Introduction

Abstract Introduction References

Mesenchymal cell hyperplasia is a central feature of pulmonary fibrogenesis following acute lung injury by a variety of occupational and environmental agents (1, 2). Myofibroblasts are the primary mesenchymal cell type that contribute to extracellular matrix deposition or "scarring" in the forming fibrotic lesion (3). The lung myofibroblast proliferative response is driven in part by platelet-derived growth factor (PDGF) secreted by activated alveolar macrophages (4, 5).

PDGF is composed of two polypeptide chains, termed A and B, that dimerize via disulfide bonds to form functional PDGF-AA, -AB, or -BB isoforms (6). PDGF isoforms bind and dimerize cell-surface tyrosine kinase receptors termed PDGF -receptor (PDGF-R) and PDGF -receptor (PDGF-R); PDGF-AA binds only to PDGF-R, whereas PDGF-B chain isoforms bind both PDGF-R and PDGF-R (7). Both of these PDGF receptors undergo autophosphorylation in response to PDGF binding and initiate signaling cascades that mediate mesenchymal cell mitogenesis and/or chemotaxis.

Recent work in our laboratory has focused on the regulation of receptors for PDGF as a possible mechanism of myofibroblast hyperplasia during lung fibrogenesis. The PDGF-R on rat lung myofibroblast (RLMF) is expressed at low levels but is inducible by interleukin (IL)-1 (8), basic fibroblast growth factor (FGF-2) (9), and bacterial lipopolysaccharide (10). Conversely, transforming growth factor-1 (TGF-1) downregulates PDGF-R (11). PDGF-R is constitutively expressed at high levels but is not inducible. PDGF-R is required for maximal mitogenic and chemotactic responses of lung myofibroblasts to PDGF in vitro (8, 12), and induction of PDGF-R precedes a fibrotic response following acute lung injury in vivo (2). We and others have postulated that upregulation of PDGF-R favors the formation of a PDGF- heterodimeric receptor complex that mediates a stronger mitogenic signal compared with the normally abundant PDGF- receptor complex (8, 13).

Prostaglandin (PG) E₂ is a lipid mediator that can be derived from cell membrane glycophospholipids through the sequential enzymatic actions of phospholipase-A₂, PGH₂ synthases (cyclooxygenases [COX]), and PGE₂ isomerase (14). Several studies have shown that IL-1 (15, 16) and other growth factors such as PDGF (17) and TGF-1 (20) stimulate the production of PGE₂ via activation of the COX pathway. Because IL-1 stimulates the production of PGE₂ and upregulates expression of PDGF-R, we postulated that IL-1 might mediate upregulation of PDGF-R through a PGE₂-dependent mechanism. Contrary to this hypothesis, we found that IL-1-induced expression of PDGF-R was indomethacin-insensitive, suggesting that PGE₂ production was not required for upregulation of PDGF-R. However, PGE₂ inhibited upregulation of PDGF-R that was stimulated by IL-1, resulting in a suppression of PDGF-stimulated mitogenesis.

	Materials and Methods

Reagents

PDGF-AA, -AB, and -BB; anti-PDGF-R; anti-PDGF-R; and IL-1 were purchased from Upstate Biotechnology (Lake Placid, NY). PGE₂ was purchased from Cayman Chemical Co. (Ann Arbor, MI). [¹²⁵I]PDGF-AA was obtained from Biomedical Technologies, Inc. (Stoughton, MA). Horseradish peroxidase (HRP)-labeled swine anti-rabbit immunoglobulin G was purchased from Dako Corp. (Carpinteria, CA). Indomethacin was from Sigma Chemical Co. (St. Louis, MO). The COX-2 selective inhibitor NS-398 was purchased from Cayman Chemical Co. [³H]thymidine was obtained from Amersham (Arlington Heights, IL).

Isolation and Characterization of RLMF

Early passage RLMF were isolated and characterized from male Sprague-Dawley rats as described previously (10). RLMF stained positively for vimentin and -smooth muscle actin and negatively for factor VIII and rat leukocyte common antigen (OX 1). In addition, examination of glutaraldehyde-fixed pellets of RLMF by transmission electron microscopy showed ultrastructural features consistent with a myofibroblast phenotype (abundant intermediate filaments and rough endoplasmic reticulum, and lack of Weibel-Palade bodies characteristic of endothelial cells). Myofibroblasts were grown to confluence in 10% fetal bovine serum (FBS)/Dulbecco's modified Eagle's medium (DMEM) before being seeded for assays of receptor expression or [³H]thymidine incorporation.

[¹²⁵I]PDGF-AA Receptor Assays

Binding of [¹²⁵I]PDGF-AA was assayed on confluent, quiescent cell cultures. RLMF in 24-well plates were grown to confluence in 10% FBS/DMEM and then rendered quiescent for 24 h in serum-free defined medium (SFDM) that consisted of Ham's F-12 with N-2-hydroxyethylpiperazine- N'-ethane sulfonic acid (Hepes), CaCl₂, and 0.25% bovine serum albumin (BSA) and supplemented with an insulin, transferrin, selenium mixture (Boehringer-Mannheim, Indianapolis, IN). Cells were then treated for 24 h with IL-1 (2 ng/ml), PGE₂ (1 µM), or a combination of IL-1 and PGE₂. PGE₂ was delivered in ethanol (final concentration of 0.1%), which did not affect experimental parameters. As a control, all other treatments, including SFDM alone, contained 0.1% ethanol. In other experiments, indomethacin (5 µM) was added to inhibit PG synthase and thereby block PGE₂ production by RLMF. The following day, cultures were chilled to 4°C and rinsed in cold binding buffer (Ham's F-12 with Hepes, CaCl₂, and 0.25% BSA). For measurements of a single concentration of radioligand bound, 1 ng/ml of [¹²⁵I]PDGF-AA was added in the absence or presence of nonradioactive PDGF-AA (500 ng/ml) to measure total and nonspecific binding, respectively. In some experiments, saturation binding analysis was performed wherein increasing concentrations of radioligand were added to the cultures (0.5 to 40 ng/ml of [¹²⁵I]PDGF-AA). Binding was allowed to occur for 3 to 4 h at 4°C on an oscillating platform. Cells were then rinsed 3 times in ice-cold binding buffer and solubilized in 1% Triton X, 0.1% BSA, and 0.1 N NaOH, and cell-associated radioactivity was counted in a -counter. The saturation binding data was subjected to Scatchard analysis (21) and linear regression analysis to obtain the maximum number of binding sites (B_max) and dissociation constant (K_d).

Western Blotting

RLMF were grown to confluence in 75-cm² flasks and rendered quiescent for 24 h in SFDM. Cultures were exposed to IL-1 (2 ng/ml), PGE₂ (1 µM), or a combination of PGE₂ and IL-1 for 24 h. Cells were washed with ice-cold phosphate-buffered saline (PBS) and 250 ml of lysis buffer (50 mM Tris-HCl; 1% Triton X-100; 150 mM NaCl; 1 mM ethyleneglycol-bis-[-aminoethyl ether]-N,N'-tetraacetic acid; 1 mM phenylmethylsulfonyl fluoride; 0.25% Na-deoxycholate; 1 µg/ml each of aprotinin, leupeptin, pepstatin; 1 mM Na₃VO₄; and 1 mM NaF) was added to cover the surface of the attached cells for 20 min. Lysates were stored at 70°C. A total of 20 µl of each sample mixed with sample buffer was boiled for 5 min before electrophoresis in a 2 to 15% Tris-glycine sodium dodecyl sulfate (SDS) polyacrylamide gel (Integrated Separation Systems, Hyde Park, MA) for 2 h at 130 V and 30 mA. The protein on the gel was transferred to a nitrocellulose membrane (Hybond; Amersham). The membrane was blocked with 3% milk/PBS for 1 h before addition of a rabbit antimouse PDGF-R or -R antibody overnight. After washing 3 times with PBS-Tween, a secondary HRP-conjugated swine antirabbit antibody was added for 90 min. An ECL luminol kit (Amersham) was used for detection of bound secondary antibody.

Northern Analysis

Total RNA was isolated with TRI reagent (Molecular Research Center, Cincinnati, OH) from confluent cultures of RLMF after being rendered quiescent in SFDM and exposed for 5 h to SFDM or SFDM supplemented with IL-1, PGE₂, or a combination of IL-1 and PGE₂. A total of 20 µg of each sample was electrophoresed in 1% agarose/ 2 M formaldehyde gels and capillary transferred onto Immobilon S membranes (Millipore Corp., Bedford, MA). A rat complementary DNA probe for the PDGF-R, kindly provided by Yutaka Kitami (Ehime University, Ehime, Japan), was labeled with [-³²P]deoxycytidine triphosphate using a Prime-It II Random primer labeling kit (Stratagene, La Jolla, CA). The autoradiographic signal was visualized with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA).

[³H]Thymidine Incorporation

Cells were grown to confluence in 24-well plates and rendered quiescent in 0.5% FBS for 24 h. The cultures were exposed to IL-1 (2 ng/ml), PGE₂ (1 µM), a combination of IL-1 and PGE₂, or 0.5% FBS alone (control) for another 24 h, after which medium containing 0.5% FBS, 5 mCi/ ml [³H]thymidine, and one of the three isoforms of PDGF (50 ng/ml) was added for a final 36 h. The medium was aspirated and the cells were rinsed three times with ice-cold PBS and fixed for 10 min in ice-cold 5% trichloroacetic acid. The precipitate was rinsed three times in ice-cold water, solubilized in 0.2 N NaOH/0.1% SDS, and diluted in Ecolume (ICN, Costa Mesa, CA). Radioactivity was measured in a scintillation counter. Cell counts were performed on all treated cultures, and [³H]thymidine data was corrected for cell number as counts per minute per 10⁶ cells.

PGE₂ Enzyme-Linked Immunosorbent Assay

Fibroblast supernatants were analyzed for PGE₂ with an enzyme-linked immunosorbert assay (ELISA) kit (Cayman Chemical Co.) according to the manufacturer's instructions.

Statistical Analysis

The Systat statistical package was used for all analyses (Systat, Evanston, IL). Two-tailed t tests were performed to compare the control group with a treatment group (IL-1 or PGE₂) or to compare two treatment groups (e.g., IL-1 versus IL-1/PGE₂). Values were considered significantly different if P < 0.05.

	Results

PGE₂ Synthesis Is Not Required for Upregulation of [¹²⁵I]PDGF-AA Binding Sites

Exposure of RLMF to IL-1 for 24 h increased [¹²⁵I]- PDGF-AA binding several-fold (Figure 1A) and stimulated the release of PGE₂ into RLMF-conditioned medium (Figure 1B). Indomethacin completely inhibited the IL-1-stimulated production of PGE₂ by RLMF, but did not inhibit IL-1-stimulated upregulation of PDGF-R. Instead, indomethacin alone stimulated a 3-fold increase in [¹²⁵I]PDGF-AA binding to RLMF cultures, and indomethacin significantly enhanced (P < 0.05) IL-1-stimulated upregulation of [¹²⁵I]PDGF-AA binding (Figure 1A).

View larger version (25K): [in this window] [in a new window]   Figure 1.   IL-1-stimulated upregulation of [125I]PDGF-AA binding sites on RLMF does not require synthesis of PGE2. (A) Induction of PDGF-R by IL-1 (1 ng/ml) was measured by an [125I]PDGF-AA binding assay as described in MATERIALS AND METHODS. Indomethacin (10 µM), an inhibitor of PGE2 synthesis, did not block the increase in [125I]PDGF-AA binding stimulated by IL-1, but instead caused a significant increase (P < 0.05) in [125I]PDGF-AA binding. (B) PGE2 secretion by RLMF into cultured supernatants was increased by IL-1 treatment as determined by ELISA, and indomethacin effectively inhibited synthesis of PGE2 by RLMF.

PGE₂ Counteracts IL-1-Induced Upregulation of [¹²⁵I]PDGF-AA Binding Sites

Exposure of RLMF to IL-1 in the presence of exogenous PGE₂ reduced the IL-1-stimulated increase in [¹²⁵I]PDGF-AA binding by 60 to 70% (Figure 2A). Another prostanoid, PGD₂, also reduced IL-1-induced upregulation of [¹²⁵I]PDGF-AA binding by ~ 70% at a concentration of 1 µM PGD₂ (data not shown). Scatchard analysis of [¹²⁵I]- PDGF-AA saturation binding data demonstrated that PGE₂ reduced B_max, but did not significantly alter the affinity of [¹²⁵I]PDGF-AA binding (K_d ~ 0.1 nM for all treatments) (Figure 2B). Experiments using a range of PGE₂ concentrations (10 nM to 1 µM) showed that 1 µM PGE₂ was necessary to maximally suppress IL-1-stimulated upregulation of PDGF-R (Figure 3). Furthermore, PGE₂ levels as high as 10 µM were not cytotoxic to RLMF (data not shown). Treatment of cells with indomethacin (10 µM) or the selective COX-2 inhibitor NS-398 (100 µM), both of which block the synthesis of endogenous PGE₂, attenuated the downregulatory effect of exogenous PGE₂ on [¹²⁵I]- PDGF-AA binding sites at 10 and 100 µM PGE₂. However, a higher concentration of PGE₂ (1 µM) overcame the effects of indomethacin and NS-398 (Figure 3).

View larger version (24K): [in this window] [in a new window]   Figure 2.   PGE2 counteracts IL-1-stimulated upregulation of PDGF-R on rat lung myofibroblasts. (A) Induction of PDGF-R by IL-1 (1 ng/ml) was measured by an [125I]PDGF-AA binding assay as described in MATERIALS AND METHODS. PGE2 (1 µM) significantly reduced (P < 0.01) the increase in [125I]PDGF-AA binding induced by IL-1 treatment. (B) Scatchard analysis of [125I]PDGF-AA saturation binding data demonstrated that PGE2 reduced the number of PDGF-AA binding sites by ~ 2-fold without affecting the affinity of PDGF-AA for PDGF-R (i.e., no change in dissociation constant, Kd 0.1 nm).

View larger version (20K): [in this window] [in a new window]   Figure 3.   Indomethacin and the selective COX-2 inhibitor NS-398 attenuate PGE2-mediated downregulation of [125I]PDGF-AA binding to RLMF. Confluent, quiescent cultures of RLMF were treated with indomethacin (10 µM) or NS-398 (100 µM) in serum-free defined medium for 24 h, then treated with increasing concentrations of PGE2 (0.01 to 1 µM) in the presence of 10 ng/ml IL-1 for 24 h. PGE2 caused a significant reduction in [125I]PDGF-AA binding at 0.1 µM (P < 0.05) and 1 µM (P < 0.01). In cultures pretreated with indomethacin (INDO) or NS-398, significant downregulation of [125I]PDGF-AA binding (P < 0.01) was observed only at 1 µM PGE2.

PGE₂ Inhibits IL-1-Stimulated Upregulation of PDGF-R Protein and mRNA without Affecting PDGF-R

Whole-cell lysates were analyzed by Western blotting using antibodies specific to either PDGF-R or PDGF-R. In agreement with the [¹²⁵I]PDGF-AA binding data, which is an indirect measure of cell-surface PDGF-R, Western blots showed that PGE₂ counteracted upregulation of PDGF-R induced by IL-1 treatment (Figure 4). Expression of PDGF-R was not affected by treatment with IL-1, PGE₂, or the combination of both agents (Figure 4). Expression of PDGF-R mRNA was induced 5-fold by IL-1, and PGE₂ reduced IL-1 upregulation of PDGF-R mRNA by ~ 70% (Figure 5).

View larger version (61K): [in this window] [in a new window]   Figure 4.   Western blotting of PDGF-R and PDGF-R using cell lysates from RLMF that were treated with IL-1 in the absence or presence of PGE2. PDGF-R was upregulated by IL-1 (1 ng/ml) and PGE2 (1 µM) counteracted the IL-1-induced increase in the expression of this receptor. Expression of the PDGF-R subtype was not affected by IL-1 or PGE2.

View larger version (31K): [in this window] [in a new window]   Figure 5.   PGE2 suppression of IL-1-induced upregulation of PDGF-R is controlled at the pretranslational level. (A) Northern blotting of PDGF-R using total RNA from RLMF that were treated for 5 h with PGE2 (1 µM), IL-1 (2 ng/ml), or PGE2 and IL-1. (B) Densitometry was used to normalize PDGF-R mRNA expression against 28s ribosomal RNA for each treatment. PDGF-R mRNA expression was increased ~ 4-fold by IL-1, and PGE2 suppressed the IL-1-mediated increase in PDGF-R mRNA by ~ 75%.

PGE₂ Inhibits IL-1-Enhanced Mitogenic Responses to PDGF Isoforms

PDGF-B chain isoforms were potent mitogens for RLMF, whereas PDGF-AA did not stimulate mitogenesis under control conditions (Figure 6). Pretreatment of RLMF for 24 h with IL-1 enhanced PDGF-AB, and PDGF-BB stimulated [³H]thymidine incorporation by twofold but increased PDGF-AA-stimulated [³H]thymidine incorporation by only ~ 50%. The effect of IL-1 pretreatment in enhancing PDGF-stimulated mitogenesis was significantly suppressed by coincubation of the IL-1 with 1 µM PGE₂ (Figure 6). In contrast to suppression of PDGF-stimulated mitogenesis, PGE₂ did not inhibit epidermal growth factor (EGF)- or FGF-2-stimulated [³H]thymidine incorporation (Figure 7). Instead, the mitogenic activity of both EGF and FGF-2 was increased ~ 50% by pretreatment with 1 µM PGE₂.

View larger version (27K): [in this window] [in a new window]   Figure 6.   IL-1 pretreatment increases PDGF-stimulated mitogenesis of RLMF, and this effect is blocked by coincubation with PGE2. Confluent, quiescent cells were pretreated for 24 h with 10 ng/ml IL-1 in the absence or presence of 1 µM PGE2 before the simultaneous addition of PDGF isoforms (50 ng/ml) and [3H]thymidine (5 µCi/ml). IL-1 pretreatment increased the mitogenic potential of PDGF isoforms by ~ 2-fold. PGE2 reduced IL-1- enhanced, PDGF-stimulated mitogenesis significantly (P < 0.05 for PDGF-AB and PDGF-BB).

View larger version (38K): [in this window] [in a new window]   Figure 7.   Differential effects of PGE2 on PDGF and EGF- or FGF-2 stimulated [3H]thymidine incorporation in RLMF. Confluent, quiescent cells were pretreated for 24 h with or without 20 ng/ml IL-1. Cultures were then treated with or without 1 µM PGE2 for 1 h before the addition of 10 ng/ml PDGF-BB, EGF, or FGF-2 in the presence of [3H]thymidine (5 µCi/ml). PGE2 decreased PDGF-BB-stimulated growth (with or without IL-1 pretreatment to upregulate PDGF-R), and the mitogenic effects of EGF and FGF-2 were enhanced ~ 50% by pretreatment with PGE2.

	Discussion

The mitogenic and chemotactic responses of myofibroblasts to PDGF depend on the relative numbers of PDGF-R and PDGF-R at the cell surface (8, 12). We recently reported that IL-1 produced by particle-activated macrophages is a major upregulatory factor for the myofibroblast PDGF-R, and that induction of this receptor increased the mitogenic response to PDGF isoforms several-fold (22). The mechanism whereby IL-1 induces PDGF-R remains unclear, although we have recently found that IL-1-stimulated upregulation of PDGF-R is transcriptionally regulated and is independent of nuclear factor-B and AP-1 signaling pathways (23). Because IL-1 is a well-known stimulator of PGE₂ production (15, 16), we tested the hypothesis that IL-1 could act through a PGE₂-dependent pathway to upregulate the PDGF receptor. However, this turned out not to be the case because IL-1 induction of PDGF-R was not inhibited by indomethacin, which completely abolished the production of PGE₂ by myofibroblasts treated with IL-1. Instead, we observed that PGE₂ functions as a negative regulator of the PDGF-R.

PGE₂ has been reported to stimulate or inhibit cell proliferation, depending on the cell type studied and the culture conditions employed. The proliferation of NIH 3T3 cells is stimulated by the addition of PGE₂ (24), but PGE₂ inhibits the growth of quiescent lung fibroblasts and smooth-muscle cells (25, 26). In the present study, PGE₂ had an inhibitory effect on PDGF-AB, and PDGF-BB stimulated mitogenesis following induction of PDGF-R by IL-1 (Figure 6). The inhibitory effect of PGE₂ on PDGF-stimulated myofibroblast mitogenesis was due, at least in part, to suppression of PDGF-R. We previously reported that maximal biologic responses to PDGF require expression of PDGF-R in combination with PDGF-R (8, 12). Thus, our data suggest that PGE₂ could attenuate the fibroproliferative response through suppression of PDGF receptor expression. This view of PGE₂ as an antifibrogenic mediator has been supported by others. For example, fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize PGE₂ and to express COX-2, as compared with lung fibroblasts from individuals with no fibrotic disease (27).

Whether PGE₂ actually functions in vivo to suppress myofibroblast mitogenesis remains unclear. In contrast to suppression of PDGF-stimulated mitogenesis by PGE₂, EGF- and FGF-2-mediated [³H]thymidine incorporation were enhanced ~ 50% by PGE₂ (Figure 7). Our observation of enhanced EGF-stimulated mitogenesis by PGE₂ is similar to reports by other investigators wherein EGF-stimulated mitogenesis of BALB/c 3T3 cells (28) or mammary epithelial cells (29) was enhanced by the addition of PGE₂. Thus, PGE₂ could either positively or negatively affect lung myofibroblast mitogenesis, depending on the specific growth factor studied. Given the differential effects of PGE₂ on various growth factors, future studies should address the role of PGE₂ in modulating a fibrogenic response. For example, examining the fibrogenic response of null mice that lack PG synthase-1 (30) or PG synthase-2 genes (31) could clarify the contribution of PGE₂ to lung fibrogenesis.

The PGE₂ concentration required for maximal suppression of IL-1-induced upregulation of PDGF-R in the present study (10⁶ M) could be physiologically relevant and is consistent with other studies reporting that concentrations above 10⁷ M are required for maximal antiproliferative effects on NIH 3T3 fibroblasts (24) and cultured lung fibroblasts (25). The concentration of PGE₂ in human airway epithelial lining fluid is 5 × 10⁸ M, and this concentration is likely an underestimate of concentrations at cell-surface receptors (32). The concentration of PGE₂ measured in RLMF cultured supernatants was 3 × 10⁸ M (Figure 1B). However, the local concentration of PGE₂ in the microenvironment of the lung interstitium could be much higher than the concentration measured in vitro, especially given the dilution of the fibroblast-derived PGE₂ in culture medium (i.e., ~ 5 × 10⁶ RLMF are cultured in a 150-cm² flask containing 30 ml of SFDM). Our study was complicated by the fact that we treated myofibroblasts with IL-1 to induce PDGF-R, yet IL-1 also induces PGE₂. Blocking PGE₂ production with indomethacin or NS-398 attenuated the effect of exogenous PGE₂ on PDGF-R downregulation (Figure 3). These data suggest that endogenous PGE₂ (or perhaps another prostanoid) contributed to the effect of exogenous PGE₂ on suppression of PDGF-R. We observed that another prostanoid, PGD₂, suppressed IL-1-induced upregulation of [¹²⁵I]PDGF-AA binding. Thus, it is possible that other prostanoids could be important in the regulation of the PDGF receptor system.

Several pulmonary cell types may contribute to local PGE₂ production during an inflammatory response. For example, activated alveolar macrophages are a source of PGE₂ and TGF-1, both of which suppress PDGF-R. Interstitial lung macrophages are perhaps better candidates than alveolar macrophages as potential sources of PGE₂ in the lung interstitium because they are in close proximity to myofibroblasts and have been reported to produce PDGF (33). Mesenchymal cells such as the myofibroblasts used in the present study could also contribute to local PGE₂ levels during lung inflammation. Finally, bronchial epithelial cells are also a source of PGE₂ (34), and both PGE₂ and TGF-1 have been identified as fibroblast antimitogenic factors in supernatants from bronchial epithelial cells (35).

We previously reported that the PDGF-R on lung myofibroblasts is downregulated by TGF-1 (11). Although both TGF-1 and PGE₂ suppress expression of PDGF-R and inhibit lung myofibroblast proliferation, TGF-1 also stimulates extracellular matrix production by myofibroblasts, leading to irreversible scarring of the lung interstitium (36, 37). Thus, TGF-1 is a potent profibrogenic agent, whereas PGE₂ appears to function in the resolution of an inflammatory response by inhibiting fibroblast growth and suppressing extracellular matrix production.

In summary, we have found that PGE₂ suppresses PDGF-stimulated RLMF proliferation by suppression of PDGF-R. Furthermore, PGE₂ counteracts IL-1-stimulated upregulation of PDGF-R. It is possible that PGE₂ functions in a negative feedback loop to suppress PDGF-R and consequently PDGF-stimulated myofibroblast proliferation following exposure to IL-1.