Regulation of TXB2 and PGE2 production by TGF-β1 in in vitro silica dust-exposed rat alveolar macrophage

We investigated the effect of transforming factor factor-β1 (TGF-β1) on thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) production in in vitro silica dust-exposed rat alveolar macrophages (AM). In the presence of 5 μg of anti-TGF-β1 antibodies, TXB2 production decreased, but PGE2 production increased. Addition of 2 ng of TGF-β1 to the culture medium potentiated TXB2 production, but PGE2 production apparently did not change. At 50 ng of TGF-β1, TXB2 production decreased, and PGE2 production varied. Our data suggest that in rat AM: (1) both endogenous and exogenous TGF-β1 regulate TXB2 production; and (2) in the absence of endogenous TGF-β1 the liberation of PGE2 increases; however, exogenous TGF-β1 does not have a regulatory effect on PGE2.


Introduction
Chronic airway inflammation and lung fibrosis are common features of prolonged exposure to mineral particles of silica in humans and in rodents. Alveolar macrophages (AM) have an essential role in lung clearance and defence mechanisms against inhaled particles. 2 They fulfil these functions via inflammatory mediators produced upon macrophage activation. Among the many mediators are arachidonic acid metabolites such as thromboxane A2 (TXA2) and prostaglandin E2 (PGE2). Thromboxane A2 (of which TXB2 is the stable metabolite) is a very potent platelet aggregating factor and pulmonary vasoconstrictor. A recent study of the regulatory effect of TXB2 on the proliferation of vascular smooth muscle cells from rats demonstrated a rapid build-up of cytoskeletal protein in these cells in hypertension, suggesting that TXB2 may play a role in the remodelling of the vascular wall in hypertension. '4 Prostaglandin E2 is one of the key inflammatory mediators with multiple functions. In the lung, it induces bronchodilation and causes an increase in epithelial cell chloride secretion. 5 PGE2 induces vasodilatation in unventilated foetal lung. 6 Another mediator that is produced by alveolar macrophages is transforming growth factor-131 (TGF-131). TGF-131 is a multifunctional peptide. Although pulmonary macrophage-derived mediators have a pivotal role in lung inflammation and fibrosis, the interrelationship between these mediators is just beginning to be understood. We have shown the very early release (15 min) of TGF-131 from silica dust-exposed rat alveolar macrophages. 1 The amount of TGF-131 released at this time was the greatest over 24h kinetic studies, and preceded the production of TXB2 and PGE2. To further unravel the interrelationship between eicosanoids and TGF-131 produced by alveolar macrophage, the modification of TXB2 and PGE2 production by TGF-131 in in vitro silica-exposed rat alveolar macrophages was investigated. We observed decreased production of TXB2 in the presence of anti-TGF-131 antibodies, or high concentration (50 ng) of TGF-131. TXB2 production increased in the presence of a low concentration of TGF-J31 (2ng) in the culture medium. PGE2 production increased in (C) 1995 Rapid Science Publishers the presence of anti-TGF-13 antibodies. Exogen-bated at 37C for 24h. After incubation the ous TGF-31 at low or high concentration did not medium containing arachidonic acid was have any effect on PGE2 production in rat alveo-removed and fresh culture medium was placed in lar macrophages, the wells. Macrophages were activated with a microcrystalline form of silica dust (Min-U Sil, 2-Materials and Methods 71.tm diameter, provided by Dr Peter Bolsitis, MIT) at a final concentration of 2001.tg/ml, at Materials.. Hanks' solution and Dulbecco's phos-37C for 24h, in the absence or presence of phate buffered saline (DPBS), and HEPES (4-(2anti-TGF-[31 antibodies, or TGF-3. Nonspecific hydroxyethyl)-l-piperazine ethanesulfonic acid) antibody as an isotype-matched control for antibuffer were prepared in our laboratory. Six-well TGF-[3 antibody was tested. Media were collected tissue culture plates were obtained from Falcon into silianized tubes for the determination of Laboratory Products (Beckton and Dickinson eicosanoids. Labware, Lincoln Park, NJ). DMSO (dimethylsulfoxide) was obtained from Calbiochem (San Radioimmunoassay for TXB2 and PGE2: Fol-Diego, CA). Allopurinol, oxypurinol, allopurinol lowing incubation, the culture medium was riboside, Penn/Strep, M199 medium (without removed and acidified to pH 3.0 with 1 N HC1. phenol red), and human serum albumin were all Eicosanoids were extracted twice with 2 ml ethyl obtained from Sigma (St. Louis, MO). Human acetate/cyclohexane (1:1). The extracts were recombinant and polyclonal anti-TGF-13 antibody stored at -20C until analysis of eicosanoids by were obtained from R&D Systems (Minneapolis, specific radioimmunoassay, as described pre-MN). RIA reagents for TXB2 and PGE2 assay viously. 12 The antibodies utilized in the assay of were purchased from Advanced Magnetics, Inc. TXB2 or PGE2 showed less than 1% cross-reac-(Boston, MA). tivity with other eicosanoids.
Isolation and culture of rat alveolar macro-Cell viability test.. The lactate dehydrogenase phages.. Sprague-Dawley rats weighing approxi-(LDH) activity in rat culture conditions was meamately 225 g were lavaged ex vivo. Rats were sured using a Sigma kit for LDH. There was no given an intramuscular injection of sodium pen-detectable LDH enzyme activity in the culture tabarbital (10mg) and the rib cage was opened medium from rat alveolar macrophages.
to expose the lungs. An incision halfway through the trachea was made and the lavage tube of the Data analysis.. Results are presented as means trachea cannula was inserted into the incision. _--t-S.E. of a number of independent experiments. The lungs were washed five times each with 10ml of lavage fluid (phosphate-buffered saline containing 0.1% EDTA, at 37C). The collected Results lavage fluid was filtered through sterile Nitex gauze and centrifuged at 250 x g for 10 min.
The effect of TGF-fll on 2XB production in rat The supernatant was discarded, and the cells alveolar macrophages: Table 1 shows the effect resuspended in M199, 10mM HEPES 0.3% of anti-TGF-[3 antibodies, or low or high conhuman serum albumin, 100U/ml penicillin and centrations of TGF-I3, on TXB2 production in 1001.tg/ml streptomycin (culture medium). Cells silica-exposed rat alveolar macrophages. In the from all rats were pooled and centrifuged again presence of 5 l.tg of anti-TGF-[3 antibodies in the at 250 x g for 10 min. Cells were enumerated in culture medium, during the process of macroa haemocytometer. Viability was determined by phage activation with silica, the production of the exclusion of trypan blue and cells were TXB2 was inhibited. The TGF-[3, at 2ng conplated at 1 x 106/ml of culture medium in 6-well centration in the culture medium, potentiated culture plates. The cells were preincubated for TXB2 production. At 50ng of TGF-[3 in the l h at 37C in a humidified atmosphere at 5% culture medium, the production of TXB2 was CO in air. The nonadherent cells were removed inhibited. by aspiration. Fresh culture medium (37C) was placed in the wells and unlabelled arachidonic The effect of TGF-fll on PGEe production in rat acid was added at a final concentration of 3 l.tM. alveolar macrophages Table 2 shows the effect This incubation with arachidonic acid was con-of anti-TGF-[3 antibodies, or low or high conducted in order to replenish endogenous arachicentrations of TGF-13, on PGE2 production in donic acid utilized during the exaggerated silica-exposed rat alveolar macrophage. In the eicosanoid production which occurs during cell presence of 5 l.tg of anti-TGF-3 antibodies in the isolation and attachment. The cells were incu-culture medium, the production of PGE2 was Modification of 7XBe and PGEe production by TGF-  potentiated. The addition of 2ng of TGF-]31 to the culture medium had no apparent effect on PGE2 production in either of two experiments performed. At 50ng of TGF-[3, the production of PGE2 varied.

Discussion
This study was designed to evaluate the regulation of TXB2 and PGE2 production by TGF-]3 in alveolar macrophages. The reason for these studies was to supplement the limited information in the literature on the interrelationship between the inflammatory mediators released by a specific inflammatory cell. TXB2, PGE2 and TGF-3 are the principal inflammatory mediators released by alveolar macrophages during the lung's defence process against pathogens or foreign matter. We have demonstrated in earlier studies that these three mediators are released from rat alveolar macrophages upon exposure to silica dust. In those studies we observed a striking difference between the kinetics of release of TGF-3 (maximum release at 15 min post-silica dust) and TXB2 (plateau release at 6 h post-silica dust), or PGE2 release (plateau release at 6h post-silica dust). Because it is thought that TGF-1 has a central regulatory role in vascular physiology and pathology, we formulated a hypothesis that perhaps TGFhas an impact on the production of either TXB or PGE2, or both, by alveolar macrophage upon silica dust-exposure.
We observed a positive effect of TGF-[I on TXB2 production in rat alveolar macrophages which had been exposed to silica dust. In the absence of TGF-[ (plus anti-TGF-l antibodies), the quantity of TXB2 released from rat alveolar macrophages to the culture medium in response to silica exposure was significantly smaller when compared to the response with silica alcne. Exogenous TGF-I, at low concentration, potentiated the action of silica dust, and the amount of TXB2 produced by rat alveolar macrophages was significantly greater than the amount of TXB2 released by alveolar macrophages activated by silica alone. High concentration of TGF-3, however, inhibited TXB2 production in rat alveolar macrophages; in response to silica, the namely PGE2, in silica-exposed rat alveolar macrophages appeared to be very different to that observed for TXB2. In the absence of TGF-131 (plus anti-TGF-131 antibodies), the amount of PGE2 produced in silica-exposed alveolar macrophages was greater than when compared to silica alone. When anti-TGF-13 antibodies were present in the culture medium during the exposure of macrophages to silica, we observed that (1) these macrophages liberated greater amount of PGE2 compared to silica alone; and (2) there was a stimulation of PGE2 production by alveolar macrophages which did not produce PGE2 after exposure to silica. Furthermore, the presence of exogenous TGF-]3, at low or high concentrations, had either no apparent effect, or a variable effect, respectively, on PGE2 production in silicaexposed rat alveolar macrophages when compared to silica alone. Our data suggest, therefore, a possible distinct biological role for endogenous, but not exogenous TGF-131 to down-regulate PGE2 production in rat alveolar macrophages after exposure to silica.
Data presented in this manuscript provide evidence of the regulatory role of TGF-[31 in the liberation of inflammatory eicosanoids, TXB2 and PGE2, in rat alveolar macrophages, when the cells are exposed to silica dust. In support of our In summary, our data suggest that in silicaexposed alveolar macrophages, both endogenous and exogenous TGF-[3 regulate TXB2 production. Our data suggest that endogenous, but not exogenous TGF-[I has a regulatory role in PGE2 production. Because both TXB2 and PGE2 were measured in the same samples, and because there is a consistent increase in silica-stimulated PGE2 synthesis in the presence of anti-TGF-/31 antibodies in the absence of an effect of authentic TGF-I, it appears that in silica-exposed alveolar macrophages the regulatory effects of TGF-131 are different between the different components of the arachidonic acid pathway.