Regulation of guinea pig macrophage collagenase production by dexamethasone and colchicine

Previous studies have demonstrated that exposure of guinea pig macrophages to a primary signal, such as lipopolysaccharide (LPS), stimulates the synthesis of prostaglandin E2 (PGE2) which, in turn, elevates cAMP levels resulting in the production of the enzyme, collagenase. The potential of regulating the biochemical events in this activation sequence was examined with the anti-inflammatory agents dexamethasone and colchicine, which suppress the destructive sequelae in chronic inflammatory lesions associated with the degradation of connective tissue. The addition of dexamethasone with LPS to macrophage cultures resulted in a dose-dependent inhibition of PGE2 and collagenase production, which was reversed by the exogenous addition of phospholipase A2. Collagenase production was also restored in dexamethasone-treated cultures by the addition of products normally produced as a result of phospholipase action, such as arachidonic acid, PGE2 or dibutyryl-cAMP. Since the effect of dexamethasone was thus linked to phospholipase A2 inhibition, mepacrine, a phospholipase inhibitor, was also tested. Mepacrine, like dexamethasone, caused a dose-dependent inhibition of PGE2 and collagenase. In addition to corticosteroid inhibition, colchicine was also found to block collagenase production. However, this anti-inflammatory agent had no effect on PGE2 synthesis. Colchicine was effective only when added at the onset of culture and not 24 h later, implicating a role for microtubules in the transmission of the activation signal rather than enzyme secretion. The failure of lumicolchicine to inhibit collagenase activity provided additional evidence that microtubules are involved in the activation of macrophages. These findings demonstrate that dexamethasone and colchicine act at specific steps in the activation sequence of guinea pig macrophages to regulate collagenase production.     

Involvement of the ornithine decarboxylase pathway in macrophage collagenase production

Definition of the cellular events involved in the production of collagenase by macrophages following activation has revealed prostaglandin E2 (PGE2)- and cAMP-dependent steps. Since ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis, is regulated by cAMP and is associated with certain aspects of protein synthesis, the potential role of this enzyme and its polyamine product, putrescine, in collagenase synthesis was examined. Lipopolysaccharide (LPS) activation of macrophages resulted in a maximal ODC response after 6 to 9 h with a 10- to 12-fold elevation in enzyme activity. This elevation in ODC appeared to be regulated by PGE2 since indomethacin inhibited LPS-induced macrophage ODC levels by 70%. Associated with the indomethacin-mediated inhibition of ODC was a loss of collagenase synthesis. Furthermore, partial restoration of collagenase production in indomethacin-inhibited cultures could be achieved by the addition of putrescine. In additional studies alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, also inhibited collagenase production when added to LPS-treated macrophages. This inhibition by DFMO could be reversed by the exogenous addition of putrescine. These findings demonstrate that the ODC pathway is an important intracellular component in the sequence of events that lead to macrophage collagenase synthesis.     

Uncoordinate regulation of collagenase,stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: Selective enhancement of collagenase gene expression in human dermal fibroblasts in culture

The degradative effects of interleukin-1 (IL-1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non-steroidal anti-inflammatory drugs. We have examined the role of prostaglandin E₂ (PGE₂) in IL-1-induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01–1.0 μg/ml) of PGE₂ led to a dose-dependent elevation of collagenase mRNA steady-state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE₂ had no additive or synergistic effect with IL-1 on collagenase gene expression. Furthermore, commonly used non-steroidal anti-inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL-1-induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE₂ did not potentiate those of IL-1 on collagenase gene expression in vitro, one could speculate that massive production of PGE₂ by connective tissue cells in vivo in response to inflammatory mediators such as IL-1 or tumor necrosis factor-α, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.     

Modulation of fibroblast functions by interleukin 1: increased steady- state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta

Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.     

Effects of prostaglandins on the production of collagenase by rabbit uterine cervical fibroblasts

Effects of prostaglandins on the production of collagenase by rabbit uterine cervical fibroblasts were investigated. Exogenous prostaglandin E2 (PGE2) and PGF2 alpha significantly stimulated the production of collagenase in a dose dependent manner, whereas PGI2 did not. Addition of arachidonic acid in the presence of absence of indomethacin to the cell culture did not show any increase in collagenase production. Recombinant human interleukin-1 (rhIL-1) also promoted the production of cervical collagenase independently of endogenous prostaglandin(s). Furthermore both exogenous PGE2 and PGF2 alpha enhanced the rhIL-1-induced collagenase production whereas PGI2 and/or indomethacin did not. These results suggested that exogenous PGE2 and PGF2 alpha but not endogenous prostaglandin(s) participate in cervical ripening and dilation by enhancing collagenase production by rabbit uterine cervical cells.     

Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes.

Activation of human monocytes results in the production of interstitial collagenase through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Inasmuch as interleukin 4 (IL-4) has been shown to inhibit PGE2 synthesis by monocytes, we examined the effect of IL-4 on the production of human monocyte interstitial collagenase. Additionally, we also assessed the effect of IL-4 on the production of 92-kDa type IV collagenase/gelatinase and tissue inhibitor of metalloproteinase-1 (TIMP-1) by monocytes. The inhibition of PGE2 synthesis by IL-4 resulted in decreased interstitial collagenase protein and activity that could be restored by exogenous PGE2 or dibutyryl cyclic AMP (Bt2cAMP). IL-4 also suppressed ConA-stimulated 92-kDa type IV collagenase/gelatinase protein and zymogram enzyme activity that could be reversed by exogenous PGE2 or Bt2cAMP. Moreover, indomethacin suppressed the ConA-induced production of 92-kDa type IV collagenase/gelatinase. These data demonstrate that, like monocyte interstitial collagenase, the conA-inducible monocyte 92-kDa type IV collagenase/gelatinase is regulated through a PGE2-mediated cAMP-dependent pathway. In contrast to ConA stimulation, unstimulated monocytes released low levels of 92-kDa type IV collagenase/gelatinase that were not affected by IL-4, PGE2, or Bt2cAMP, indicating that basal production of this enzyme is PGE2-cAMP independent. IL-4 inhibition of both collagenases was not a result of increased TIMP expression since Western analysis of 28.5-kDa TIMP-1 revealed that IL-4 did not alter the increased TIMP-1 protein in response to ConA. These data indicate that IL-4 may function in natural host regulation of connective tissue damage by monocytes.     

Arachidonic acid metabolites regulate interleukin-1 production

We have investigated the role of arachidonic acid metabolites in the regulation of interleukin-1 production by murine peritoneal macrophages. Indomethacin a potent inhibitor of prostaglandin synthesis caused a dose-dependent augmentation of lipopolysaccharide induced interleukin production (up to 7-fold at 5 microM). In contrast, lipoxygenase inhibitors, nordihydroguarietic acid and nafazatrom had no effect at doses that did not significantly decrease prostaglandin synthesis. Added to lipopolysaccharide stimulated cultures, PGE2 was also augmented by indomethacin but unlike lipopolysaccharide treated cultures was suppressed by nordihydroguarietic acid. These data suggest that arachidonate metabolites may be potent autoregulators of macrophage interleukin-1 production.     

Interactive roles of progesterone, prostaglandins, and collagenase in the ovulatory mechanism of the ewe

Interrelationships between production of progesterone (P4), prostaglandin (PG) E2 and PGF2 alpha, and collagenase by periovulatory ovine follicles and their possible involvements in the ovulatory process were investigated. Follicles were isolated from ovaries at intervals (0 to 24 h) after the initiation of the preovulatory surge of luteinizing hormone (LH). Progesterone and PGs within follicles were determined by radioimmunoassay. Digestion of radioactive collagen during coincubation with tissue homogenates was used to assess the production of a bioactive follicular collagenase(s). Follicular accumulation of PGs and P4 increased at 12 and 16 h, respectively, after the onset of the surge of LH; PGE2 then decreased at 20 h. Collagenolytic activity of follicular tissue increased at 20 h and was maximal at 24 h (during the time of follicular rupture). An inhibitor of synthesis of P4 (isoxazol) or PGs (indomethacin) was injected into the follicular antrum at 8 h. Isoxazol did not prevent the initial rise in PGs, but inhibited synthesis of PGF2 alpha at 16 h and therafter. Isoxazol negated the decline in PGE2 and increase in collagenolysis. Indomethacin did not influence synthesis of P4; however, it suppressed collagenolytic activity of follicular tissue. Ovaries with treated follicles were left in situ and observed for an ovulation point at 30 h. Isoxazol or indomethacin was a potent inhibitor of ovulation. The blockade of ovulation by isoxazol was reversed by systemic administration of P4 or PGF2 alpha, but not by PGE2. Reversal of the blockade by indomethacin was accomplished with PGE2 or PGF2 alpha. Collagenolytic activity of follicular tissue was likewise restored by such treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the effect of nonsteroidal and steroidal antiinflammatory agents on prostaglandin production during ovulation in the rabbit

The antiinflammatory agents diclofenac, fenoprofen and aspirin were tested to determine how well they inhibit the pre-ovulatory elevation in prostaglandin (PG) production in rabbit follicles in comparison to indomethacin. In addition, the antiinflammatory agent dexamethasone and the antipyretic agent acetaminophen were tested. The agents were administered 8 h after the ovulatory process was stimulated by hCG (50 I.U./kg). At 10 h after hCG (i.e., at the expected time of ovulation) control follicles had PGF and PGE levels of 370.0 and 582.6 pg/mg of follicle, respectively. Diclofenac inhibited PG production the most-reducing PGF and PGE to 22.8 and 53.6 pg/mg, respectively. Indomethacin reduced the PGF and PGE levels to 27.4 and 76.6 pg/mg, respectively. Fenoprofen was less effective, reducing the PGF and PGE to 77.8 and 222.4 pg/mg, respectively. Aspirin reduced the PGF and PGE to 123.4 and 174.6 pg/mg, respectively. Dexamethasone and acetaminophen did not inhibit PG production. Ovulation was completely inhibited by diclofenac and indomethacin, partially inhibited by fenoprofen, and unaffected by aspirin, acetaminophen, or dexamethasone. The results suggest that any potent antiinflammatory agent can inhibit ovulation provided it adequately reduces PG production; whereas antiinflammatory agents are ineffective. The anti-inflammatory agent must completely abolish the preovulatory elevation in PGs in mature follicles in order to totally inhibit ovultion.     

Prostaglandins as endogenous mediators of interleukin 1 production

We examined the role of cyclooxygenase (CO)-derived metabolites of arachidonic acid (AA) in the regulation of interleukin 1 (IL 1) production by lipopolysaccharide (LPS)-stimulated murine resident peritoneal macrophages. The use of LPS proved to be an efficacious probe, because it stimulated both IL 1 production and AA metabolism via only the CO pathway. The production of the CO metabolites prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2; measured as its stable metabolite 6-Keto prostaglandin F1 alpha) by LPS-stimulated macrophages was demonstrated by high pressure liquid chromatography and radioimmunoassay. The addition of exogenous PGE2 or PGI2 resulted in a dose-dependent suppression of macrophage IL 1 production. Inhibitors of the CO pathway (indomethacin, piroxicam, and ibuprofen) caused a dose-dependent augmentation in the LPS-induced IL 1 response. This augmentation directly correlated with the efficacy of the compounds as CO inhibitors. Similar results were found when macrophage-derived fibroblast growth factor was assessed. The addition of exogenous IL 1 to macrophage cultures caused an increase in the levels of PGE2, over a narrow dose range (0.05 to 0.6 IL 1 units). These studies provide detailed evidence that AA metabolites synthesized via the CO pathway can modulate the production of growth factors by LPS-stimulated macrophages. In addition, our data support the concept that IL 1, as with classical hormones, can regulate its own production through a self-induced inhibitor, PGE2.     

Cytostatic and cytolytic activities of macrophages regulation by prostaglandins

Murine peritoneal macrophages (M phi), activated in vivo or in vitro, remarkably inhibited the uptake of thymidine by a lens epithelial cell line, while resident M phi, or M phi induced by thioglycollate, exhibited much lower or no cytostatic capacity. The target cells were partially protected from the cytostatic activity by the anti-inflammatory agents indomethacin, aspirin, and dexamethasone, but not by lipoxygenase inhibitors. The protective activity of indomethacin and aspirin, but not of dexamethasone, was completely counteracted by prostaglandin E2 (PGE2). Yet, PGE2 alone has no effect on the uptake of [3H]thymidine by lens epithelial cells. PGE1 resembled PGE2 in its effect on this system, whereas PGA2, PGB2, or PGF2 alpha had no detectable activity. The counteracting effect of PGE2 was mimicked by dibutyryl cAMP or by cholera toxin, an agent which increases cAMP levels. These findings suggest that PGEs are not direct cytostatic agents, but rather, are essential mediators for the development of the cytostasis. Activated M phi did not lyse cells of the original lens epithelial cell line, but caused substantial cytolysis of cells of a subline derived from it. In contrast to its aforementioned effect on the cytostasis, PGE2 inhibited the cytolytic activity of M phi. Thus, this study provides a first demonstration in a single system of the opposite effects of PGEs on M phi activity on target cells, i.e., mediating the cytostasis and inhibiting the cytolysis.     

Mediation of macrophage collagenase production by 3'-5' cyclic adenosine monophosphate

The production of collagenase by lipopolysaccharide-(LPS) activated guinea pig macrophages is mediated by prostaglandins (PG) of the E series. After stimulation of guinea pig macrophages with LPS, extracellular PGE levels and cellular cAMP levels are elevated. Indomethacin inhibits not only PG synthesis, but also cAMP and collagenase production in LPS-stimulated macrophage cultures. In these indomethacin-inhibited cultures containing LPS, dibutyryl (dB) cAMP, or cholera toxin can restore macrophage collagenase production but not PG synthesis. Moreover, dBcAMP and cholera toxin enhance collagenase production in LPS-activated cultures. Initial activation of the macrophages by an agent such as LPS is a prerequisite for synthesis of collagenase, since in the absence of LPS, dBcAMP or cholera toxin alone are ineffective stimuli. These findings clearly demonstrate a role for PG-induced elevations of cAMP in the production of collagenase by LPS-activated macrophages.     

Regulation by PGE2 of the production of oxygen intermediates by LPS-activated macrophages

The regulation by prostaglandin E2 (PGE2) of production of oxygen radicals by bacterial lipopolysaccharide-(LPS) activated macrophages was studied in vitro. A 48-hr incubation of murine thioglycollate-elicited macrophages with LPS (0.1 micrograms/ml) resulted in an enhanced ability of these cells to produce oxygen radicals when challenged with phorbol myristate acetate (PMA). Macrophages incubated for 48 hr without LPS did not produce measurable amounts of oxygen radicals when exposed to this triggering stimulus. Thus, PMA-triggered production of oxygen radicals was the result of macrophage activation by LPS. The PMA-triggered production of oxygen radicals by the LPS-activated macrophages was inhibited when PGE2 (10(-5) to 10(-9) M) was present during the incubation with LPS. Inhibition by PGE2 occurred during the early stages of macrophage activation, since the addition of PGE2 24 hr after LPS no longer inhibited the production of oxygen radicals by the macrophages. This inhibitory effect of PGE2 on the LPS-induced activation of macrophages could be reproduced by cyclic-adenosine-monophosphate (cAMP) agonists, such as isoproterenol and cholera toxin as well as by the cAMP analog dibutyryl-cAMP, suggesting a cAMP-mediated mechanism for the inhibitory effect of PGE2 on macrophage activation by LPS. Previous reports have implicated prostaglandins as mediators of destructive processes associated with chronic inflammation. Our findings suggest that PGE2 may, on the other hand, reduce tissue damage in a chronic inflammatory site by inhibiting the production of oxygen radicals by macrophages activated in the sera.     

Rapid changes in the activities of the enzymes of cyclic AMP metabolism after addition of A23187 to macrophages

The ionophore A23187 stimulated adenylate cyclase activity in intact macrophages within 1 min. This action was blocked by pretreatment with indomethacin (25 μmol/l) suggesting the involvement of a prostaglandin (PG). PGE₂ (500 nmol/l) also stimulated adenylate cyclase activity in intact cells, but this was not prevented by indomethacin pretreatment. Colchicine (100 μmol/l) potentiated the increases in macrophage cyclic AMP production seen after addition of PGE₂ or A23187. The high affinity form of cyclic AMP phosphodiesterase (PDE) was activated within 1 min of the addition of A23187 to intact macrophages. The data suggest that the increase in macrophage cyclic AMP production after A23187 is a consequence of adenylate cyclase activation and not PDE inhibition. The endogenous production of a prostaglandin probably mediates this effect of A23187, emphasizing the importance of arachidonic acid metabolites in the regulation of macrophage functions.     

Glucocorticoid effect on arachidonic acid metabolism in vivo

Glucocorticoids have been shown in in vitro systems to inhibit the release of arachidonic acid metabolites, namely prostaglandins (PGs) and leukotrienes, apparently, via the induction of a phospholipase A2 inhibitory protein, called lipocortin. On the basis of these in vitro results, it has been suggested that inhibition of eicosanoid production is, at least partially, responsible for the well-known anti-inflammatory effect of glucocorticoids. There is, however, no firm evidence proving that glucocorticoids also inhibit prostaglandin or leukotriene synthesis in vivo. In a series of studies, we have investigated the effects of anti-inflammatory steroids on the production of six different cyclo-oxygenase products in vivo. Urinary prostaglandin (PG) E2(1), PGF2 alpha, thromboxane B2 (TxB2), 6-keto-PGF1 alpha, and the major urinary metabolites of the E and F PGs, PGE-M and PGF-M, respectively, were determined by radioimmunoassay and by GC-MS. Administration of pharmacological doses of dexamethasone to rabbits failed to inhibit urinary excretion rates of PGE2, TxB2, 6-keto-PGF1 alpha and that of PGE-M and PGF-M. In contrast, urinary PGF2 alpha was slightly reduced by dexamethasone. In further experiments the effect of dexamethasone was studied in humans. Urinary excretion rates of PGE2, PGE-M, PGF-M, 2,3-dinor TxB2 and 2,3-dinor 6-keto-PGF1 alpha were not suppressed by dexamethasone. Collagen-induced platelet TxB2 formation and platelet aggregation was also unaltered. To test one possible explanation for the apparent discrepancy between in vitro and in vivo effects of glucocorticoids on arachidonic acid metabolites we investigated the effects of dexamethasone in vivo on basal and on antidiuretic hormone-stimulated renal PG synthesis. Dexamethasone treatment failed to inhibit both basal and antidiuretic hormone-stimulated PGE2 and PGF2 alpha production. We conclude that glucocorticoids in vivo do not decrease the basal rate of total body, kidney and platelet prostanoid synthesis, and that dexamethasone does not inhibit renal PG production when it is elevated by antidiuretic hormone, a physiological stimulus. Thus, a differential effect of glucocorticoids on basal vs stimulated PG synthesis cannot account for the discrepancy between in vivo and in vitro effects.     

Regulation of human peripheral blood monocyte collagenase by prostaglandins and anti-inflammatory drugs

Macrophages, which produce the collagenolytic enzyme collagenase, are commonly found at sites of connective tissue destruction in chronic inflammatory lesions. Since tissue macrophages are derived from circulating peripheral blood monocytes, we used these less-differentiated, more readily available cells to examine the production and regulation of collagenase. Human monocytes, isolated in large quantities by counterflow centrifugal elutriation, were shown to produce substantial amounts of collagenase when stimulated by concanavalin A (Con A) and to a lesser extent with lipopolysaccharide, while unstimulated monocyte cultures produced negligible collagenase. Collagenase was detected in the culture media within the first 24 hr of culture after activation with peak production at 48 hr. Analysis of the intracellular regulation of collagenase revealed that synthesis of this enzyme required a prostaglandin (PGE₂)-dependent step since indomethacin-inhibited enzyme synthesis was reversed by PGE₂. Additionally, dibutyryladenosine cyclic monophosphate (dBcAMP) restored collagenase synthesis in indomethacinblocked cultures, indicating a PGE₂-dependent generation of cAMP requirement for collagenase production similar to that demonstrated in experimental animals systems. In additional studies, anti-inflammatory drugs which are known to modulate connective tissue destruction were analyzed for their influence on monocyte-derived collagenase. Dexamethasone, colchicine or retinoic acid all inhibited collagenase synthesis by monocytes in a dose-dependent manner although the effect of these drugs on monocyte PGE₂ synthesis differed. Dexamethasone inhibited PGE₂ synthesis, which resulted in the suppression of collagenase. However, PGE₂ production was unaffected by colchicine whereas retinoic acid caused a significant increase in PGE₂ levels. Inhibition of collagenase synthesis by dexamethasone, but not colchicine or retinoic acid, could be reversed by PGE₂ or phospholipase A₂. These findings provide insight into the intracellular events regulating monocyte collagenase synthesis and also implicate monocytes as a target of anti-inflammatory agents which ameliorate connective tissue degradation associated with chronic inflammatory lesions.     

Glucocorticoid stimulation of amnion cell prostaglandin synthesis: suppression by protein kinase C inhibitors and independence of phorbol ester-sensitive protein kinase C.

Glucocorticoids stimulate the prostaglandin E2 production of confluent amnion cell cultures, but have no stimulatory effect on the PGE2 output of freshly isolated human amnion cells. Since protein phosphorylation may modify the responsiveness of target cells to steroids, and activators of protein kinase C (PKC), as well as corticosteroids, promote amnion cell PGE2 output by stimulating the synthesis of prostaglandin endoperoxide H synthase (PGHS), we investigated the possibility that PKC is involved in the glucocorticoid-induction of PGE2 synthesis in cultured amnion cells. The dexamethasone-induced PGE2 output of arachidonate-stimulated cells was blocked by the protein kinase inhibitors staurosporine, K-252a, H7, HA1004, and sphinganine, in a manner consistent with their effect on PKC. However, dexamethasone increased the PGE2 production of cultures treated with maximally effective concentrations of the PKC-activator compound TPA. Moreover, dexamethasone stimulated PGE2 synthesis in cultures which were desensitized to TPA-stimulation by prolonged phorbol ester treatment. Concentration-dependence studies showed that staurosporine completely (greater than 95%) blocked glucocorticoid-provoked PGE2 synthesis at concentrations which did not inhibit TPA-stimulated prostaglandin output, and that K-252a inhibited the effect of TPA by more than 95% at concentrations which decreased the effect of dexamethasone only moderately (approximately 40%). Dibutyryl cyclic AMP had no influence on the basal- or dexamethasone-stimulated PGE2 production, and on the staurosporine inhibition of the steroid effect. These results show that glucocorticoids and phorbol esters control amnion PGE2 production by separate regulatory mechanisms. It is suggested that the response of human amnion cells to glucocorticoids is modulated by protein kinase(s) other than phorbol ester-sensitive PKC and cyclic AMP-dependent protein kinase.     

Prostaglandin E(2) mediates inhibition of insulin secretion by interleukin-1beta.

Interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic beta cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1beta effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1beta induces cyclooxygenase-2 (COX-2) gene expression and PGE(2) synthesis in islet beta cells, we have reassessed the possibility that PGE(2) mediates IL-1beta effects on beta function. By using two cell lines (HIT-T15 and betaHC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1beta inhibition of insulin secretion. Both drugs prevented IL-1beta from inducing PGE(2) synthesis and inhibiting insulin secretion; adding back exogenous PGE(2) re-established inhibition of insulin secretion in the presence of IL-1beta. We also found that EP3, the PGE(2) receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE(2) mediates the inhibitory effects of exogenous IL-1beta on beta cell function.     

Regulation of B cell tolerance by macrophage-derived mediators: antagonistic effects of prostaglandin E2 and interleukin 1

A role for prostaglandins in the mechanism of B cell tolerance induction in normal adult mouse spleen cells was examined. Two inhibitors of the cyclooxygenase pathway of arachidonic acid metabolism, indomethacin and acetylsalicylic acid, abrogated hapten-specific B cell tolerance induction by trinitrophenyl-human gamma-globulin. Tolerance was fully restored by the addition of prostaglandin E2 (PGE2) at a concentration of greater than or equal to 6 nM. T cell-depleted spleen cells produced comparable amounts of PGE2 in culture, indicating that the tolerance promoting activity of PGE2 occurred with physiologically relevant concentrations. Depletion and reconstitution experiments indicated that macrophages in the spleen cell preparations completely accounted for both PGE2 production and the effects of indomethacin and acetylsalicylic acid on B cell tolerance induction. The macrophage product interleukin 1 (IL 1) was also found to alter B cell susceptibility to tolerance induction. Thus, human IL 1 containing monocyte supernatants and purified IL 1 were found to interfere with B cell tolerance induction when added to macrophage- and T cell-depleted splenic B cells. Tolerance was restored in such cultures by the addition of 10 nM PGE2. These experiments demonstrate that within mixed lymphoid populations macrophages through the release of mediators modulate B cell susceptibility to tolerance induction.