Introduction

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PAF is a phospholipid that acts as a potent proinflammatory mediator and elicits its biological effects via binding to specific receptors on responsive cells (Prescott et al., 1990). These receptors have been identified in a variety of tissues and cell types (Hwang, 1990), including lung tissue and lung-resident cells such as alveolar macrophages (Schaberg et al., 1991) and epithelial cells (Stoll et al., 1994). In virtually all cell types examined to date, interaction of PAF with specific PAF receptors activates heterotrimeric GTP-binding proteins, which triggers the activation of various protein kinases, such as protein kinase C, tyrosine kinase and MAP kinase (Franklin et al., 1995; Honda et al., 1994), and mobilization of intracellular free calcium. Signal transduction initiated by PAF supposedly mediates diverse cellular effects, including activation of monocytes/macrophages to produce inflammatory mediators such as eicosanoids, TNF-, IL-1 and IL-6, stimulation of eosinophils and basophils to adhere to vascular endothelial cells before their degranulation and up-regulation of adhesion molecules on neutrophils (McCall and O'Flaherty, 1995). A number of studies have suggested that PAF plays a role in modulating acute and chronic lung injury (McCall and O'Flaherty, 1995), such as bronchoconstriction, increased vascular permeability and alveolitis. PAF is also known to be associated with several human diseases, such as asthma (Chung and Barnes, 1991), adult respiratory distress syndrome (Worthen et al., 1983), pulmonary hypertension (Caplan et al., 1990) and sarcoidosis (Scappaticci et al., 1992). It was recently shown that PAF receptors were up-regulated in rat alveolar macrophages by O₃ inhalation (Pendino et al., 1993), which is known to produce lung fibrosis after chronic exposure (Last et al., 1993). Recent studies from our laboratory demonstrated that the PAF receptor antagonist WEB2086 significantly inhibited BL- and amiodarone-induced lung fibrosis in hamsters (Giri et al., 1993a, 1995). These findings prompted us to investigate the role of PAF receptors in the pathophysiology of BL-induced lung fibrosis.

BL, an extensively used antitumor drug, causes interstitial pneumonitis leading to pulmonary fibrosis, a major side effect of this drug. The lung pathological changes are initially characterized by type II epithelial cell proliferation, edema of alveolar walls and an inflammatory exudate of predominantly mononuclear cells (monocytes/macrophages) in the alveolar walls and spaces, followed by excessive deposition of collagen in the lung interstitium (Hay et al., 1991). However, the mechanisms responsible for BL-induced lung injury are not clearly understood. Studies from several laboratories indicated that multiple factors, including generation of reactive oxygen species and lipid peroxidation (Buettner and Moseley, 1992) and cytokines such as TNF-, IL-1 and TGF- (Giri et al., 1993b; Piguet et al., 1989, 1993) produced by lung macrophages (Raghow et al., 1989; Scheule et al., 1992), could be involved in BL-induced lung toxicity. These actions of BL are similar, to some degree, to PAF-induced lung interstitial inflammation, at least in the early stages, and therefore could be inhibited by the PAF receptor antagonist WEB2086 (Giri et al., 1995). Thus, it is possible that increased PAF production and/or up-regulation of PAF receptors after BL treatment could be one of the underlying mechanisms for the pathogenesis of lung fibrosis. To test this hypothesis, we have investigated PAF receptors in lung and the functional activities of PAF receptors in alveolar macrophages from BL-treated hamsters. Our results indicate that BL treatment in vivo up-regulates PAF receptors in the lung and also up-regulates functional PAF receptors in alveolar macrophages in hamsters. It is concluded that the up-regulation of PAF receptors in the lung and alveolar macrophages might constitute one of the important mechanisms for BL-induced lung toxicity.

	Materials and Methods

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Treatment of animals. Golden Syrian hamsters (males weighing 100-120 g) were purchased from Simonsen, Inc. (Gilroy, CA). Hamsters were housed in groups of four in facilities with filtered air and constant temperature and humidity. All care was in accordance with National Institutes of Health guidelines for animal welfare. The hamsters were allowed to acclimate in the facilities for 1 week before all treatments. A 12-hr/12-hr light/dark cycle was maintained, and hamsters had access to water and rodent laboratory chow ad libitum. Hamsters were instilled intratracheally with 7.5 U/kg BL or its vehicle saline (5 ml/kg), under pentobarbital anesthesia, as routinely used in our laboratory (Giri et al., 1986). The animals were sacrificed by decapitation at different time points after the treatment. The hamster lungs were excised, immediately frozen in liquid nitrogen and stored at 80°C until used for radioligand binding studies and hydroxyproline measurement. For collection of alveolar cells, the control and BL-treated hamsters were first anesthetized and then subjected to bronchoalveolar lavage, as described below.

Pulmonary lavage and cell culture. BL- and saline-treated hamsters were anesthetized with pentobarbital and subjected to pulmonary lavage with Ca⁺⁺-free HBSS containing 15 mM HEPES. The cells were pooled from BL- and saline-treated hamsters separately. After being washed twice (200 × g for 10 min at 4°C), the cells were resuspended in Ca⁺⁺-containing HBSS with HEPES. Cell viability in all experiments was >95%, as determined by trypan blue dye exclusion assay. The cell numbers were counted by hemocytometer. For measurement of intracellar Ca⁺⁺ mobilization in lung lavage cells, the cell suspensions were immediately loaded with the fluorescent calcium indicator fura-2/acetoxymethyl ester, as described below. Otherwise, the cells were resuspended in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 15 mM HEPES, 100 U/ml penicillin and 50 µg/ml streptomycin and were plated on coverslips, followed by incubation at 37°C for 1.5 hr to produce alveolar macrophage monolayers (Mosier, 1984).

Lung receptor preparation. The lungs from BL- and saline-treated hamsters were homogenized with a Polytron homogenizer in 25 mM HEPES buffer (pH 7.4) containing 10 mM MgCl₂, 100 µM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin. An aliquot (1 ml) of the homogenate was taken for measurement of hydroxyproline (Woessner, 1961), and the remaining homogenate (9-10 ml) was centrifuged at 110,000 × g at 4°C for 1 hr. After one wash in homogenization buffer, the resultant pellet was resuspended in 25 mM HEPES/10 mM MgCl₂ buffer with a Dounce homogenizer, dispensed in 1-ml aliquots and stored at 80°C until used for radioligand-receptor binding assays. Protein concentration in the receptor preparation was determined by the method of Lowry et al. (1951), after trichloroacetic acid precipitation.

Radioligand binding studies. PAF receptors in lung homogenate from control and BL-treated hamsters were determined by radioligand binding studies using [³H]WEB2086, a potent PAF receptor antagonist (Dent et al., 1989). Briefly, the binding reaction with lung receptor preparations was performed in assay buffer (25 mM HEPES, pH 7.4, 10 mM MgCl₂, 0.1% BSA) in the presence of single or increasing concentrations of [³H]WEB2086. Nonspecific binding was determined by addition of 10 µM unlabeled WEB2086 to the reaction in parallel. The reaction mixture was incubated at 25°C for 1.5 hr, with shaking, and the reaction was terminated by filtration through GF/C glass filter membranes , followed by three rinses with ice-cold assay buffer. Radioactivity retained on the filters was extracted with scintillation cocktail and counted in a scintillation counter. Specific [³H]WEB2086 binding was calculated by subtraction of nonspecific binding (measured in the presence of 10 µM unlabeled WEB2086) from total binding (measured in the absence of unlabeled WEB2086) and is expressed as femtomoles of WEB2086 bound per milligram of protein of receptor preparation.

[Ca⁺⁺]_i measurement. Mobilization of intracellular free calcium in lung lavage cells and alveolar macrophage monolayers in response to PAF was measured with the fluorescent calcium indicator fura-2. Briefly, approximately equal numbers of cells (1 × 10⁵), either in suspension or used to prepare monolayers, were loaded with 1.5 µM fura-2/acetoxymethyl ester in HBSS containing 15 mM HEPES, 0.1% BSA and 30 µg/ml Pluronic F-127, by incubation at room temperature for 30 min. The cells were then washed twice and kept in the same buffer (without fura-2/acetoxymethyl ester) at room temperature. Immediately before [Ca⁺⁺]_i measurements, the cells were placed in Ca⁺⁺-free assay buffer [Ca⁺⁺-free HBSS containing 15 mM HEPES, 0.1% BSA, 0.5 mM ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid]. Fluorescence in the cells was measured with an Hitachi F-2000 spectrofluorometer, with emission at 510 nm and excitation at 340 and 380 nm. [Ca⁺⁺]_i was calculated by using the equation (Grynkiewicz et al., 1985) [Ca⁺⁺]_i = K_d(R  R_min)/(R_max  R)Sf₂/Sb₂, where K_d = 224 nM, the dissociation constant of the fura-2-Ca⁺⁺ complex; R is the measured 340/380 fluorescence ratio; R_max is the maximal fluorescence ratio when the cells are permeated with 0.2 mg/ml digitonin, allowing Ca⁺⁺ to saturate all intracellular fura-2; R_min is the minimal fluorescence ratio after chelation of Ca⁺⁺ by addition of 10 mM ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid; and Sf₂/Sb₂ is the ratio of the fluorescence at 380 nm with fura-2 free and saturated by Ca⁺⁺. The [Ca⁺⁺]_i response of the cells to tested compounds was expressed as a change in peak [Ca⁺⁺]_i caused by Ca⁺⁺ release from internal Ca⁺⁺ stores or extracellular Ca⁺⁺ influx, where the dose-response curves were plotted against different concentrations of PAF.

Materials. [³H]WEB2086 (specific activity, 14.1 Ci/mmol) was obtained from DuPont-NEN. WEB2086 was a gift from Boehringer Ingelheim, and L659,989 from Dr. William Parsons of Merck & Co., Inc. (Rahway, NJ). Stock solutions (10 mM) of WEB2086 and L659,989 were freshly made in 30% ethanol and in dimethylsulfoxide, respectively, and stored at room temperature. PAF-C₁₈, lyso-PAF, HBSS, HEPES, Dulbecco's modified Eagle's medium, fetal bovine serum and BSA were purchased from Sigma Chemical Co. (St. Louis, MO). PAF-C₁₈ and lyso-PAF were dissolved in 100% ethanol to 1 mM and stored at 20°C. Fura-2/acetoxymethyl ester and Pluronic F-127 were from Molecular Probes, Inc. (Eugene, OR). BL sulfate (Blenoxane) was generously supplied by Mr. S. J. Lucania of Bristol-Myers Squibb Pharmaceutical Research Institute (Princeton, NJ).

Statistical analysis. The data are expressed as mean ± S.D. The data were analyzed using the Student-Newman-Keuls test for multiple comparisons among the groups. A value of P  .05 was considered to be the minimal level of statistical significance.

	Results

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BL-induced lung fibrosis and increases in PAF receptor binding. In initial studies, we determined whether BL treatment would induce changes in PAF receptor binding in lungs in the BL-hamster model of lung fibrosis, in a time-dependent manner. The lung homogenate (receptor preparation) was prepared from control and BL-treated hamsters at 3, 7, 14 and 21 days after the treatments, as described in "Materials and Methods." Hydroxyproline, as an index of collagen deposition, and PAF receptor binding were measured in the lung receptor preparations and are shown in figure 1. The receptor binding was carried out by incubation of the lung homogenate with 25 nM [³H]WEB2086, in the presence or absence of 10 µM unlabeled WEB2086 (for determination of nonspecific binding). As shown in figure 1A, BL caused increases in lung collagen deposition at all time points except day 3, as reported in our earlier study (Giri et al., 1986). The specific [³H]WEB2086 binding in the lung receptor preparation from BL-treated hamsters was significantly increased over saline control at all time points, including day 3 (fig. 1B). This suggests that the alteration in PAF receptors could have occurred at an early stage of BL-induced lung fibrosis. Because receptor-ligand interaction in most cases initiates signal transduction events which precede the detectable biological response, the subsequent receptor-related studies were carried out at the earliest time, 3 days after BL or saline treatment.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Effects of intratracheal instillation of BL on hydroxyproline content and specific [3H]WEB2086 binding in hamster lung. A single dose of BL (7.5 U/kg/5 ml) or an equivalent volume of saline (as control) was instilled by the transoral route under pentobarbital anesthesia. Hamsters were sacrificed at different days after the treatment, and their lungs were excised and homogenized. An aliquot of the homogenate was used for measurement of hydroxyproline (Hyp) and its value expressed as micrograms per lung (A). The remaining homogenate was used for receptor preparation, as described in "Materials and Methods." The [3H]WEB2086 binding was performed by incubating the receptor preparation with 25 nM [3H]WEB2086 in the presence or absence of 10 µM unlabeled WEB2086. The specific binding (B) was calculated by subtracting nonspecific binding from total binding and is expressed as femtomoles of [3H]WEB2086 per milligram of protein. Data for both control and BL-treated groups are presented as the mean ± S.D. of five hamsters.

Equilibrium PAF receptor binding. Lung receptor preparations from control and BL-treated hamsters were incubated with various concentrations of [³H]WEB2086 in the presence (for nonspecific binding) or absence (for total binding) of 10 µM unlabeled WEB2086, as described in "Materials and Methods." The equilibrium binding curve (fig. 2A) and its Scatchard plot analysis (fig. 2B) indicated specific and saturable binding of [³H]WEB2086 to the lungs of both BL- and saline-treated hamsters, with a single binding site. The dissociation constant (K_d) of [³H]WEB2086 was 45 nM in control and 41 nM in BL-treated hamster lungs. Although the binding affinity (K_d) of [³H]WEB2086 for the receptor preparations was not different between the two groups, an approximately 2-fold increase in the maximal specific [³H]WEB2086 binding (receptor density) was found in BL-treated hamster lungs (B_max = 202.4 fmol/mg), compared with saline-treated control (B_max = 116.9 fmol/mg). The specific [³H]WEB2086 binding in both cases was competitively inhibited by PAF, in a dose-dependent manner (fig. 2C), suggesting that the increase in specific [³H]WEB2086 binding to lungs after BL treatment was due to increased density of the receptors for PAF.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Characteristics of [3H]WEB2086 binding to PAF receptor preparations from BL-treated and control hamster lungs at 3 days after treatment. The animal treatment and lung receptor preparation were as described in the legend to figure 1 and in "Materials and Methods." The receptor preparation was incubated in triplicate with increasing concentrations of [3H]WEB2086 at 25°C, with shaking, for 1.5 hr. Specific binding was determined as described for figure 1. A, representative equilibrium binding curve of three experiments; B, Scatchard plot analysis of the equilibrium data, showing Kd = 41 nM and Bmax = 202.4 fmol/mg protein in BL-treated hamsters and Kd = 45 nM and Bmax = 116.9 fmol/mg protein in control; C, competitive binding curve generated by incubating 20 nM [3H]WEB2086 in the presence of increasing concentrations of PAF. Competition by PAF was expressed as a percentage of the specific binding in the absence of PAF.

PAF-stimulated intracellular Ca⁺⁺ mobilization in alveolar macrophages. It is well known that PAF-induced intracellular Ca⁺⁺ signaling is mediated by the specific PAF receptor and it is an important component of the PAF signal transduction pathway responsible for many of its cellular effects (Chao and Olson, 1993). Therefore, intracellular Ca⁺⁺ mobilization in PAF-responsive cells can be an ideal index to monitor the functional activity of PAF receptors. To test whether the up-regulated PAF receptors in BL-treated hamster lung were functional, we measured PAF-stimulated intracellular Ca⁺⁺ mobilization in total lung lavage cells and alveolar macrophages, which are considered one of the major factors in BL-induced lung injury. [Ca⁺⁺]_i was measured with the fluorescent calcium indicator fura-2, using a Ca⁺⁺-free/Ca⁺⁺-reintroduction protocol (Clementi et al., 1992) to dissociate and thus precisely quantify Ca⁺⁺ release from internal Ca⁺⁺ stores (in Ca⁺⁺-free buffer) and extracellular Ca⁺⁺ influx (after Ca⁺⁺ reintroduction to the buffer).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effects of intratracheal instillation of BL on functional activity of PAF receptors in lung lavage cells. Hamster treatment was the same as described for figure 1. Alveolar cells were collected by bronchoalveolar lavage at day 3 after the treatment. The cells were loaded with fura-2/acetoxymethyl ester, and [Ca++]i was measured in Ca++-free HBSS/15 mM HEPES/0.1% BSA at 37°C, as described in "Materials and Methods." The concentrations of PAF or lyso-PAF are indicated on the right. Arrows, time of PAF addition to stimulate Ca++ release from internal Ca++ stores and reintroduction of Ca++ to the assay buffer to demonstrate Ca++ influx. This is a representative tracing of three to five different experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Dose-response curve for PAF-induced Ca++ release in lung lavage cells from control and BL-treated hamster lungs. The animal treatment and [Ca++]i measurement were the same as described for figures 1 and 3, respectively. The peak [Ca++]i values above basal levels were measured and plotted against the different concentrations of PAF. Each value represents the mean ± S.D. of three to five experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effects of the PAF antagonist WEB2086 on PAF-induced intracellular Ca++ mobilization in lung lavage cells from control and BL-treated hamsters. The alveolar cells were collected from hamster lungs at 3 days after treatment (see fig. 1 for animal treatment), and the [Ca++]i measurement was performed as described in the legend to figure 3. The cells in suspension were preincubated with different concentrations of WEB2086 (as indicated on the right) for 1 min before stimulation by PAF (10 nM). A, representative tracing showing changes in PAF-induced intracellular Ca++ mobilization by pretreatment with WEB2086; B, dose-response curve for the inhibitory effect of WEB2086 on PAF-induced intracellular Ca++ release. Each point represents the mean of three to five experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Effects of the PAF antagonist L659,989 on PAF-induced intracellular Ca++ mobilization in lung lavage cells from control and BL-treated hamsters. The alveolar cells were collected from hamster lungs at 3 days after treatment (see fig. 1 for animal treatment), and the [Ca++]i measurements were performed as described in the legend to figure 3. The cells in suspension were preincubated with different concentrations of L659,989 (as indicated on the right) for 1 min before stimulation with PAF (10 nM). A, representative tracing showing changes in PAF-induced intracellular Ca++ mobilization by pretreatment with L659,989; B, dose-response curve for the inhibitory effect of L659,989 on PAF-induced intracellular Ca++ release. Each point represents the mean of three to five experiments. DMSO, dimethylsulfoxide.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effects of intratracheal instillation of BL on functional activity of PAF receptors in alveolar macrophages. Lung lavage cells from control and BL-treated hamsters at 3 days after treatment (see fig. 1) were plated on coverslips and incubated at 37°C for 1.5 hr in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, to allow alveolar macrophages to attach. The resultant macrophage monolayer was loaded with fura-2/acetoxymethyl ester, and the [Ca++]i response to different concentrations of PAF (indicated on the right) was measured as described in "Materials and Methods." Arrows, time of addition of different concentrations of PAF to stimulate Ca++ release and reintroduction of Ca++ to the assay buffer to demonstrate Ca++ influx. This is a representative tracing of three to five experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Dose-response curve for PAF-induced Ca++ release in alveolar macrophages from control and BL-treated hamster lungs. See figure 7 for animal treatment and [Ca++]i measurement. The peak [Ca++]i values with PAF-induced Ca++ release were determined and plotted against the different concentrations of PAF. Each point represents the mean of three to five experiments.

	Discussion

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In this study, we demonstrated that BL treatment in vivo increased PAF receptor binding in the hamster lung and sensitized the functional activity of PAF receptor in alveolar macrophages. These results suggest the up-regulation of functional PAF receptors in hamster lung after BL treatment in vivo. Considering these findings in conjunction with our previous reports that the PAF antagonist WEB2086 attenuated both BL-induced (Giri et al., 1995) and amiodarone-induced (Giri et al., 1993a) lung fibrosis in hamsters, the present study provides strong evidence that the up-regulation of functional PAF receptors might be associated with the mechanism underlying the BL-induced lung fibrogenic response.

Macrophages are one of the major cell types responsive to PAF. PAF can stimulate the cells to produce several cytokines, such as TNF- (Ruis et al., 1991), IL-1 (Poubelle et al., 1991), IL-6 (Thivierge and Rola-Pleszczynski, 1994) and other inflammatory mediators such as leukotrienes (Fauler et al., 1989). These cytokines have been demonstrated to be involved in BL-induced lung fibrosis (Piguet et al., 1989, 1993; Scheule et al., 1992). For instance, neutralization of TNF- in vivo by anti-TNF- antibody (Piguet et al., 1990) or soluble TNF- receptor (Piguet et al., 1993) significantly inhibited BL-induced lung fibrosis. Similarly, IL-1 receptor antagonist was found to prevent and cure BL- or silica-induced lung fibrosis (Piguet et al., 1993). Therefore, it is possible that the up-regulation of functional PAF receptors in alveolar macrophages after BL treatment might stimulate these cells to excessively produce various fibrogenic cytokines in response to endogenous PAF. The availability of these cytokines would, in turn, modulate fibroblast proliferation and excess collagen synthesis, a hallmark of fibrosis. Cytokines such as TNF-, IL-1 and IL-6 also control PAF production in macrophages (McCall and O'Flaherty, 1995) by paracrine or autocrine pathways. Obviously, up-regulated PAF receptors and/or induced PAF production may play a key role in the cytokine network of BL-induced lung fibrosis.

Several cell types in the lung, including macrophages, fibroblasts, epithelial cells and endothelial cells, are capable of producing PAF, and the presence of PAF receptors in these cells has been well established (McCall and O'Flaherty, 1995). It has been suggested that, besides pulmonary macrophages, other cell types (such as fibroblasts, epithelial cells and neutrophils) are involved in BL-induced lung fibrosis (Hay et al., 1991). Therefore, increased PAF receptor density in BL-treated hamster lung tissue, as found in the present study, might be contributed by these cell types as well, particularly the lung fibroblasts. Recent studies have indicated that PAF can act as a mitogen to stimulate differentiation and proliferation of noninflammatory cells, including fibroblasts. For example, PAF was reported to stimulate proliferation and transformation of human skin fibroblasts in vitro (Bennet and Birnboim, 1995) and induce expression of the growth-related early-response genes c-jun and c-fos in human fibroblasts (Roth et al., 1995) and of IL-6 and IL-8 in human fibroblasts (Roth et al., 1995). PAF was also found to stimulate the mouse embryonic fibroblast cell line L929, producing IL-6 in a dose-dependent manner (Braquet et al., 1991). In addition, PAF is a potent activator of MAP kinase and MAP kinase kinase in guinea pig PAF receptor cDNA-transfected Chinese hamster ovary cells (Honda et al., 1994). Also, PAF receptor-mediated activation of tyrosine kinase has been found in several cell types (Dhar et al., 1990; Gomez-Cambronero et al., 1991). These kinases have been considered as key factors in the modulation of cell growth and differentiation (Yarden and Ullrich, 1988). Therefore, further studies will determine whether up-regulated PAF receptors in other cell types, in addition to alveolar macrophages, may be important in the cytokine network implicated in the pathogenesis of BL-induced lung fibrosis.

Several cytokines involved in BL-induced lung fibrotic response, as discussed above, are also known to directly up-regulate PAF receptors in vitro in some cells. TGF-, the most significant one, was found to induce PAF receptor expression in monocytic and B cell lines (Parent and Stankova, 1993). TGF-, one of the major cytokines associated with BL-induced lung fibrosis, was significantly increased after BL treatment in vivo in lungs (Madtes et al., 1994; Raghow et al., 1989) and in vitro in pulmonary macrophages (Denholm and Rollins, 1993; Kelley et al., 1991b), lung fibroblasts (Kelley et al., 1991a,b) and endothelial cells (Phan et al., 1991). The anti-TGF- antibody studied in our laboratory (Giri et al., 1993) blocked in vivo BL-induced lung injury, including lipid peroxidation and collagen deposition. Therefore, it is possible that up-regulation of PAF receptors in lung cells could be a mechanism underlying TGF--modulated lung inflammatory responses in BL-treated animals.

Alternatively, BL may directly modulate the functional PAF receptors in macrophages through interaction with PAF receptor or interruption of the plasma membrane. These interactions may then change the PAF receptor conformation or functional status. Specific binding sites for [³H]BL were found in rat alveolar macrophages (Denholm and Phan, 1990). Although signal transduction mediated by the binding is not known, it is possible that BL binding may communicate with PAF receptors at some level of the signal transduction pathway. On the other hand, free radical reactions and lipid peroxidation initiated by BL in the lung (Buettner and Moseley, 1992, 1993) could change biophysical characteristics of the plasma membrane and thus may interrupt the membrane lipid environment associated with functional activities of PAF receptors. It was recently reported that in vivo acute exposure to O₃, a potent oxidant, was associated with the induction of functional PAF receptors in rat alveolar macrophages (Pendino et al., 1993); the development of lung fibrosis is one of the consequences of chronic exposure to O₃. It is likely that the up-regulation of PAF receptors at the early stage of lung fibrosis constitutes a common denominator in the pathogenesis of a variety of fibrogenic agents of multifactorial origins.

Intracellular calcium mobilization is a universal second message of PAF-induced signal transduction in all responsive cells, and this is accomplished by Ca⁺⁺ release from internal Ca⁺⁺ pools and extracellular Ca⁺⁺ influx (Chao and Olson, 1993). In this study, we demonstrated that PAF significantly induced both Ca⁺⁺ release from internal Ca⁺⁺ pools and extracellular Ca⁺⁺ influx in alveolar macrophages, with much higher potency in BL-treated hamsters than in control hamsters (figs. 4 and 8). The different potencies in the two groups were also shown in the inhibition of PAF-induced Ca⁺⁺ responses by PAF receptor antagonists (figs. 5 and 6). This indicates that functionally active PAF receptors on alveolar macrophage surfaces were up-regulated by BL treatment; the blockade of these functional receptors by specific antagonists might have beneficial effects against BL-induced lung fibrosis, as reported in our earlier paper (Giri et al., 1995).