Enhancement by cyclo-oxygenase inhibitors of platelet-activating factor production in thapsigargin-stimulated macrophages

1. Thapsigargin stimulated the accumulation of cell-associated platelet-activating factor (PAF) and extracellular prostaglandin E2 (PGE2) in rat peritoneal macrophages. PAF in the conditioned medium was less than the detectable amount. To obtain further insight into the mechanism of PAF accumulation, the role of PGE2 in PAF accumulation was investigated. 2. When macrophages were incubated in medium containing thapsigargin (30 ng ml(-1), 46.1 nM) and cyclo-oxygenase inhibitors such as indomethacin, naproxen or ibuprofen, the PAF content of the cells at 10 min was increased in a concentration-dependent manner in accordance with inhibition of PGE2 production. The stimulation by thapsigargin, cyclo-oxygenase inhibitors did not increase PAF accumulation. 3. In thapsigargin-stimulated macrophages, when PGE2(10(-7) M) was added to the medium, the cyclo-oxygenase inhibitor-induced stimulation of PAF accumulation at 10 min was markedly inhibited. 4. The accumulation of PAF induced by thapsigargin alone at 10 min was inhibited by exogenous PGE2 (10(-8) and 10(-7) M), or arachidonic acid (10(-6) and 10(-5) M) in accordance with the increase in PGE2 production. 5. The accumulation of PAF induced by thapsigargin alone or by thapsigargin and indomethacin (10(-6) M) was inhibited by dibutyryl cyclic AMP. 6. These results indicate that the concurrently produced PGE2 in thapsigargin-stimulated macrophages down-regulates PAF accumulation by increasing intracellular cyclic AMP levels, and that cyclo-oxygenase inhibitors increase PAF accumulation by inhibiting PGE2 production.     

Pituitary adenylate cyclase-activating polypeptide (PACAP38) modulates lymphocyte and macrophage functions: stimulation of adherence and opposite effect on mobility

The effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) in a concentration range from 10(-13) to 10(-6) M were studied, in vitro, on two functions of peritoneal rat lymphocytes and macrophages: adherence and mobility (spontaneous and chemotaxis). The results show that PACAP38 raised the adherence of the two cell types, increased the mobility of macrophages and decreased the mobility of lymphocytes. The maximal effects were observed at 10(-10) M in macrophages and at 10(-9) M in lymphocytes. Moreover, incubation with increasing concentrations of phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, resulted in a progressive enhancement of adherence and chemotaxis of both macrophages and lymphocytes. In contrast, retinal, a PKC inhibitor, significantly decreased these capacities. Incubation of macrophages with both PMA and PACAP38 did not have a synergistic effect on chemotaxis and adherence whereas, with lymphocytes, adherence was increased and chemotaxis was partially decreased. On the other hand, incubation with forskolin (an enhancer of intracellular cyclic AMP [cAMP] levels) caused inhibition and stimulation of chemotaxis and adherence, respectively, in both cell types. PACAP38 prevented the inhibitory effect of forskolin on chemotaxis of macrophages but not of lymphocytes, whereas the simultaneous presence of PACAP38 and forskolin was synergistic for adherence of both peritoneal cells. In addition, PACAP38 was chemoattractant for macrophages but not for lymphocytes. Furthermore, a VIP receptor antagonist was able to partially reverse the modulatory effects of PACAP38 on lymphocytes, but not on macrophages. These data suggest that PACAP38 exerts its action through the binding to type I PACAP receptors and PKC activation in macrophages and through the elevation of intracellular cAMP levels by binding to type II PACAP receptors in lymphocytes. The present work reveals an additional link between neuropeptides and the immune system and suggests that the peptide PACAP modulates the immunological function of macrophages and lymphocytes.     

Macrophage oxidative modification of low density lipoprotein occurs independently of its binding to the low density lipoprotein receptor

The oxidative modification of low density lipoproteins (LDL) by arterial wall cells is thought to contribute to atherogenesis. Monocyte/macrophages, among other arterial wall cells, oxidatively modify LDL to a form that is recognized by scavenger/oxidized LDL receptors. It has recently been suggested that LDL binding to the LDL receptor (B/E receptor) is essential for macrophage-mediated oxidation of LDL. In the present study, we compared the ability of resident peritoneal macrophages from LDL-R-deficient (LDLR-/-) mice to oxidize LDL with that of resident peritoneal macrophages from C57B6 mice. The LDLR-/- macrophages oxidized LDL at least as rapidly as did the C57B6 macrophages both in F-10 medium and in Dulbecco's modified Eagle's medium supplemented with 1 microM copper (DMEM-Cu2+). Studies were also conducted to examine the effect of preincubation of LDLR-/- and C57B6 macrophages with 10% lipoprotein-deficient serum (LPDS), which up-regulates LDL receptors, or with acetylated LDL (Ac-LDL), which increases cellular cholesterol and down-regulates LDL receptors. Preincubation with 10% LPDS had no significant effect on subsequent LDL oxidation by either type of cells in F10 medium, but the C57B6 cells did show a small (18%) but significant increase in LDL oxidation in DMEM-Cu2+. Preincubation with 50 micrograms/ml Ac-LDL in F10 medium had no effect on LDL oxidation by either LDLR-/- or C57B6 macrophages. Preincubation with 100 micrograms/ml Ac-LDL had no effect on subsequent LDL oxidation by C57B6 cells but, unexpectedly, caused a modest (26%) fall in LDL oxidation by the receptor-negative cells. Using DMEM-Cu2+ medium, preincubation with Ac-LDL reduced LDL oxidation substantially (50-66%) but the effect was just as great in LDL-R negative cells (59-66%) as in C57B6 cells (50-58%), suggesting that the effect is not due to changes in LDL receptor density. It may be related somehow to the Ac-LDL-induced increase in cell cholesterol content. The data demonstrate that the absence of LDL receptors does not reduce the ability of macrophages to oxidize LDL and that LDL binding to LDL receptors is not an essential requirement for macrophage oxidation of LDL.     

Perinatal exposure to morphine: reactive changes in the brain after 6-hydroxydopamine

The decapeptide H2N-Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-COOH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of the human immunoglobulin G1 Eu heavy chain and displaying a 43% identity with the antigenic determinant of beta-endorphin was synthesized. Immunorphin was found to compete with 125I-beta-endorphin for high-affinity receptors on murine peritoneal macrophages (K = 2.5 +/- 0.9 x 10(-9) M) and with 3H-morphin for receptors on murine thymocytes (Ki = 2.7 +/- 0.6 x 10(-9) M) and murine macrophages (Ki = 5.9 +/- 0.7 x 10(-9) M). In particular two types of receptors to 125I-beta-endorphin with Kd1 = 6.1 +/- 0.6 x 10(-9) M and Kd2 = 3.1 +/- 0.2 x 10(-8) M were revealed on macrophages. The second type of receptors interacted with 125I-beta-endorphin, 3H-Met-enkephalin, 3H-Leu-enkephalin and 3H-morphin; the first displayed reactivity with 125I-beta-endorphin, 3H-morphin and immunorphin. The first type receptors are not present on murine brain cells nor are inhibited by naloxone. A minimum fragment of immunorphin practically completely retaining its inhibitory activity in the competition tests with 125I-beta-endorphin for common receptors on thymocytes was found to correspond to the tetrapeptide H2N-Lys-Gly-Phe-Tyr-COOH (Ki = 5.6 +/- 0.5 x 10(-9) M).     

DNA polymerase-beta from the pupal ovaries of Bombyx mori

The action of vasoactive intestinal peptide (VIP) on macrophages has not yet been studied, although there are studies that show an inhibitory action of VIP on lymphocyte functions. The present study shows that VIP in a range from 10(-12) to 10(-7) M increased significantly the phagocytosis and digestion capacities of rat peritoneal macrophages. The most effective concentration of VIP was 10(-9) M followed by 10(-8) M. With respect to the phagocytic capacity, the ingestion of cells (Candida albicans) or inert particles (latex beads) was stimulated significantly with all the concentrations used. The digestion capacity was analyzed through the production of superoxide anion, measured by the reduction of nitroblue tetrazolium (NBT). As with phagocytic capacity, superoxide anion production was increased by VIP in non-stimulated macrophages (incubated without latex beads) and even more in stimulated cells (incubated in the presence of latex beads). The study of the mechanism of action of this neuropeptide showed that protein kinase C (PKC) was activated in the presence of VIP concentrations from 10(-10) to 10(-8) M in a similar way to that found with a specific PKC activator such as phorbol myristate acetate (PMA, 50 ng/ml). PMA also stimulated significantly the phagocytosis and digestion capacities of rat macrophages. By contrast, a PKC inhibitor, retinal (20 microM), decreased significantly the phagocytosis and digestion capacities. These data show that VIP could stimulate these macrophage functions through PKC activation.     

The perceptions of line and senior managers in relation to occupational health issues

The present study demonstrated the change in interleukin-1 (IL-1) production of peritoneal macrophages during the estrous cycle in golden hamsters and discussed its possible roles in ovarian function. Macrophages were collected from the peritoneal cavity at 0900 h on various days of the estrous cycle and incubated for 6 h in the presence of ovine pituitary LH (500 ng/ml). The IL-1 concentration in the media was measured by bioassay with the A375S2 human melanoma cell line. The number of macrophages significantly (P < 0.01) increased on estrus and proestrus compared with diestrus 1 or diestrus 2. LH-induced production of IL-1 was also greater (P < 0.01) on proestrus (292 +/- 36 pg/10(6) cells/ ml) and estrus (222 +/- 30 pg/10(6) cells/ml) than on diestrus 1 (34 +/- 15 pg/10(6) cells/ml) or diestrus 2 (117 +/- 16 pg/10(6) cells/ml). To clarify the factor inducing the changes in peritoneal macrophages, hamsters were ovariectomized on diestrus 1, and 3 weeks later the animals were treated with s.c. injections of progesterone (200 micrograms/day), testosterone (100 micrograms/day), estradiol (10 micrograms/day) or sesame oil for three days. The hamsters were killed 24 h after the last injection, and the number and IL-1 producing capacity of macrophages were determined. The number of macrophages and their response to LH to produce IL-1 were increased significantly (P < 0.01) by estradiol treatment but not by progesterone or testosterone treatment. It was concluded that the peritoneal macrophages became more sensitive to LH to produce IL-1 on proestrus and estrus in cyclic hamsters, and that these changes in macrophages, probably induced by estradiol, would play important roles in ovarian function.     

Corticosteroids inhibit murine macrophage Ia expression and interleukin 1 production

Corticosteroids have profound effects on functions of the macrophage associated with antigen presentation to T cells. The drugs inhibited the expression of surface I-region-associated (Ia) antigens by peritoneal macrophages both in vitro and in vivo, reduced the production of IL 1, and inhibited antigen presentation for T cell proliferation by macrophages. The doses of hydrocortisone and prednisolone that inhibited by 50% Ia expression in cultured macrophages ranged around 2 to 5 x 10(-8) M. These results could explain one mechanism by which corticosteroids suppress the induction of immune responses.     

Protein translocation functions of Escherichia coli SecY: in vitro characterization of cold-sensitive secY mutants

Activated macrophages are among the major constituents of the periapical granuloma. Their state of activation may persist for long periods after the local irritant is removed and may delay resolution and repair of the lesion. The effect of activated macrophages on fibroblast growth was studied in vitro. Circular fibroblast colonies were formed using a drop containing 7.5 x 10(5) murine dermal fibroblasts and allowed to grow for 7 days. When peritoneal exudate macrophages were added (0.5-3.0 x 10(6) cells/dish) and activated in vitro by LPS (1 microgram/ml), the fibroblast colony's growth was suppressed. LPS alone, at the concentration used, had no effect on the fibroblast growth. Hydrocortisone (> or = 10(-7) M) totally reversed the suppression, when added either simultaneously with or 6, 24, or 48 h after the LPS. The efficacy of late hydrocortisone treatment suggests that its effect was through prevention of the expression of the LPS activation of the macrophages. These findings may provide a possible clue to a pharmacological modulation of the healing processes that occur in the periapical lesion once its infective source had been eliminated.     

Isolation and characterization of a proteoglycan variant from human aorta exhibiting a marked affinity for low density lipoprotein and demonstration of its enhanced expression in atherosclerotic plaques

Proteoglycans (PG) are implicated in the pathophysiology of atherosclerosis due to their ability to complex with plasma low density lipoproteins (LDL). Studies were conducted to determine whether human aorta contains PG subclasses that exhibit enhanced LDL binding ability. PG were isolated from normal and atherosclerotic aortas by a combination of dissociative extraction and ion-exchange chromatography. The PG were further subfractionated on an LDL affinity column based on their binding affinity to LDL. Two PG fractions exhibiting high-affinity binding to LDL, as evidenced by their elution at 1.0 and 1.5 M NaCl, respectively, were isolated from both normal and atherosclerotic tissue. Compared with normal tissue, atherosclerotic tissue showed a twofold increase in the high-affinity PG that eluted at 1.5 M NaCl. Gel filtration of the high-affinity PG from normal tissue yielded two peaks (nPG2 and nPG3), while the high-affinity PG from plaque tissue was resolved into three peaks (pPG1, pPG2, and pPG3). pPG1 eluted at the void volume of the column, indicating that it was of very large molecular size. The hydrodynamic size of pPG2 was larger than that of the corresponding nPG2 (Kav = 0.44 versus 0.51), while pPG3 had the same hydrodynamic size as nPG3 (Kav = 0.86). The high-affinity PG subfractions from normal aorta contained varying proportions of chondroitin sulfates, dermatan sulfates, and heparan sulfate. In contrast, the PG subfractions from plaque tissue contained predominantly chondroitin sulfates and heparan sulfate. In vitro complexes of LDL and the high-affinity PG fractions from normal aorta and plaque tissue stimulated cholesteryl ester synthesis in human monocyte-derived macrophages. However, the LDL-plaque PG complex was significantly more potent than the LDL-normal aorta PG complex in this respect. These results indicate that PG subclasses with enhanced binding affinity to LDL occur in the normal human aorta and that their concentration increases significantly in atherosclerotic lesions. In addition, the high-affinity PG in plaque tissue have altered characteristics and increased ability to stimulate LDL-mediated cholesterol ester synthesis in macrophages. This could lead to increased lipid deposition during atherogenesis.     

Substance P regulates somatostatin expression in inflammation

Substance P (SP) and somatostatin (SOM) are made at mucosal surfaces and sites of inflammation. There is a SP/SOM immunoregulatory circuit that modulates the IFN-gamma response in murine schistosomiasis. SP enhances, while SOM decreases, IFN-gamma secretion. Various inflammatory mediators induce macrophages to make SOM, but no known factor limits this expression. It was discovered that SP regulates SOM synthesis. Splenocytes from normal, uninfected mice cultured with LPS, IFN-gamma, or IL-10 for 4 h strongly expressed SOM mRNA, but failed to do so in the presence of SP. The inhibition with 10(-9) M SP was > 85% shown by quantitative PCR. Also, splenocyte SOM content decreased from 1048 +/- 275 to < 10 pg/4 x 10(8) cells following SP exposure. Immunohistochemistry identified SOM solely within splenic macrophages following cytokine stimulation. Mice infected with Schistosoma mansoni form granulomas in the liver and intestines resulting from deposition of parasite eggs in these organs. The granulomas contain macrophages that make SOM constitutively. SP at 10(-8) M decreased SOM mRNA expression > 90% in dispersed granuloma cells cultured for 4 h or longer. Specific SP receptor antagonists blocked SP suppression of SOM expression in splenocytes and dispersed granuloma cells, showing that an authentic SP receptor mediated the regulation. Additional studies revealed that IL-4 antagonized the SP effect in the spleen. It is concluded that in granulomas and splenocytes from mice with schistosomiasis and in splenocytes from uninfected animals that 1) SP inhibits macrophage SOM induction and ongoing expression at the mRNA and protein levels acting through the SP receptor, and 2) IL-4 can antagonizes this SP effect.     

Involvement of reactive oxygen intermediates in tumor necrosis factor alpha-dependent bacteriostasis of Mycobacterium avium

We studied the involvement of reactive oxygen intermediates and reactive nitrogen intermediates in the bacteriostasis of two Mycobacterium avium strains differing in virulence by resident peritoneal macrophages. We found that both the highly virulent strain (25291) and the low-virulence strain (1983) of M. avium induced superoxide production but inhibited nitrite production in vitro. This inhibition was due to the production of superoxide, a nitric oxide scavenger. The stimulation of superoxide production was two- to fivefold higher in strain 1983-infected than in strain 25291-infected resident peritoneal macrophages and was independent of contaminating T cells or NK cells. Superoxide secretion was dependent on the tumor necrosis factor (TNF) produced endogenously by the macrophages. This was also true when macrophages were isolated from infected mice. Addition of TNF to the infected resident peritoneal macrophages caused only a slight, albeit significant, increase in superoxide production by strain 25291-infected macrophages. Incubation of resident peritoneal macrophages with different scavengers of reactive oxygen intermediates showed that strain 1983 was susceptible to hydrogen peroxide produced by resident peritoneal macrophages. Strain 25291 was shown to decrease superoxide secretion inside heavily infected bone marrow-derived macrophages. This strain was also shown to be a better trigger for production of reactive oxygen intermediates than strain 1983. In summary, strain 1983 induced high levels of TNF synthesis that acted in an autocrine fashion to stimulate production of reactive oxygen intermediates by macrophages leading to growth restriction mediated by hydrogen peroxide. The highly virulent strain 25291 induced low levels of TNF synthesis, and therefore little reactive oxygen intermediate production, and could also inhibit superoxide production by the infected macrophages.     

Direct exposure to gallium arsenide upregulates costimulatory activity of murine macrophages

Gallium arsenide (GaAs) is an intermetallic semiconductor compound used in the electronics industry. Acute exposure of animals to GaAs systemically suppresses several immune functions while paradoxically causing inflammation at the exposure site. We investigated the effect of GaAs on costimulatory activity of murine peritoneal macrophages, 5 days after ip exposure. Costimulation by macrophages was determined by activation of CD4(+) helper T cell hybridomas to secrete interleukin-2 in the presence of immobilized monoclonal anti-CD3 antibody. Both peritoneal exudate cells (PEC) and resident peritoneal cells exposed to GaAs provided greater costimulation to the T cells than vehicle control cells. Resident peritoneal cells exposed to GaAs were also more efficient than latex bead-exposed cells, indicating that phagocytosis alone did not cause the GaAs effect. Double immunofluorescence staining and flow cytometric analysis revealed that GaAs-exposed PEC had increased cell surface expression of costimulatory B7-1 and B7-2 molecules and intracellular adhesion molecule-1 (ICAM-1) compared to controls. In addition to these molecules, resident peritoneal macrophages exposed to GaAs also expressed significantly higher levels of heat-stable antigen (HSA). Monoclonal antibodies specific for these costimulatory molecules significantly inhibited T cell activation, demonstrating that the molecules on GaAs-exposed cells were functional. In contrast, GaAs did not upregulate costimulatory molecules on splenic macrophages. These findings suggest that direct GaAs exposure improves macrophage costimulatory activity, possibly by activating the cells, which may contribute to respiratory inflammation caused by inhalation of GaAs particles.     

Prostaglandin synthesis by cumulus-oocyte complexes: effects on in vitro fertilization in mice

Cumulus-oocyte complexes, obtained from superovulated Balb/C virgin female mice, released to the incubation media significant amounts of PGE1, PGE2 and PGF2 alpha, as estimated by bioassay. Fertilization rates in vitro decreased sharply when cumulus-oocyte complexes were treated with indomethacin (10(-6) M) and then inseminated with 5000 sperm per oocyte. In order to explore if the reduced prostaglandin (PG) concentration was responsible for diminished fertilization rates, PGE1, PGE2 and PGF2 alpha (10(-9) M) were added to the fertilization media of treated oocytes. PGE1 and PGE2 but not PGF2 alpha returned fertilization rates to control levels. Besides, PGE1 (10(-9) M) enhanced fertilization rates with reduced sperm numbers (1000 sperm per oocyte) of untreated cumulus-oocyte complexes. In conclusion, PG synthesis and release of mouse cumulus-oocyte complexes affects fertilization in vitro, and it is suggested that PGs of the E series modulate sperm function at the moment of fertilization.     

Synergistic cooperation between phorbol ester and IFN-gamma for induction of nitric oxide synthesis in murine peritoneal macrophages

The role of protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages was examined. Phorbol ester, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity. When phorbol ester was used in combination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA, as determined by Northern blotting. The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma. Phorbol ester also induced the release of NO to the incubation medium by bacillus Calmette-Guerin-infected peritoneal macrophages. Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma. In addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by IFN-gamma plus phorbol ester. When the cells were treated with both actinomycin D and phorbol ester after IFN-gamma stimulation, more NO was produced and more iNOS mRNA was expressed than in the cells treated with actinomycin D alone. On the basis of these observations, we conclude that PKC might not be directly involved in the expression of NO synthase, but, instead, might be involved in the stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma.     

On the limited value of fine-needle aspiration for the diagnosis of benign melanocytic proliferations of the skin

Ligation of the alpha2-macroglobulin signalling receptor (alpha2MSR) with alpha2-macroglobulin (alpha2M)-methylamine or a cloned and expressed receptor binding fragment (RBF) stimulates DNA synthesis. To examine the possible role of the Ras pathway in the mitogenic effects observed on ligating alpha2MSR, we studied the formation of p2 Ras-GTP in murine peritoneal macrophages upon treatment with alpha2M-methylamine and RBF, respectively. Both alpha2M-methylamine (50 pM) and RBF (50 pM) stimulated a 2-3-fold increase in the formation of the p21Ras-GTP complex compared with unstimulated cells. p21Ras-GT32P complex formation was both time and RBF concentration dependent and was comparable to p21Ras-GT32P complex formation induced by EGF (200 ng/mL) and platelet derived growth factor (50 mg/mL). Up-regulation of cells with phorbol dibutyrate prior to stimulation with RBF had no effect on p2 Ras-GT32P formation. However, treatment of macrophages with the tyrosine kinase inhibitor genestein drastically reduced RBF-induced formation of the p21 Ras-GT32P complex. Wortmannin, an inhibitor of phosphatidylinositol-3'-kinase (PI3K), had no effect on p21Ras-GT32P complex formation. It is concluded that the mitogenic effects of ligating alpha2MSR are mediated through a Ras-dependent pathway.     

Tumor growth alters T cell and macrophage production of and responsiveness to granulocyte-macrophage colony-stimulating factor: partial dysregulation through interleukin-10

Tumor growth induces phenotypic and functional changes among splenic T cells and macrophages (M phi) that contribute to the immunosuppression observed in tumor-bearing hosts (TBH). These changes partly arise through alterations in immune cell production of and responsiveness to cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important T cell- and M phi-derived cytokine that is produced during normal host immunogenic challenge, but it's involvement during cancer is poorly defined. In contrast, interleukin-10 (IL-10) is an inhibitory cytokine that is produced by immune cells as a deactivation factor. IL-10 can disrupt GM-CSF synthesis and may be associated with tumor-induced changes in cytokine synthesis. We determined if tumor growth alters T-cell and M phi synthesis of and responsiveness to GM-CSF, and if these alterations occur because tumor growth heightens immune cell sensitivity to IL-10. Tumor growth significantly decreased T-cell synthesis of GM-CSF during activation by concanavalin A, and TBH T cells were more susceptible to GM-CSF synthesis inhibition by IL-10 than their normal host (NH) counterparts. This suppression was observed using both unseparated splenic lymphocyte preparations and purified CD4+ and CD8+ T cells. Similarly, TBH M phi (both splenic and peritoneal) produced less GM-CSF than NH M phi during activation by lipopolysaccharide. Tumor growth also altered major histocompatibility complex (MHC) class II- M phi GM-CSF synthesis. TBH M phi were more susceptible to GM-CSF synthesis inhibition by IL-10 than their NH counterparts. Although TBH T cells demonstrate less proliferation than NH T cells during activation, tumor growth did not compromise T-cell responsiveness to GM-CSF. However, tumor growth did increase TBH T-cell susceptibility to inhibition of proliferation by IL-10. Tumor growth suppressed M phi responsiveness to GM-CSF, and IL-10 further decreased M phi responsiveness to GM-CSF. Collectively, these results suggest that T cell and M phi production of and responsiveness to GM-CSF is disrupted during tumor growth, and that TBH T cells and M phi are more susceptible to the suppressor activity of IL-10 than their NH counterparts.     

Azole antifungals: weak inhibitors of inducible nitric oxide synthase in mouse and human cells

The effect of three azole antifungals on inducible nitric oxide (iNOS) activity in different mouse and human cells was evaluated. The iNOS activity was determined by L-citrulline and nitrite measurement. In the murine macrophage cell line RAW 264.7, in mouse peritoneal macrophages (MPM) and in human colorectal adenocarcinoma cells (DLD-1), iNOS activity could be induced with lipopolysaccharides and cytokines. Under similar conditions, no iNOS induction was found in human monocytes/macrophages. The concentration of itraconazole, ketoconazole or miconazole needed to inhibit iNOS activity by 50% in RAW 264.7 cells, MPM and DLD-1 cells was > or = 10 mumol l-1. This is at least 100 times more than the concentrations of these azole antifungals required to produce a 50% inhibition of yeast growth and ergosterol synthesis of, for example, Candida albicans after the same incubation period. These results show that azole antifungals are weak inhibitors of iNOS in intact cells.     

Is there a role for nitric oxide in regulation of T cell secretion of IL-2?

Nitric oxide (NO) can have both effector (cytotoxic) and regulatory roles in immune function. In this study, we have re-examined the potential role of nitric oxide in mediating the macrophage-dependent suppression of IL-2 synthesis. In our model, TNP-specific CD4+ T cells are cocultured with Ag and either peritoneal or alveolar macrophages. Both populations of macrophages after in vitro stimulation with IFN-gamma can inhibit IL-2 release. In vitro stimulation also induces substantial levels of NO release by these macrophages, as well as high levels of prostaglandin E2 (PGE2). However, there was no correlation between NO levels and inhibitory activity. Furthermore, NG-monomethyl-L-arginine monoacetate, a specific inhibitor of NO release had no effect on IL-2 release, while indomethacin, which blocked prostaglandin synthesis, largely abrogated the suppressor activity of both macrophage populations. Although the addition of exogenous NO donors at high concentrations could inhibit IL-2 release by T cells, our data does not support the hypothesis that NO is a major macrophage mediator of suppression in this model.