Induction of Functionally Active Platelet-activating Factor Receptors in Rat Alveolar ~ a c r o p ~ a g e s *

of platelet-activating These receptors to be functionally inert in that PAF failed to induce intracellular calcium mobilization in the cells. of rats (2 ppm, 3 h), a pulmonary irritant and inflammatory agent that is rapidly converted to molecular oxy- gen, expression of PAF receptors was markedly up-reg-ulated on lung phagocytes (13.1 x 10" binding sites/cell), with no significant effect on receptor affinity (Kd = 2.0 nM). In these cells, PAF, but not lyso-PAF', an inactive analog, caused a rapid and transient rise in intracellular calcium demonstrating that the PAF receptors were functionally active. The calcium response to PAF, which was largely due to mobilization of calcium from intracellular stores, was found to be dose-dependent in the nanomolar concentration range and inhibitable by the PAF receptor antagonist triazolam. The results of these studies may be important in elucidating mechanisms of lung inflammation induced by this model irritant. Platelet-activating animals, >97.1% of the radioactivity was found to be associated with PHIPAF. These results are similar to those observed in rat hepatic macrophages (36). Measurement of Intracellular Calcium Mobilization-Intracellular calcium mobilization was monitored using the calcium-sensitive indi- cator dye Indo-l/AM (39). Cells were inoculated into Nunc coverglass chambers (2 x lo6 celldchamber). After 2 h at 37 "C, the cells were washed with HBSS containing 0.3% BSA and incubated with 1 PM Indo-l/AM. Thirty min later, the cells were washed three times and 1 ml of HBSS containing 0.3% BSA was added. Calcium mobilization was quantified on a Meridian 570 anchored cell analysis system (ACAS) (Meridian, Okemos, MI) by scanning single 130 x 130-pm fields containing alveolar macrophages (10-20 celldfield) every 30 s for a total of 10 min and collecting the emissions at 500 nm (calcium-free Indo-1) and 400 nm (calcium-saturated Indo-1). In some experiments, cells were treated with triazolam or TMB-8 for 2 min prior to the addition of PAF.

In the lungs, PAF is thought to contribute to a variety of inflammatory conditions including allergic asthma, pulmonary hypertension, and adult respiratory distress syndrome 117-24).
Inhalation of pulmonary irritants such as ozone ( 0 3 j , a highly reactive oxidant, results in airway inflammation and lung injury (25)(26)(27). Both in vivo and in tpitro studies have suggested that one mechanism underlying tissue injury induced by this irritant involves the production of cytotoxic mediators by activated lung phagocytes (28)(29)(30)(31). In this regard, we have recently demonstrated that acute exposure of rats to Os results in increased production of hydrogen peroxide and nitric oxide by these cells.2 A number o f studies have shown that in vitro exposure to O3 results in increased production and release of PAF by macrophages and epithelial cells, suggesting that this inflammatory mediator may be important in the actions of this irritant (32). This prompted us to investigate the role of PAF in lung i n f l a m m a t o~ responses following inhalation of 03.
We specifically chose O3 as a model irritant since it is rapidly converted to molecular oxygen (33) and, thus, does not persist in the lung. We found that O3 exposure in vivo up-regulates PAF receptors on rat lung phagocytes and induces receptor functional activity. These findings may be important in understanding mechanisms of lung inflammation induced by pulmonary irritants.

MATERIALS AND METHODS
Reagents-Indo-l-acetoxymethyl ester (Indo-l/AM) and Indo-1 free acid were purchased from Molecular Probes (Eugene, OR) and stored as 1 mM stock solutions in dimethyl sulfoxide (Me2SO) at -20 "C. PAF and lyso-PAF (Calbiochem) and TPA (LC Services, Woburn, M A ) were dissolved in ethanol and stored at -20 "C as 10 and 170 mill stock solutions, respectively. All other reagents were from Sigma. "iazolam was stored a t room temperature as a 30 m M stock solution in Me2S0. 3,4,5-Trimethylbenzoic acid 8-(diethylamino)octyl ester (TMB-8) was stored a t -20 "C as a 23 mM stock solution in ethanol. All working solutions were prepared fresh for each experiment.
Animals and Exposures-Female specific pathogen-free Sprague-Dawley rats (200-225 g, 6-8 weeks) were obtained from Taconic Farms, Germantown, N Y . The animals were maintained on food and water ad libitum and were housed in microisolator cages. Exposures were conducted in 5.5-ft3 airtight Plexiglass chambers. Rats were exposed to either 2 ppm O3 or ultra-pure air for 3 h. We have prcviously found that this exposure protocol induces a n inflammatory response characterized by an increase in the number of macrophages and polymorphonuclear leukocytes in the lung.2 O3 was generated from oxygen gas via a W light O3 generator (Orec Corp., Phoenix, A Z ) and was mixed with the inlet air of the exposure chamber. 0, concentrations within the chamber were maintained by adjusting both the intensity of the W light and the flow rate of O3 into the chamber. Concentrations were continuously monitored using a n O3 monitor (model 1008AH, Dasibi Environmental Corp., Glendale, CA).
Cell isolation-Cells were harvested from isolated perfused lungs 2 days following exposure to air or 0, by serial hronchoalveolar lavage as previously described (28,34). Cells from air-or 03-exposed rats were pooled, washed twice with HBSS (350 x g, 7 min, 4 "CJ and resuspended in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 pg/ml penicillin, 100 unitsiml streptomycin, and 10 mgiml gentamicin. In all experiments, viability, as determined by trypan blue dye exclusion, was >go%. Differential staining revealed that the cells from air-exposed animals were greater than 99% macrophages and from 0,-exposed rats, approximately 90% macrophages and 10% neutrophils.z

19165
PAF Receptor Binding-Binding of PAF to lung phagocytes was quantified as described previously (35). Briefly, cells (2 x 106) were inoculated into 60-mm tissue culture dishes and incubated at 37 "12. After 2 h, the cells were washed and incubated for 60 min at 25 "C with increasing concentrations of [3H]PAF (141.6 Ci/mmol; Du Pont-New England Nuclear) in HBSS containing 1.3 m M CaC12, 0.5 m M MgCIZ, and 0.1% BSA, in the absence or presence of 3 PM unlabeled PAF. The binding reaction was terminated by rinsing the cells with ice-cold phosphate-buffered saline containing 0.1% BSA. Cells were then lysed in 2.0 ml of 1 N NaOH containing 0.025% Triton X-100. Aliquots of the cell lysates (0.5 ml) were then counted for radioactivity. Specific [3H]PAF binding was calculated by the difference between total binding (measured in the absence of 3 p~ unlabeled PAF) and nonspecific binding (measured in the presence of 3 p~ unlabeled PAF). The average number of PAF binding sites per cell and the dissociation constant were quantified by Scatchard analysis. To exclude the possibility that [3H]PAF was metabolized by the cells during the binding assay, PH]PAF metabolism experiments were performed as previously described for rat macrophages (36,37). Briefly, at the conclusion of the binding assays, cellular lipids were extracted using the method of Bligh and Dyer (38) and separated by thin layer chromatography on Silica Gel IB-F plasticbacked plates (J. T. Baker Inc.) using a solvent system consisting of chlorofordmethanolater (65:35:7, v/v/v). The plate was then cut into 0.5-cm sections and counted for radioactivity. In macrophages from control and 0,-treated animals, >97.1% of the radioactivity was found to be associated with PHIPAF. These results are similar to those observed in rat hepatic macrophages (36).
Measurement of Intracellular Calcium Mobilization-Intracellular calcium mobilization was monitored using the calcium-sensitive indicator dye Indo-l/AM (39). Cells were inoculated into Nunc coverglass chambers (2 x lo6 celldchamber). After 2 h at 37 "C, the cells were washed with HBSS containing 0.3% BSA and incubated with 1 PM Indo-l/AM. Thirty min later, the cells were washed three times and 1 ml of HBSS containing 0.3% BSA was added. Calcium mobilization was quantified on a Meridian 570 anchored cell analysis system (ACAS) (Meridian, Okemos, MI) by scanning single 130 x 130-pm fields containing alveolar macrophages (10-20 celldfield) every 30 s for a total of 10 min and collecting the emissions at 500 nm (calcium-free Indo-1) and 400 nm (calcium-saturated Indo-1). In some experiments, cells were treated with triazolam or TMB-8 for 2 min prior to the addition of PAF. Trypan blue dye exclusion revealed that these compounds had no effect on cell viability (data not shown). Intracellular calcium concentrations were determined by generating a standard curve using known quantities of calcium in 20 m M MOPS buffer (pH 7.4) containing 115 m M KCl, 20 m M NaCl, 1 m M MgS04, 1 m M EGTA, 10% ethanol, and 2 p~ Indo-1 free acid. All data were analyzed utilizing Meridian ACAS statistical software. Each experiment was repeated three to four times with similar results.

RESULTS AND DISCUSSION
In initial studies, we characterized PAF binding to freshly isolated rat lung phagocytes. Fig. 1 shows that specific binding of PAF to the cells was concentration-dependent and saturable. Treatment of the macrophage cell line IC-21 for 5-8 h with lipopolysaccharide has been reported to result in up-regulation of PAF receptors (35). Lipopolysaccharide is a potent inflammatory agent, the biological activity of which is thought to be mediated, at least in part, by PAF released from activated were stimulated (arrows) with 10 m PAF or lyso-PAF.
461, which in a number of cell types, including rat Kupffer cells, irritant unmasks membrane associated PAF receptors. Alteris associated with up-regulation of PAF binding sites (37). The natively, O3 may act indirectly by increasing production of cymechanisms underlying 03-induced up-regulation of PAF re-tokine(s) such as interleukin-4, which, as indicated above, upceptors in rat alveolar macrophages are unknown. O3 is a PO-regulates cell surface PAF receptors (40). tent oxidant that exerts many of its toxic effects on the cell To determine if PAF receptors on rat lung phagocytes are membrane resulting in lipid peroxidation of polyunsaturated Eunctionally active, we analyzed PAF-induced calcium mobilifatty acids (47). It is possible that inhalation of this pulmonq zation. This is important in the signal transduction pathway of P M Receptors on Rat Alveolar Macrophages this mediator (4). Unexpectedly, we found that 10 nM PAF had no effect on calcium mobilization in cells from control animals (Fig. 2). This suggests that the PAF receptors on alveolar macrophages from rats are inactive. Alternatively, a critical number of PAF receptors may be required to elicit functional responses such as calcium mobilization in these cells. We have previously demonstrated that 10 nM PAF readily induces calcium mobilization, as well as oxidative metabolism in hepatic macrophages (5). Our findings that resident alveolar macrophages do not respond to this cytokine provide support for the concept that macrophages develop specialized functions in response to their microenvironment. In contrast to the results obtained with control alveolar macrophages, 10 nM PAF was found to induce calcium mobilization in lung phagocytes from rats exposed to 0 3 (Figs. 2 and 3). At this concentration, PAF had no effect on cell viability as determined by trypan blue dye exclusion (not shown). Mobilization of calcium in response to PAF was rapid and transient, occurring within 1-2 min and returning to base-line levels after 3 min (Fig. 2). PAF-induced calcium mobilization was also found to be dose-dependent in the range of 0.1-10 nM (Fig. 4A), and to be inhibited by triazolam, a PAF receptor antagonist (Fig. 4B). In contrast, lyso-PAF (10 nM), a biologically inactive analog of PAF, had no effect on calcium mobilization (Fig. 4C). These results demonstrate that the response of macrophages from Os-treated rats t o PAF is receptor mediated. In further studies, we found that PAFinduced calcium mobilization was markedly reduced when the cells were pretreated with TMB-8, an inhibitor of intracellular calcium stores (48) (Fig. 4B). In contrast, in the absence of calcium in the culture medium, PAF-induced calcium mobilization was, for the most part, unaffected (Fig. 4B 1. These data suggest that the PAF-induced increases in intracellular calcium were largely derived from intracellular sources. Similarly, in both bovine and human vascular endothelial cells, the transient component of calcium mobilization in response to PAF reflects the release of this ion from internal stores (49, 50). In contrast, calcium mobilized in response to PAF in hepatic macrophages is apparently derived from the extracellular environment (5). Thus, PAF-induced responsiveness appears to be tissue-specific and provides additional evidence for the unique attributes of macrophages from different compartments.
The actions of PAF have been most clearly defined in the lung and include bronchoconstriction, increased vascular permeability with protein exudation, and platelet aggregation (17-24). PAF has also been implicated in the release of cytokines such as tumor necrosis factor from lung cells (24). Interestingly, similar physiological responses are observed in the lung following 0 3 exposure (51,52), suggesting that PAF may be directly involved in at least some of these responses. PAF may also act indirectly, e.g. by mediating Os-induced increases in eicosanoid release (11,12,53). Taken together, our data suggest that PAF may be an important mediator in Os-induced lung injury/ inflammation. Our results demonstrating up-regulation of PAF receptors following Os exposure support this model and are similar t o results for the peritoneal macrophage cell line IC-21, which showed up-regulation of PAF receptors following exposure to inflammatory stimuli in vitro (35).