TOXICOLOCiY

AND

APPLIED

PHARMACOLOGY

Modulation

SUSANNE

15 ( 199 1)

110,403-4

of Human Alveolar Macrophage by Ozone Exposure in Vitro’

BECKER,* MICHAEL C. MADDEN,? ROBERT B. DEVLIN,~ AND HILLEL

Properties

SIMON L. NEWMAN,$ S. KOREN~

*ABB Environmental Services, Inc., 6320 Quadrangle Drive, Chapel Hill, North Carolina 27514; tcenterfor Environmental Medicine, University of North Carolina, Chapel Hill, North Carolina 27599; #Department of Internal Medicine, Division of Infectious Diseases, University of Cincinnati Medical Center, 231 Bethesda Avenue, Cincinnati, Ohio 4.5267; and §U.S. Environmental Protection Agency, Health Eflects Research Laboratory, Research Triangle Park, North Carolina 2771 I

Modulation of Human Alveolar Macrophage Properties by Ozone Exposure in Vitro. BECKER, M. C., NEWMAN, S. L., DEVLIN, R. B., AND KOREN, H. S. (1991). Toxicol. Appl. Pharmacol. 110,403-4 15. We have investigated changes in human alveolar macrophage (HAM) function after exposure in vitro to ozone (0,) (O.l- 1.O ppm for 2-4 hr). The functions studied reflect concern that Or is detrimental to host defense mechanisms in the bronchoalveolar spaces. Exposure of HAM to O3 caused a concentration-dependent increase in release of prostaglandin E2 (PGE,), an important modulator of inflammation, phagocytosis, and oxidative burst. Although phagocytosis of particulate immune complexes was decreased by OX, we found no change in the quantity of Fc receptors and complement receptors on the HAM surface. Superoxide (0;) production in response to phorbol ester was reduced atIer exposure of HAM to O3 while the basal 0; release in response to plastic adherence was not affected.Growth inhibition of the opportunistic yeast Cryptococcus neoformans by HAM was not affected by 0, exposure. The production of inflammatory mediators and immune modulators such as tumor necrosis factor-a, interleukin 1, and interleukin 6 were not induced by exposure to 03. However, compared to controls, Ojexposed HAM produced significantly lower levels of these cytokines when stimulated with bacterial lipopolysaccharide (LPS). Two-dimensional gel electrophoretic analysis of proteins made by HAM following in vitro exposure to O9 identified 11 proteins whose rate of synthesis was significantly altered. Thus, these studies show that exposure to O9 alters the functional competence of HAM. While there is a minimal effect on protein expression or synthesis, the responses of HAM to particulate immune complexes, to bacterial LPS, and to PMA are impaired. The release of arachidonic acid and PGEl suggest that the effect of O3 is primarily targeted to the HAM cell membrane. These changes may ultimately result in increased susceptibility to inhaled infectious agents in the Or-exposed individual. 0 1991 Academic PISS. IX S., MADDEN,

Exposure of experimental animals to ozone (0,) followed by infection of the animals with bacteria (Ehrlich et al., 1979; Ehrlich, 1980; Illing et al., 1980; Gardner, 1982, 1984; Goldstein et al., 1970, 1974) has indicated that de’ The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. 403

fense mechanisms against microorganisms are impaired by the pollutant. The inhibition of bacterial clearance could be on the level of mucociliary function (Rombout et al., 1982) or on immune cell-mediated defenses (EPA, 1986). With the exception of a study by Van Loveren et al. ( 1988) who found O3 effects on macrophage and T cell-mediated host defense mechanisms in Listeriu monocytogenes-infected rats, increased susceptibility to infection has been monitored using mortality or bacterial burden as endpoints. In separate exper0041008x/91

$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

404

BECKER ET AL.

iments alveolar macrophages from O+xposed imal macrophages, are impaired by O3 expouninfected mice, rats, and rabbits have been sure and that these defects may translate into tested for phagocytic activity (Driscoll et al., less efficient elimination of pathogens from the 1986a, 1987; Prasad et al., 1988; Wenzel and lung. Morgan, 1983), release of lysosomal enzymes (Hurst and Coffin, 197 1), lysozyme levels (KiMATERIALS AND METHODS mura and Goldstein, 198 1; Sherwood et al., 1986) production of reactive oxygen interBronchoalveolar Lavage (BAL) and Isolation of HAM mediates (Amuruso et al., 198 1; Ryer-Powder et al., 1988), interferon production (Ibrahim BAL was performed over a 1.5-year period on healthy et al., 1976; Shinghu et al., 1980), and release nonsmoking volunteers, age 25-35 years, as described of arachidonic acid metabolites (Driscoll et al., previously (Koren et al., 1989). Thirty-five individuals of 1986b, 1988; Madden et al., 1990). All these both sexes participated in the study. BAL samples were functions have been shown to be altered by kept on ice during the procedure and the cells then were pelleted by centrifugation at 4”C, 33Og. and washed twice O3 in a manner which would suggest that imwith RPM1 1640 containing 2 pg/ml gentamycin. Morpairment of macrophage antimicrobial activ- phological analysis of cytocentrifuge preparations stained ities may be a cause of increased sensitivity to with Leukostat (Fisher Scientific, Orangeburg, NY) indicated that 80-95% of the cells were macrophages, lo- 15% infection in Orexposed animals. were lymphocytes, and the remainder (~3%) was made Controlled chamber exposure studies with up of neutrophils, eosinophils, and epithelial cells. HAM human volunteers, meanwhile, have investiwere enriched by adherence to 24-well tissue culture plates gated effects of exposure to environmental (Costar, Cambridge, MA) at 3 X 10’ cells/well, or to 35levels of O3 in vivo by in vitro analysis of mm tissue culture grade petri dishes (Costar) at 5 X lo5 cells/dish. Nonadherent cells were removed after 60 min changes in function of cells involved in microbial defenses (Petersen et al., 1978, 1979; and the adherent cells were then cultured in RPM1 1640 (GIBCO, Grand Island, NY) containing 2% fetal bovine Orlando et al., 1988; Koren et al., 1989; Devlin serum (FBS, J. R. Scientific, Woodland, CA). et al., 1990). Minimal effects have been found on human alveolar macrophage (HAM) phagocytic function, lysosomal enzyme levels, Ozone Exposure and production of 0, (Koren et al., 1989; The tissue culture dishes or plates were placed in a rockDevlin et al., 1990). Lymphocytes from OXing tissue culture box (Bellco, Vineland, NJ) with conexposed individuals showed a significant de- trolled temperature and humidity placed in an incubator crease in their mitogen response (Petersen et kept at 37°C. The tissue culture box was equipped with al., 1978; Orlando et al., 1988). In parallel with $ in. tube fittings for entry and removal of humidified 5% CO*, air, and 0,. Oj was produced by pumping dry HEPAthe in vivo exposures, cells from unexposed individuals have been exposed to O3 in vitro filtered air past an ultraviolet (uv) lamp where it entered the exposure chamber. The 0, concentration in the culture and a decrease in immune mechanisms in- box was monitored with a Dashibi 1003 AH 0, analyzer volving T, B, and NK cells have been mea- connected to the lowest port and the concentration adsured (Becker et al., 1989; Harder et al., 1990; justed with a shield over the uv lamp. An identical box Becker et al., 1990). In the present study we receiving only filtered air (control exposure) was placed have investigated the effects of in vitro expo- in a separate but identical incubator. HAM were exposed to O3 in 0.2 ml of phosphate-buffsure to O3 on several HAM functions which ered saline (PBS)-glucose (PBS-G) in 24-well tissue culture are important in host defense. The functions wells or in 0.5 ml PBS-glucose in 35-mm dishes. This studied were selected to gain an understanding amount of PBS in the wells covered the cells and maintained >90% viability while maximizing the exposure to of OX effects on HAM-mediated antimicrobial activity under nontoxic conditions, as well as 0,. The HAM were exposed to Or for 2 hr, or for 4 hr if no effect of exposure to 1 ppm was seen at 2 hr. Viability to enable a comparison of results with human was similar for control and 03-exposed cells for at least 3 cells to those obtained from animal experi- days of culture after either exposure time. Thus any changes ments. The results suggest that HAM, like an- in HAM function can be attributed to effectsother than

MODULATION

OF HAM

PROPERTIES

loss of HAM viability. The cells were analyzed for the various functions immediately after exposure as described below. Prostaglandin Ez (PGE,) Production The PBS-G exposure medium was removed from control and O,-exposed cultures, centrifuged (SOOg, 10 min, at 4”C), and then stored at -70°C until analysis. PGEl levels in supernatants were analyzed by a commercial radioimmunoassay kit (NEN, Boston, MA; 1”‘-tracer) sensitive to 1 pg/ml. Binding and Phagocytosis of Antibody-Coated Erythrocytes (EA)

Sheep

EAs were prepared with sheep erythrocytes and rabbit anti-E IgG from Cordis (Miami, FL) according to a previously described procedure (Newman et al., 1985). Attachment of >3 EA/HAM (percentage rosettes), phagocytosis (percentage cells ingesting one or more EA), and the phagocytic index (number of EA ingested/l00 cells) were determined on 03-exposed and unexposed HAM as described previously (Newman et a/., 1985). Immunojlaorescence Control and O,-exposed HAM were removed from the culture wells by incubation in Versene buffer (GIBCO), followed by vigorous pipetting, which resulted in recovery of 85-90% of the cells. Viability of the recovered HAM was >80% by trypan-blue dye exclusion. HAM (2 X lo5 cells/tube) were incubated for 45 min at 4°C with the monoclonal antibodies mab 32.2 (anti FcRl) and mab IV3 (anti FcR2) (Anderson et al., 1986) kindly supplied by Dr. Fanger (Darthmouth Medical School, VT); and anti-Leull (antiFcR,,), anti-CR3, anti-CR I, and antiLeuM5 (antiCR4) (Beckton-Dickinson, Mountainview, CA). The cells were washed twice with PBS containing 5% human serum and then incubated with a second FITCconjugated rabbit anti-mouse lg (Becton Dickinson) for 30 min. The cells were washed, then fixed in 1% paraformaldehyde, and were stored at 4°C until analysis by flowcytometry (FACS Analyzer, Becton-Dickinson). Superoside Anion Production Macrophages were adhered and cultured in 1 ml RPM1 1640 containing 2% FBS for 2 hr before exposure. This design was based on the observation that in the process of adherence, the HAM “spontaneously” released 0, and did not respond when stimulated with phorbol-13-myrislate-12-acetate (PMA). After the HAM have been cultured

BY 0, EXPOSURE

405

for 2 hr and exposed to OJ for 2 hr the adherence-induced release had diminished and the HAM had gained responsivenessto PMA. (After culture for 16- 18 hr the cells are maximally responsive to PMA). Release of 0; was assayed as previously described by Pick and Keisari ( 198 1). Immediately after exposure to Ox or air, 1 ml of cytochrome c (3.6 mg/ml), in the presence or absence of PMA (100 t&ml), and in the presence or absence of superoxide dismutase (SOD) (300 U/ml), was added to the cells. 0; production was determined at 30, 60, and 90 min by measuring the OD550 of 0.1 ml of converted ferricytochrome c in an EIA spectrophotometer (Bio-Tee Instruments, Winooski, VT). To obtain the concentration of 0; produced, the OD550 for the test samples treated with SOD were subtracted from the OD550 values for test samples stimulated in the absence of SOD (AOD550). Then the following formula was used (Pick and Keisari, 198 1): PM 0:

=

AOD550 x 100 6.3

Growth Inhibition of Cvptococcus neoformans Intracellular and extracellular growth inhibition of C. neoformans by HAM were quantified as previously described (Weinberg et al., 1987). HAM were exposed to O3 or filtered air and removed from the exposure wells by treatment with Versene buffer and vigorous pipetting. These cells were then tested for extracellular growth inhibition at a HAM:C. neoformans (unopsonized) ratio of 5: 1 in polypropylene test tubes in the absence of serum. Intracellular growth inhibition was tested in the exposure plates at an effector:opsonized fungus ratio of 1:5. Inhibition of replication of the fungus was quantified with a Coulter counter by counting the number of fungal cells in tubes or in wells after lysis of the HAM by 0.5% deoxycholate. Colony forming units were determined on Sabouraud agar plates at the same time points.

Assaysfor Cytokine Production One milliliter of RPM1 + 2% FBS was added to wells containing the HAM immediately after exposure for 2 hr to 1.O ppm O3 or filtered air. Half of the cells were stimulated with 1 pg/ml LPS for 18 hr and then the cell supernatants were collected and were stored at -20°C until assayed. Immunoreactive interleukin- 1-o (IL- l-p) in the HAM supematants was determined by ELISA assay(Cistron, Pine Brook, NJ). Tumor necrosis factor-a (TNF-a) in the supematants was determined by a biological cytotoxicity assay using Wehi 164 cells treated with actinomycin-D (1 fig/ml) and varying dilutions of supematant as previously described (Becker et al., 1989). Units of TNF activity was determined from a standard curve obtained

406

BECKER

with human recombinant TNF-(U (kindly provided by Cetus Corp., Emeryville, CA). IL-6 was determined in a proliferation assaywith the IL-6-dependent 7YTD cell line, using the hexosaminidase assay for quantitation of cell number (Van Snick et al., 1987). Human recombinant IL-6 (Genzyme. Boston, MA) was used to construct the standard curve. CSF- 1 was determined by radioimmunoassay using ‘151labeled human recombinant CSF- 1 and a rabbit anti CSF1 antibody. The assaywas kindly performed by Cetus Corp. (Emeryville, CA). Two-Dimensional

Gel Electrophoresis

Cells were exposed to 1.O ppm O3 or to filtered air for 2 hr and then incubated for an additional 2 hr in methionine-free RPM1 containing 500 &i/ml [35S]methionine. The cells were washed three times in PBS and lysed in a 0.1 ml 0.3% SDS/S% mercaptoethanol. After a brief digestion with DNase and RNase, the lysate was collected and stored at -70°C until analysis. Two-dimensional gel electrophoresis and quantitation of individual protein spots were oerformed exactlv as described (Devlin and Koren.

ET AL.

shown in Fig. 1 an O3 concentrationdependent increase in PGE;! release was induced during the exposures to 0.1,0.3, and 1.O ppm. The PGE2 concentration after exposure of HAM to 0.3 and 1.Oppm O3 was 1.8-fold and 2.2-fold, respectively, that of the PGE2 concentration in control supernatants. After the initial incubation with O3 HAM were stimulated with Ca2+ ionophore A23 187 or RPM1 as a control for up to 4 hr. There was no further difference in the amount of PGE2 released by O3 and control-exposed cells either in the presence or absence of A23 187 (data not shown). Efect of Ozone on Fc-Receptor Function

HAM were exposed to 0.18, 0.5, and 1.0 ppm of O3 and to filtered air for 4 hr and im1990). mediately thereafter binding and ingestion of EA were quantified. Figure 2a shows the percentage rosette forming cells after each expoStatistical Analysis sure. There was no change in the percentage cells binding the immune complexes although Our hypothesis was that exposure of HAM to O3 in there appeared to be fewer EAs bound per vitro would affect specific functions of HAM that are reHAM (not shown). However, a concentrationquired for protection of the lung from infectious disease. dependent effect of O3 was found on the perThus, an increase in PGE2 production, and a decrease in

phagocytosis, 0; production, and secretion of inflammatory cytokines would impair the ability of HAM to initiate inflammatory responses and phagocytose and kill microorganisms. The experimental design was to divide each individual HAM samples into two parts, one which was exposed to air and one which was exposed to 03. Therefore, statistical analysis of the data was done by paired t test with the measurements of air and O3 from each subject making up each pair. For all variables except for Fcmediated functions, the LOG 10 transform was applied to the data before the paired t test was done.

300 83 25w 00 B m?5 8

150-

1 Laa

'W-

iz?

RESULTS

* *

50. 0 0.1

Eflect of Ozone on the Production HAM

of PGE, by

PGE2 produced by HAM during control and O3 exposures was determined in the exposure media which was collected immediately after a 2-hr exposure to O3 or air. As

0.3

Ozone concentration

1.0

(ppm)

FIG. 1. Effect of ozone on the production of PGEl PGEz levels were quantified in the exposure media (PBS-glucose) of control and O,-exposed HAM. A significant increase in PGEz was found after exposure of the cells for 2 hr to 0.3 ppm (p = 0.013) and to 1.0 ppm (p = 0.032). The bars represent the mean f SEM of 7 (0.1 ppm O&9 (0.3 ppm 0,) and 17 ( 1.O ppm 0,) experiments.

MODULATION

OF HAM

PROPERTIES

407

BY 0, EXPOSURE

‘00 b

Ozone concentration

Ozone

(ppm)

Ozone

concentration

concentration

(ppm)

(ppm)

FIG. 2. Effects of ozone on Fc-receptor function. Rosette formation, phagocytosis, and ingestion of EA were determined in HAM exposed for 4 hr to various concentrations of Ox. The percentage of cells forming rosettes (a) was the same in air- and Os-exposed HAM. The percentage of phagocytic cells (b) was significantly decreased at I.0 ppm (p = 0.009), and the phagocytic index (2~) was inhibited at 0.5 ppm (p = 0.003) and 1.0 ppm (p < 0.001). The bars represent the mean k SEM of three experiments performed in duplicate. In (a) all three experiments with cells exposed to 0.18 ppm 100% of the cells formed rosettes with a SEM of 0.

centage of cells phagocytozing EAs (Fig. 2b). There was a 30% reduction in phagocytic cells at 1 ppm and a 16% reduction at 0.5 ppm. The phagocytic index was the most sensitive measure of O3 effects on FcR-mediated function (Fig. 2c) with a 52% decrease at 1 ppm, and a 24% decrease at 0.5 ppm. No effect of OX was observed at 0.18 ppm. Eflect of Ozone on Fc- and Complement-Receptor Expression HAM were detached from the wells after a 4-hr exposure to 1 ppm O3 or air and analyzed for FcRl, FcR2, FcR,,, CRl, CR3, and CR4 expression by immunofluorescence and flowcytometry. No change in receptor expression

was found in the exposed cells in three separate experiments (Table 1). Eflect of Ozone on HAM Superoxide Production in Response to PMA HAM were exposed for 2 hr to 1.O ppm O3 or filtered air and then stimulated with PMA. Spontaneous release (induced by adherence) of 0; was not affected by 03, while PMAinduced 0; production was decreased by 27% by the pollutant (Fig. 3). Eflect of Ozone on Growth Inhibition neoformans by HAM

of C.

Growth inhibition of C. neoformans by HAM is mediated by an intracellular process

BECKER ET AL.

408

TABLE 1 EF’FEC~OF OZONE ON Fc AND COMPLEMENT RECEPTOR EXPRESSIONONHUMANALVEOLARMACROPHAGES Fluorescence intensity Y Receptors

Antibody

Air

Ozone

FcRl FcR2

32.3 IV3

130.4 152.2

131.8 147.8

F&o

anti&u 11 anti-CR 1 anti-OKM 1 anit-Le.uMS

115.0 121.1 137.1 130.4

117.0 119.0 130.0 132.2

CR1 CR3 CR4

a Mean channel number of fluorescence histogram minus mean fluorescence channel number of background fluorescence. The samples were run on log scale and the mean channel number converted to linear scale before subtraction of background.

after opsonization of the organism and by an extracellular process at high HAM:C. neofirmans ratios in the absence of opsonization. Neither growth inhibitory mechanism has been identified, but growth inhibition is not mediated by O;/H202 or TNF (Weinberg et al., 1987). In the present study, HAM were exposed to 1 ppm O3 or filtered air for 4 hr and then incubated with opsonized C. neoformans for 24 and 60 hr. No difference in the growth inhibitory capacity of control versus Orexposed macrophages was observed (Fig. 4) at either of the time points. The HAM were also tested for opsonin-independent growth inhibition of the yeast. Again, O3 exposure did not alter the efficiency of the inhibitory activity (not shown).

. 1 Stimulus

FIG. 3. Effect of ozone on superoxide production. 0; production was determined in HAM exposed to O9 for 2 hr, and then stimulated with PMA or medium for an additional 60 min. The hatched bars represent data obtained with OS-exposed HAM and the open bars represent data obtained with HAM exposed to filtered air. Spontaneous production of 0; was not affected by the 0, exposure while PMA-induced production was significantly decmased (p = 0.002). The results shown are the mean r SEM of five experiments performed in duplicate.

shown). However, exposure to O3 altered the ability of the HAM to produce IL- 1, IL-6, and TNF in response to bacterial endotoxin. When HAM were exposed to 1.0 ppm O3 for 2 hr and then cultured for 18 hr in the presence of 1 pg/ml LPS, there was a 34% decrease in the production of TNF, a 32% decrease in IL-l,

Hours

Effect of Ozone on the Production IL-l /3, IL-6, and CSF-1

of TNF-a,

Macrophages produce IL- 1, IL-6, and TNF, three cytokines important in inflammation and host defense (Le and Vilcek, 1987, 1989). O3 exposure by itself (1 ppm for 2 or 4 hr) did not induce production of these cytokines (not

of culture

FIG. 4. Effect of ozone on intracellular growth inhibition of C. neoformans by human alveolar macrophages. After exposure of HAM to 1.0 ppm O9 for 4 hr (hatched bars) or to filtered air (open bars), the cells were cocultured with serum opsonized C. neoformans for 24 and 60 hr. The number of intracellular yeast was quantified by coulter counting. O+xposed HAM were equal to air-exposed HAM in inhibiting replication of the yeast. The data are presented as the mean f SEM of five experiments done in duplicate.

MODULATION

OF HAM

PROPERTIES

409

BY 0s EXPOSURE

and a 37% decrease in IL6 (Fig. 5). Production of CSF-1, a macrophage colony stimulating factor produced in equal amounts by unstimulated and LPS-stimulated HAM, was not altered by 03. Efect of Ozone on the Synthesis of HAM Proteins In order to understand the extent to which in vitro O3 exposure perturbs HAM processes, two-dimensional gel electrophoresis was used to analyze more than 900 different proteins for changes induced by the pollutant. Exposure to O3 did not result in general cellular activation of protein synthesis as determined by identical amount of radioactivity incorporated in control and 03-exposed cells. It is possible, though unlikely, that O3 may change the specific activity of the methionine pool, in which case changes in the absolute rate of protein synthesis may not be detected. There were, however, differences in the relative rate of synthesis of several individual proteins after exposure to OX. Representative autoradiographs of cellular proteins from cells exposed to air and O3 are shown in Fig. 6. Computer analysis of the autoradiographs revealed that the relative rate of synthesis of 11 proteins was altered significantly; these are shown numbered in Fig. 6. Table 2 lists the relative abundance of each protein as well as the fold change in synthesis of each protein after O3 exposure. Changes in 9 proteins were quantitative; i.e., the proteins were made in both air- and 03exposed cells but at different rates in each instance. One protein (No. 6), however, was not synthesized at a detectable rate in HAM exposed to 03, and one protein (No. 7) was not synthesized at a detectable rate in HAM exposed to air. DISCUSSION In the lower airways, HAM represent the most important host defense cells against inhaled infectious agents (Goldstein et al., 1974;

50 0t

CSF-1

FIG. 5. Effect of ozone on production of cytokines by endotoxin-stimulated human alveolar macrophages. HAM were stimulated with LPS after exposure for 2 hr to 1 ppm O9 (hatched bars) or filtered air (open bars). Supematants were collected after 18 hr and various cytokines quantified. A significant decrease in TNF (p = O.OOS),IL- 1 (p = 0.003), and IL-6 (p = 0.026) was found while release of CSF-1 was not affected. IL- 1 levels are given in pg/ml while the other cytokines are given in units/ml. The data are expressed as the mean f SEM of five to seven experiments.

Sibille and Reynolds, 1990). In addition to their extensive phagocytic ability, HAM release a number of cytokines which direct the influx of polymorphonuclear leukocytes, modulate the immune response, and initiate repair processes in response to injury. Perturbation of any of these activities by O3 may lead to altered efficiency in controlling airway infections and inflammation. In this study we demonstrate that in vitro exposure of HAM to O3 results in a variety of changes in HAM function that are detrimental to their ability to protect the lung. As a result of a decrease in the ability to phagocytose, in combination with an Orinduced decrease in the efficiency of mucociliary clearance (Rombout et al., 1982), a larger burden of inhaled infectious organisms, as well as particulate antigens and dusts, are likely to end up in the bronchoalveolar region. Furthermore decreased production of inflammatory mediators would add to the harm caused by increased infection and particle burden, resulting in higher infectivity rates. The O3 exposure conditions chosen for this study minimize the effect of O3 on HAM viability and are similar to those described by Valentine (1985). Viability of the pollutant-

410

BECKER ET AL.

1

F

FIG. 6. Proteins made by HAM following exposure to air or ozone. Macrophages were exposed to 0s and labeled with [?S]methionin, and cellular proteins were fractionated by two-dimensional gel electrophoresis. (A) Proteins made by HAM after exposure to filtered air. (B) Proteins made by HAM after a 2-hr exposure to 1.Oppm 09. Proteins whose rate of synthesis changed are circled and numbered. Bars designating isoelectric points are on the bottom of (A) and are (from left to right): 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. Bars designating molecular weight are to the left of (A) and are from bottom to top: 15. 20, 30,40, 50, 60, 80, and 100 kDa.

exposed cells was identical to that of filtered air-exposed cells for at least 48 hr after exposure. Thus the observed changes in HAM function are related to molecular changes in the cells due to reactivity with O3 rather than to direct cytotoxicity. It is not known how the in vitro O3 concentrations in the exposure chamber and the delivered dose to the cells relate to the dose delivered to the HAM in in vivo exposure studies (Koren et al., 1989;

Devlin et al., 1990). However, this dosimetry issue is currently being investigated in our laboratory in both in vivo and in vitro exposures using the nonradiolabeled ‘*OX isotope. Exposure of HAM to O3 resulted in the release of increased amounts of arachidonic acid (not shown) and in increased production of PGE2. These results are in agreement with previous work with rat and rabbit alveolar macrophages which were induced to release

MODULATION

OF

HAM

FIG.

PROPERTIES

BY

Ox EXPOSURE

411

ti-Continued

PGEz by exposure to O3 both in vivo and in vitro (Driscoll et al., 1986b; Madden et al., 1990), as well as with studies with 03-exposed humans who showed increase in PGEz in their lavage fluids (Seltzer et al., 1987; Koren et al., 1989). It is possible that O3 stimulates phospholipase activity as has been shown with other oxygen-derived free radicals (Au et al., 1985) or that OX activates cyclooxygenase via a membrane lipid ozonization process which generates H202 (Bailey, 1958). Release of PGE2 may be a sensitive indicator of effects on the cell membrane and membrane alterations may be responsible for 03-induced de-

fects in other HAM functions. PGE2 has been shown to downregulate macrophage phagocytosis, inhibit production of reactive oxygen intermediates, and inhibit antigen presentation by reducing Ia antigen expression. Phagocytosis of EA was inhibited by 03. This process is mediated mainly by FcRl on the HAM cell surface, with minor involvement of FcR2 (Unkeless, 1989). However, the expression of these receptors on OX-exposed cells was not changed from that of control cells, suggesting that either the affinity or avidity of the binding of immune complexes was altered by O3 or that the signal pathway leading to

412

BECKER TABLE

CHANGES IN HUMAN OZONE

Spot number 1 2 3 4 5 6 I 8 9 10 11

2

IN THE RATE OF SYNTHESIS OF 11 PROTEINS ALVEOLAR MACROPHAGES EXPOSED TO

Percentage abundance” 0.04 0.08 0.82 0.12 0.11 0.04 0.05 0.12 0.07 0.10 0.13

Fold change’ 2.3 -3.9 -2.3 -1.9 4.6 -2.9 5.1 2.5 2.8 1.6 3.1

,J Percentage abundance was obtained by dividing the cpm present in each spot by the total cpm present in the gel. b Fold change reflects the relative increase or decrease in the rate of synthesis of individual proteins in 0,exposed macrophages compared with changes in the rate of synthesis of the same proteins in air-exposed macrophages. Negative numbers refer to proteins whose relative rate of synthesis decreased after Or exposure; positive numbers refer to those whose rate of synthesis increased.

EA ingestion was compromised. Membrane proteins have been shown to also be sensitive to O3 oxidation, and -SH groups are especially vulnerable (Mudd and Freeman, 1977; Menzel, 1984) which could offer an explanation for decreased Fc receptor function despite unaltered expression of immunoreactive receptors. Phagocytosis of opsonized C. neofirmans was not affected by 03, nor was there a change in the number of membrane complement receptors which are required for phagocytosis of the yeast. In addition, neither intra- nor extracellular growth inhibition of the yeast was inhibited by exposure of HAM to 03. Freshly isolated HAM are relatively poor producers of oxygen radicals (Fels et al., 1987). We found that if the cells were adhered to tissue culture dishes they “spontaneously” released 0; and did not respond to stimulation with PMA by additional 0; release. Upon

ET

AL.

short term (2 hr) culture of the cells the “spontaneous” 0; production subsided and the cells became responsive to stimulation with PMA. Maximum responsiveness to PMA appeared after overnight culture ( 16- 18 hr). Therefore. we exposed HAM to O3 immediately after isolation and after 2 and 18 hr of culture. O3 only affected 0; production in the cells cultured for 2 hr that were in transition from a PMA unresponsive to responsive state. The negative results with HAM exposed immediately after isolation and after 18 hr of culture are not shown. We interpret this observation to indicate that O3 affects reorganization of the HAM membrane which occurs during their adaptation to culture, rather than to specific enzymatic processes which result in 0; release. Several investigations with various cell types have indicated that O3 affects membrane fluidity and permeability (Goldstein et al., 1977; Witz et al., 1983; Rietjens et al., 1987). The production of reactive oxygen intermediates by in vitro and in vivo Orexposed rodent macrophages has previously been shown to be significantly reduced (Goldstein et al., 1970; Amoruso et al., 1981; Ryer-Powder et al., 1988). Upon stimulation by microorganisms, injury, or interaction with toxic particles, macrophages release a number of inflammatory mediators which interact in recruitment and activation of other inflammatory cells. IL- 1, TNF, and IL-6 are polypeptide mediators with pleiotropic effects on a wide variety of host defense processes which ensue upon infection or injury (Le and Vilcek, 1987, 1989). HAM can be induced to produce high concentrations of TNF and IL-6 as compared to other cells, while IL- 1 appears to be produced less by lung macrophages than by blood monocytes (Becker et al., 1989). We were interested to see if injury caused by O3 could induce the production of these cytokines by HAM. Low to undetectable levels of these cytokines were found in both 03-exposed and control HAM supernatants. However, 03-exposed HAM produced decreased levels of cytokines in response to a strong activating signal such as

MODULATION

OF HAM

PROPERTIES

bacterial LPS. The release of CSF- 1, which is constitutively produced by HAMS and not induced by LPS, was not affected by 03. Although O3 may attack and cause alterations in the cell membrane (Mudd and Freeman, 1975; Menzel, 1984) it appears that in vitro O3 exposure of HAM causes very few changes in HAM proteins. This can be seen by two-dimensional gel analysis, in which we found that the relative rate of synthesis of only 11 proteins was altered by the exposure. These results contrast with those obtained with HAM removed from humans exposed to 0.4 ppm O3 in vivo. In these studies the relative rate of synthesis of 123 different proteins was altered (Devlin and Koren, 1990). Therefore it is possible that most of the protein changes seen after an in vivo O3 exposure are secondary effects on the HAM resulting from airway injury and inflammation, rather than a direct effect of O3 on the HAM. It remains to be determined if the 11 proteins which are altered after in vitro exposure of HAM are membrane proteins, and therefore in a position to be directly attacked by the pollutant. In summary these data suggest that exposure to O3 has direct modulatory effects on HAM in the absence of an effect on HAM viability. We hypothesize that under the experimental conditions described, these changes in HAM are mainly due to alterations in the cell membrane, where both lipids and receptor proteins are likely to be affected (Mudd and Freeman, 1977; Menzel, 1984; Veninga and Evelyn, 1986). The production of PGE2 may be the result of lipid ozonization/peroxidation while the decreased response of exposed HAM to stimuli like immune complexes and LPS may be the result of altered signal transduction since protein synthesis appears to be marginally affected. The results with in vitro 03-exposed HAM suggest that the effects on HAM seen after in vivo O3 exposure (Koren et al., 1989; Devlin et al., 1990) are the results of a complex cascade of events in response to tissue injury rather than the direct effect of the pollutant on the cells.

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ACKNOWLEDGMENT The writers thank Mrs. Velva Millholland for expert assistance in flowcytometric analysis of our samples.

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