Resident Rat Peritoneal Macrophages Research Articles

Screening New Drugs for Immunotoxic Potential: I. Assessment of the Effects of Conventional Nonsteroidal Anti-Inflammatory Drugs and Selective COX-2 Inhibitors on In Vitro and In Vivo Phagocytic Activity

Although both experimental and clinical literature contain reports suggestive of associations between enhanced susceptibility to soft tissue infections and nonsteroidal anti-inflammatory drug (NSAID) use, the immunotoxicological potential of this class of therapeutic agents has not been thoroughly investigated. In consideration of the widespread clinical use of these agents, we have initiated studies of the interaction between NSAIDs (both nonselective and selective COX-2 inhibitors) and the immune system. This communication describes the conduct and results of assessments of the effects of NSAIDs on the in vitro phagocytic activity of rat macrophages and canine neutrophils and on the functional activity of the intact murine mononuclear phagocytic system. During in vitro experiments 0.1 to 10 μM concentrations of naproxen, indomethacin, and experimental selective COX-2 inhibitors, SC-236, SC-245 and SC-791, caused marginal, but statistically significant, reductions in phagocytic activity of resident rat peritoneal macrophages. The effects were consistently small and there was no evidence of concentration-response relationships. An in vitro concentration of 10 μM of either SC-236 or SC-791 was required to decrease phagocytosis by dog neutrophils. Repeated oral doses of either naproxen or SC-236 (3, 10, or 30 mg/kg twice daily for 3 days) were without effect on the intact phagocytic system of the mouse. A potential immunotoxicologic effect based on direct impairment of phagocytic processes seems an unlikely explanation for drug-induced susceptibility to infection reported earlier. However, the results of these experiments do not support an unequivocal conclusion relative to immunotoxicological potential of either conventional NSAIDs or selective COX-2 inhibitors. Further studies on other components of the immune system are needed to fully explore possible immunomodulatory effects of NSAIDs.

Methionine-Enkephalin Stimulates Hydrogen Peroxide and Nitric Oxide Production in Rat Peritoneal Macrophages: Interaction of μ, δ and κ Opioid Receptors

Objective: Methionine-enkephalin (MET) modulates various functions of macrophages related to both immune and inflammatory reactions in a naloxone reversible manner, suggesting that opioid receptors are involved in the regulation of macrophage activity. Since an endogenous opioid ligand might interact with more than one type of opioid receptor, the receptor interaction determines its effect on a particular function. Methods: In the present study we have investigated the involvement of different opioid receptor types/subtypes in MET-induced modulation of H₂O₂ and NO production in macrophages. Thioglycollate-elicited or resident rat peritoneal macrophages were treated in vitro with MET and/or specific antagonists of δ_1,2, δ₁, δ₂, µ and ĸ opioid receptors. Results: MET increased H₂O₂production inphorbol myristate acetate-stimulated rat peritoneal macrophages mainly through δ₁ opioid receptor. MET also enhanced NOproduction in rat peritoneal macrophages stimulated with lipopolysaccharide through δ₁ and µ opioid receptors. The blockade of µ and ĸ receptor facilitated a potentiating effect of MET on H₂O₂ release, and blockade of ĸ receptor further raised the MET-induced increase of NO production in macrophages. Conclusion: It is concluded that both negative and positive functional interaction between δ, µ and ĸ opioid receptors regulate the influence of MET on H₂O₂ and NO production in rat peritoneal macrophages.

Prolonged lipopolysaccharide inhibits leukotriene synthesis in peritoneal macrophages: mediation by nitric oxide and prostaglandins

Resident rat peritoneal macrophages synthesize a variety of prostanoids and leukotrienes from arachidonic acid. Overnight treatment with lipopolysaccharide (LPS) induces the synthesis of cyclooxygenase-2 (COX-2) and an altered prostanoid profile that emphasizes the preferential conversion of arachidonic acid to prostacyclin and prostaglandin E 2. In these studies, we report that exposure to LPS also caused a strong suppression of 5-lipoxygenase but not 12-lipoxygenase activity, indicated by the inhibition of synthesis of both leukotriene B 4 and 5-hydroxyeicosatetraenoic acid (5-HETE), but not of 12-HETE. Inhibition of 5-lipoxygenase activity by LPS was both time- and dose-dependent. Treatment of macrophages with prostaglandin E 2 partially inhibited leukotriene synthesis, and cyclooxygenase inhibitors partially blocked the inhibition of leukotriene generation in LPS-treated cells. In addition to COX-2, nitric oxide synthase (NOS) was also induced by LPS. Treatment of macrophages with an NO donor mimicked the ability of LPS to significantly reduce leukotriene B 4 synthesis. Inhibition of NOS activity in LPS-treated cells blunted the suppression of leukotriene synthesis. Inhibition of both inducible NOS and COX completely eliminated leukotriene suppression. Finally, macrophages exposed to prolonged LPS demonstrated impaired killing of Klebsiella pneumoniae and the combination of NOS and COX inhibitors restored killing to the control level. These results indicate that prolonged exposure to LPS severely inhibits leukotriene production via the combined action of COX and NOS products. The shift in mediator profile, to one that minimizes leukotrienes and emphasizes prostacyclin, prostaglandin E 2 and NO, provides a signal that reduces leukocyte function, as indicated by impaired killing of Gram-negative bacteria.

Respiratory Burst Responses of Rat Macrophages to Microsporidian Spores

Leiro, J., Iglesias, R., Paramá, A., Sanmartin, M. L., and Ubeira, F. M. 2001. Respiratory burst responses of rat macrophages to microsporidian spores. Experimental Parasitology98, 1–9. This study investigated the respiratory burst responses of rat resident peritoneal macrophages and of peritoneal macrophages stimulated 5 days previously with viable spores of the fish infecting microsporidian Microgemma caulleryi. Nitric oxide production by resident macrophages and prestimulated macrophages in response to viable microsporidian spores was significantly lower than in response to Escherichia coli lipopolysaccharide (LPS) (nitrite concentration in medium 57 ± 1 μM for resident macrophages stimulated with LPS versus 31 ± 1 μM for resident macrophages stimulated with microsporidian spores and 36 ± 4 μM for M. caulleryi prestimulated macrophages; P < 0.05). Extracellular release of reactive oxygen species (ROS) by resident macrophages in response to microsporidian spores was similar to that in response to Kluyveromyces lactis yeast cells and to that in response to phorbol myristate (a stimulator of protein C kinase). Intracellular ROS production by resident macrophages in response to microsporidian spores was similar to that produced in response to yeast cells. Both extracellular ROS production and intracellular ROS production (in response to all stimuli) were significantly lower after in vivo prestimulation of macrophages with microsporidian spores. These results demonstrate that microsporidian spores of species other than those that habitually infect mammals are capable of modulating the respiratory burst of rat peritoneal macrophages. Such modulation may contribute to avoidance by the microsporidian of cytotoxic responses associated with the respiratory burst.

Activation of rat macrophages by Betafectin PGG-glucan requires cross-linking of membrane receptors distinct from complement receptor three (CR3).

PGG-glucan (Betafectin) is a soluble, highly purified yeast (1,3)-beta-glucan with broad anti-infective and immunomodulatory activities. These studies evaluated the ability of PGG-glucan to directly elicit O2- and tumor necrosis factor alpha (TNF-alpha) production by rat leukocytes in vitro. Particulate beta-glucan stimulated O2- production by the rat NR8383 alveolar macrophage cell line and resident rat peritoneal macrophages, but soluble PGG-glucan did not. In contrast, presentation of PGG-glucan to cells after covalent immobilization to a plastic surface caused a direct stimulation of O2- and TNF-alpha production. The O2- response of rat leukocytes to immobilized PGG-glucan was inhibited by soluble PGG-glucan, indicating that cellular responses to both immobilized and soluble PGG-glucan occur via common cell surface receptors. Because complement receptor type three (CR3) has been proposed as a beta-glucan receptor on human leukocytes, NR8383 cells were evaluated for the presence of CR3. Indirect immunofluorescence and flow cytometric analysis showed that despite being responsive to both particulate and immobilized beta-glucans, NR8383 cells expressed no detectable CR3. These results indicate that the beta-glucan receptors on NR8383 cells are not CR3 and suggest that physical presentation plays an important role in inducing pro-inflammatory leukocyte responses to PGG-glucan.

Lipoprotein(a) induces cell growth in rat peritoneal macrophages through inhibition of transforming growth factor-β activation

To elucidate the atherogenicity of lipoprotein(a) (Lp(a)), we examined its growth-stimulating activity in rat resident peritoneal macrophages. When macrophages were incubated with Lp(a), cell numbers were increased 1.5-fold as compared with control macrophages. Furthermore, apolipoprotein(a) (apo(a)), a plasminogen-like glycoprotein which is covalently attached to a low density lipoprotein-like particle (Lp(a-)), also induced macrophage growth, while the growth-stimulating effect of Lp(a-) was negligible. These results suggest that apo(a) plays an active role in the mitogenic activity of Lp(a). Lp(a)-induced macrophage growth was inhibited by exogenously added active transforming growth factor-β (TGF-β) dose-dependently, and also by the addition of plasmin, which converts latent TGF-β to an active form. Moreover, the amounts of endogenous active TGF-β in the medium were significantly reduced by the incubation with Lp(a). It is evident from these results that Lp(a) induces macrophage growth by inhibiting TGF-β activation. The capacity of Lp(a) to stimulate macrophage growth shown here could be a novel atherogenic function of Lp(a).

Effect of degree of unsaturation in dietary fatty acids on arachidonic acid mobilization by peritoneal macrophages

Cells from rats fed with a tripalmitin diet showed a depletion of phospholipid arachidonate and n-3 fatty acids such as eicosapentaenoic and docosahexaenoic acids (EPA and DHA). In rats fed fish oil diet, a significant reduction in arachidonic acid (AA) content was observed whereas EPA and DHA were incorporated into membranes lipids. These changes in lipid composition of membranes did not affect cellular adherence, phagocytic capability, or [3H]AA incorporation. However, both tripalmitin and fish oil diets induced a decrease in [3H]AA mobilization stimulated by 4 beta-phorbol-12-myristate 13-acetate, A23187, or opsonized-zymosan in rat peritoneal macrophages. These results demonstrate that the antiinflammatory effects of essential fatty acids deficiency or n-3 enrichment diets may be associated with a decreased AA mobilization in resident rat peritoneal macrophages treated with proinflammatory agents.

Expression of vasoactive intestinal peptide binding sites in rat peritoneal macrophages is stimulated by inflammatory stimulus

Vasoactive intestinal peptide (VIP) binding to resident and stimulated-rat peritoneal macrophages was studied. No specific VIP binding was obtained with resident rat peritoneal macrophages. In contrast, VIP bound specifically to casein-elicited macrophages. The Scatchard analysis of binding data was consistent with the presence of two classes of VIP binding sites, but may represent a receptor site and internalized VIP. Both specific VIP binding and number of specific high affinity binding sites for VIP augmented progressively after sodium caseinate injection, reaching maximum at days 4–5. Macrophages obtained 1 day after injection showed a minimal specific VIP binding (0.3 ±0.1% of total), but cells obtained 4 days after injection showed a maximal binding to the peptide (3.1 ± 0.2% of total). The number of high affinity binding sites per cell raised also progressively after sodium caseinate injection: 2650 ±301 at day 2, 4939 ± 723 at day 3, 6684 ± 903 at day 4 and 9636± 1626 at day 5 ( P = 0.0035). The number of low affinity binding sites per cell exhibited the same changes. In contrast, the K d values of both high and low affinity VIP binding sites did not vary significantly ( P > 0.05). These results demonstrate that VIP binding sites are only displayed by stimulated macrophages, suggesting that VIP binding sites could be considered to be a pre-activation marker in macrophages and could be used to recognize inflammatory or stimulated macrophages.

INCREASED PHOSPHOLIPID MASS WITH DECREASED ARACHIDONOYL MOLECULAR SPECIES IS ASSOCIATED WITH DECREASED EICOSANOID SYNTHESIS AND INCREASED TUMOR NECROSIS FACTOR SYNTHESIS BY THIOGLYCOLLATE-ELICITED RAT PERITONEAL MACROPHAGES

Because the activation state of macrophages may alter their response to endotoxin, we compared phospholipid arachidonic acid content, and synthesis of eicosanoids and tumor necrosis factor by resident and thioglycollate-elicited rat peritoneal macrophages. Thioglycollate elicitation increased macrophage phospholipid mass twofold, increased the relative percentages of 16:0-20:4 diacylglycerophosphocholine (PtdCho) and 18:0-20:4 diacylglycerophosphoethanolamine (PtdEtn), and decreased the relative percentages of 18:0-20:4 alkenylacylglycerophosphoethanolamine (PlsEtn) and 18:0-20:4 alkylacylglycerophosphocholine (PakCho) compared with resident peritoneal macrophages. Thioglycollate-elicited macrophages synthesized significantly less thromboxane B2, 6-keto-prostaglandin F1 alpha, and prostaglandin E2 and more tumor necrosis factor (TNF) activity in response to both endotoxin and A23187 than did resident macrophages. These results suggest that thioglycollate elicitation decreases specific arachidonic acid-containing molecular species in PlsEtn and PakCho, which may, in part, explain the decrease in eicosanoid and increase in TNF synthesis by thioglycollate-elicited macrophages. The differences between resident and thioglycollate-elicited macrophages in the synthesis of the eicosanoids and TNF activity was not altered by increasing either the concentration of either stimulus or the incubation time.

Selective induction of PDGF gene expression in peritoneal macrophages by interleukin-2

We have previously reported that culture of human peripheral blood leukocytes with interleukin-2 (IL-2) triggers the secretion of mediators which induce fibroblast proliferation and collagen synthesis. In addition, fibrogenic cytokines (transforming growth factor-β 1 (TGFβ 1) and platelet-derived growth factor (PDGF) A and B chain) are present in the peritoneal fluid of patients undergoing intraperitoneal immunotherapy (IL-2-activated killer cells and IL-2) who go on to develop peritoneal adhesions. To determine the role of IL-2 in the formation of these adhesions, we chose to investigate whether IL-2 can induce the expression of fibrogenic cytokine genes in resident rat peritoneal macrophages. Cells were cultured with or without IL-2 or lipopolysaccharide (LPS) and expression of PDGF A chain, PDGF B chain, and TGFβ 1 mRNAs was determined. PDGF A and B chain mRNAs are minimally expressed in macrophages prior to stimulation and are induced within 2 hours of treatment with IL-2. In contrast, TGFβ 1 mRNA is constitutively expressed and can not be upregulated. The studies suggest that peritoneal macrophage-derived PDGF plays a critical role in the production of adhesions in patients receiving intra-abdominal immunotherapy.

Arachidonic acid stimulates interleukin-6 release from rat peritoneal macrophages in vitro: Evidence for a prostacyclin-dependent mechanism

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (Mø) in vitro. AA (0.5–16 μM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 μM AA generating a peak of IL-6 release (3-5-fold). AA (0.5–16 μM) also induced an increasing release of the AA metabolite thromboxane B 2 (TXB 2). The AA-induced release of IL-6 occurred within 1–2 h of incubation, whereas TXB 2 concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 μM and 40.0 μM) effectively blocked the generation of TXB 2, but increased prostacyclin (PGI 2) generation and potentiated the release of IL-6. In addition, PGI 2, as well as the PGI 2 agonists iloprost and cicaprost, stimulated IL-6 release from Mø by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI 2 (but not TXA 2) is involved in AA-induced IL-6 release from peritoneal Mø.

A single exogenous stimulus activates resident rat macrophages for nitric oxide production and tumor cytotoxicity.

Based on experiments with mouse cells, it appears that macrophage activation for cytotoxicity requires two exogenous signals. One signal primes or sensitizes the cells, while the second activates them for killing. The present studies were designed to analyze the capacity of rat macrophages to be activated for nitric oxide production and for cytotoxicity by different inflammatory stimuli. We found that both resident alveolar macrophages (AMs) and resident peritoneal macrophages (PMs) from Sprague-Dawley rats produced nitric oxide in response to relatively low doses of a single exogenous activating stimulus [interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS)]. Resident PMs treated with either of these agents alone also exhibited nitric oxide-mediated cytotoxicity toward xenogeneic and allogeneic tumor targets. In contrast to results reported previously with both resident and elicited PMs from mice, tumor necrosis factor-alpha (TNF-alpha) exerted only a small enhancing effect on IFN-gamma-induced nitric oxide production by resident rat PMs. In addition, although some level of cooperativity between IFN-gamma and LPS was observed at low concentrations of LPS (< 10 ng/ml), IFN-gamma did not augment the effects of higher concentrations of LPS (> or = 10 ng/ml) on nitric oxide production by PMs. In contrast to PMs, AMs had a strong synergistic response to combinations of IFN-gamma and LPS or TNF-alpha but also only at relatively low concentrations of IFN-gamma and LPS. Furthermore, maximum nitric oxide production induced by IFN-gamma in these cells could be enhanced by TNF-alpha or low doses of LPS. However, as observed with PMs, combinations of IFN-gamma and higher doses of LPS did not significantly augment maximum nitric oxide production induced in AMs by LPS alone. Thus, our data suggest that resident rat PMs and AMs resemble elicited mouse PMs in their ability to respond to a single activating stimulus. However, PMs and in some respects AMs differ from the latter by their reduced responsiveness to combinations of activators. Taken together, our results suggest that two exogenous stimuli are not required for full activation of resident macrophages from Sprague-Dawley rats.

Modulation of glucose metabolism in macrophages by products of nitric oxide synthase

Nitric oxide (NO) is a product of L-arginine metabolism that suppresses cellular oxidative metabolism through the inhibition of tricarboxylic acid cycle and electron transport chain enzymes. The impact of NO synthase (NOS) activity on specific pathways of glucose metabolism in freshly harvested and overnight-cultured rat resident peritoneal macrophages, at rest and after stimulation with zymosan, was investigated using radiolabeled glucose. NOS activity was modulated through the L-arginine concentration in culture media and the use of its specific inhibitor, NG-monomethyl-L-arginine, and quantitated using radiolabeled L-arginine. Results demonstrated that NOS activity was associated with increased glucose disappearance, glycolysis, and hexose monophosphate shunt activity and, in line with the known inhibition of oxidative metabolism associated with the production of NO, with a decrease in the flux of glucose and butyrate carbon through the tricarboxylic acid cycle. In addition, the relative increase in glucose utilization that follows zymosan stimulation was enhanced by treatments that suppressed NOS activity. These results demonstrate that the characteristics of glucose metabolism by macrophages are, to a significant extent, determined by products of NOS.

Beta-VLDL-induced cholesterol ester deposition in macrophages may be regulated by neutral cholesterol esterase activity.

We examined the role of enzyme activities involved in cholesterol ester metabolism in the accumulation of cholesterol ester in macrophages. When [3H]cholesterol linoleate-beta-very low density lipoproteins (beta-VLDLs) were incubated with rat resident peritoneal macrophages (RPMs), thioglycolate-elicited macrophages (TEMs), or alveolar macrophages (AMs), cholesterol ester accumulation was the greatest in TEMs and the least in AMs. The uptake of [3H]cholesterol linoleate-beta-VLDL into AMs was four times that into RPMs, and the uptake into TEMs was twice that into RPMs. The [3H]cholesterol released from [3H]cholesterol linoleate-beta-VLDL-loaded AMs was five times higher than from TEMs and RPMs, whereas those from RPMs and TEMs were almost the same. In studies using cell homogenates, the acid cholesterol esterase activity in AMs was 1.7 times that in TEMs and six times that in RPMs. The acyl-coenzyme A:cholesterol acyltransferase (ACAT) activities in AMs and TEMs were similar but were higher than that in RPMs. The neutral cholesterol esterase activity was seven times higher in AMs than in RPMs and TEMs. In the study using intact cells, the hydrolysis of loaded [3H]cholesterol linoleate-beta-VLDL in the presence of the ACAT inhibitor CL277,082 and the cholesterol [14C]oleate synthesis from [14C]oleate were enhanced in AMs about twofold compared with RPMs and TEMs. The reduction of de novo synthesized cholesterol [14C]oleate in the presence of CL277,082 was enhanced in AMs four times compared with TEMs or RPMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane association of active 5-lipoxygenase in resting cells. Evidence for novel regulation of the enzyme in the rat alveolar macrophage.

The enzyme 5-lipoxygenase (5-LO) catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes, substances which play pivotal roles both in normal host defense and in pathologic states of inflammation. Recent studies in granulocytic cells have shown that activation of 5-LO involves its Ca(2+)-dependent translocation from cytosol to membrane compartments. However, little information exists about the molecular regulation of 5-LO in macrophages, even though these cells comprise the resident effector cell population of most organs. We therefore examined the levels of 5-LO activity and immunoreactive protein in cytosol and membrane fractions of resident rat alveolar (AM) and peritoneal macrophages (PM) and compared them with the well studied human neutrophil (polymorphonuclear leukocyte). In the resting state, PM resembled polymorphonuclear leukocyte in that most of their cell-free 5-LO activity, as well as protein content, were localized to the cytosol fraction. By contrast, resting AM contained most of their activity and almost half of their immunoreactive protein in the crude membrane fraction. The inability of the drug MK-886 to reverse this membrane association suggested that the 5-LO-activating protein was not the site of binding in the resting cell; however, this drug completely inhibited leukotriene B4 synthesis in ionophore A23187-stimulated AM, indicating that an interaction between 5-LO and 5-LO-activating protein was nonetheless required for product synthesis upon stimulation. Translocation of cytosolic 5-LO protein could not be convincingly demonstrated in A23187-stimulated AM, suggesting that the pool of 5-LO enzyme responsible for product formation originated in the membrane rather than the cytosol fraction of the resting cell. The AM therefore represents the first mammalian cell in which 5-LO has been recovered from the membrane fraction (a) of a resting cell and (b) in active form. These novel findings extend our understanding of the molecular regulation of 5-LO and may be of importance in designing strategies to limit inflammation in the lung and other sites.

Habituation of the acetylcholine-induced formation of inobitol 1,4,5-trisphosphate in human neuroblastoma SH-SY5Y cells

Leiro, J., Iglesias, R., Paramá, A., Sanmartin, M. L., and Ubeira, F. M. 2001. Respiratory burst responses of rat macrophages to microsporidian spores. Experimental Parasitology98, 1–9. This study investigated the respiratory burst responses of rat resident peritoneal macrophages and of peritoneal macrophages stimulated 5 days previously with viable spores of the fish infecting microsporidian Microgemma caulleryi. Nitric oxide production by resident macrophages and prestimulated macrophages in response to viable microsporidian spores was significantly lower than in response to Escherichia coli lipopolysaccharide (LPS) (nitrite concentration in medium 57 ± 1 μM for resident macrophages stimulated with LPS versus 31 ± 1 μM for resident macrophages stimulated with microsporidian spores and 36 ± 4 μM for M. caulleryi prestimulated macrophages; P < 0.05). Extracellular release of reactive oxygen species (ROS) by resident macrophages in response to microsporidian spores was similar to that in response to Kluyveromyces lactis yeast cells and to that in response to phorbol myristate (a stimulator of protein C kinase). Intracellular ROS production by resident macrophages in response to microsporidian spores was similar to that produced in response to yeast cells. Both extracellular ROS production and intracellular ROS production (in response to all stimuli) were significantly lower after in vivo prestimulation of macrophages with microsporidian spores. These results demonstrate that microsporidian spores of species other than those that habitually infect mammals are capable of modulating the respiratory burst of rat peritoneal macrophages. Such modulation may contribute to avoidance by the microsporidian of cytotoxic responses associated with the respiratory burst.

Basal activation of protein kinase C in rat alveolar macrophages: implications for arachidonate metabolism.

Alveolar macrophages (AM) exhibit numerous functional differences from other mononuclear phagocyte populations, even though they are derived from a common circulating monocytic precursor. Yet no differences in fundamental signaling mechanisms uniquely expressed by AM have been elucidated to date. Protein kinase C (PKC) is one signal transduction mechanism thought to have an important role in regulating macrophage function and about which little information exists for AM. This study was undertaken to assess the state of activation of PKC in cultured resident rat AM compared with resident rat peritoneal macrophages (PM) and the means by which active PKC regulates arachidonic acid (AA) metabolism in the two cell types. As assessed by a histone phosphorylation assay, resting AM, in contrast to PM, exhibited constitutive activation of PKC as evidenced by localization of a majority of PKC activity to the membrane fraction. Ionophore A23187-stimulated release and metabolism of AA were attenuated by depletion of or inhibition of cellular PKC activity in AM but not in PM. In contrast, A23187-stimulated AA metabolism was augmented by activation of PKC to a greater extent in PM than in AM. Results from both cell types indicated that the 5-lipoxygenase pathway was particularly upregulated by PKC activation. We conclude that activation of PKC occurs uniquely during macrophage residence in the alveolar space and that this property as well as the downregulation of PKC which results have profound consequences for the regulation of at least one important macrophage function, the synthesis of bioactive eicosanoids.

Endotoxin-induced arachidonic acid metabolism requires de novo protein synthesis and protein kinase C activation

The mechanisms whereby bacterial endotoxins stimulate arachidonic acid metabolism in macrophages are uncertain. Both protein kinase C activation and de novo protein synthesis occur in macrophages in response to endotoxin. In this study we evaluated the time course and role of protein kinase C and de novo protein synthesis in endotoxin stimulated arachidonic acid metabolism in resident rat peritoneal macrophages. Thromboxane (TX) B2 was measured as the representative arachidonic acid metabolite synthesized in response to Salmonella enteritidis endotoxin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate (PMA). The effect of inhibition of protein kinase C by 1-(5-isoquinolinsulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine on endotoxin- and A23187-induced TXB2 synthesis was examined. The potential roles of transcriptional and translational events in endotoxin- and A23187-stimulated TXB2 synthesis were determined by utilizing the transcriptional inhibitors camptothecin (10 microM) or actinomycin D (0.08 microM), and the translational inhibitor cycloheximide (0.1 microM). Whereas, A23187 stimulated maximal TXB2 synthesis within 15 min, endotoxin showed a more prolonged time course with a 12-fold increase in TXB2 synthesis above basal levels after 3 h (P less than 0.05). PMA induced an approx. 8-fold increase above basal TXB2 levels that was blocked by inhibition of transcription with actinomycin D. H-7 (10 microM to 50 microM) inhibited endotoxin- and A23187-stimulated eicosanoid synthesis. Staurosporine (0.2 microM) produced a selective 66% inhibition of endotoxin, but not A23187-stimulated TXB2 synthesis. Endotoxin-induced TXB2 production was significantly (P less than 0.05) inhibited by staurosporine, camptothecin, actinomycin D or cycloheximide at intervals from 30 min prior to, through 60 min after endotoxin stimulation. These studies suggest a role for protein kinase C activation and de novo protein synthesis in endotoxin signal transduction events leading to increased macrophage arachidonic acid metabolism. These intracellular events are essential in sustaining the prolonged inflammatory response to endotoxin.

A thrombin receptor in resident rat peritoneal macrophages

Resident rat peritoneal macrophages possess 6 × 102 high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 × 104 low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 °C, and after 12 min at 4 °C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min−1 at 4 °C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radioactivity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor Size of Mr 120,000.

Effect of anti-leprosy drugs on superoxide anion production by rat peritoneal macrophage with special reference to light exposed clofazimine

The present study describes the in vitro effect of anti-leprosy drugs on superoxide anion (O 2 −) production by rat resident peritoneal macrophages. Of the three drugs tested i.e. clofazimine, rifampicin and dapsone, the first was most effective in increasing O 2 − production in a dose dependent manner, while rifampicin had some stimulatory effect and dapsone exhibited minimal action. Furthermore, when clofazimine and dapsone were added together it was observed that the increase of O 2 − production by macrophages due to clofazimine was not significantly altered by the addition of dapsone. Moreover, it was found that killed Mycobacterium leprae could induce a lesser amount of O 2 − production in comparison to that of Staphylococcus aureus and the enhancement of O 2 − release due to clofazimine was stimulus dependent. This increase of O 2 − release after addition of clofazimine was inhibited by the addition of p-bromophenacyl bromide. Another interesting finding was that the enhancement of O 2 − production by clofazimine gradually decreased as clofazimine was exposed to light for days. On further investigation it was found that ultraviolet, NMR, infrared and mass spectra of the light unexposed and exposed drug were similar, but the diffusion current of the polarogram of light exposed drug was remarkably more than that observed in light unexposed drug, indicating, thereby, a possible increase in the electron accepting capacity of the light reacted molecule. As far as we know this is the first report describing the effect of light exposed clofazimine on the respiratory burst activity of macrophages.