METODOLOGÍA propuesta PARA EL ESTUDIO DE CONFIABILIDAD [8]

Very few studies have examined a role for PGE2 in the pathogenesis o f pulmonary fibrosis, despite its inhibitory effects on fibroblast function. The following section will review current evidence supporting a role for PGE2 in the pathogenesis of pulmonary fibrosis.

1.5.1 In vitro evidence

Only one in vitro study has examined the regulation o f PGE2 synthesis in fibroblasts fi'om patients with pulmonary fibrosis (Wilbom et aL, 1995). This study investigated basal and stimulated PGE2 release by fibroblasts isolated from normal and fibrotic lung. Levels of PGE2 synthesis were dramatically reduced in fibroblasts cultured fi"om patients with pulmonary fibrosis under basal conditions and when induced. Basal PGE2 synthesis was almost seven-fold lower in fibroblasts from fibrotic lung. Radiolabelling studies confirmed the release of endogenous arachidonic acid by both groups o f fibroblasts, therefore excluding a defect in PLA2-mediated arachidonic acid release. To eliminate the possibility of a defect in PGE synthase, the intermediate endoperoxide, PGH2 was supplied exogenously thus bypassing the COX pathway and this resulted in PGE2 production by both groups of fibroblasts. The difference in basal PGE2 synthesis could not be attributed to aberrant COX-1 or COX-2 protein expression either, although COX activity was lower in fibroblasts from fibrotic lung. Furthermore, fibroblasts isolated fi'om fibrotic lung failed to induce PGE2 synthesis upon stimulation with IL-lp, PMA or lipopolysaccharide (LPS). In normal fibroblasts, these agonists upregulated PGE2 synthesis by two to three-fold with a concomitant increase in COX activity. The increase in COX activity was susceptible to inhibition by the glucocorticoid, dexamethasone implicating the COX-2 isoform in mediating this response. In fibroblasts fi'om patients with pulmonary fibrosis, COX activity did not increase with any agonists. Immunoblot analysis confirmed the increase in COX-2 activity in normal fibroblasts but the enzyme was undetectable in fibroblasts from fibrotic lung. Finally, RT-PCR analysis o f RNA

from both groups of fibroblasts failed to detect an increase in COX-2 mRNA levels in fibroblasts from patients with pulmonary fibrosis, suggesting that the lack o f COX-2 activity was due to a pre-translational defect. The functional significance o f these findings was not investigated.

1.5.2 In vivo evidence

Intratracheal instillation o f bleomycin sulphate is used extensively to induce pulmonary fibrosis in animal models o f the disease. The model is characterised by an acute lung injury response consisting o f an initial inflammatory phase followed by a fibrotic reaction. A doubling in lung collagen content occurs by about two- three weeks post-injury (Goldstein et aL, 1979; Mutsaers et aL, 1998b).

A number of studies support a role for prostaglandins o f the E series in modulating the fibrotic response to bleomycin. Plasma levels of PGE2 are increased four days after instillation of bleomycin (Chandler and Giri 1983) and remain elevated for at least 28 days (Chandler et aL, 1983). However, the COX isoform responsible for this increase in PGE2 synthesis, following bleomycin-induced lung injury is not known. Conditioned medium from lung explants obtained from bleomycin- exposed hamsters increases fibroblast PGE2 production and intracellular cAMP (Clark et aL, 1982). Functionally, this resulted in a dose-dependent decrease in fibroblast proliferation and a reduction in collagen production. Other studies have examined the effect o f enhancing PGEi levels prior to bleomycin injury. Dietary supplementation of animal feed with y-linoleic acid, the fatty acid substrate for PGEi synthesis results in a marked increase in lung PGEi levels and increases in lung collagen in response to bleomycin is suppressed (Ziboh et aL, 1997). PGEi can also affect the early inflammatory response to bleomycin. In hamsters, continous i.v. infusion of PGEi for 22h after bleomycin instillation results in reduced neutrophil influx and plasma leak (Grant et aL, 1991). However, other studies combining indomethacin, an inhibitor o f prostaglandin synthesis with bleomycin sulphate have yielded conflicting results, suggesting that indomethacin reduces lung collagen and histological evidence o f fibrosis (Thrall et aL, 1979; Mall et aL, 1991). In the first study, rats treated with bleomycin alone or in

combination with indomethacin exhibited an equivalent increase in lung collagen content, 14 days post-injury (Thrall et aL, 1979). By day 60, a dramatic reduction was seen in levels of collagen in rats treated with bleomycin and indomethacin compared to those receiving bleomycin alone. However, the study did not mention whether total collagen content increased between days 14 and 60 and if so whether indomethacin inhibited the formation of newly synthesized collagen. In the second study, histological evidence and morphometric analysis were provided suggesting that indomethacin prevented bleomycin-induced pulmonary fibrosis (Mall et aL, 1991). However, animals treated with bleomycin and indomethacin lost over 50% in body weight and demonstrated reduced lung volumes. The changes in these animals were greater than in those rats receiving bleomycin alone.

PGE] is normally present at much higher concentrations in the lung than in plasma (Ozaki et aL, 1987). However, levels of PGE] in BALF recovered fi'om patients with pulmonary fibrosis are 50% lower than in normal individuals (Borok et aL,

1991), despite elevated levels of TGF-pi and other pro-fibrotic mediators which induce PGE] synthesis (Kuroki et aL, 1995, Ludwicka et aL, 1995). This suggests that the cells responsible for synthesising PGE] in response to injury fail to upregulate levels of PGE] in patients with pulmonary fibrosis. The functional consequences o f such a defect are not known, however, as mentioned earlier, PGE] regulates proliferation and collagen synthesis in normal fibroblasts in the presence o f pro-inflammatory mediators. Therefore, in the absence of PGE], and in the presence o f pro-inflammatory mediators, the potential exists for uncontrolled fibroblast activity. To investigate this concept, the specific interactions between PGE] and a major fibrogenic cytokine, TGF-P] will be discussed in detail.