EFFECTS OF NO2 ON THE RESPONSE OF BABOON ALVEOLAR MACROPHAGES TO MIGRATION INHIBITORY FACTOR Nathan D. Greene, Sandra L. Schneider Immunology Department, Southwest Foundation for Research and Education, San Antonio, Texas

Pulmonary alveolar macrophages (PAM) were obtained by lavage from baboons exposed for 6 mo to 2 ppm /VO2 for 8 h/d, 5 d/wk, and the response of these cells to autologous migration inhibitory factor (MIF) was determined. PAM from two of three antigen-sensitized, NO2-exposed animals failed to respond to MIF derived from antigen-stimulated autologous lymphocytes. Similarly, PAM from three of the four NO2-exposed animals had diminished responsiveness to MIF obtained by phytohemagglutinin stimulation of their own lymphocytes. The altered responsiveness resulted from an effect on the macrophages and not on the lymphocytes used to prepare the MIF, as shown by the normal blastogenic responsiveness of the lymphocytes and the normal activity of the MIF thus produced on guinea pig peritoneal macrophages. These results demonstrate that inhalation of 2 ppm NO2 may have important subtle effects on pulmonary cells, which may result in altered immune capabilities within the lung.

INTRODUCTION Inhalation of air contaminated with various concentrations of NO2 has been shown to result in altered susceptibility to both bacterial and viral infectious agents (Ehrlich and Henry, 1968; Henry et al., 1969; Fenters et al., 1973; Goldstein et al., 1973, 1974). The findings of altered serological responses in mice (Ehrlich et al., 1975) and squirrel monkeys after viral challenge after NO2 exposure (Fenters et al., 1971) suggest that this decreased resistance to infection is related to alterations in functional capabilities of immunologically relevant cells in the lung or to systemic alterations. Further indications of such an effect are seen in the report of Valand et al. (1970), in which NO2 inhalation inhibited interferon production by rabbit alveolar macrophages in response to in vivo challenge with parainfluenza-3 virus. Resistance to subsequent in vitro challenge with rabbit pox virus was shown to be lost in rabbits that had previously been exposed to air containing 25 ppm NO2 for 3 h. We thank Dennis Mynarcik and Jorge Padilla for expert technical assistance. This work was supported by National Institutes of Health grant ES-01153. Requests for reprints should be sent to Nathan D. Greene, Southwest Foundation for Research and Education, San Antonio, Texas 78284.

869 Journal of Toxicology and Environmental Health, 4:869-880,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/040869-12$2.25

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Interference with functional capabilities of alveolar macrophages resulting from inhalation of other air pollutants has been reported. Of particular relevance to this report are the findings of Warr and Martin (1973, 1974) and Martin and Laughter (1976) that alveolar macrophages obtained by lavage from cigarette smokers have enhanced responsiveness to a chemotactic stimulus, impaired responsiveness to macrophage migration inhibitory factor (MIF), and impaired capability to substitute for blood monocytes in the mediation of mixed lymphocyte reactions. This indicates that inhalation of certain pollutants may interfere with functional capabilities of cells that are important in the mediation of local immunologie responses in the lung. In the studies reported here, the ability of alveolar macrophages from baboons exposed intermittently for 6 mo to 2 ppm NO2 to respond to MIF derived from specific antigen and mitogen stimulation of autologous lymphocytes was examined. METHODS

Animals and Sensitization Baboons {Papio cynocephalus) of both sexes, ranging in age from 3 to 4 yr, were used. The animals were obtained from commercial sources and were quarantined for 4 wk before the initiation of the study, during which time they were shown to be free of infectious or other diseases. Four of the six baboons used in the study were sensitized by infection with the human blood parasite Schistosoma mansoni, by using procedures commonly employed in our laboratory for this agent and known to result in a high degree of immediate and delayed hypersensitivity and circulating antibodies with specificity for antigens derived from the parasite (Damián et al., 1972, 1974; Greene and Damián, 1972; Greene, 1974). The animals, housed in individual squeeze cages to facilitate periodic handling and collection of blood samples, were subsequently placed in exposure chambers. Inhalation Exposures Four baboons, three sensitized and one control, housed in one chamber, were exposed to 2 ppm NO 2 . The exposure schedule consisted of 8 h inhalation of NO2 followed by 16 h of clean, filtered air for 5 d/wk. Two animals, one sensitized and one control, were housed in identical chambers in squeeze cages and breathed only filtered air. Exposures were monitored continuously when NO2 was present in the chamber, using a NO2-NOX monitor. The actual concentration of NO2 inside the chamber during a typical exposure period, as determined by comparison with a permeation tube and by the wet chemistry method of Saltzman, was 2.01 ± 0.04 ppm. The

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animals were exposed for 6 mo, after which they were sacrificed and necropsies were performed for immunologie and histopathologic examination. Lymphocyte Isolation and Culture The animals were sedated with ketamine im (10 mg/kg), and 10-20 ml blood was taken from the femoral vein and collected in sterile evacuated tubes containing 10 units heparin (preservative-free) per milliliter blood. Plasma was removed from the heparinized blood by centrifugation at 265 Xg for 20 min at 22°C and replaced with a volume of sterile phosphate-buffered saline (PBS), pH 7.2, sufficient to yield a 1:2 dilution of the original blood volume. The thoroughly mixed diluted blood was layered onto a 15-ml cushion of Ficoll-Hypaque, specific gravity 1.007-1.079 (Pharmacia, Piscataway, N.J.), in a 50-ml conical centrifuge tube. After centrifugation (265 Xg, 30 min, 22°C) the cells at the interface were removed with a sterile Pasteur pipette and washed three times with PBS by centrifugaron (385 Xg, 10 min, 22°C). The washed cells were resuspended in RPMI 1640 (Grand Island Biological Co., Grand Island, N.Y.) containing 25 m/W HEPES buffer, 2 mg/ml sodium bicarbonate, 10% heat-inactivated fetal calf serum, and supplemented with 200 m/W L-glutamine and penicillin and streptomycin, hereafter referred to as a complete medium, following the method of Greene et al. (1977) as modified from Thurman et al. (1973). A sample of cells was removed for viability assessment as determined by the ability to exclude 0.2% Erythrocin B, and for differential counting using Turks solution. The final cell concentration was adjusted to 3 X 10s viable lymphocytes per milliliter contained in complete medium. To elicit release of macrophage Ml F from sensitized lymphocytes, 1-ml portions of the stock cell suspension containing 3 X 105 cells were dispensed into 13 X 75 mm culture tubes. To each tube was added selected amounts of antigen (preparation described below). In addition, lymphocytes from normal and sensitized animals were similarly stimulated with the mitogen phytohemagglutinin (PHA-P; Difco, Detroit, Mich.), at the optimal concentration of 8 jug/ml, as previously determined (unpublished). Controls consisted of cells treated identically, but without the addition of antigen or mitogen. The tubes were lightly capped to facilitate gas exchange, and incubated for 48 h (37°C, 5% CO 2 , 100% humidity). The culture supernatants were then harvested by centrifugation (265 Xg, 10 min), supplemented with 10% complete medium, and assayed for MIF activity. Antigenic Stimulation of Lymphocytes The antigen used for stimulation of mediator release from sensitized lymphocytes was a PBS extract of the infecting organism prepared as described previously (Greene and Damián, 1972). Briefly, 200-300 adult

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parasites were homogenized in 2 ml PBS in an ice bath and extracted overnight at 4°C, after which the sample was centrifuged at 10,000 Xg for 30 min at 4°C. The resulting supernatant was sterilized by filtration, divided into 0.1-ml volumes, and stored at — 70°C until used. Cultures were stimulated with 2 concentrations of antigen: 30 and 50 y.g protein per milliliter of culture containing 3 X 105 lymphocytes. Mitogenic Stimulation of Lymphocytes with Phytohemagglutinin (PHA) The ability of peripheral lymphocytes from normal and NO2-exposed animals to respond to mitogenic stimulation with PHA was determined by using procedures that we have found to be optimal for baboon lymphocytes (Greene et al., 1977). Lymphocytes were adjusted to a concentration of 10 6 /ml in medium RPMI 1640 containing 10% fetal calf serum and supplemented as above, and dispensed in 0.2-ml portions containing 2 X 10s cells into flat-bottomed microtissue culture plates (Linbro, Hamden, Conn.). To triplicate cultures was added 25 jig PHA-P in 0.05 ml medium to obtain a total incubation volume of 0.25 ml. Control cultures received an equal volume of complete medium. After 96 h incubation (37°C, humidified atmosphere of 5% CO2) each culture received 1 juCi [3 H] thymidine (Schwarz-Mann, Orangeburg, N.Y.; specific activity 1.9 Ci/m/W). Cultures were harvested after an additional 18 h incubation with a sem¡automated multiple-sample harvester (Brandel, Rockville, Md). The cells were deposited onto glass fiber filters and rinsed with PBS; the macromolecular constituents were precipitated with cold 10% trichloroacetic acid; and the precipitates were again rinsed with PBS. The precipitates were solubilized with Soluene (Packard, Downers Grove, III.), and the incorporated radioactivity was determined by scintillation counting. Recovery and Purification of Alveolar Macrophages Pulmonary alveolar macrophages (PAM) were recovered from the lungs of all experimental and control baboons by lavage. At necropsy, one lung lobe was removed aseptically and washed free of externally contaminating blood with sterile physiological saline solution (PSS), after which the lobe was filled with PSS at 37°C and placed in a water bath containing PSS at 37°C. After 15 min incubation the lavage fluid was removed by gravity into sterile centrifuge tubes. The lavage procedure was then repeated an additional 6 times, omitting the 15-min incubation period. The lavage fluids were pooled and centrifuged (265 X g , 4°C, 10 min) to sediment the cells. The cell pellet was resuspended in PBS and washed by centrifugaron an additional two times. The washed cells were resuspended in Eagle's minimal essential medium (MEM) containing 10% fetal calf serum, dispensed into 75-cm2 plastic tissue culture flasks (Corning, Corning, N.Y.), and allowed to attach at 37°C for 1 h. Nonadherent cells were then removed by gently agitating the flasks and removing the suspending

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medium by aspiration. The adherent cells were washed an additional two times with MEM to remove additional nonadherent cells, and detached into MEM by placing the flask on wet ice and scraping with a rubber policeman. Lung lavage cells prepared in this manner have been found to be an essentially pure population of alveolar macrophages, with viability, as judged by dye exclusion, to be near 100%. Guinea Pig Peritoneal Exúdate Cells For induction of a peritoneal exúdate, young adult random-bred Hartley guinea pigs (weighing 300-450 g) were injected ip with 15 ml sterile mineral oil. Three days later the exudates were harvested through a ventral incision in the abdominal wall, and the recovered cells were washed three times with PBS. Macrophage Migration Inhibition Assay The agarose droplet method of Harrington and Stastny (1973) was used. Indicator cells, either baboon alveolar macrophages or guinea pig peritoneal exúdate cells, were suspended in an equal volume of agarose solution consisting of 0.4% Seakim agarose (MCI Biomédical, Rockland, Maine) in 2 volumes of tissue culture medium 199 (Gibco, Grand Island, N.Y.). While the temperature of the cell-agarose mixture was maintained at 37°C, 2 /xl of suspension was dispensed onto the bottom of flat-bottomed 6-mm microtissue culture plates (Linbro, Hamden, Conn.), which had been precoated with 1 jul 0.8% agarose and allowed to dry. The resulting droplets were chilled at 4°C for 5 min, and 0.1 ml of the supernatant from control or antigen-stimulated lymphocyte cultures was carefully added to the wells containing the droplets. Each supernatant was assayed in quadruplicate. The plates were incubated for 24 h at 37°C in a humidified atmosphere of 5% CO 2 . The distance of migration from the margin of the droplet was measured in arbitrary units with the aid of an inverted phase-contrast microscope equipped with an ocular grid. Percentage inhibition was calculated from the mean of the distance migrated in the presence of MIF-containing supernatants compared with control nonstimulated supernatants. In this assay inhibition of migration by 20% or more is usually considered to indicate the presence of Ml F in the medium (Harrington and Stastny, 1973; Harrington, 1974; McCoy et al., 1977). RESULTS The PAM from the normal, nonsensitized baboon breathing clean air as well as the cells from the nonsensitized NO2-exposed animal were indifferent to the presence of supernatant media from their autologous lymphocytes cultured in the presence of the sensitizing antigen. Their migratory activity was not different from those cultures containing supernatant media from nonstimulated lymphocyte cultures (Fig. 1). The

N. D. GREENE AND S. L. SCHNEIDER

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FIGURE 1. Migration of pulmonary alveolar macrophages (PAM) from sensitized (hatched bars) and normal baboons exposed to clean air (*) or 2 ppm NO 2 , in response to supernatants from autolögous lymphocytes incubated with the sensitizing antigen.

response of PAM from the sensitized, clean air control baboon was as expected in that migration was inhibited. However, cells from two of the three sensitized NO2-exposed animals did not respond to the presence of the MIF-containing supernatant media with the expected migration inhibition. On the contrary, migration of the PAM from these two animals was somewhat greater than that of the control supernatants from nonantigen-stimulated lymphocyte cultures. To determine whether the observed loss of Ml F responsiveness of PAM from some NO2-exposed animals was caused by an alteration in the macrophages themselves or a loss of the ability of the animals' lymphocytes to produce or release the lymphokine, we examined the responsiveness of heterologous macrophages to the same lymphocyte supernatant media. The response of guinea pig peritoneal exúdate cells is shown in Fig. 2. With the exception of one animal, the lymphocytes were capable of releasing Ml F on stimulation with the sensitizing antigen, and the lymphokine thus released was capable of inhibiting the migration of heterologous macrophages. As additional confirmation that the baboons' lymphocytes were capable of producing and releasing MIF, the cells were stimulated with the nonspecific mitogen PHA, an agent known to induce blastogenesis in baboon lymphocytes (Greene et al., 1977). It also has been shown to stimulate the production and/or release of MIF from human lymphocytes (Papageorgiou et al., 1974; Lomnitzer et al., 1975). When alveolar macrophages from the animals that had breathed only clean air were assayed with the PHA-stimutated lymphocyte culture supernatant media, inhibition of migration was observed as expected (Fig. 3). Inhibition, but to a considerably less extent, was also observed with the PAM from three

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FIGURE 2. Migration of guinea pig peritoneal exúdate cells in response to supernatants from baboon lymphocyte cultures. Lymphocytes were obtained from sensitized (hatched bars) or normal baboons exposed to air (*) or 2 ppm NO 2 , and were incubated in the presence of the sensitizing antigen.

of the four NO2-exposed animals, there being no apparent difference between the nonsensitized and the sensitized animals. This apparently reduced responsiveness of PAM from the NO2-exposed animals further supports the concept that such exposure may interfere with the normal reactivity of the cells to the lymphocyte product. The same supernatant media from the PHA-stimulated lymphocytes were assayed for Ml F activity on guinea pig peritoneal exúdate cells (Fig. 4). It is clear that MIF was produced and released from all the animals, with no apparent differences between normal and NO2-exposed animals. Further confirmation of the normal reactivity of the lymphocytes from

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FIGURE 3. Migration of PAM from baboons in response to supernatants from autologous lymphocytes incubated with phytohemagglutinin (PHA). Other conditions as in Fig. 1.

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FIGURE 4. Migration of guinea pig peritoneal exúdate cells in response to supernatants from PHA-stimulated baboon lymphocytes. Other conditions as in Fig. 2.

the two groups of animals is provided by the results of the lymphocyte blastogenesis experiment, in which the degree of mitogenic response following PHA stimulation was quantitated (Table 1). While it is evident that there is wide variability in blastogenic response among individual animals, as we have routinely seen with baboons (Greene et al., 1977), it is clear that the lymphocytes from all the animals were capable of responding to the mitogenic stimulus.

DISCUSSION We have shown that the alveolar macrophages of normal baboons respond to antigen- and mitogen (PHA)-induced M1F, and that this response is reduced or lost in some animals after intermittent exposure to NO 2 . Although the numbers of animals used in these studies were small and considerable individual variability was seen, the implications of TABLE 1. Blastogenic Response of Peripheral Lymphocytes Stimulated with Phytohemagglutinin

Animal Air control Air sensitized NO2 control NO2 sensitized, 1 NO2 sensitized, 2 NO2 sensitized, 3

Counts per minute"

14,511 56,230 18,434 16,000 99,487 17,723

Mean of triplicate cultures.

877

impaired immunologie function in the lung after inhalation of NO2 are sufficiently important to warrant further consideration. These implications of potential hazards to pulmonary defense processes must be considered in the context of current knowledge of the pulmonary immune system. Interactions between macrophages and other cells involved in immunologie defense processes occur at a variety of levels including direct cell-to-cell contact and through soluble mediators. In simplistic terms, macrophages are recruited into areas of infection or inflammation by means of chemotactic stimuli emanating from the infecting organism or arising from interactions of other cells, especially lymphocytes, with the inflammatory agent. This interaction may produce such chemotactic stimuli as antigen-antibody complexes and complement fragments, which are effective in recruiting phagocytic cells. Once present, the macrophages respond to inhibitory factors, particularly macrophage Ml F produced by sensitized lymphocytes in response to interaction with the sensitizing antigen. The MIFs thus assist in maintaining an effective phagocyte population in the localized area of infection or inflammation. The capacity of PAM to respond to MIF in a manner similar to that of other mononuclear phagocytic cells has been controversial. Leu et al. (1972) studied the responsiveness of guinea pig alveolar and peritoneal exúdate cells to homologous MIF and concluded that the cells from the lungs were unresponsive to the lymphokine. Moreover, on the basis of absorption experiments in which the peritoneal cells were demonstrated to possess MIF receptors capable of quantitatively removing MIF from solution, the alveolar cells were judged to lack such receptors. Others, however, have presented evidence to support the concept that PAM from the guinea pig and other species do not differ from macrophages from other body sites with respect to MIF responsiveness. Bartfeld and Atoynatan (1969) found that cells recovered from tracheo-alveolar washings of guinea pigs were inhibited by MIF of homologous origin. Later, Bartfeld and Antoynatan (1970) showed similar inhibition of human lung lavage cells by human MIF derived from purified protein derivative-stimulated lymphocytes from tuberculin-positive donors. Galindo and Myrvik (1970) studied the responsiveness of BCG-induced pulmonary granulomatous cells of rabbits and concluded that the migratory activity of the PAM of this species is inhibited by some factor produced by the interaction of the lavage cells, which included 5-15% lymphocytes, with purified protein derivative. In addition, autoinhibition in the absence of specific antigen was commonly seen with cells from animals with extensive granulomatous disease, suggesting the possible production of an inhibitory factor with properties similar to MIF by the interaction of activated macrophages with sensitized lymphocytes, or release of such a factor during the assay after in vivo stimulation of the cells. Other reports of the MIF responsiveness of PAM are available. Alveolar wash cells from rabbits immunized with a thermophilic actinomycete

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antigen were inhibited in the presence of the immunizing antigen, presumably through the production and/or release of MIF by lymphocytes also present in the lavage cell population (Kawai et al., 1973). Similarly, sensitization of rabbits with the same agent {Micropolyspora faeni) (Harris et al., 1976) or with a soluble protein antigen (BSA) (Salvaggio et al., 1975) by way of the respiratory tract caused lung lavage cells to become able to respond to antigen-induced MIF. Ml F responsiveness occurred to both sensitizing antigens, even in the absence of pulmonary lesions, as was the case with primary BSA sensitization. Secondary stimulation with BSA resulted in mild focal mononuclear infiltration with increasing sensitivity to the stimulating antigen, while primary sensitization with M. faeni led to marked progressive alveolar and interstitial infiltrates with heightened MIF responsiveness of the alveolar cells to the sensitizing agent. The studies of Warr and Martin (1973, 1974) and Martin and Laughter (1976) on the PAM of cigarette smokers and normal individuals confirm earlier observations that normal human PAM can respond to MIF and that this response is impaired in cigarette smokers. Further, the number of macrophages recoverable from the lungs of smokers was approximately four times greater than the number in normal lungs, and these cells displayed a marked increase in both random migratory and chemotactic activity. Also noted was a three-to-five fold increase in "small mononuclear cells," many of which were probably lymphocytes. The mechanism whereby these cells are recruited into the lung is not known, but the fact that such recruitment occurs in combination with altered functional capacities of other ¡mmunologically important cells suggests that inhaled pollutants may have a significant impact on local immune responses in the lung. The cell population of the lung after inhalation of tobacco smoke is activated, that is, the metabolic rate is higher than in cells from nonsmokers (Harris et al., 1970) and the cells are more actively motile in culture (Warr and Martin, 1973). The phagocytic stimulus presented by smoke particulates may be responsible for the increased activation of the PAM and subsequent loss of responsiveness of the cells to MIF. Such a result must suggest the existence of a balanced steady-state relationship between stimulatory and inhibitory influences in which the action of either can predominate if the stimulus is of sufficient strength. However, our finding that NO2 alone may cause a loss of MIF responsiveness suggests that mechanisms other than generalized activation of the cells may be responsible for decreased responsiveness to the lymphokine. In further studies designed to confirm these initial findings, and to examine other aspects of possible impairment of immunologie function in the lung by inhaled pollutants, we are using guinea pigs as experimental models. In addition, we are performing in situ lavage of baboons to permit repeated sampling of normal and exposed animals. This will allow compilation of data on the time-course of changes in alveolar macrophage responsiveness seen in these preliminary investigations, will provide an

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increased sample size, and may reduce the individual animal variability observed here. REFERENCES Bartfeld, H. and Atoynatan, T. 1969. Cytophilic nature of migration inhibitory factor associated with delayed hypersensitivity. Proc. Soc. Exp. Biol. Med. 130:497-501. Bartfeld, H. and Atoynatan, T. 1970. Cellular immunity. Activity and properties of human migration inhibitory factor. Int. Arch. Allergy Appl. Immunol. 38:549-553. Damian, R. T., Greene, N. D., and Fitzgerald, K. 1972. Schistosomiasis mansoni in baboons. The effect of surgical transfer of adult Schistosoma mansoni upon subsequent challenge infection. Am. J. Trop. Med. Hyg. 21:951-958. Damian, R. T., Greene, N. D., and Fitzgerald, K. 1974. Schistosomiasis mansoni in baboons. 11. Acquisition of immunity to challenge infection after repeated small exposures to cercariae of Schistosoma mansoni. Am. J. Trop. Med. Hyg. 23:78-80. Ehrlich, R. and Henry, M. C. 1968. Chronic toxicity of nitrogen dioxide. I. Effect on resistance to bacterial pneumonia. Arch. Environ. Health 17:860-865. Ehrlich, R., Silverstein, E., Maigetter, R., and Fenters, j . D. 1975. Immunologic response in vaccinated mice during long-term exposure to nitrogen dioxide. Environ. Res. 10:217-223. Fenters, J. D., Ehrlich, R., Findlay, J., Spangler, J., and Tolkacz, V. 1971. Serologic response in squirrel monkeys exposed to nitrogen dioxide and influenza virus. Am. Rev. Respir. Dis. 104:448-451. Fenters, J. D., Findlay, J. C., Port, C. D., Ehrlich, R., and Coffin, D. L. 1973. Chronic exposure to nitrogen dioxide. Immunologie, physiologic, and pathologic effects in virus-challenged squirrel monkeys. Arch. Environ. Health 27:85-89. Galindo, B. and Myrvik, Q. N. 1970. Migratory response of granulomatous alveolar cells from BCG-sensitized rabbits. J. Immunol. 105:227-237. Goldstein, E., Eagle, M. D., and Hoeprich, P. D. 1973. Effect of nitrogen dioxide on pulmonary bacterial defense mechanisms. Arch. Environ. Health 26:202-204. Goldstein, E., Warshauer, D., Lippert, W., and Tarkington, B. 1974. Ozone and nitrogen dioxide exposure. Murine pulmonary defense mechanisms. Arch. Environ. Health 28:85-90. Greene, N. D. 1974. Schistosoma mansoni: IgG and IgE skin-sensitizing antibodies in infected baboons. Exp. Parasitol. 35:219-224. Greene, N. D. and Damian, R. T. 1972. Skin-sensitizing antibodies in Schistosoma mansoni infected baboons: Evidence for the presence of two types. Proc. Soc. Exp. Biol. Med. 141:291-294. Greene, N. D., Schneider, S. L., and Meltz, M. L. 1977. Response of baboon peripheral blood lymphocytes to phytohemagglutinin: Time course and dose-response relationships. J. Med. Primatol. 6:298-308. Harrington, J. T., Jr. 1974. Macrophage migration from an agarose droplet: A micromethod for assay of delayed hypersensitivity in the mouse. Cell Immunol. 12:476-480. Harrington, J. T., Jr. and Stastny, P. 1973. Macrophage migration from an agarose droplet: Development of a micromethod for assay of delayed hypersensitivity. J. Immunol, 110:752-759. Harris, J. O., Swenson, E. W., and Johnson, J. E., III. 1970. Human alveolar macrophages: Comparison of phagocytic ability, glucose utilization, and ultrastructure in smokers and nonsmokers. J. Clin. Invest. 49:2086-2096. Harris, J. O., Bice, D., and Salvaggio, J. E. 1976. Cellular and humoral bronchopulmonary immune response of rabbits immunized with thermophilic actinomyces antigen. Am. Rev. Respir. Dis. 114:29-43. Henry, M. C., Ehrlich, R., and Blair, W. H. 1969. Effect of nitrogen dioxide on resistance of squirrel monkeys to Klebsiella pneumonlae infection. Arch. Environ. Health 18:580-587. Kawai, T., Salvaggio, J., Harris, J. O., and Arquembourg, P. 1973. Alveolar macrophage migration inhibition in animals immunized with thermophilic actinomycete antigen. Clin. Exp. Immunol. 15:123-130.

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Leu, R. W., Eddleston, A. L. W. F., Hadden, J. W., and Good, R. A. 1972. Mechanism of action of migration inhibitory factor (M1F). I. Evidence for a receptor for MIF present on the peritoneal macrophage but not on the alveolar macrophage. J. Exp. Med. 136:589-603. Lomnitzer, R., Rabson, A. R., and Koornhof, H. J. 1975. Production of leucocyte inhibitory factor (LIF) and macrophage inhibitory factor (MIF) by PHA-stimulated lymphocytes. Clin. Exp. Immunol. 22:522-527. McCoy, J. L., Dean, J. H., and Herberman, R. B. 1977. Human cell-mediated immunity to tuberculin as assayed by the agarose micro-droplet leukocyte migration inhibition technique: Comparison with the capillary tube assay. J. Immunol. Methods 15:355-371. Martin, R. R. and Laughter, A. H. 1976. Pulmonary alveolar macrophages can mediate immune responses: Cigarette smoking impairs these functions. Fed. Proc. 35:716. Papageorgiou, P. S., Sorokin, C. F., and Glade, P. R. 1974. Similarity of migration inhibitory factor(s) produced by human lymphoid cell line and phytohemagglutinin and tuberculinstimulated human peripheral lymphocytes. J. Immunol. 112:675-682. Salvaggio, J., Phanuphak, P., Stanford, R., Bice, D., and Claman, H. 1975. Experimental production of granulomatous pneumonitis. Comparison of immunological and morphological sequelae with particulate and soluble antigens administered via the respiratory route. J. Allergy Clin. Immunol. 56:364-380. Thurman, G. B., Strong, D. M., Ahmed, A., Green, S. S., Sell, K. W., Hartzman, R. J., and Bach, F. H. 1973. Human mixed lymphocyte cultures. Evaluation of a microculture technique utilizing the multiple automated sample harvester (MASH). Clin. Exp. Immunol. 15:289-302. Valand, S. B., Acton, J. D., and Myrvik, Q. N. 1970. Nitrogen dioxide inhibition of viral-induced resistance in alveolar monocytes. Arch. Environ. Health. 20:303-309. Warr, G. A. and Martin, R. R. 1973. In vitro migration of human alveolar macrophages: Effects of cigarette smoking. Infect. Immun. 8:222-227. Warr, G. A. and Martin, R. R. 1974. Chemotactic responsiveness of human alveolar macrophages: Effects of cigarette smoking. Infect. Immun. 9:769-771. Received October 14, 1977 Accepted November 23, 1977

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Effects of NO2 on the response of baboon alveolar macrophages to migration inhibitory factor.

EFFECTS OF NO2 ON THE RESPONSE OF BABOON ALVEOLAR MACROPHAGES TO MIGRATION INHIBITORY FACTOR Nathan D. Greene, Sandra L. Schneider Immunology Departme...
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