Effect of Ozone Exposure on Alveolar Macrophage-Mediated Immunosuppressive Activity in Rats

TOXICOLOGICAL SCIENCES 41, 217-223 (1998) ARTICLE NO. TX972417

Eiko Koike,* Takahiro Kobayashi,f' Delia J. Nelson,$ Andrew S. McWilliam,$ and Patrick G. HoltJ ^Department of Medical Sciences, Tsukuba University, Tsukuba 305, Japan; tEnvironmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba 305, Japan; ami $TVW Telethon Institute for Child Health Research, West Perth, Western Australia, Australia Downloaded from https://academic.oup.com/toxsci/article/41/2/217/1733894 by guest on 27 September 2021

Received June 20, 1997; accepted December 12, 1997 this stimulation is required in order to prevent excessive im- Effect of Ozone Exposure on Alveolar Macrophage-Mediated mune responses. Alveolar macrophages (AM)2 function poorly Immunosuppressive Activity in Rats, Koike, E., Kobayashi, T., as accessory cells for T-cell activation and as antigen-present- Nelson, D. J., McWilliam, A. S., and Holt, P. G. (1998). ToxicoL ing cells (APC) (Bilyk and Holt, 1993; Holt, 1980). In many Sci. 41, 217-223. situations, AM suppress the antigen- or mitogen-induced pro- Ozone (O3), a major component of photochemical air pollution, is liferation of T-cells (Bilyk and Holt, 1993; Strickland et al., a strong oxidizing agent and highly toxic Resident alveolar macro- 1993, 1994; Upham et al, 1995). AM also suppress the anti- phages (AM) play an important immunomodulatory role in the lung gen-presenting activity of lung dendritic cells (DC) in situ via suppression of lymphocyte proliferation, thus limiting the mag- (Holt et al, 1993). These AM-mediated immunosuppressive nitude and duration of local immune responses. Nitric oxide (NO) activities have been proposed to play an important role in plays a crucial role in the immunosuppressive activity of AM. How- maintaining immunological homeostasis in the normal lung ever, during inununoinflammatory responses, microenvironmental changes within the alveoli inhibit this AM function, permitting full (Bilyk and Holt, 1993; Holt et al, 1993; Strickland etal, 1993, expression of local T-cell-mediated immune responses. We hypothe- 1994; Upham et al, 1995). During lung inflammation induced size that similar changes in AM function may occur during inflam- by exposure to bacterial lipopolysaccharide (LPS), soluble mation induced by exposure to inorganic air pollutants, such as O3. In factors released into the lung microenvironment have been order to test this hypothesis, in the present study, we investigated (1) shown to inhibit the AM-mediated suppressive activity of whether O3 exposure of rats might affect the immunosuppressive T-cell proliferation (Bilyk and Holt, 1993). Several mediators activity and NO production of bronchoalveolar lavage cells (BAL have been suggested as potential causes of the macrophage- cells) and (2) whether changes in the microenvironment of the alveoli mediated suppression of T-cell proliferation, including nitric induced by O3 exposure can affect the immunosuppressive activity oxide (NO) and tumor necrosis factor-a (TNF-a) (Kawabe et and NO production of AM. AM-mediated immunosuppressive activ- al, 1992;Metzgere?a/., 1980; Schleifer and Mansfield, 1993). ity was measured as inhibition of concanavalin A (Con A>induced However, in relation to the present model, it has been shown proliferation of lymph node cells (LNC). Bronchoalveolar lavage was used to sample the alveolar microenvironment, and the resulting fluid that AM-mediated suppression of lymphocyte proliferation in (BALF) was tested for capacity to modulate AM activity in the the rat can be almost entirely inhibited by the use of agents cultures. BALF and BAL cells from rats exposed to 1 ppm O3 or which block enzyme-inducible NO synthase (Kawabe et al, filtered air for 3 days were used. The present results demonstrate that 1992; Upham et al, 1995), indicating that the principle immu- BAL cells isolated from O3-exposed rats suppressed Con A-induced nosuppressive agent is NO. It is significant to note in this LNC proliferation and produced NO in the same manner as BAL context that the loss of immunosuppressive activity of AM cells (AM) from air-exposed rats. AM-mediated suppressive activity of LNC proliferation and NO production were markedly inhibited by 2 Abbreviations used: AM, alveolar macrophages; APC, antigen-presenting BALF from 03-exposed but not from air-exposed rats. These results cells; DC, dendritic cells; LPS, lipopolysaccharide; NO, nitric oxide; TNF-a, suggested that AM-mediated immunosuppressive activity in vivo may tumor necrosis factor-a; O3, ozone; igE, immunoglobulin E; BAL cells, be inhibited by the O3-induced release of soluble factors which inhibit bronchoalveolar lavage cells; LNC, lymph node cells; Con A, concanavalin A; NO production by AM. e 1998 society <* Toxicokw BALF, bronchoalveolar lavage fluid; PBS, phosphate-buffered saline; FBS, fetal bovine serum; 2-ME, 2-mercaptoethanol; H3PO4, phosphoric acid; NaNO2, sodium nitrite; ELISA, enzyme-linked immunosorbent assay; BrdU, 5-bromo-2'-deoxyuridine; RIO, RPMI 1640 medium plus 10% heat-inacti- 3 The tissues of the lung are constantly contact with incoming vated FBS, 100 fig/ml penicillin, 100 U/ml streptomycin, and 5 X 10~ M 2-ME, Air-BALF, BALF from air-exposed rats; O -BALF, BALF from O - antigens from the atmospheric environment during gas ex- 3 3 exposed rats; NOJ, nitrite; LCM, lung-conditioned medium; GM-CSF, gran- change. Precise regulation of the response of local T-ceOs to ulocyte/macrophage colony-stimulating factor, IFN-y, interferon-y, IL, inter- leulcin; PG, prostaglandin; TGF-0, transforming growth factor-^; SP-A, 1 To whom correspondence should be addressed. Fax: +81-298-50-2439. surfactant-associated protein A.

induced by soluble factors released from LPS-exposed lung METHODS tissue (Bilyk and Holt, 1993) was associated with loss of capacity to produce NO (Bilyk and Holt, 1995). Reagents. The following reagents were obtained: RPMI 1640 medium, Dulbecco's calcium- and magnesium-free phosphate-buffered saline (PBS Asthma and allergic rhinitis appear to be increasing in many (—)), fetal bovine serum (FBS), and pentobarbital sodium from Dainippon countries (Leikauf et al, 1995; Wardlaw, 1995). Previous Pharmaceutical Co. (Osaka, Japan); 2-mercaptoethanol (2-ME) from Wako reports have shown that the prevalence rate of asthma (Koren, Pure Chemical Industries (Osaka, Japan); penicillin-streptomycin solution and 1995; Leikauf et al, 1995) and allergic rhinitis (Ishizaki et al., Con A from Sigma (St. Louis, MO); trypan blue from Gibco Laboratories 1987; Kaneko et al., 1980) in air-polluted areas is higher than (NY); phosphoric acid (H3PO4), sulfanilamide, AKl-naphthyl)ethylenediamine dihydrochloride and sodium nitrite (NaNOj) from Nacalai Tesqe, Inc. that in nonpolluted areas. Controversial epidemiological re- (Kyoto, Japan); and cell-proliferation enzyme-linked immunosorbent assay sults, however, have also been reported (Nicolai and von- (F.I ISA) and the 5-bromo-2'-deoxyuridine (BrdU) (colorimetric) Kit from Mutius, 1996, 1997). Therefore, the relationship between con- Boehringer Mannheim (Tokyo, Japan). Downloaded from https://academic.oup.com/toxsci/article/41/2/217/1733894 by guest on 27 September 2021 centration and/or composition of air pollutants and prevalence Animals and ozone exposure. Male, specific pathogen-free Wistar strain of allergic disorders remained to be elucidated. Ozone (O3), a rats (8-12 weeks old) were obtained from Japan Charles River Inc. (Yoko- major component of photochemical air pollution, is a strong hama, Japan) and were housed under conventional conditions until used. The oxidizing agent and highly toxic. Lung inflammation is caused rats were placed in stainless-steel cages and exposed to 1 ±0.1 ppm O3 or filtered air for 3 days. Food or water was provided to the animals during the by exposure to air pollutants, such as O3 (Chen et al., 1995; exposure period. The conditions in the chamber (volume 1.16 m3) were Laskin et al., 1994; Peden et al., 1995; Pendino et al., 1996). (temperature) 25 ± 1°C; (humidity) 55 ± 10%; (pressure) -5 mm H2O 3 A contributory role of O3 to the development of asthma was relative to atmospheric pressure; (total air flow rate) 90-100 m /h. The suggested by the results of epidemiological studies (Balmes, concentrations of O3 were continuously monitored with an O3 analyzer (Model 1993; Bascom, 1996; Koren, 1995; Leikauf et al, 1995). 8410, Monitor Lab. Inc., U.S.A.) using a chemiluminescence method. Preparation of bronchoalveolar lavage fluid and bronchoalveolar lavage Therefore, it is necessary to determine whether O3 might affect cellular mechanisms which are known to play a role in the cells. Air- or O3-exposed rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally) and exsanguinated from the aorta abdominalis. induction of asthma and allergic rhinitis. BALF and BAL cells were obtained via in situ BAL. An incision was made Immunoglobulin E (IgE) antibody production and airway between the cartilaginous rings of the trachea, and a cannula was placed into hyperresponsiveness are important factors in respiratory aller- the trachea and secured with suture. Lungs and trachea were lavaged with 37°C gic diseases. It has been reported that O exposure enhances the RPMI 1640 medium (10 ml/300 g body weight). The lavage was done 10 times 3 by slowly instilling and withdrawing the instillation with the same solution. activity of IgE-containing cells in lung (Gershwin et al, 1981) The lavage fluid recovered was centrifuged at 400g for 10 min and cells were and also airway hyperresponsiveness to chemical mediators removed. The acellular lavage fluid was then sterilized by filtration with released by antigen-antibody reactions (Kobayashi, 1996). MILLEX-HA (0.45 urn: Millipore, Bedford, MA). Ten percent heat-inacti- 3 There are many reports on the nature of the mechanisms of vated FBS, 100 /i.g/ml penicillin, 100 U/ml streptomycin, and 5 X 10" M 2-ME were added to each filtrate and this final solution was designated the O3-induced airway hyperresponsiveness. The mechanism of BALF. There was no significant difference in pH of the BALF from O3- or enhancement of IgE production by O3 exposure, however, air-exposed rats. In order to prepare BAL cells, the lungs and trachea were remains to be elucidated. In this context, it has been shown that lavaged twice with 10 ml 37°C PBS (-). BAL cells were collected by abolition of the AM-mediated immunosuppressive pathway in centrifugation and resuspended in RPMI 1640 medium plus 10% heat-inacti- 3 vivo by in situ depletion of AM enhances the functions of DC vated FBS, 100 /ig/ml penicillin, 100 U/ml streptomycin, and 5 X 10~ M 2-ME (R10). The numbers of viable cells were determined using the trypan and T-cells in the lung (Holt et al., 1993; Strickland et al, blue exclusion method. 1993), both of which play an important role in IgE production. In addition, animals lacking functional AM display markedly Preparation of lymph node cells. LNC were derived from a pool of superficial, facial, and posterior mediastinal lymph nodes in air-exposed rats. enhanced IgE responses to aerosolized antigen (Thepen et al, The lymph nodes were minced with a set of fine-tipped scissors and then 1992). pushed through a sterile wire mesh in order to disperse LNC from tissue, and Therefore, in the present studies, we investigated (1) the resulting cells were suspended in warm PBS (-). The suspension was rapidly filtered through a nylon mesh to sieve a mass of residual tissue (200 whether O3 exposure of rats might affect the immunosuppressive activity and NO production of bronchoalveolar lavage mesh, Abe Science, China, Japan). Cells were collected by centrifugation at 400g for 10 min and resuspended in R10. The numbers of viable cells were cells (BAL cells) and (2) whether changes in the microenvi- determined using the trypan blue exclusion method. ronment of alveoli induced by O exposure can affect the 3 Differential bronchoalveolar lavage cell counts. The lavage fluid was immunosuppressive activity and NO production of AM. Rats centrifuged at 400g for 10 min and resuspended in R10, and total cells were were exposed to O3 or filtered air, and AM-mediated immu- counted with a hemacytometer. Cell viability was assayed by the trypan blue nosuppressive activity was measured as inhibition of the pro- exclusion method. Differential cell counts were determined by Diff-Quik liferation of lymph node cells (LNC) induced by concanavalin (International Reagents Co., Kobe, Japan) stain after cytospin preparation. A (Con A). Bronchoalveolar lavage fluid (BALF) was used as Protocol of the effect of O3 exposure on BAL cell-mediated suppression. a sample of the microenvironment of alveoli. Our present BAL cells were prepared from rats exposed to air or 1 ppm O3 for 3 days (Air-BALF and O3-BALF, respectively). All LNC were prepared from rats results suggest that AM-mediated immunosuppressive activity 3 exposed to air for 3 days. Aliquots of 4 X 10 LNC were placed (in triplicate) is inhibited by the O3-induced changes in the microenviron- into wells of a 96-well flat-bottom plate (Nunclon, Denmark) in 190 fil R10 ment of alveoli. containing 10 /ig/ml Con A, and 10-/J aliquots of various numbers (0.075 to 03 INHIBITS SUPPRESSIVE ACTIVITY OF AM 219

TABLE 1 H3PO4 in the wells of a 96-well flat-bottom plate and incubated for 10 min at Differential Bronchoalveolar Lavage Cell Counts room temperature. Absorbance was measured in an F.I .ISA reader at a wavelength of 540 run. The values measured were compared with the standard Air Ozone curves, which were established with known amounts of NaNO2 in R10.

Alveolar macrophages >99° (2.28 X 106*) 72 ± 6° (3.42 ±0.29 x 106*) RESULTS Neutrophil 22 ± 5" (1.04 ±0.24 x 10**) Eosinophil 4 ± l"(0.18 ±0.04 x 10"") Differential Bronchoalveolar Lavage Cell Counts Lymphocyte 2 ± l"(0.07 ± 0.04 X 10**) Table 1 shows that the number of AM collected after exposure Note. Differential bronchoalveolar lavage cell counts. Rats were exposed to to O3 for 3 days increased by approximately 50% over the number air or 1 ppm O3 for 3 days. Bronchoalveolar lavage cells were collected after in control rats. The differential counts of BAL cells revealed that

this exposure. Differential cell counts were determined by Diff-Quik stain after Downloaded from https://academic.oup.com/toxsci/article/41/2/217/1733894 by guest on 27 September 2021 cytospin preparation. Results are the mean ± SE of data from four individual cells in O3-BALF consisted of 72 ± 6% AM, 22 ± 5% neutro- rats. " and * represent percentages of total cells and cell number, respectively. phils, 4 ± 1% eosinophils, and 2 ± 1% lymphocytes, whereas BAL cells from control rats were >99% AM.

20% vs LNC) of BAL cells in R10 were added to each well. These cell cultures Effect of O3 Exposure on BAL Cell-Mediated Suppression of were incubated for 72 h in a humidified atmosphere at 37°C in a 5% CO2 LNC Proliferation incubator. Cell proliferation was measured by incorporation of BrdU added 24 h before the measurement After incubation, culture supernatant was col- Graded numbers of BAL cells were added to LNC cultures. lected for measurement of nitrite content as an indicator of NO production. Figure 1 shows that Con A-induced LNC proliferation was Protocol of the effect of BALF on AM-mediated suppression. BALF suppressed by addition of BAL cells from both air- and O3- were prepared from rats exposed to air or 1 ppm O3 for 3 days. All LNC and exposed rats. This suppression depended on the number of 3 AM were prepared from rats exposed to air for 3 days. Aliquots of 4 X 10 BAL cells added to the LNC cultures. BAL cells, at numbers LNC were placed (in triplicate) into wells of a 96-well flat-bottom plate in 190 equivalent to between 5 and 20% of the LNC, almost com- fid R10 or BALF containing 10 ^.g/ml Con A, and 10-p.l aliquots of 5% vs LNC of AM in R10 were added to each well. Our preparatory experiments pletely suppressed Con A-induced LNC proliferation. BAL show this AM vs LNC ratio is optimum to examine the effect of BALF. These cells from O3-exposed rats also suppressed Con A-induced cell cultures were incubated for 72 h. Cell proliferation was measured by LNC proliferation in the same manner as BAL cells from incorporation of BrdU added 24 h before the measurement. After incubation, air-exposed rats. culture supernatant was collected for measurement of nitrite content

Measurement of cell proliferation. Cell proliferation was measured by Effect of O3 Exposure on NO Production by BAL Cells using the Cell-Proliferation-ELISA, BrdU (colorimetric) Kit. This technique is based on the incorporation of pyrimidine analogue BrdU instead of thymidine To confirm that suppression of Con A-induced LNC prolif- into the DNA of proliferating cells. BrdU incorporated into DNA is measured eration in this system is associated with release of NO into the by a sandwich-type enzyme immunoassay using a monoclonal anti-BrdU antibody. This technique is at least as sensitive as the traditional counting of [3H]thymidme (Porstmann et al., 1985). Cell proliferation was measured by adding 20 /JJ of 100 yM BrdU to each well 24 h before the measurement to allow the pyrimidine analogue BrdU to be incorporated in the place of thymidine in the DNA of proliferating cells. The plate was centrifuged at 35Og for 10 min, and the culture medium was removed. Then the cells were fixed and DNA denatured by adding FixDenat (200 /xl/well) and incubating the plate for 30 min at room temperature. FixDenat solution was decanted from wells and residual solution was removed thoroughly by tapping the plate on clean paper toweling, anti-BrdU-peroxidase (100 jil/well) added, and the plate incubated for 90 min at room temperature. Excess antibody conjugate was removed by decantation and wells were rinsed three times with washing solution (PBS) (200 jU/well). Washing solution was decanted from wells and residual solution was removed thoroughly by tapping die plate on clean paper toweling, substrate (letramethylbenzidine) (100 fil/well) was added, and the plate was incubated for 30 min at room temperature. Substrate reaction was stopped by adding 1 M sulfuric acid (25 /il/well) and mixing. Absorbance of 0 0.075 0.3 1.25 5 20 the samples was measured in an ELISA reader (ImmunoReader NJ-2000, Inter Med, Tokyo, Japan) at a wavelength of 450 nm (reference wavelength: Number of AM added to LNC cultures (%) 620 nm). FIG. 1. BAL cells-mediated suppression of LNC proliferation. LNC (4 X Measurement of NO production. LNC were cultured with Con A in R10, lO'/well) from rats exposed to air were cultured with 10 fig/ml Con A in R10, 2 Air-BALF, or O3-BALF in the presence or the absence of AM as described in in the presence of graded numbers (0.075 to 20% for LNC: 3.1 x 10 to 8 X the protocols. Culture supernatant was collected to measure of NO production. lOVwell) of BAL cells from rats exposed to air (•) or 1 ppm O3 (•) for 3 days. NO was measured as nitrite (NO^") in the culture supernatant using the Griess Plates were incubated for 72 h, and BrdU was added 24 h before measuring cell reagent Culture supernatant (50 yX) was mixed with 50 /J of 1% sulfanil- proliferation. Data represent the mean ± SE of triplicate samples from one of amide, 0.1% /V-{l-naphthyl)ethylenediamine dihydrochloride, and 2.5% four similar experiments. 220 KOIKE ET AL.

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0 0 0.075 0.3 1.25 5 20 Air-BALF O3-BALF Number of AM added to LNC cultures (%) FIG. 3. Effect of BALF on AM-mediated suppression of LNC proliferation. After exposure for 3 days, BALF were prepared from rats exposed to air FIG. 2. NO production by BAL cells. LNC (4 X lOVwell) from rats or 1 ppm O3; LNC and AM were prepared from rats exposed to air. LNC (4 X exposed to air were cultured with 10 jig/ml Con A in R10, in the presence of 1 Orwell) were cultured with 10 jig/ml Con A in R10, Air-BALF, or O3-BALF graded numbers (0.075 to 20% for LNC: 3.1 X 102 to 8 X lOVwell) of BAL in the presence (•) or the absence (D) of AM (5% for LNC: 2 X lCrVwell). cells from rats exposed to air (•) or 1 ppm O3 (•) for 3 days. After incubation Plates were incubated for 72 h, and BrdU was added 24 h before measuring cell for 72 h, 50 fii of supernatant was taken from each well. NO was measured as proliferation. Data represent the mean ± SE of triplicate samples from one of NOJ using the Griess reagent. Data represent the mean ± SE of triplicate four similar experiments. samples from one of four similar experiments.

nosuppressive activity. Suppression of Con A-induced LNC cultures, we measured the NO2 content in the supematants of proliferation depended on the number of BAL cells added to the cultures. Figure 2 shows NO production in the LNC:BAL the LNC cultures (Fig. 1). NO production was directly related cell cocultures. The NO production was increased by addition to the number of BAL cells present (Fig. 2). Recently, several of BAL cells from both air- and O3-exposed rats. The level of mediators have been suggested as potential causes of macro- NO production depended on the number of BAL cells added to phage-mediated suppression of lymphocyte proliferation, in- the LNC cultures. cluding NO (Kawabe et al., 1992; Metzger et al., 1980; Schleifer and Mansfield, 1993; Upham et al., 1995). The Effect of BALF on AM-Mediated Suppression of LNC Proliferation In R10, AM suppressed LNC proliferation induced by Con 50-. A (Fig. 3). AM-mediated suppression of LNC proliferation was inhibited by O -BALF, but not by Air-BALF. Figure 3 also 3 40-1 shows that Con A-induced proliferation of LNC was directly suppressed by O3-BALF by approximately 40%. Air-BALF, however, had no effect on Con A-induced proliferation of |30-l LNC. In the presence of O3-BALF, the addition of AM to the coculture resulted in no further suppression of LNC prolifera- •C 20-1 tion. 10- Effect of BALF on NO Production by AM We examined whether changes in the microenvironment of 0- the alveoli by O3 exposure potentially affected NO production by AM. Figure 4 shows that in vitro NO production by AM R10 Air-BALF O3-BALF from air-exposed rats was markedly inhibited by O3-BALF. FIG. 4. Effect of BALF on NO production by AM. After exposure for 3 Air-BALF did not inhibit NO production by AM. days, BALF and AM were prepared from rats exposed to air or 1 ppm O3; LNC and AM were prepared from rats exposed to air. LNC (4 X 103/well) were cultured with 10 ng/ml Con A in R10 (•), Air-BALF (•), or O3-BALF (•) DISCUSSION in the presence of AM (5% for LNC: 2 X 1 Orwell). After incubation for 72 h, 50 y.\ of supernatant was taken from each well. NO was measured as NOJ In this study, we demonstrated that O3-induced changes in using the Griess reagent. Data represent the mean ± SE of triplicate samples the microenvironment of alveoli affected AM-mediated irnmu- from one of four similar experiments. O3 INHIBITS SUPPRESSIVE ACTIVITY OF AM 221

present results are consistent with a major role for NO in irritant known to induce pulmonary inflammation or lung AM-mediated suppressive activity. It should also be noted that injury in humans and experimental animals (Balmes, 1993; O3 exposure increased the number of neutrophils in BALF Chen et al, 1995; Koren, 1995; Laskin et al, 1994; Peden (Table 1). It has been also shown that high-density cells which et al., 1995; Pendino et al., 1996) and O3 exposure increases look like monocytes are increased in BAL by O3 exposure GM-CSF in BALF (Balmes et al., 1996; Scannell et al., (Mochitate and Miura, 1989). It is accordingly possible that 1996). Therefore, GM-CSF could be one of the factors such changes in the BAL cell population may affect the overall underlying the inhibitory effect of O3-BALF on AM-medi- level of suppressive activity and NO production in the lung. ated immunosuppressive activity. Cytokines and mediators released from neutrophils and other NO production from macrophages is regulated by various types of cells also could affect suppressive activity and NO factors. Inducible NO synthesis in macrophages is up-regulated production of AM. To our knowledge, however, there are no

by interferon-y (IFN-y), TNF-a, interleukin (IL)-l/3, and IL-2 Downloaded from https://academic.oup.com/toxsci/article/41/2/217/1733894 by guest on 27 September 2021 reports in the literature which compare the suppressive activity and down-regulated by EL-4, IL-10, prostaglandin (PG) Ej, and and NO production of AM with those of neutrophils or mono- transforming growth factor-/3 (TGF-/3) (Al-Ramadi et al, cytes. Figures 1 and 2 also show that BAL cells from O3- 1992; Cunha et al, 1992; Gutierrez et al, 1995; Modolell et exposed rats suppressed Con A-induced LNC proliferation and al, 1995; Schleifer and Mansfield, 1993; Warner et al, 1995; produced NO in the same manner as AM from air-exposed rats, Bilyk and Holt, 1995). One or more of those cytokines may but direct change in the BAL cells induced by O3 exposure did also be involved in the effects of O3 in this system. Without not have major effects on suppressive activity and NO produc- LNC, AM from air- or O3-exposed rats did not produce NO tion. Figure 1 and Table 1 also suggested that AM from (data not shown). Therefore, some cytokines such as IFN-y 03-exposed rats might be more suppressive than those from released from LNC could induce NO production by AM. Our air-exposed rats, since the absolute number of AM in under- preliminary study shows that O3-BALF inhibited both IFN-y loading BAL cells from O3-exposed rats was less than that of production from LNC induced by Con A and NO production air-exposed rats. from AM induced by EFN-y (data not shown). Air-BALF did not inhibit the AM-mediated suppression of It has also been shown that phospholipid (Bartmann et al., LNC proliferation (Fig. 3). Figure 3 also shows that Con 1992) and pulmonary surfactant-associated protein A (SP-A) A-induced proliferation of LNC was directly suppressed by suppress phytohemagglutinin-activated lymphocyte prolifera- O3-BALF by approximately 40%, but not by Air-BALF. How- tion. In contrast, Con A-activated lymphocyte proliferation is ever, in the presence of O3-BALF, no further suppression of slightly augmented by SP-A (Borron et al., 1996). Dipalmi- LNC proliferation was observed by addition of AM to the toyl-L-a-phosphatidylcholine and Survanta as phospholipids cocultures, whereas normal AM in the absence of O3-BALF suppress Con A-induced lymphocyte proliferation, which is cause complete suppression of proliferation (Fig. 3). Therefore, reversed somewhat by addition of SP-A (Kremlev et al, 1994). our results suggested that AM-mediated suppressive activity of It has been shown that TGF-0 also decreased both Con A-in- Con A-induced LNC proliferation was inhibited by O3-BALF duced T-cell proliferation and IL-2 mRNA expression (Ahmad completely. It has been reported that in the presence of NO et al., 1997). Lung inflammation, such as that which is O3 synthase inhibitor, A^-monomethylarginine acetate, NO^" lev- exposure-induced, may alter the relative amounts of surfactant els were completely reduced, and in accordance with this, the components and cytokines, such as TGF-/3. These possibilities suppression of T-cell proliferation was almost completely in- will be investigated in subsequent experiments. hibited (Upham et al., 1995). Figures 1 and 2 also show that if It has been shown that the AM suppress the antigen-present- NO production was inhibited under 10 jiM NO^~ in the culture ing activity of lung DC (Holt et al, 1993), antigen-induced medium, proliferation of LNC was not suppressed. Figure 4 proliferation of T-cells (Bilyk and Holt, 1993), as well as Con shows that NO production was inhibited by O3-BALF under 7 A-induced proliferation of T-cells. Moreover, in vivo elimina- /xM NO^" in the culture medium. These results also supported tion of AM increases IgE responses to inhaled antigen (Thepen the finding that AM-mediated suppressive activity was inhib- et al, 1992). Therefore, inhibition of AM-mediated immuno- ited by O3-BALF completely. suppressive activity by O3 exposure might be linked with an It is important to identify the factor in O3-BALF which enhancement in IgE and IgG production. The number of IgE- inhibits AM-mediated immunosuppressive activity and also containing cells following allergen inhalation in O3-exposed Con A-induced LNC proliferation. In this regard, it has been mouse lung was larger than that in air-exposed mouse (Gersh- shown that LPS-stimulated lung-conditioned medium win et al, 1981). Conflicting results, however, also have been (LCM) inhibits the AM-mediated suppressive activity of shown previously (Ozawa et al, 1985). This disparate obser- T-cell proliferation (Bilyk and Holt, 1993). This activity of vation depends on the experimental design used (duration and LCM is blocked by an antibody specific for granulocyte/ concentration of O3 exposure, animal species, and so on). macrophage colony-stimulating factor (GM-CSF) and is re- Therefore, systematic studies are necessary to clarify the rela- produced by recombinant GM-CSF, which inhibits NO pro- tionship between IgE production and inhibition of AM-medi- duction (Bilyk and Holt, 1995). O3 is a potent respiratory ated suppressive activity by O3 exposure. 222 KOIKE ET AL.

Localization of AM, T-cells, and DC in peripheral lung is pressive phenotype of pulmonary alveolar macrophage populations. Immu- important in understanding how T-cell activation in the lung in nology 86, 231-237. vivo may be controlled by the microenvironment of the alveoli. Borron, P., Veldhuizen, R. A., Lewis, J. F., Possmayer, F., Caveney, A., Lung digests contained significant numbers of Ia+ DC and Inchley, K., McFadded, R. G., and Fraher, L. J. (1996). Surfactant associated protein-A inhibits human lymphocyte proliferation and IL-2 production. T-cells, which were rarely found in BAL cells. A large popu- Am. J. Respir. Cell MoL Biol. 15, 115-121. lation of T-cells was identified within the alveolar septa (Holt Chen, X., Gavett, S. H., and Wills-Karp, M. (1995). CD4+ T lymphocyte and Schon-Hegrad, 1987). The T-cell population is at least five modulation of ozone-induced murine pulmonary inflammation. Am. J. times larger than the peripheral blood T-cell pool (Holt et al, Respir. Cell MoL Biol. 12, 396-403. + 1985). A DC-like population and also Ia , but differing from Cunha, F. Q., Moncada, S., and Liew, F. Y. (1992). Interleukin-10 (IL-10) the airway DC were identified within the alveolar septal walls. inhibits the induction of nitric oxide synthase by interferon-y in murine Therefore, major populations of T-cells and DC present in rat macrophages. Biochem. Biophys. Res. Commun. 182, 1155-1159. Downloaded from https://academic.oup.com/toxsci/article/41/2/217/1733894 by guest on 27 September 2021 lung were in close proximity to AM located in alveoli. These Gershwin, L. J., Osebold, J. W., and Zee, Y. C. (1981). Immunoglobulin observations suggest that T-cell activation by interactions be- E-containing cells in mouse lung following allergen inhalation and ozone exposure. Int. Arch. Allergy Appl. Immunol 65, 266-277. tween DC and AM in the lung in vivo may be controlled by Gutierrez, H. H., Pitt, B. R., Schwarz, M., Walkins, S. C, Lowenstein, C, changes in the microenvironment in the alveoli. Caniggia, I., Chumley, P., and Freeman, A. (1995). Pulmonary alveolar In summary, AM-mediated immunosuppressive activity epithelial inducible NO synthase gene expression: Regulation by inflamma- plays an important role in maintaining immunological ho- tory mediators. Am. J. Physiol. 268, L501-L508. meostasis in the lung. Our present results suggest that AM- Holt, P. G. (1980). Alveolar macrophages. IV. Interspecies differences in mediated immunosuppressive activity is inhibited by changes activity in proliferating lymphocyte cultures. Cell. Immunol. 50, 210-215. in the microenvironment of the alveoli induced by O3 expo- Holt, P. G., Degebrodt, A., O'Leary, C, Krska, K., and Plozza, T. (1985). T sure. Therefore, the mechanisms underlying lung inflammation cell activation by antigen-presenting cells from lung tissue digests: Suppres- sion by endogenous macrophages. Clin. Exp. Immunol. 62, 586-593. induced by O3 exposure may involve not only damage of host tissue induced by the direct toxic effects of O or lung stromal Holt, P. G., and Schon-Hegrad, M. A. (1987). Localization of T cells, macro- 3 phages and dendritic cells in rat respiratory tract tissue: Implications for and/or mesenchymal cells, but also indirect tissue damage immune function studies. Immunology 62, 349-356. caused by disruption of control mechanisms which regulate the Holt, P. G., Oliver, J., Bilyk, N., MacMenamin, C, MacMenamin, P. G., Kraal, local activation of T-cells. G., and Thepen, T. (1993). Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. J. Exp. Med 177, 397-407. ACKNOWLEDGMENTS Ishizaki, T., Koizumi, K., Ikemori, R., Ishiyama, Y., and Kushibiki, E. (1987). Studies of prevalence of Japanese cedar pollinosis among the residents in a The writers thank Dr. Masataka Murakami, Tsukuba University, and Drs. densely cultivated area. Ann. Allergy 58, 265-270. Ryozo Fujii and Noriko Ohsima, Toho University, for encouragement. This study was supported by the Science and Technology Agency. Kaneko, S., Shimada, K., Horiuchi, H., Endo, A., Kodama, M., Shinoda, N., Imai, T., and Takiguchi, K. (1980). Nasal allergy and air pollution. Oto. Rhino. LaryngoL 23(Suppl. 4), 270-281. REFERENCES Kawabe, T., Isobe, K. I., Hasegawa, Y., Nakashima, I., and Shimokata, K. (1992). Immunosupprcssive activity induced by nitric oxide in culture Ahmad, S., Choudhry, M. A., Shankar, R., and Sayeed, M. M. (1997). supernatant of activated rat alveolar macrophages. Immunology 76, 72—78. Transforming growth factor-beta negatively modulates T-cell responses in Kobayashi, T. (1996). Air pollutants and airway hyperresponsiveness. Environ. sepsis. FEBS. Lett, 402, 213-218. Sci. 27, 195-202. AJ-Ramadi, B. K., Meissler, J. J., Huang, D., and Eisenstein, T. K. (1992). Koren, H. S. (1995). Associations between criteria air pollutants and asthma. Immunosuppression induced by nitric oxide and its inhibition by interleu- Environ. Health Perspect. 103(Suppl. 6), 235-242. kin^J. Eur. J. Immunol. 22, 2249-2254. Kremlev, S. G., Umatead, T. M., and Phelps, D. S. (1994). Effects of surfactant Balmes, J. R. (1993). The role of ozone exposure in the epidemiology of protein A and surfactant lipids lymphocyte proliferation in vitro. Am. J. asthma. Environ. Health Perspect. SuppL 101(Suppl. 4), 219-224. Physiol. 267, L357-L364. Balmes, J. R., Chen, L., Scannell, C, Tager, I., Christian, D., Heame, P. Q., Laskin, J. D., Pendino, K. J., Punjabi, C. J., Rodriguez, M., and Laskin, D. L. Kelly, T., and Aris, R. M. (1996). Ozone-induced decrements in FEV1 and (1994). Pulmonary and hepatic effects of inhaled ozone in rats. Environ. FVC do not correlate with measures of inflammation. Am. J. Respir. Crit. Health Perspect. 102, 61-64. Care Med 153, 904-909. Leikauf, G. D., Kline, S., Alvert, R. E., Baxtar, C. S., Bernstein, D. I., Bartmann, P., Gortner, L., Pohlandt, F., and Jaeger, H. (1992). In vitro Bernstein, J., and Buncher, R. (1995). Evaluation of a possible association lymphocyte functions in the presence of bovine surfactant and its phospho- of urban air toxics and asthma. Environ. Health Perspect. 103(Suppl. 6), lipid fractions. J. Perinat. Med 20, 189-196. 253-271. Bascom, R. (1996). Environmental factors and respiratory hypersensitivity: Metzger, Z., Hoffeld, J. T., and Oppenheim, J. J. (1980). Macrophage-medi- The Americas. Toxicol. Lett. 86, 115-130. ated suppression. I. Evidence for participation of both hydrogen peroxide Bilyk, N., and Holt, P. G. (1993). Inhibition of the immunosuppressive activity and prostaglandins in suppression of murine lymphocyte proliferation. J. Im- of resident pulmonary alveolar macrophages by granulocyte/macrophage munol X2A, 983-988. colony-stimulating factor. J. Exp. Med 177, 1773-1777. Modolell, M., Corraliza, I. M., Link, F., Soler, G., and Eichmann, K. (1995). Bilyk, N., and Holt, P. G. (1995). Cytokine modulation of the immunosup- Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse O3 INHIBITS SUPPRESSIVE ACTIVITY OF AM 223

bone marrow-derived macrophages by TH1 and TH2 cytokines. Eur. J. Im- inflammatory responses in subjects with asthma. Am. J. Respir. Crit. Care munol. 25, 1101-1104. Med. 154, 24-29. Mochitate, K., and Miura, T. (1989). Metabolic enhancement and increase of Schleifer, K., and Mansfield, J. (1993). Suppressor macrophages in African alveolar macrophages induced by ozone. Environ. Res. 49, 79-92. trypanosomiasis inhibit T-cell proliferation responses by nitric oxide and Nicolai, T., and von-Mutius, E. (1996). Respiratory hypersensitivity and en- prostaglandins. J. Immunol. 151, 5492-5503. vironmental factors: East and West Germany. Toxicol. Lett. 86, 105-113. Strickland, D. H., Kees, U. R., and Holt, P. G. (1994). Suppression of T-cell Nicolai, T., and von-Mutius, E. (1997). Pollution and the development of activation by pulmonary alveolar macrophages: Dissociation of effects on allergy: The East and West Germany story. Arch. Toxicol. Suppl. 19, TcR, IL-2R expression, and proliferation. Eur. Respir. J. 7, 2124-2130. 201-206. Strickland, D. H., Thepen, T., Kees, U. R., Kraal, G., and Holt, P. G. (1993). Ozawa, M., Fujimaki, H., Itami, T., Honda, Y., and Watanabe, N. (1985). Regulation of T-cell function in lung tissue by pulmonary alveolar macro- Suppression of IgE antibody production after exposure to ozone in mice. Int. phages. Immunology 80, 266-272. Arch. Allergy Appl. Immunol 76, 16-19. Thepen, T., McMenamin, C, Gim, B., Kraal, G., and Holt, P. G. (1992). Peden, D. B., Setzer, R. W., Setzer, R. W. Jr., and Devlin, R. B. (1995). Ozone Regulation of IgE production in pre-sensitized animals: in vivo elimination Downloaded from https://academic.oup.com/toxsci/article/41/2/217/1733894 by guest on 27 September 2021 exposure has both a priming effect on allergen-induced responses and an of alveolar macrophages preferentially increases IgE responses to inhaled intrinsic inflammatory action in the nasal airways of perennially allergic allergen, din. Exp. Allergy 11, 1107-1114. asthmatics. Am. J. Respir. Crit. Care Med. 151, 1336-1345. Upham, J. W., Strickland, D. H., Bilyk, N., Robinson, B. W. S., and Holt, P. G. Pendiao, K. J., Gardner, C. R., Quinones, S., and Laslcin, D. L. (1996). (1995). Alveolar macrophages from humans and rodents selectively inhibit Stimulation of nitric oxide production in rat lung lavage cells by anti- T-cell proliferation but permit T-cell activation and cytokine secretion. Mac- 1/3 antibody: Effect of ozone inhalation. Am. J. Respir. Cell Mol. Biol. Immunology 84, 142-147. 14, 327-333. Wardlaw, A. J. (1995). Air pollution and allergic disease: Report of a working Porstmann, T., Temynck, T., and Avrameas, S. (1985). Quantitation of 5-bro- party of the British Society for Allergy and Clinical Immunology. Clin. Exp. mo-2-deoxyuridine incorporation into DNA: An enzyme immunoassay for Allergy 25(Suppl. 3), 6-8. the assessment of the lymphoid cell proliferative response. J. Immunol Warner, R. L., Paine, R., Christensen, P. J., Marietta, M. A., Richards, M. K., Methods 82, 169-179. Wilcoxen, S. E., and Ward, P. A. (1995). Lung sources and cytokine Scannell, C, Chen, L., Aris, R. M., Tager, I., Christian, D., Ferrando, R., requirements for in vivo expression of inducible nitric oxide syrithase. Am. J. Welch, B., Kelly, T., and Balmes, J. R. (1996). Greater ozone-induced Respir. Cell Mol Biol. 12, 649-661.