PGE2 exerts its effect on the LPS-induced release of TNF-alpha, ET-1, IL-1alpha, IL-6 and IL-10 via the EP2 and EP4 receptor in rat liver macrophages

Lipopolysaccharide (LPS) induces a release of tumor necrosis factor (TNF)-alpha, endothelin (ET)-1, interleukin (IL)-1alpha, IL-6 and IL-10 in rat liver macrophages (Kupffer cells). Prostaglandin (PG)E2 inhibits the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and enhances the release of the anti-fibrogenic mediators IL-6 and IL-10. This effect of PGE2 is mimicked by specific agonists for the PGE2 receptors EP2 and EP4; whereas, agonists for the PGE2 receptors EP1 and EP3 are inactive. Rat liver macrophages express mRNA encoding the PGE2 receptors EP2 and EP4 but not the PGE2 receptors EP1 and EP3. These data suggest that PGE2 exerts its anti-fibrogenic effect through the EP2 and EP4 receptor by inhibiting the release of the fibrogenic mediators TNF-alpha, ET-1 and IL-1alpha, and by enhancing the release of the anti-fibrogenic mediators IL-6 and IL-10 in liver macrophages.     

Relationship between membrane potential changes and superoxide-releasing capacity in resident and activated mouse peritoneal macrophages

In an attempt to understand better the molecular basis for the enhanced respiratory burst of activated macrophages (M phi), we investigated the relationship between stimulus-induced changes in membrane potential and release of superoxide anion (O2-) in mouse peritoneal M phi. Resident M phi and M phi elicited by injection of lipopolysaccharide (LPS-M phi) or obtained from animals infected with bacille Calmette-Guérin (BCG-M phi) were used. LPS-M phi and BCG-M phi showed more pronounced changes in membrane potential (depolarization) and greater release of O2- on contact with phorbol myristate acetate (PMA) than did resident macrophages. The lag time between addition of stimulus and onset of release of O2- was reduced in activated compared with resident cells. Membrane potential changes began 60 to 90 sec before release of O2- could be detected in each cell type. The dose-response curves for triggering of membrane potential changes and O2- release by PMA were identical. The magnitude of membrane potential changes and of O2- release in LPS-M phi and BCG-M phi declined progressively during in vitro culture, and values on day 3 approached those in resident macrophages ("deactivation"). Extracellular glucose was required for effective stimulated change in membrane potential and O2- release. These findings indicate that membrane potential changes are closely associated with O2- -releasing capacity in macrophages, and that the systems that mediate membrane potential changes and production of O2- develop or decline concomitantly during activation or deactivation of the cells. Although the plasma membrane was highly depolarized by high extracellular K+ or by the sodium ionophore gramicidin, O2- release was not induced by these maneuvers, indicating that changes in membrane potential by themselves are not sufficient to trigger the respiratory burst in macrophages. Release of O2- was not impaired in buffers in which Na+ was completely replaced with equimolar concentrations of K+ or choline+; thus, induction or maintenance of the respiratory burst in M phi does not require an influx of Na+.     

Tumor modulation of autoreactivity: decreased macrophage and autoreactive T cell interactions

The autologous mixed lymphocyte reaction (AMLR) is an in vitro measure of autoreactivity, a key mechanism in immune homeostasis. In this system, macrophages (M phi) act as accessory cells to autoreactive L3T4+ T cells by presenting self-Ia and releasing soluble modulators. During tumor growth, changes occur in M phi and T cells. Tumor-bearing host (TBH) M phi have a reduced ability to act as accessory cells. In fact, TBH M phi suppressed autoreactivity by 60-70%. The decrease in TBH M phi or T-cell abilities was not due to differences in cell numbers or incubation time. Because tumor growth causes increased prostaglandin E2 (PGE2) production by M phi, indomethacin was used to assess the contribution of prostaglandins. Normal and TBH T-cell reactivity increased nearly 50% when stimulated by normal host M phi, while normal and TBH T-cell reactivity increased nearly 100% when stimulated by TBH M phi. Thus increased prostaglandin production is partly responsible for the increased TBH suppressor M phi activity and in the normal host, suppressor M phi may be responsible for maintaining immune regulation. To assess the direct role of prostaglandins in T-cell hyporesponsiveness, PGE2 was titrated into the cultures. PGE2 suppressed normal and TBH T-cell responsiveness in a dose-dependent manner. Normal host T cells were suppressed to a greater extent than TBH T cells by PGE2 (66% versus 42% suppression, respectively). Reduced Ia expression and active suppressor mechanisms are not the only mechanisms mediating hypoautoreactivity during tumor growth. TBH autoreactive L3T4+ T cells were less responsive to self-Ia; they were only 60-80% as reactive as their normal counterparts. To address whether the helper T (TH)-cell defect involved cytokines, T cells were treated with interleukin (IL)-1, IL-2, and IL-4. In all cases, the TBH T-cell response to the factors was decreased (only 60-75% as reactive as normal T cells). Because TBH M phi-mediated suppression can override the addition of IL-1, IL-2, and IL-4, indomethacin was also added with the exogenous interleukins. This coaddition significantly enhanced normal host autoreactivity above control levels while TBH autoreactivity (the combination of TBH T cells and TBH M phi) only returned to normal host unstimulated levels. Tumor growth modulates the immune response at least by (i) decreasing the accessory cell abilities of TBH M phi through decreased Ia expression and increased production of suppressive molecules such as prostaglandins; and (ii) decreasing the responsiveness to immune enhancing factors by TH cells.     

IL-1-induced prostacyclin production by cerebral vascular endothelial cells inhibits myelin basic protein-specific lymphocyte proliferation.

Addition of cerebral vascular endothelial cells (EC) to myelin basic protein (MBP) immune lymph node cells (LNC) cultured in the presence of MBP resulted in the inhibition of MBP-specific proliferative responses. Proliferation was not inhibited in cultures containing indomethacin (IM), suggesting a possible role for prostaglandins. Significant levels of 6-KPGF1 alpha, the stable hydrolysate product of PGI2, but not PGE2 were observed in culture (LNC + EC) supernatants but not in supernatants from cultures containing only LNC or EC. The levels of PGI2 release were proportional to the concentration of exogenous EC present in culture and synthesis of PGI2 could be blocked by IM. These results indicate the requirement for coculture in the generation PGI2. Additional experiments indicated that EC were required for the generation of PGI2 and that either macrophages (M phi), or recombinant murine IL-1 were able to replace LNC in cocultures with EC in order to generate PGI2. The ability of IL-1 to stimulate EC-derived PGI2 synthesis was dose dependent with maximal stimulation observed at 50 U/ml IL-1. The IL-1-induced production of PGI2 by EC as well as PGI2 production in cultures containing EC and either LNC or M phi was inhibited by treatment with anti-IL-1 antibody. These results indicate that EC are capable of inhibiting antigen-specific lymphocyte proliferation by producing PGI2, which can be induced by the lymphokine, IL-1.     

Differing signal requirements for the activation of macrophages from C3H/HeJ and C3H/OuJ mice

Abstract Endotoxin-associated protein (EP) from Salmonella typhi stimulated the release of prostaglandin E2 (PGE2), interleukin-1 (IL-1), and interferon (IFN) activity in macrophages from the lipopolysaccharide (LPS) responder C3H/OuJ mouse strain. However, only PGE2 and IL-1 were stimulated by EP in macrophages from the LPS nonresponder C3H/HeJ mouse strain. LPS stimulated the release of PGE2, IL-1 and IFN activity in C3H/OuJ macrophages, but not in C3H/HeJ macrophages. The protein kinase C (PKC) activator phorbol myristic acid (PMA) stimulated PGE2 production in both strains but not IL-1 production, suggesting that signalling pathways other than PKC may be involved in IL-1 production. The calcium ionophore ionomycin stimulated PGE2 production in C3H/OuJ but not C3H/HeJ macrophages, suggesting a defective calcium-related pathway in the C3H/HeJ macrophages as compared to the C3H/OuJ cells.     

Heregulin-alpha and heregulin-beta expression is linked to a COX-2-PGE2 pathway in human gastric fibroblasts

We have previously shown heregulin (HRG)-alpha expression in human gastric fibroblasts and its stimulation of gastric epithelial cell growth. Although cyclooxygenase (COX)-2 has also been shown to stimulate growth factor production in these cells, the interaction between COX-2 and HRG remains unknown. Conditioned media (CM) from gastric fibroblasts incubated with PGE(2) or interleukin (IL)-1beta, a well known COX-2 inducer, were analyzed for their effect on erbB3 tyrosine phosphorylation in MKN28 gastric epithelial cells. HRG protein expression in fibroblast lysates and CM was also examined by western blot. HRG-alpha and HRG-beta mRNA expression in gastric fibroblasts and human gastric tissue was examined by real-time quantitative PCR. HRG and COX-2 expressions in surgical resections of human gastric ulcer tissue were examined immunohistochemically. CM from fibroblasts incubated with PGE(2), or IL-1beta, stimulated erbB3 phosphorylation in MKN28 cells. Preincubation of the fibroblasts with celecoxib, a selective COX-2 inhibitor, suppressed CM-induced erbB3 phosphorylation. This inhibition was reversed by exogenous PGE(2). As with erbB3 phophorylation, IL-1beta stimulated both HRG-alpha and HRG-beta mRNA expression, as well as HRG release into gastric fibroblast CM. IL-1beta-stimulated HRG expression and release were also inhibited by celecoxib, and exogenous PGE(2) restored this inhibitory effect, suggesting the activation of an IL-1beta-COX-2-PGE(2) pathway that culminates in the release of HRG from fibroblasts. HRG-alpha and HRG-beta mRNA levels were significantly higher in gastric ulcer tissue than in normal gastric mucosa. HRG immunoreactivity was found in interstitial cells of the gastric ulcer bed and coexpressed with COX-2. These results suggest that HRG might be a new member of the growth factor family involved in the COX-2-dependent ulcer repair process.     

Interferon-gamma reduces macrophage-suppressive activity by inhibiting prostaglandin E2 release and inducing interleukin 1 production

Normal peritoneal M phi of C3H/HeN mice were able to suppress lymphocyte proliferation in a dose-dependent fashion when added to Con A-pulsed spleen cell cultures. However, M phi-suppressive activity could be partially or completely reduced by in vitro pre-exposure to nonimmune IFN-alpha or immune recombinant IFN-gamma. For both IFN-alpha and IFN-gamma, reduction of M phi suppression was marginal at 10(1) U/ml and became highly significant at 10(2) to 10(3)/ml. The ability of IFN-alpha and IFN-gamma to modulate M phi suppression appears to be related to distinct mechanisms. In fact, impairment of M phi suppression by IFN-alpha occurred in parallel to the decrease of M phi capacity to produce PGE2 and the oxygen intermediate O2-, two molecules responsible for M phi-suppressive activity. In contrast, M phi exposed to IFN-gamma showed only impairment of PGE2 production, whereas O2- release was not significantly affected. Furthermore, at variance with IFN-alpha, IFN-gamma directly stimulated M phi to synthesize and release IL 1, a monokine known to promote lymphocyte proliferation.     

Mechanisms of enhanced macrophage-mediated prostaglandin E2 production and its suppressive role in Th1 activation in Th2-dominant BALB/c mice

PGE(2) has been known to suppress Th1 responses. We studied the difference in strains of mice in PGE(2) production by macrophages and its relation to Th1 activation. Macrophages from BALB/c mice produced greater amounts of PGE(2) than those from any other strains of mice, including C57BL/6, after LPS stimulation. In accordance with the amount of PGE(2) produced, macrophage-derived IL-12 and T cell-derived IFN-gamma production were more strongly suppressed in BALB/c macrophages than in C57BL/6 macrophages. When macrophages were treated with indomethacin or EP4 antagonist, Th1 cytokines were more markedly increased in cells from BALB/c mice than in those from C57BL/6 mice. Although cyclooxygenase-2 was expressed similarly after LPS stimulation in these mouse strains, the release of arachidonic acid and the expression of type V secretory phospholipase A(2) mRNA were greater in BALB/c macrophages. However, exogenous addition of arachidonic acid did not reverse the lower production of PGE(2) by C57BL/6 macrophages. The expression of microsomal PGE synthase, a final enzyme of PGE(2) synthesis, was also greater in BALB/c macrophages. These results indicate that the greater production of PGE(2) by macrophages, which is regulated by secretory phospholipase A(2) and microsomal PGE synthase but not by cyclooxygenase-2, is related to the suppression of Th1 cytokine production in BALB/c mice.     

Up-regulation of interleukin-6 induced by prostaglandin E from invading macrophages following nerve injury: an in vivo and in vitro study

The mechanisms underlying neuropathic pain caused by nerve injury are not well understood. Inflammatory responses in injured nerves are likely to be key contributing factors in the generation and maintenance of neuropathic pain. The pro-inflammatory cytokine interleukin-6 (IL-6) is up-regulated in invading macrophages and has been implicated in the development of neuropathic pain. We previously demonstrated that invading macrophages up-regulate cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) receptors EP1 and EP4, suggesting that PGE2 may affect macrophage function via autocrine or paracrine mechanisms. This study was undertaken to determine whether PGE2 is involved in the up-regulation of IL-6 in invading macrophages. Two weeks following partial sciatic nerve ligation, numerous IL-6 immunoreactive (IR) cell profiles were present in injured nerves. Colocalization of IL-6 with the invading macrophage marker ED1 or with COX2 was frequently observed. IL-6-IR, COX2-IR and ED1-IR cells were present only in cultures derived from injured nerve segments. PGE2 and IL-6 release from cultured cells derived from injured nerves was increased significantly compared with uninjured nerves. Non-selective and selective COX2 inhibitors suppressed PGE2 and IL-6 release. Treatment with PGE2 further enhanced IL-6 release in a concentration- and time-dependent manner. A selective EP4 receptor antagonist L-161982 was able to suppress IL-6 release, whereas an EP1 receptor antagonist, SC19220, was ineffective. Moreover, a protein kinase C inhibitor, calphostin C, dramatically suppressed IL-6 release, whereas a protein kinase A inhibitor H-89 and a Ca2+ chelator EGTA failed. Taken together, our data suggest that PGE2 is involved in mediating the up-regulation of IL-6 occurring in invading macrophages. This action is mediated through an EP4 receptor and the protein kinase C signaling pathway.     

Antagonistic and additive effects of IL-4 and interferon-gamma on human monocytes and macrophages: effects on Fc receptors, HLA-D antigens, and superoxide production

During an immune response a multitude of lymphokines are produced which modulate the function of mononuclear phagocytes. In this study, we investigated possible additive, synergistic, or antagonistic effects of three lymphokines, IL-4 (1-100 U/ml, 0.01-1 ng/ml), interferon-gamma (IFN) (1-100 U/ml) and IL-2 (30-300 U/ml) on Fc receptors (FcR1 and FcR2), complement receptors (CR3 and CR4), and HLA-D antigens (HLA-DR and HLA-DQ) on human monocytes and macrophages. Exposure of monocytes to IL-4 alone resulted in changes in the expression of all these receptors. Both FcR1 and FcR2 were downregulated in a dose-dependent manner while the expression of CR3, CR4, HLA-DR, and HLA-DQ was increased. Antagonistic effects of IL-4 and IFN were observed on FcR1 and FcR2; IL-4-induced downregulation of the FcR1 and FcR2 was inhibited by IFN, and vice versa, IFN-induced upregulation of FcR1 and FcR2 was inhibited by IL-4. Phagocytosis of particulate immune complexes (EAs) as well as production of superoxide (O2-) in response to EAs were inhibited by IL-4, and the inhibition was reversed by IFN. Antagonistic effects of IL-4 and IFN were also observed on CR3 and CR4 expression. Additive effects of IL-4 and IFN were on the other hand seen on HLA-DR and HLA-DQ expression as well as on O2- production in response to stimulation with phorbol ester (PMA). The addition of IL-2 to IL-4 and/or IFN-containing cultures had no further modulatory effect on receptor expression or O2- production. In vitro matured macrophages (M phi) had a similar response pattern to IL-4 and IFN as the freshly isolated monocytes. Alveolar macrophages (AM phi), on the other hand, did not modulate FcR1 and HLA-DQ in response to IL-4, and downregulated FcR2 in response to IFN. Antagonistic effects of the two factors were only seen on CR expression. These results imply that FcR expression and function on monocytes and inflammatory macrophages may be in sensitive balance with the relative concentrations of IL-4 and IFN in the immune environment. FcRs on AM phi are less responsive to modulation by these lymphokines.     

Platelet-activating factor and prostaglandin E2 impair esophageal ACh release in experimental esophagitis

ACh is a neurotransmitter in cat esophageal circular muscle, as atropine nearly abolishes contraction of in vitro circular muscle strips in response to electric field stimulation (EFS) (5, 12). Experimental esophagitis reduced EFS- but not ACh-induced contraction of esophageal circular muscle, suggesting that esophagitis impairs neurotransmitter release. Because IL-1beta and IL-6 are produced in esophagitis and reproduce these changes in normal esophageal muscle (12), we examined the role of IL-1beta and IL-6 in this motor dysfunction. IL-1beta, IL-6 (12), H2O2, PGE2, and platelet-activating factor (PAF) were elevated in esophagitis specimens. Normal muscle incubated (2 h) in IL-1beta and IL-6 had increases in H2O2, PGE2, and PAF levels. H2O2 contributed to increased PGE2 and PAF, as the increase was partially (60-80%) reversed by the H2O2 scavenger catalase. EFS-induced [3H]ACh release from muscle strips significantly (42%) decreased in esophagitis and after 2 h incubation in PGE2 and in PAF C-16. Similarly, EFS-induced but not ACh-induced muscle contraction decreased in esophagitis and after incubation in PGE2 and PAF C-16. Finally, in normal muscle strips treated with IL-1beta electrical field stimulation (EFS)-induced contraction was partially restored by indomethacin or by the PAF antagonist CV3988 and was completely restored by the combination of CV3988 and indomethacin, whereas in strips treated with IL-6, EFS-induced contraction was partially restored by the PAF antagonist CV3988 and not affected by indomethacin. We conclude that IL-1beta-induced production of H2O2 causes formation of PGE2 and PAF that inhibit ACh release from esophageal cholinergic neurons without affecting ACh-induced contraction of esophageal circular muscle. IL-6 causes production of H2O2, PAF, and other unidentified inflammatory mediators.     

Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide.

Although IL-1 is an important modulator of chondrocyte metabolism, the postreceptor events triggered by IL-1 remain obscure. The present study shows that IL-1 induces the biosynthesis of nitric oxide (.N = O) by articular chondrocytes. Synthesis of .N = O is also induced by LPS. Other inflammatory mediators such as IFN-gamma, fibroblast growth factor, and TNF-alpha fail to provoke the production of .N = O, but they increase the potency of IL-1. A combination of IL-1, LPS, and TNF-alpha was shown to induce maximal production of 355 +/- 51 nmol/10(6) cells/72 h of nitrite (NO2-), which was measured as a stable end-product of .N = O generation. The biosynthesis of .N = O requires an induction period of approximately 6 h and continues for at least 72 h. Inhibition of .N = O production with the competitive inhibitor NG-monomethyl-L-arginine (NMA) leads to a suppression of gelatinase and PGE2 synthesis by chondrocytes activated with IL-1 alone. In contrast, NMA enhances the synthesis of both gelatinase and PGE2 after activation with a combination of IL-1, LPS, and TNF-alpha. An increase of PGE2 synthesis from 42.0 +/- 21.0 to 174.0 +/- 33.5 ng/10(6) cells/72 h resulted from the addition of NMA when these stimulatory agents were combined. Exposure of IL-1 and fibroblast growth factor-stimulated chondrocytes to authentic, exogenous .N = O led to an increase of PGE2 synthesis from 5.6 +/- 1.7 of untreated cells to 15.8 +/- 6.8 ng/10(6) of .N = O treated cells within the 1st h. This was followed by a suppression of PGE2 synthesis within the next 2 h.     

Regulation of oxygen radical release from murine peritoneal macrophages by pharmacologic doses of PGE2

The ability of pharmacologic doses of PGE2 to alter the release of superoxide (O2-) and hydrogen peroxide (H2O2) from elicited peritoneal macrophages (M theta) was studied. Twice-daily administration of 200 or 100 micrograms of PGE2 to mice during accumulation of peritoneal M theta resulted in a significant reduction in M theta recovery and in the triggered release of H2O2, but not O2-. Cultivation of elicited M theta from normal mice with concentrations of PGE2 in excess of 10(-7) M for 24-48 h resulted in a significant reduction in the triggered release of H2O2, but not O2-. Cultivation for shorter periods of time or with lower concentrations of PGE2 failed to alter H2O2 release. This effect of PGE2 was reproduced by the phosphodiesterase inhibitor theophylline. The ability of PGE2 to inhibit H2O2 release in the presence of normal production of O2- was not prevented by the addition of superoxide dismutase. Cultivation of peritoneal M theta with 10(-5) M PGE2 for 48 h failed to increase intracellular catalase, although increased H2O2 scavenger activity was demonstrated. The inhibition of extracellular release of H2O2, but not O2-, by pharmacologic doses of PGE2 may be one mechanism for the anti-inflammatory action of this compound.     

In vitro cytokine release by activated murine peritoneal macrophages: role of prostaglandins in the differential regulation of tumor necrosis factor alpha, interleukin 1, and interleukin 6

Distinct subpopulations of macrophages or differently activated macrophages display various functions in immune reactions. Some of their activities depend on specific sets of factors (i.e., cytokines and eicosanoids) produced by activated macrophages. We have studied the ability of murine (CBA/ca) peritoneal macrophages to selectively release tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and IL-6. We have found that the priming of cells (Mo) with different stimulants (thioglycolate vs. LPS) induces the release of particular cytokines by reactivated macrophages. The increased release of TNF-alpha correlates with lower levels of IL-1 and IL-6. We have also found that prostaglandin E2 (PGE2) and prostacyclin (PGI2) have opposing effects on the production of two of these cytokines. The release of TNF-alpha is inhibited by prostaglandins, whereas increased levels of PGE2 and PGI2 correlate with higher levels of IL-6.     

Peritoneal macrophages from patients on continuous ambulatory peritoneal dialysis show a differential secretion of prostanoids and interleukin-1 beta.

In vitro secretion of the prostanoids PGE2 and PGI2 and of the cytokine IL-1 beta by peritoneal macrophages obtained from CAPD patients during episodes of peritonitis and infection free periods, was determined, after culturing with or without 5 micrograms/ml of LPS. The release of PGE2 and PGI2 as measured by its stable metabolite 6-keto-PGF alpha was determined in 10 episodes of peritonitis and 10 infection free periods. IL-1 beta release was determined in 14 episodes of peritonitis and 20 infection free periods. PGI2 release from macrophages declined sharply during peritonitis both in the absence and presence of LPS in the culture medium (p less than 0.005). A tendency to decreased PGE2 release was found during peritonitis, when macrophages were cultured in the absence of LPS. In the presence of LPS, the same amounts of PGE2 were released during peritonitis and during an infection free period. On the other hand, peritoneal macrophages released significantly more IL-1 beta during peritonitis as compared to an infection free period, provided that the cells were in vitro stimulated with LPS. In view of the interregulatory effects between prostanoids and macrophage cytokines in their production, these findings may indicate that the impaired release of PGI2 during peritonitis has allowed the macrophages to secrete more IL-1 beta after in vitro stimulation with LPS. This implies that PGI2 and PGE2 may play a distinct role in the regulation of cytokine secretion by these cells.     

IL-4 inhibits superoxide production by human mononuclear phagocytes

The activation of mononuclear phagocytes (M phi) and their generation of oxidative products is influenced by various cytokines as well as by normal maturational changes. We examined the effects of IL-4 on superoxide (O2-) production (cytochrome c reduction) by cultured M phi and the modulation of these effects by IFN-gamma and IL-1. Incubation of IL-4 (200 U/ml) with M phi inhibited M phi PMA (100 ng/ml)-stimulated O2-. production by 23% at 24 h, 34% at 48 h, and 70 to 85% at 72 to 96 h. IL-4 similarly inhibited M phi O2-. production in response to zymosan. IL-4 did not affect M phi viability, adherence to microtiter plates, or ability to phagocytose boiled yeast. In comparison with M phi, neutrophil O2-. production was not inhibited after 4 to 20 h incubation with IL-4. When IL-4 was washed out as early as 1 h after the initiation of M phi culture, significant inhibition of O2-. production was observed 4 days later. Sequential addition of either IL-4 or IFN-gamma to cultures demonstrated reciprocal cytokine effects on M phi; IL-4 partially inhibited O2-. production by M phi previously treated with rIFN-gamma whereas rIFN-gamma partially augmented O2-. production by M phi previously treated with IL-4. Because IL-4 has been reported to inhibit IL-1 production, add-back experiments were performed; addition of IL-1 only partly reconstituted O2-. production in IL-4-treated cells. Further characterization showed that although M phi protein synthesis was enhanced by both rIFN-gamma and IL-4 treatment, acid phosphatase, a marker of maturation to the macrophage phenotype, was markedly increased at an earlier time point in IL-4-treated M phi, and correlated with a decline in O2-. production. The ability of IL-4 to suppress M phi O2-. production implicates IL-4 as an important regulator of this aspect of the inflammatory response.     

Recombinant granulocyte-macrophage colony-stimulating factor down-regulates expression of IL-2 receptor on human mononuclear phagocytes by induction of prostaglandin E

Recent studies have shown that normal human alveolar macrophages and blood monocytes, as well as HL-60 and U937 monocyte cell lines, newly express IL-2R after stimulation with rIFN-gamma or LPS. In addition, macrophages transiently express IL-2R in vivo during immunologically mediated diseases such as pulmonary sarcoidosis and allograft rejection. We therefore investigated in vitro factors that modulate macrophage expression of IL-2R. IL-2R were induced on normal alveolar macrophages, blood monocytes, and HL-60 cells using rIFN-gamma (24 to 48 h at 240 U/ml), and cells were cultured for an additional 12 to 24 h with rIL-2 (100 U/ml), recombinant granulocyte-macrophage CSF (rGM-CSF, 1000 U/ml), rGM-CSF plus indomethacin (2 X 10(-6) M), PGE2 (0.1 to 10 ng/ml), 1 X 10(-6) M levels of caffeine, theophylline, and dibutyryl cyclic AMP, or medium alone. IL-2R expression was quantitated by cell ELISA (HL-60 cells) or determined by immunoperoxidase staining (alveolar macrophages, blood monocytes, and HL-60 cells), using anti-Tac and other CD25 mAb. PGE production was assayed by RIA. We found greater than 95% of alveolar macrophages, monocytes, and HL-60 cells expressed IL-2R after rIFN-gamma treatment and remained IL-2R+ in the presence of IL-2R or medium alone. By comparison, greater than 95% of cells induced to express IL-2R became IL-2R- after addition of rGM-CSF, and the culture supernatants from GM-CSF-treated cells contained increased levels of PGE. This inhibition of macrophage IL-2R expression by rGM-CSF was blocked by indomethacin, and IL-2R+ macrophages became IL-2R- after addition of PGE2 alone. These findings indicate GM-CSF down-regulates IL-2R expression by human macrophages via induction of PGE synthesis. Moreover, a similar down-regulation of IL-2R expression was seen after stimulation with caffeine, theophylline, or dibutyryl cyclic AMP. Hence, GM-CSF, PGE, and other pharmacologic agents that act to increase intracellular levels of cAMP may play a modulatory role, antagonistic to that of IFN-gamma on cellular expression of IL-2R by human inflammatory macrophages in vivo.     

Cyclic-AMP inhibits neither A23187-stimulated [14C]-arachidonic acid release from prelabelled lipids nor phospholipase A2 activity in resident rat peritoneal macrophages

8-Bromo cyclic AMP inhibited A23187-stimulated PGE2 production in adherent resident rat peritoneal macrophages by 50% when this was assessed by radioimmunoassay. In contrast, neither exogenous 8-bromo cyclic AMP nor elevation of endogenous cyclic AMP with cholera toxin inhibited 14C-arachidonic acid release or labelled prostaglandin formation by [1-14C]-arachidonic acid-prelabelled macrophages stimulated with either A23187 or melittin. Inhibition by cyclic AMP appears to be confined to PGE2 originating from a pool of endogenous phosphoglyceride that does not readily exchange with isotopically-labelled arachidonic acid. Phospholipase A2 activity, assessed as calcium-dependent [1-14C]-arachidonic acid release from exogenous 1-stearoyl, 2-[1-14C]-arachidonyl phosphatidyl choline at pH 8.6, was activated by melittin but not by A23187 in 1000 x g supernates from sonicated cells. Neither melittin nor calcium activation of phospholipase A2 was inhibited by preincubation of the cells prior to breakage with 8-bromo-cyclic AMP, nor by inclusion of either 8-bromo cyclic AMP or the catalytic subunit of cyclic AMP-dependent kinase in the assay. The results are inconsistent with the hypothesis that inhibition of A23187-stimulated PGE2 production by cyclic AMP in peritoneal macrophages is due to inhibition of a calcium-stimulated phospholipase A2.