSC-MS selectively suppressed interferon gamma (IFNg) induction of four macrophage functional capacities: enhanced phagocytosis of immunoglobulin-opsonized sheep red blood cells, TPA-induced H2O2 production, class II major histocompatibility complex expression, and nitric oxide synthesis. In contrast, two macrophage functions that are not induced by IFNg, basal electron transport and LPS-induced TNFa production, were enhanced by treatment with CSC-MS. These results suggest that the suppressive effects of CSC-MS on macrophage responsiveness were selective and were not due to nonspecific inhibition of general functions such as RNA or protein synthesis. Since macrophage responsiveness to IFNg can result in induction of functional capacities that are fundamental to immunity, the data suggest that CSC-MS maybe deleterious to the general health of the smoker. © 1998 Academic Press
Transient effects of short-term exposure to environmental chemicals on human health are difficult to assess. Yet such exposures may cause immediate or delayed deleterious effects to the subject. A case in point is cigarette smoke. The chemical constituents of cigarette smoke, including some polycyclic aromatic hydrocarbons (PAH), clearly exert potent long-term effects on the smoker as initiators and promoters of cancer (IARC, 1976; Hoffmann and Wynder, 1971). Less well defined are the short-term effects of acute and transient exposure to 1
Present address: Department of Biochemistry, University of Wisconsin, Madison, WI 53706. 2 Present address: Neuroscience Program, University of Illinois, Urbana, IL 61801. 3 To whom correspondences should be addressed at Department of Biology, Bradley University, Peoria, IL 61625. E-mail: [email protected]. 0041-008X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
tobacco smoke. Of particular concern are the immediate effects on the host’s immune defenses against infectious diseases and cancer. A potential target for functional modulation by tobacco smoke is the macrophage. Smokers who inhale have higher incidences of infection and cancer of the respiratory tract as well as a higher predisposition to cardiovascular diseases (IARC, 1976). Recently, environmental tobacco smoke (ETS) also became a subject of concern, as data emerged from studies indicating higher health risks for those who do not smoke but live in the same household as smokers compared with those who do neither (U.S. Surgeon General, 1986). The products of burning cigarettes are airborne and thus are apt to enter the human airways and eventually deposit in the alveoli of the lungs. Since alveoli are lined with macrophages, the lung is a potential site where macrophages encounter cigarette smoke. Lung macrophages would encounter constituents of cigarette smoke more frequently, and in larger quantities, than those resident in the rest of the body. In fact, macrophages recovered from smokers but not nonsmokers contain numerous granules that fluoresce upon illumination with blue light (Skold et al., 1989; Streck et al., 1994). The granules are thought to be ingested polycyclic aromatic hydrocarbons (PAH), a major component of the condensate fraction of cigarette smoke (CSC), primarily because PAH fluorescence has similar spectral characteristics as the granules (Skold et al., 1989). Macrophages can help prevent infectious diseases and cancer. Directly, macrophages can ingest and destroy infectious agents (Lepay et al., 1985), and they can kill neoplastic cells by a variety of mechanisms (Evans, 1986; Adams and Hamilton, 1988); indirectly, macrophages can initiate and regulate immune responses against infectious agents (Unanue and Askonas, 1968). Many of these functions are not constitutively expressed but are induced in response to stimuli such as interferon gamma (IFNg), bacterial lipopolysaccharide (LPS), or combinations of various stimuli (Adams and Hamilton, 1988). Thus, macrophages must maintain their responsiveness to a full range of stimuli and their capacity to perform their full range of functions for the host organism to thrive. By extension, environmental factors that modulate macrophages’ capacity to
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respond to stimuli or to perform functions would impair the host organism’s well being. Using cultured murine peritoneal macrophages as a model, we have investigated systematically the effects of short-term exposure to CSC prepared from mainstream smoke (CSC-MS) on basal and inducible functional capacities of macrophages. We report here that macrophages ingest CSC-MS. We also report that CSC-MS selectively suppresses macrophage functional capacities that are inducible by IFNg, while functional capacities that are basal to the macrophages or are inducible by LPS are either not affected or are enhanced.
METHODS Cigarette smoke condensates. Since CSC-MS are complex mixtures (Grimmer et al., 1987), obtaining a standardized reagent is improbable. Instead, we prepared large lots of CSC-MS, so that many experiments can be performed with a single CSC-MS preparation to make the results directly comparable within a trial. CSC-MS were collected from lit cigarettes (regular size, national brand) as crude smoke condensates by simulating periodic 2-s puffs. Condensates were the components of smoke that condense on glass Erlenmeyer flasks that had been baked at 280°C for 4 h to destroy bacterial LPS. Condensates were subsequently eluted-off with methanol, evaporated to dryness by roto-evaporation under reduced pressure, and then redissolved in methanol at five cigarette-equivalents per milliliter. This constitutes the 100% stock solution, which was diluted to 25% with methanol and stored at 4°C. Further dilutions for experimental use were in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech, Herndorn, VA) supplemented with 5% newborn bovine serum (NBS, Hyclone, Logan, UT). All data presented in this report refer to a single lot (the most recent) of CSC-MS (from mainstream smoke). However, similar results were obtained with two other preparations of CSC-MS for many of the functional capacities described here. The lot used in the reported experiments was intermediate in potency (i.e., modulatory activity) of the three lots that we have worked with. Macrophages. The experimental system is well characterized (Marino and Adams, 1980; Fan et al., 1991). Female C57BL/6 mice (Trudeau Institute, Saranac Lake, NY) were kept in a PHS-certified mouse housing unit at Bradley University and were fed tap water and Teklad LM-485 M/R Diet (Harlan Teklad, Madison, WI) ad libitum. They were used at between 7 to 15 weeks of age. Peritoneal macrophages were obtained by peritoneal lavage with Hank’s balanced salt solution (HBSS, GIBCO, Grand Island, NY) following established procedures (Marino and Adams, 1980; Fan et al., 1991). To increase yield, peritoneal macrophages were obtained from mice injected peritoneally with 1 ml 1.2% aqueous casein (nach Hammerstein) 3 days prior to harvest. Typically, each mouse yielded about 1 3 107 peritoneal exudate cells, of which about 95% were morphologically similar to macrophages. Harvested macrophages were suspended in DMEM with 5% NBS and dispensed into 96 flat-well microtiter plates (Falcon) at 2 3 105/well in 100 ml. After 2– 4-h incubation at 37°C in a 95% air, 5% CO2 environment, nonadherent cells were removed by washing with DMEM with 5% NBS, yielding macrophage monolayers. Fluorescence microscopy. Macrophage ingestion of CSC-MS was monitored by observing macrophages that were exposed to 0.05% CSC-MS, fixed in 0.1% formaldehyde in Dulbecco’s phosphate-buffered saline (DPBS), and mounted in 90% glycerol/10% 103 DPBS under blue (490 nm) illumination. Photomicroscopy was carried-out at 10003 magnification using an Olympus BX 40 microscope. Spectrophotometry. All spectrophotometry was performed in polystyrene microwell plates in a BioTek 311S microplate reader equipped with 405-, 450-, 570-, and 600-nm filters.
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Electron transport activity. Macrophage metabolic activity was assessed by the cells’ ability to produce intermediates of the electron transport chain, reflected by reduction of an orange dye (MTT, Molecular Probes, Eugene, OR) to yield a purple precipitate. The soluble dye was removed along with detached macrophages, and remaining precipitate was then dissolved in acidified isopropanol and quantified by spectrophotometry at 600 nm (Slater et al., 1963; Mosmann, 1983). This assay is commonly used to evaluate cellular viability, based on the assumption that mammalian cells that are unable to effect electron transport are nonviable. Class II major histocompatibility complex (class II MHC). The responsiveness of experimentally treated macrophages to stimulation by IFNg was assessed in part by the cells’ ability to express class II MHC molecules after treatment with IFNg. Macrophage monolayers were either treated with IFNg or left untreated. After a 36- to 40-h incubation, media in the wells were removed, and 50 ml 0.1% formaldehyde was added. The wells were washed twice with blocking buffer (DPBS containing 2% nonfat dry milk). Sixty microliters of diluted monoclonal antibody from the hybridoma 28-16-8S (Ozato and Sachs, 1981; IgM anti-Iab,d, culture fluids diluted 1/5 in blocking buffer) was added to each well and incubated for 1 h. The wells were washed several times with blocking buffer, and 60 ml peroxidase-conjugated goat anti-mouse immunoglobulin antibodies (Southern Biotechnology, Birmingham, AL) diluted 1/2000 in blocking buffer was added to each well. The plates were incubated for 45 min. The plates were washed four times in DPBS, and 100 ml of substrate solution (1 mg/ml 2,29-azino bis[3-ethylbenzthiazolinesulfonic acid] (ABTS), 0.012% H2O2 in 25 mM citrate/50 mM Na2HPO4, pH 9.0) was added to each well. Positive reactions were indicated by color changes in the substrate solution from colorless to green and were quantified by spectrophotometry at 405 nm. Respiratory burst. Another indication of macrophage responsiveness to IFNg is an increase in the macrophage’s capacity to mount respiratory bursts in response to stimulation by tetradodeconyl phorbol acetate (TPA). Respiratory bursts result in H2O2 production, which can be monitored by oxidation of phenol red (Pick and Mizel, 1981). Media were removed from IFNg-treated macrophage monolayers and replaced by 100 ml of assay mixture (0.01% phenol red, 100 U/ml horseradish peroxidase [Sigma, St. Louis, MO], 200 ng/ml TPA in DMEM 1 5% NBS). The cells were incubated for 1 h at 37°C before 20 ml of 1N NaOH was added, and the color product was quantified by spectrophotometry at 600 nm and compared to a H2O2 standard curve. Fc-mediated phagocytosis. Yet another indication of macrophage responsiveness to IFNg is an increase in the macrophages’ capacity to phagocytize antibody-coated sheep red blood cells (EA). Sheep red blood cells (E) in Alsever’s solution were washed in DMEM 1 5% NBS and diluted in DMEM 1 5% NBS to 8 3 107/ml. Anti-E (Cappel, Durham, NC) antibodies were then added to a final concentration of 1/2000 (forming EA). The media were removed from the macrophage monolayers, and 50 ml of diluted EA was added to each well to yield a EA/macrophage ratio of 20. Macrophages and EA were incubated together at 37°C for 30 min and then washed twice with DMEM with 5% NBS to remove unbound EA. Bound but uningested EA were then lysed by a brief treatment with 0.82% NH4Cl in 10 mM Tris, pH 8.0. The monolayers were then washed once with DMEM 1 5% NBS and lysed in 100 ml 1% Triton X-100 to solubilize intracellular hemoglobin, which is an indirect indication of the number of E present in the sample. Released hemoglobin was assessed by spectrophotometry at 405 nm and was expressed as percentage phagocytosis, which is the estimated amount of hemoglobin ingested normalized to the amount of hemoglobin in the EA inoculum. Tumor necrosis factor alpha (TNFa). The macrophages’ responsiveness to bacterial LPS is reflected in their ability to produce TNFa. TNFa produced over 18 h was assayed by ELISA (Perceptive Bioresearch Products, Cambridge, MA). Nitric oxide. Macrophages’ responsiveness to synergistic stimulation by IFNg 1 LPS was assessed by their production of NO in response to low concentrations of both agents. Nitrite, the stable degradatory product of NO, which accumulated over 16 –18 h in culture, was assessed by the Griess
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FIG. 1. Macrophage monolayers incubated with 0.05% CSC-MS for 2 h. Fluorescent micrograph under blue (490 nm) excitation, showing orange fluorescent granules.
reaction (Drapier and Hibbs, 1988). The final pink chromophore was quantified by spectrophotometry at 570 nm and was compared to a standard curve of NaNO2. Statistical analysis. All data presented were from single experiments representative of three to 10 separate experiments performed in quadruplicate, except for the experiments measuring TNFa production, which were performed in triplicate. Data points indicate means and standard deviation of the measurements. Data presented were analyzed for significant differences among points at the p , 0.05 level by Duncan’s New Multiple Range Test (Dowdy and Wearden, 1983). While the test indicates all significantly different points, we mark with asterisks only those points that differed significantly from controls that were not exposed to CSC-MS.
RESULTS
Macrophage Ingestion of CSC-MS As early as 2 h after CSC-MS was added to macrophage monolayers, granules that fluoresced orange under blue (490 nm) illumination began to appear within many, but not all, macrophages in the monolayer (Fig. 1). Control macrophages, characteristic of casein-elicited macrophages and in contrast to those elicited with Brewer’s thioglycollate broth (another common macrophage-eliciting agent, not used in this study), contained few granular inclusions that fluoresced green, not orange, under our observational conditions. In both control and CSC-MS-treated macrophages, the usual faint green background fluorescence was present.
Macrophage Basal Metabolism We first determined whether treatment with CSC-MS at the concentrations used, and in the duration of the intended assays, affects macrophage viability. As assessed by MTT reduction over 2 h, macrophages treated with CSC-MS for 18 h prior to assay exhibited increased mitochondrial electron transport activities compared to untreated control macrophages (Fig. 2). Macrophage TNFa Production in Response to Stimulation by LPS Macrophages produce cytokines in response to stimulation by LPS. One of these is TNFa. CSC-MS enhanced TNFa production in response to LPS over 18 h at both concentrations used (Fig. 3). Macrophage Fc-Dependent Phagocytic Capacity Induced by IFNg Macrophages are phagocytic, and their phagocytic activity can be further enhanced by treatment with IFNg. CSC-MS inhibited increase in phagocytosis in response to IFNg over 18 h at the highest concentration used, 0.1% (Fig. 4). Macrophage Respiratory Burst Capacity Induced by IFNg and Elicited by TPA Casein-elicited macrophages produce H2O2, a product of respiratory burst, in response to stimulation by TPA without
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ergistic induction of iNOS at physiological concentrations of IFNg. CSC-MS markedly inhibited NO production induced by low concentrations of IFNg and LPS over 18 h in a dosedependent manner, with a reduction of over 80% at 0.1% CSC-MS (Fig. 7). DISCUSSION
FIG. 2. Macrophage basal mitochondrial electron transport activity is modulated by CSC-MS. Macrophage monolayers were treated with varying concentrations of CSC-MS for 18 h, then MTT reduction was assessed over 2 h. CSC-MS-treated and control untreated macrophages both remained active in mitochondrial electron transport with the CSC-MS-treated macrophages increasing in activity. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
We demonstrate here that macrophages exposed to CSC-MS in vitro form granules that were not present in unexposed controls, and that were fluorescent under blue illumination (Fig. 1). This is consistent with prior suggestions that macrophages ingest components of CSC-MS to form these granules (Skold et al., 1989; Streck et al., 1994). This observation is also consistent with the observation that alveolar macrophages from smokers, to a much greater extent than those from nonsmokers, contain numerous granules that were fluorescent under blue illumination (490 nm, Skold et al., 1989; Streck et al., 1994). The authors of those studies also speculated, as we do, that the granules contain PAH, substantial constituents of CSC-MS. More importantly, our data also suggest that exposure of macrophages to CSC-MS has functional consequences. The most striking of these functional consequences was the macrophages’ failure to acquire functional capacities in response to IFNg, while their basal metabolic activities and response to
additional induction. TPA-stimulated H2O2 production can be further enhanced by treatment with IFNg. CSC-MS inhibited increase in TPA-stimulated H2O2 production in response to IFNg in a dose-dependent manner. Maximal inhibition was at 0.1% CSC-MS and was about 75% of maximum (Fig. 5). Macrophage Surface Class II MHC Expression in Response to Stimulation by IFNg Unlike human monocytes, murine macrophages express very little Class II MHC on their surfaces until stimulated by such cytokines as IFNg. Forty hours after macrophages were stimulated by IFNg, a substantial increase in surface Class II MHC was detected. CSC-MS markedly inhibited this increase in surface Class II MHC in a dose-dependent manner. Maximal inhibition was at 0.1% CSC-MS and was about 90% of maximum (Fig. 6). Macrophage Nitric Oxide Production Induced Synergistically by IFNg and LPS While high levels of IFNg will elicit the inducible NO synthase (iNOS) and result in NO production, small amounts of a second signal, such as bacterial LPS, is needed for syn-
FIG. 3. Macrophage LPS-induced TNFa production is modulated by CSC-MS. Macrophages were treated with 200 ng/ml LPS with varying concentrations of CSC-MS, and culture fluids were assayed by ELISA for TNFa. TNFa production increased with the amount of CSC-MS used. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
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FIG. 4. Macrophage IFNg-induced Fc-dependent phagocytic capacity is inhibited by CSC-MS. Macrophages were treated for 18 h with 5 U/ml IFNg with varying concentrations of CSC-MS and then incubated with antibodycoated SRBC for 30 min to assess phagocytic capacities. Ingestion of antibodycoated SRBC decreased to uninduced levels (indicated by the dotted line) at the highest concentration of CSC-MS used, 0.1%. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisk indicates the point that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
LPS not only remained intact but were enhanced. Indicators of macrophage responsiveness to IFNg that were suppressed by CSC-MS were as follows: (1) phagocytosis of antibody-coated SRBC (Fig. 4), (2) respiratory burst stimulated by TPA (Fig. 5), (3) surface expression of class II MHC (Fig. 6), and (4) production of NO with costimulation by low doses of LPS (Fig. 7). While these are important functions, our interest in them is first and foremost as indicators of macrophage responsiveness, and only secondarily as mediators and effectors of immunity. Thus, among the indicators, we included NO, although its induction in human macrophages and the possible pathophysiological consequences are not completely resolved (Granger et al., 1996). The observed suppression of macrophage responsiveness to IFNg was not due to general toxicity of CSC-MS to macrophages. Basal electron transport activity and LPS-induced TNFa production were not suppressed in CSC-MS-treated macrophages but were enhanced. The observed enhancement of TNFa production was not due to LPS contamination—all glassware used to collect CSC-MS was baked to remove LPS; also, NO production was not enhanced by CSC-MS, as would be expected if CSC-MS were contaminated by LPS. In putting these results within the context of the smoker, two considerations are of foremost concern: whether the murine
peritoneal macrophage is a suitable model system for human lung macrophages and whether the doses of CSC-MS used in this study are reasonable representations of that encountered in the lung and elsewhere in the body. While these experiments could be carried out with human or murine alveolar macrophages rather than murine peritoneal macrophages, they would require either eight to 10 times the mice used in this study or the acquisition of human tissues. At the time we initiated this study we did not have adequate rationale to undertake either of these approaches. Further, those models still would not obviate the arguments that macrophages, whether from the peritoneum or the lung (Brain, 1986; Lehnert, 1992), are heterogeneous in characteristics, or that culturing macrophages alters their responsiveness to stimuli. Our results on CSC-MS modulation of macrophage responsiveness to LPS are consistent with results from studies comparing alveolar macrophages from human smokers and nonsmokers, suggesting that murine peritoneal macrophages and human alveolar macrophages share some characteristics. Alveolar macrophages from smokers and nonsmokers did not differ significantly in either basal TNFa production or their capacity to produce TNFa in response to LPS (Saulty et al., 1994). Both Soliman and Twigg (1992) and Brown et al. (1989) showed that while LPS-induced IL-1 secretion in mac-
FIG. 5. Macrophage IFNg-induced and TPA-elicited respiratory burst capacity is modulated by CSC-MS. Macrophages were treated for 18 h with 5 U/ml IFNg and with graded concentrations of CSC-MS, and then assayed for TPA-induced H2O2 production over 1 h. Hydrogen peroxide production was inhibited by CSC-MS in a dose-dependent manner. Maximal inhibition was at 0.1% CSC-MS and was about 75% of maximum. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
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FIG. 6. Macrophage IFNg-induced surface Class II MHC expression is inhibited by CSC-MS. Macrophage monolayers were treated for 40 h with 5 U/ml IFNg with varying concentrations of CSC-MS and then fixed with 0.1% formaldehyde for ELISA determination of surface class II MHC expression. Surface class II MHC expression was inhibited by CSC-MS in a dosedependent manner, with maximal inhibition of about 90% of maximum at 0.1% CSC. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
rophages from smokers were lower than that from nonsmokers, intracellular IL-1 levels in macrophages from smokers were higher than that in macrophages from nonsmokers. This evidence suggests that smoking may inhibit macrophage IL-1 secretion but not IL-1 synthesis. Since IL-1 and TNFa production are similarly regulated (Collart et al., 1986; Koide and Steinman, 1987), these data corroborate our observation that CSC-MS enhanced LPS-induced TNFa production by murine peritoneal macrophages. Thus, the responses of murine peritoneal macrophages and human alveolar macrophages to CSC-MS seem to differ at a late step in the response cascade, secretion of peptides, rather than at the early steps where we propose modulation by CSC-MS takes place. The difference may be attributable to chronic smoke exposure by human smokers and the relatively short-term exposure suffered by our macrophages. This possible difference between human alveolar macrophage and murine peritoneal macrophage may not be relevant in our main observations here, since none of the four parameters of macrophage responsiveness to IFNg depended on protein secretion. Higashimoto et al. (1992), using murine lung macrophages, and Dubar et al. (1993), using guinea pig lung macrophages, showed respectively, that cigarette smoke trapped in aqueous
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solution and cigarette smoke gas phase decreased LPS-induced TNFa production. Both studies differed from ours in the sources of macrophages and toxicant used and thus are not directly comparable. In addition, the LPS concentration used by Higashimoto et al. was substantially higher than ours. The literature does not enable us to perform similar analysis on the effects of CSC-MS on macrophage responsiveness to IFNg. Sherman et al. (1991) have shown that macrophages from smokers, compared to those from nonsmokers, exhibit increased capacity for TPA-stimulated production of reactive oxygen intermediates but decreased capacity for phagocytosis and killing of Candida albicans. Ortega et al. (1992) also showed that murine lung macrophages exposed to whole cigarette smoke phagocytized latex beads less well than controls. Davis et al. (1988) showed that oxidation-mediated cytotoxicity of alveolar macrophages were higher in smokers than nonsmokers. All these studies exposed hosts to unfractionated cigarette smoke and then assessed basal, uninduced functions and are not directly comparable to the effects of CSC-MS on IFNg-induced capacity for respiratory burst and phagocytosis that we report here. Further, both Sherman et al. (1991) and Davis et al. (1988) used human alveolar macrophages that were chronically exposed to mainstream smoke, while our exposure protocol might be considered short term. However,
FIG. 7. Macrophage IFNg and LPS-induced NO production is inhibited by CSC-MS. Macrophages monolayers were treated for 18 h with 5 U/ml IFNg and 100 ng/ml LPS with varying concentrations of CSC-MS, and culture fluids were removed for measurement of nitrite by the Griess reaction. Nitrite accumulation was inhibited by CSC-MS in a dose-dependent manner, with maximal inhibition of about 80% of maximum at 0.1% CSC. The means and standard deviations of quadruplicate samples in one experiment are shown. Asterisks indicate points that differed significantly from controls that were not exposed to CSC-MS. Samples in one representative experiment are shown.
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the observation that TPA increased oxidation-mediated cytotoxicity in alveolar macrophages from nonsmokers but not smokers (Davis et al. 1988) may bear on our observation that exposure to CSC-MS reduced the capacity of murine peritoneal macrophage, in response to IFNg to mount TPA-stimulated H2O2 production. Murine peritoneal macrophages elicited by a mild inflammatory stimulus such as casein could be a reasonable model for human lung macrophages. Humans, unlike experimental rodents, do not live under controlled animal care conditions. Humans, particularly those resident outside rural areas, are constantly exposed to air containing inflammatory material, and their lung macrophages would therefore consist of mixed populations of resident lung macrophages and macrophages that migrate from the circulation in response to inflammatory stimuli. Our model system harvested peritoneal macrophages from mice after an inflammatory stimulus and should consist of a mixture of resident and inflammatory macrophages as well. Because mainstream smoke is inhaled by the smoker and then some components are absorbed through the respiratory tract, our results must be interpreted first within the context of the respiratory tract where primary exposure and absorption occur, and then the rest of the body where absorbed toxicants are distributed. The progression of CSC-MS from the cigarette to the alveoli is a complex process, making determination of exact doses delivered to alveolar macrophages a difficult proposition. We estimate that if a single cigarette dose of CSC-MS were to deposit completely into alveolar spaces of one human lung, then the macrophages would have been exposed to a CSC-MS dose equivalent to 0.1% of our preparation. We expect fractional loss of CSC-MS due to trapping and dilution as the smoke progress down the respiratory tract, so that a 0.1% CSC-MS dose would require exposure to several cigarettes. Note, however, that substantial suppression of macrophage responsiveness to IFNg occurred at CSC-MS doses less than 0.1% (Figs. 4 – 6). CSC-MS levels would clearly diminish still further as the absorbed CSC-MS distribute throughout the body, even though elevated concentration of some CSC components in selected parts of the body remains a possibility. Thus our data do not substantiate a role for CSC-MS in modulating macrophage responsiveness to IFNg outside of the lung, although selective absorption of large quantities of some immunomodulatory components of CSC-MS remains a possibility. While these studies ideally would be performed by exposing healthy human individuals to cigarette smoke and then examining their lung macrophages, our model system does offer some conveniences not available with the ideal system. We were able to isolate defined populations of macrophages from healthy and syngeneic mice in sufficient quantities to determine their responses to individual stimuli. We were also able to determine the effects of toxicants on macrophage responses to stimuli and to provide information on dose responses.
CSC-MS may exert their effects on macrophage responses to IFNg at many points over the stimulus–response process: signal transduction, transcription initiation, mRNA synthesis and processing, translation and posttranslational peptide processing, transport, and secretion. Our results showed that macrophage basal metabolism and production of TNFa in response to LPS was not disrupted by CSC-MS, but enhanced, suggesting that general processes such as RNA or protein synthesis remained intact and highly functional after exposure to CSCMS. Therefore, CSC-MS likely affect processes that permit some level of selectivity, such as signal transcription or transcription initiation, both early events. Results from this study suggest that low levels of CSC-MS can suppress macrophage responsiveness to interferon g. While alterations in responsiveness to cytokines may have both beneficial and deleterious consequences in general, we believe that in this particular case, the consequences are not beneficial. We have not yet identified all functional capacities that macrophages acquire in response to interferon g. However, decreased capacities to phagocytose opsonized antigens, to produce reactive oxygen and nitrogen intermediates to kill phagocytosed organisms, and to present processed antigen to T-helper lymphocytes via surface class II MHC molecules cannot be conducive to effective host defense against infection. ACKNOWLEDGMENTS This work was supported by grants from the American Heart Association, Illinois Affiliate (GB/CGB-01) and the National Science Foundation (DUE9650507).
REFERENCES Adams, D. O., and Hamilton, T.A. (1988). Phagocytic cells: Cytotoxic activities of macrophages. In Inflammation: Basic Principles and Clinical Correlates (J. I. Gallin, I. M. Goldstein, and R. Snyderman, Eds.), pp. 471– 492. Raven Press, New York. Brain, J. D. (1986). Toxicological aspects of alterations of pulmonary macrophage function. Annu. Rev. Pharmacol. Toxicol. 26, 547–565. Brown, G. P., Iwamoto, G. K., Monick, M. M., and Hunninghake, G. W. (1989). Cigarette smoking decreases interleukin 1 release by human alveolar macrophages. Am. J. Physiol. 256, C260-C264. Collart, M. A., Belin, D., deCossodo, S., and Vassalli, P. (1986). g-Interferon enhances macrophage transcription of tumor necrosis factor/cachectin, interleukin-1, and urokinase genes which are controlled by short-lived repressors. J. Exp. Med. 164, 2113–2118. Davis, W. B., Pacht, E. R., Spatafora, M., and Martin, W. J., II. (1988). Enhanced cytotoxic potential of alveolar macrophages from cigarette smokers. J. Lab. Clin. Med. 111, 293–298. Drapier, J. C., and Hibbs, J. B. (1988). Interferon-g and tumor necrosis factor induce the l-arginine-dependent cytotoxic effector mechanism in murine macrophages. Eur. J. Immunol. 18, 1587–1592. Dowdy, S., and Wearden, S. (1983). Statistics for Research. Wiley, New York. Dubar, V., Gosset, P., Aerts, C., Voisin, C., Wallaert, B., and Tonnel, A. B. (1993). In vitro acute effects of tobacco smoke on tumor necrosis factor-
SMOKE CONDENSATES SUPPRESS MACROPHAGE RESPONSES alpha and interleukin 6 production by alveolar macrophages. Exp. Lung Res. 19, 345–359. Evans, R. (1986). The immunological network at the site of tumor rejection. Biochim. Biophys. Acta 865, 1–11. Fan, S., Fehr, H.-G., and Adams, D. O. (1991). Activation of macrophages for ADCC in vitro: Effects of IL-4, TNF, interferons-ab, interferon-g, and GM-CSF. Cell. Immunol. 135, 78 – 87. Grimmer, G., Naujack, K. W., Dettbarn, G. (1987). Gas chromatographic determination of polycyclic aromatic hydrocarbons, aza-arenes, aromatic amines in the particle and vapor phase of mainstream and sidestream smoke of cigarettes. Toxicol. Lett. 35, 117–124. Higashimoto, Y., Shimada, Y., Fukuchi, Y., Ishida, K., Shu, C., Teramoto, S., Sudo, E., Matsuse, T., and Orimo, H. (1992). Inhibition of mouse alveolar macrophage production of tumor necrosis factor alpha by acute in vivo and in vitro exposure to tobacco smoke. Respiration 59, 77-80. Hoffmann, D., and Wynder, E. L. (1971). A study of tobacco carcinogenesis. XI. Tumor initiators, tumor accelerators, and tumor promoting activities of condensate fractions. Cancer 27, 848 – 864. International Agency for Research on Cancer. (1976). Coumarin. IARC Monogr. 10, 113–119. Koide, S., and Steinman, R. M. (1987). Induction of murine interleukin-1. Stimuli and responsive primary cells. Proc. Natl. Acad. Sci. USA 84, 3802–3806. Lehnert, B. E. (1992). Pulmonary and thoracic macrophage subpopulations and clearance of particles from the lung. Environ. Health Perspect. 97, 17– 46. Lepay, D. A., Steinman, R. M., Nathan, C. F., Murray, H. W., and Cohn, Z. A. (1985). Liver macrophages in murine listeriosis: Cell-mediated immunity is correlated with an influx of macrophages capable of generating reactive oxygen intermediates. J. Exp. Med. 161, 1503–1512. Marino, P. A., and Adams, D. O. (1980). Interaction of bacillus CalmetteGuerin-activated macrophages and neoplastic cells in vitro. I. Conditions of binding and its selectivity. Cell. Immunol. 54, 11–25. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63. Ortega, E., Hueso, F., Collazos, M. E., Pedrera, M. I., Barriga, C., and
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Rodriguez, A. B. (1992). Phagocytosis of latex beads by alveolar macrophages from mice exposed to cigarette smoke. Comp. Immunol. Microbiol. Infect. Dis. 15, 137-142. Ozato, K., and Sachs, D. H. (1981). Monoclonal antibodies to mouse major histocompatibility complex antigens: 3. Hybridoma antibodies reacting to antigens of the H-2b haplotype reveal genetic control of isotype expression. J. Immunol. 126, 317-321. Pick, E., and Mizel, D. (1981). Rapid microassays for measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J. Immunol. Methods 46, 211–226. Sauty, A., Mauel, J., Philippeaux, M. M., and Leuenberger, P. (1994). Cytostatic activity of alveolar macrophages from smokers and nonsmokers: role of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha. Am. J. Respir. Cell Mol. Biol. 11, 631– 637. Sherman, M. P., Campbell, L. A., Gong, H., Roth, M. D., Tashkin, D. P. (1991). Antimicrobial and respiratory burst characteristics of pulmonary alveolar macrophages recovered from smokers of marijuana alone, smokers of tobacco alone, smokers of marijuana and tobacco, and nonsmokers. Am. Rev. Resp. Dis. 144, 1351–1356. Sko¨ld, C. M., Eklund, A., Hallde´n, G., and Hed, J. (1989). Autofluorescence in human alveolar macrophages from smokers: Relation to cell surface markers and phagocytosis. Exp. Lung Res. 15, 823– 835. Slater, T. F., Sawyer, B., and Strauli, U. (1963). Studies on succinate-tetrazolium reductase systems. III. Points of coupling of four different tetrazolium salts. Biochim. Biophys. Acta 77, 383–393. Soliman, D. M., and Twigg, H. L., III. (1992). Cigarette smoking decreases bioactive interleukin-6 secretion by alveolar macrophages. Am. J. Physiol. 263, L471–L478. Streck, R. J., Jezewski, H. M., Rodriguez, M. I., Hurley, E. L., Rich, G. A., Braun, K. M., and Pauly, J. L. (1994). A method for isolating human lung macrophages and observations of fluorescent phagocytes from the lungs of habitual cigarette smokers. J. Immunol. Methods 174, 67– 82. Unanue, E., and Askonas, B. (1968) The immune response of mice to antigen in macrophages. Immunology 15, 287–296. U.S. Surgeon General. (1986). The health consequences of involuntary smoking. DHHS (CDC) 87-8398. Washington, DC.