Thioglycollate-elicited Peritoneal Macrophages Research Articles

Ship is a positive regulator of nitric oxide production

Studies to date with the hemopoietic-specific inositol 5-phosphatase, SHIP, have established that it is a negative regulator of hemopoietic cell proliferation, survival, and end cell activation. It appears to carry out this inhibitory function, at least in part, by hydrolysing the PI-3 kinase-generated second messenger, PI-3,4,5-P3, to PI-3,4-P2. However, recent studies in our laboratory with SHIP +/+ and −/− macrophages have established a hitherto unexpected role for SHIP as a positive regulator of nitric oxide (NO) synthesis. Both resident and thioglycollate-elicited peritoneal macrophages as well as retrovirally-immortalized bone marrow-derived macrophage cell lines (J2M) from SHIP −/− mice are impaired in their induction of the inducible isoform of NO synthase (iNOS) in response to LPS/IFN-γ co-stimulation, and produce 5 to 10-fold less NO than wild-type cells. Furthermore, upon LPS stimulation alone, wild-type J2M macrophages induce significantly more iNOS mRNA and protein and 5 to 10-fold more NO than SHIP −/− J2M cells. Consistent with these results, we found that the luciferase activity of a wild type iNOS promoter/luciferase construct in SHIP+/+ J2M cells, following 18 h with LPS, was substantially higher than that in SHIP−/− cells. We are currently testing various mutant iNOS promoter/luciferase constructs to hone in on the transcription factors that are regulated by SHIP and these results will be presented.

Tumor necrosis factor induces resistance of macrophages to Legionella pneumophila infection.

Legionella pneumophila is an ubiquitous opportunistic intracellular pathogen that replicates readily in thioglycollate-elicited peritoneal macrophages from genetically susceptible A/J mice. Treatment of macrophage cultures in vitro with tumor necrosis factor-alpha (TNF-alpha) induced resistance of the macrophages to infection by Legionella as compared with control macrophages treated with medium alone. Addition of small amounts of monoclonal antibody to TNF-alpha restored susceptibility of the macrophages. Furthermore, antibody to the proinflammatory cytokine interleukin-1 (IL-1) alpha/beta increased resistance, but recombinant IL-1 had little effect. Such decreased susceptibility to Legionella growth in anti-IL-1 antibody-treated cultures corresponded with enhanced levels of TNF-alpha in the supernatants of the treated cells. An antibody to another proinflammatory cytokine with known immunoregulatory properties (i.e., IL-6) had little or no effect on the ability of the macrophages to be infected by Legionella and, furthermore, treatment with recombinant IL-6, similar to recombinant IL-1, did not modify the ability of the cells to be infected in vitro. These results indicate that TNF-alpha is important in controlling L. pneumophila replication, and IL-1 can regulate TNF-alpha levels, affecting susceptibility of macrophages to infection with an intracellular opportunistic pathogen like Legionella.

Mouse and Human GTPBP2, Newly Identified Members of the GP-1 Family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-γ in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-γ in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.

Somatostatin and Substance P Induced in vivo by Lipopolysaccharide and in Peritoneal Macrophages Stimulated with Lipopolysaccharide or Interferon-Gamma Have Differential Effects on Murine Cytokine Production

We have investigated whether lipopolysaccharide (LPS) induces substance P (SP) and somatostatin (SOM) in popliteal lymph nodes in vivo and whether macrophages are a source of SP and SOM in vitro. We have also investigated the effect of SP and SOM treatment on the production of cytokines. SP reached a maximum 3 days after injection of LPS (100 μg/footpad) and then declined. SOM expression after LPS injection reached a maximum at 5–7 days. Stimulation of thioglycolate-elicited peritoneal macrophages with LPS (20 μg/ml), recombinant interferon-γ (rIFN-γ, 100 U/ml), and LPS plus rIFN-γ induced SOM and SP. Thioglycolate-elicited, unstimulated peritoneal macrophages also synthesized these peptides. SOM (10^–12–10^–8 M) significantly inhibited IL-6 and IFN-γ production, whereas SP at those concentrations enhanced cytokine production by activated lymphocytes and macrophages. These findings suggest that neuropeptides which originate from macrophages and nerve fibers act as immunomodulators to mediate changes in the pattern of cytokine production.

LIVER-INFILTRATING T LYMPHOCYTES ARE ATTRACTED SELECTIVELY BY IFN-INDUCIBLE PROTEIN-10

We have demonstrated that interferon-inducible protein-10 (IP-10) is produced in hepatocytes surrounded by infiltrative mononuclear cells in chronic hepatitis. To clarify the role of IP-10 in hepatitis, we examined the chemoattractive activity of IP-10 on liver-infiltrating lymphocytes in experimental animal models of hepatitis. IP-10 was specifically induced in the livers of mice treated intravenously (i.v.) with Con A, while monocyte chemotactic protein-1 (MCP-1) showed a much lower level of induction and neither RANTES nor macrophage inflammatory protein-1α (MIP-1α) was detected. The liver-infiltrating lymphocytes in Con A-induced hepatitis were attracted only by IP-10, and not by other chemokines such as RANTES, MCP-1 and MIP-1α. The chemoattractive effect of IP-10 was dose-dependent and was neutralized by monoclonal antibodies to IP-10. The specific effect of IP-10 on liver-infiltrating lymphocytes was also seen on those obtained from rat livers with fulminant hepatitis induced by sequential treatment with killed Propionibacterium acnes (P. acnes) and LPS. Peripheral blood lymphocytes were slightly attracted by IP-10 as well as RANTES and MIP-1α, while hepatic resident lymphocytes were not. On the other hand, thioglycolate-elicited peritoneal macrophages did not respond to IP-10, although they did show a response to RANTES, MCP-1 and MIP-1α. These results indicated that IP-10 is a specific chemoattractant for T lymphocytes in the inflammatory liver tissues and may play a specific role in the development of hepatitis.

Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages.

The human MD-2 molecule is associated with the extracellular domain of human Toll-like receptor 4 (TLR4) and greatly enhances its LPS signaling. The human TLR4-MD-2 complex thus signals the presence of LPS. Little is known, however, about cell surface expression and LPS signaling of the TLR4-MD-2 complex in vivo. We cloned mouse MD-2 molecularly and established a unique mAb MTS510, which reacted selectively with mouse TLR4-MD-2 but not with TLR4 alone in flow cytometry. Mouse MD-2 expression in TLR4-expressing cells enhanced LPS-induced NF-kappaB activation, which was clearly inhibited by MTS510. Thioglycolate-elicited peritoneal macrophages expressed TLR4-MD-2, which was rapidly down-regulated in the presence of LPS. Moreover, LPS-induced TNF-alpha production by peritoneal macrophages was inhibited by MTS510. Collectively, the TLR4-MD-2 complex is expressed on macrophages in vivo and senses and signals the presence of LPS.

A Rab11-containing rapidly recycling compartment in macrophages that promotes phagocytosis.

Macrophages are specialized cells of the immune system that exhibit a prodigious capacity for phagocytosis. The ability of macrophages to internalize a substantial proportion of their plasma membrane during phagocytosis indicates that they possess a mechanism for the rapid renewal of plasma membrane. We examined the role of endocytic membrane recycling in promoting phagocytosis. In contrast to many other cell types, macrophages lack a morphologically distinct peri-centriolar recycling compartment but instead demonstrate an extensive network of transferrin receptor-positive tubules and vesicles that participated in recycling. The rate of transferrin recycling in thioglycollate-elicited murine peritoneal macrophages (thio-macrophages) was exceedingly rapid, with exocytic rate constants that were 2- to 3-fold higher than those of most other cells. Because the GTPase Rab11 has been implicated in transferrin recycling in other cells, we determined its role in transferrin recycling and phagocytosis in macrophages. Macrophages expressing epitope-tagged Rab11 demonstrated the presence of Rab11 in several intracellular membrane compartments, including endosomes and nascent phagosomes. Expression of Rab11 25N, a GTP binding-deficient allele of Rab11, led to a decreased rate of transferrin efflux and impaired Fc(gamma)R-mediated phagocytosis, where Fc(gamma)R is the receptor for the Fc portion of IgG. In contrast, expression of Rab11 70L, a GTPase-deficient allele of Rab11, led to an increased rate of transferrin efflux and enhanced phagocytosis. We conclude that macrophages have adapted a rapidly mobilizable, endocytic compartment to enhance phagocytosis. Rab11 participates in the recruitment of this compartment to the macrophage cell surface.

Surface expression and rapid internalization of macrosialin (mouse CD68) on elicited mouse peritoneal macrophages.

Macrosialin, the mouse homolog of human CD68, is a heavily glycosylated transmembrane protein found almost exclusively in macrophages. Its function remains uncertain. It has a high affinity for oxidized low-density lipoprotein (LDL) in ligand blots and antibodies against the human homolog, CD68, inhibit the binding of oxidized LDL to a human monocyte-derived cell line (THP-1). However, there is still controversy as to whether macrosialin, found predominantly in late endosomes, is expressed at all on the plasma membrane. The present studies, done in thioglycollate-elicited peritoneal macrophages, confirm that macrosialin is predominantly intracellular but show clearly that 10-15% of it is expressed on the cell surface. Exchange with intracellular pools occurs at an extremely high rate. The results are compatible with a surface function, including internalization of bound ligands or adhesion to surfaces.

Effect of solid lipid nanoparticles (SLN) on cytokine production and the viability of murine peritoneal macrophages

The purpose of this study was to investigate possible immunomodulatory and cytotoxic effects of solid lipid nanoparticles (SLN) on murine peritoneal macrophages. Immunomodulatory effects of SLN composed of either a lipid(glycerol-behenate) or a wax (cetylpalmitate) matrix stabilized by the surfactant Poloxamer 188 were analysed by detection of proinflammatory and downregulatory cytokines in supernatants of thioglycollate-elicited peritoneal macrophages using enzyme-linked immunosorbent assay (ELISA). Cytotoxicity of SLN was assessed using the MTT test. Incubation of macrophages with either SLN at low concentrations did not increase production of interleukin (IL)-6, IL-12, and tumour necrosis factor (TNF)-alpha. At higher SLN concentrations, a concentration-dependent decrease in IL-6 secretion was observed compared to background production of IL-6 by untreated macrophages. IL-12 and TNF-alpha production was neither detected in supernatants of macrophages treated with SLN at any concentration nor in those of untreated cells. The decrease in IL-6 secretion was paralleled by concentration-dependent cytotoxicity of SLN on these cells. In contrast, incubation with polystyrene reference particles neither resulted in decreased IL-6 production nor in a loss of viability. SLN-treated macrophages were found to up-regulate their cytokine production following stimulation with Pansorbin, despite the concentration-dependent cytotoxicity induced by SLN. Down-regulatory effects on SLN-treated macrophages by IL-10 were not observed. In conclusion, incubation of SLN with murine peritoneal macrophages did not induce the production of proinflammatory and down-regulatory cytokines. At high concentrations of SLN, cytotoxic effects on these cells were observed. Cytotoxicity appears to be the main cause of decreased cytokine production by these cells.

Sterol synthesis is up-regulated in cholesterol-loaded pigeon macrophages during induction of cholesterol efflux

The extent to which cholesterol synthesis is modulated in macrophage foam cells by changes in cholesterol influx and efflux was determined using thioglycollate-elicited peritoneal macrophages from normal and cholesterol-fed White Carneau (WC) and Show Racer (SR) pigeons. In peritoneal macrophages from normocholesterolemic pigeons, sterol synthesis from [(14)C]-acetate was down-regulated by more than 90% following incubation in vitro with beta-VLDL. Sterol synthesis was increased when the cellular free cholesterol concentration was decreased in response to stimulation of cholesterol efflux with apoHDL/phosphatidylcholine vesicles and cyclodextrin. Peritoneal macrophages isolated from hypercholesterolemic pigeons were loaded with cholesterol to levels similar to foam cells from atherosclerotic plaques (375-614 microg/mg cell protein), and had an extremely low rate of sterol synthesis. When cholesterol efflux was stimulated in these cells, sterol synthesis increased 8 to 10-fold, even though the cells remained grossly loaded with cholesterol. Cholesterol efflux also stimulated HMG-CoA reductase activity and LDL receptor expression. This suggests that only a small portion of the total cholesterol pool in macrophage foam cells was responsible for regulation of sterol synthesis, and that cholesterol generated by hydrolysis of cholesteryl esters was directed away from the regulatory pool by efflux from the cells. When the increase in sterol synthesis was blocked with the HMG-CoA reductase inhibitor mevinolin, there was no difference in the cholesterol content of the cells, or in the mass efflux of cholesterol into the culture medium.Thus, under these conditions, the increase in cholesterol synthesis during stimulation of cholesterol efflux does not appear to contribute significantly to the mass of cholesterol in these macrophage foam cells. Whether a similar situation exists in vivo is unknown.

Inhibition by a CD14 monoclonal antibody of lipopolysaccharide binding to murine macrophages

We have established an anti-CD14 mAb named 4C1 against murine macrophages. 4C1 can bind to thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages and casein-induced peritoneal neutrophils. Immunostaining with 4C1 was inhibited by treatment of the cells with phosphatidylinositol specific phospholipase C, suggesting that the antigen is GPI-anchored. Immunoprecipitates from biotin-labeled RAW264.7 cell lysate with 4C1 were around 55 kDa and were visualized with rmC5-3, the only commercially available anti-murine CD14 mAb. 4C1 positively stained COS7 cells transfected with an expression vector containing cDNA of murine CD14. Pretreatment of macrophages with 4C1 reduced LPS-mediated production of TNFα, IL-6, and nitrite. The binding of FITC-LPS to RAW264.7 cells was blocked by pretreatment with 4C1 but not with rmC5. Pretreatment of cells with unlabeled 4C1 mAb but not unlabeled rmC5-3 reduced binding of FITC-4C1. These results suggest that the 4C1 epitope on murine CD14 plays an important role in LPS binding and is distinct from the rmC5-3 epitope.

Interferon-γ, Bacterial Lipopolysaccharide, and Tumor Necrosis Factor-α Induce CD11a mRNA and Protein via Na+/H+Exchange and Protein Kinase C-Dependent Mechanisms in Tissue Macrophages

Previously CD11a or leukocyte function-associated antigen α-1 was found to be induced at the surface protein level in thioglycolate-elicited peritoneal macrophages by bacterial lipopolysaccharide and interferon-γ. To investigate this induction further, Northern blotting and enzyme-linked immunosorbent assays were used to examine the role of second messengers in CD11a gene product induction by these agents. Here I report that CD11a RNA and cell surface protein induced by bacterial lipopolysaccharide and tumor necrosis factor-α are sensitive to inhibition of protein kinase C, while insensitive to inhibition of Na+/H+exchange. CD11a induction by interferon-γ conversely is sensitive to inhibition of Na+/H+exchange and insensitive to inhibition of protein kinase C. These observations indicate that CD11a may be induced by multiple and separate second messenger systems in primary macrophages.

Thermal injury-induced enhancement of oxidative metabolism by mononuclear phagocytes.

C57BL/6 female mice were subjected to full-thickness scald thermal injuries covering 25% of their total body surface areas, and thioglycollate-elicited peritoneal macrophages were isolated 4 days later. Macrophages from burned mice displayed a 2-fold increase in phorbol ester-stimulated superoxide production, whereas superoxide production in response to zymosan or immune complexes was unaltered. Maximal levels of superoxide production by thermal injury macrophages were comparable for all stimuli. Reduction of injury size to 12.5% total body surface area abrogated the enhancement of superoxide production. Protein kinase C activity and protein phosphorylation was not altered after thermal injury. These results indicate that the enhancement of macrophage-oxidative metabolism after thermal injury is independent of changes in reduced nicotinamide adenine dinucleotide phosphate oxidase or protein kinase C activity and may in part explain the increased susceptibility to multiple organ failure after this form of trauma.

Dietary omega-3 polyunsaturated fatty acids reduce IFN-gamma receptor expression in mice.

Enrichment of immune cells in vivo or in vitro with omega-3 polyunsaturated fatty acid (n-3 PUFA) has been reported to diminish their response to interferon-y (IFN-gamma). We hypothesized that the n-3 PUFA-induced hyporesponsiveness to IFN-gamma is mediated, in part, by a reduction in the number of IFN-gamma receptors (IFNGR) expressed on the surface of these cells. To test this hypothesis, we fed mice experimental diets containing low or high amounts of n-3 PUFA. Thioglycollate-elicited peritoneal macrophages (PEC) were collected and tested for binding and internalization of [125I]-labeled recombinant murine IFN-gamma. High n-3 PUFA intake was associated with a significant (n = 2, p < 0.01) reduction in [125I]-IFN-gamma binding without affecting binding affinity (Kd). When studies were performed at 37 degrees C, high n-3 PUFA intake reduced internalization of [125I]-rmIFN-gamma by 20%-30% (n = 2,p < 0.001). Results from flow cytometric analysis of IFNGR-1 expression on the surface of murine splenocytes were in agreement with the binding studies. Further, total cellular IFNGR-1 from PEC and splenocytes was examined via immunoprecipitation and Western blotting. High n-3 PUFA diet was associated with a 50% decline (n = 3-6, p < 0.05) in total IFNGR-1 in both immune cell populations studied. These data suggest that reduced IFNGR expression may be responsible for immune cell hyporesponsiveness to IFN-gamma, which may, in part, explain some of the immunomodulatory and anti-inflammatory effects associated with the consumption of diets high in n-3 PUFA.

Effects of interleukin-10 on chemokine KC gene expression by mouse peritoneal macrophages in response to Candida albicans

Chemokine KC has been considered to be a murine homologue of human GRO/MGSA and was identified as chemoattractant for monocytes and neutrophils. This study examined the expression of KC mRNA in thioglycollate-elicited mouse peritoneal macrophages that were stimulated in vitro with Candida albicans (CA). Also examined were the inhibitory effects of IL-10 on the CA-induced expression of KC gene by Northern blot analysis. CA was found to induce chemokine gene expression in a gene-specific manner, CXC chemokine IP-10 mRNA expression was not detected in CA-stimulated macrophages. Maximum KC mRNA expression was observed approximately 2 hr after adding CA. The inhibitory action of IL-10 to CA-induced KC mRNA expression on mouse peritoneal macrophages was independent on concentration and stimulation time of IL-10 and was observed approximately one hour after adding IL-10 and CA simultaneously. IL-10 produced a decrease in the stability of KC mRNA, and CA-stimulated macrophages with cycloheximide blocked the suppressive effect of IL-10. These results suggest that CA also induces chemokine KC from macrophages, and IL-10 acts to destabilize CA-induced KC mRNA and de novo synthesis of an intermediate protein is a part of the IL-10 suppressive mechanism.

Tinospora tuberculata suppresses nitric oxide synthesis in mouse macrophages.

We have obtained evidence that co-incubation of thioglycollate-elicited peritoneal macrophages with an aqueous extract of Tinospora tuberculata inhibits lipopolysaccharide-stimulated excessive production of nitric oxide (NO) in vitro. This effect is concentration-dependent and appears to involve suppression of both inducible nitric oxide synthase (iNOS) activity and NADPH-diaphorase activity, thus altering NO production. As NO is one of the critical mediators in various disorders and iNOS inhibitors may have therapeutic potential, these results may explain some aspects of the multifunctional properties of Tinospora tuberculata, which has been used in various folk remedies in southeast Asia and China.

Macrophage Formation of Angiostatin during Inflammation: A BYPRODUCT OF THE ACTIVATION OF PLASMINOGEN

Angiostatin is a potent inhibitor of tumor angiogenesis and the growth of metastatic foci. Recent studies have indicated that neoplastic cells can generate angiostatin directly or in cooperation with tumor-associated macrophages. In studies reported here, we determined whether angiostatin is generated in mice under non-neoplastic settings. Utilizing murine RAW264.7 macrophages and thioglycollate-elicited peritoneal macrophages, we demonstrate that angiostatin-like fragments are generated as a byproduct of the proteolytic regulation of membrane-bound plasmin. Plasmin proteolysis and subsequent loss in membrane-bound plasmin activity requires active plasmin but was unaffected by inhibitors of metalloproteinases. Lysine binding fragments of plasmin, isolated from macrophage-conditioned media utilizing affinity chromatography, appeared as a major (48 kDa) and two minor bands (42 and 50 kDa) in SDS-polyacrylamide gel electrophoresis and were immunoreactive with anti-kringle 1-3 IgG. Each peptide begins with Lys77 and contains the entire sequence of angiostatin. The affinity isolated plasmin fragments inhibited bFGF-induced endothelial cell proliferation. Lavage fluid recovered from the peritoneal cavities of mice previously injected with thioglycollate contained angiostatin-like plasmin fragments similar to those generated in vitro. This is the first demonstration that angiostatin-like plasmin fragments are generated in a non-neoplastic inflammatory setting. Thus, in addition to regulating pericellular plasmin activity, proteolysis of plasmin generates inactive kringle-containing fragments expressing angiostatic properties.

Exogenous and endogenous catecholamines inhibit the production of macrophage inflammatory protein (MIP) 1 alpha via a beta adrenoceptor mediated mechanism.

Noradrenaline (NA) and adrenaline (Ad) are modulators of cytokine production. Here we investigated the role of these neurotransmitters in the regulation of macrophage inflammatory protein (MIP)-1alpha expression. Pretreatment of RAW 264.7 macrophages with NA or Ad decreased, in a concentration-dependent manner (1 nM-100 microM), MIP-1alpha release induced by bacterial lipopolysaccharide (LPS 10 ng ml(-1) LPS). The effect of NA was reversed by the selective beta-adrenoceptor antagonist propranolol (10 microM), but not by the alpha-adrenoceptor antagonist phentolamine (10 microM). In the concentration range of 10 nM-10 microM, isoproterenol, a beta-adrenoceptor agonist, but not phenylephrine (a selective alpha1-adrenoceptor agonist) or UK-14304 (a selective alpha2-adrenoceptor agonist) mimicked the inhibitory effects of catecholamines on MIP-1alpha production. Increases in intracellular cyclic adenosine monophosphate, elicited either by the selective type IV phosphodiesterase inhibitor rolipram (0.1 - 10 microM), or by prostaglandin E2, (10 nM-10 microM) decreased MIP-1alpha release, suggesting that increased cyclic AMP may contribute to the suppression of MIP-1alpha release by beta-adrenoceptor stimulation. Northern blot analysis demonstrated that NA (100 nM-10 microM), Ad, isoproterenol, as well as rolipram (100 nM-10 microM) decreased LPS-induced MIP-1alpha mRNA accumulation. NA and Ad (1-100 microM) also decreased MIP-1alpha production in thioglycollate-elicited murine peritoneal macrophages. Pretreatment of mice with either isoproterenol (10 mg kg(-1), i.p.) or rolipram (25 mg kg(-1), i.p.) decreased LPS-induced plasma levels of MIP-1alpha, while propranolol (10 mg kg(-1), i.p.) augmented the production of this chemokine, confirming the role of a beta-adrenoceptor mediated endogenous catecholamine action in the regulation of MIP-1alpha production in vivo. Thus, based on our data we conclude that catecholamines are important endogenous regulators of MIP-1alpha expression in inflammation.

Lipopolysaccharide and Ceramide Use Divergent Signaling Pathways to Induce Cell Death in Murine Macrophages

Ceramide is a well-known apoptotic agent that has been implicated in LPS signaling. Therefore, we examined whether LPS-induced macrophage cytotoxicity is mediated by mimicking ceramide. Both LPS and the cell-permeable ceramide analogue, C2 ceramide, induced significant cell death in IFN-gamma-activated, thioglycollate-elicited peritoneal macrophages after 48 and 24 h, respectively. Ceramide-induced cell death was neither accompanied by DNA fragmentation nor phosphatidyl serine externalization, characteristics of apoptosis. In contrast, LPS induced a significant fraction of cells to undergo apoptosis, as demonstrated by DNA fragmentation and quantified by DNA analysis on FACS, yet the majority of the cells died in a necrotic fashion. C3H/HeJ Lps(d) macrophages were resistant to LPS-induced cell death and less sensitive to C2 ceramide-evoked cytotoxicity, when compared with Lps(n) macrophages. C2 ceramide plus IFN-gamma failed to activate release of nitric oxide (NO.), whereas LPS-induced cell death, but not C2-induced cytotoxicity, was blocked by an inhibitor of inducible NO. synthase (iNOS), NG-monomethyl-L-arginine. Macrophages from IFN regulatory factor-1 (-/-) mice shown previously to respond marginally to LPS plus IFN-gamma to express iNOS mRNA and NO., were refractory to LPS plus IFN-gamma-induced cytotoxicity and apoptosis. These data suggest that although LPS may mimic certain ceramide effects, signal transduction events that lead to cytotoxicity, as well as the downstream mediators, diverge.

Gallium Arsenide Modulates Proteolytic Cathepsin Activities and Antigen Processing by Macrophages

Gallium arsenide (GaAs) is a semiconductor utilized in the electronics industry. Chemical exposure of animals causes a local inflammatory reaction, but systemic immunosuppression. Mice were administered i.p. 200 mg/kg GaAs crystals or latex beads, or vehicle. Five days after exposure, splenic macrophages were defective, whereas thioglycolate-elicited peritoneal macrophages (PEC) were more efficient in processing the Ag, pigeon cytochrome c, than vehicle control macrophages. Various aspects of the MHC class II Ag-processing pathway were examined. Both macrophage populations normally presented a peptide fragment to the CD4+ T cells. Surface MHC class II expression on the PEC was up-regulated, but splenic cells had normal MHC class II expression. PEC had elevated levels of glutathione and cysteine, major physiologic reducing thiols. However, the cysteine content of splenic macrophages was diminished. Proteolytic activities of aspartyl cathepsin D, and thiol cathepsins B and L were decreased significantly in splenic macrophages. On the other hand, thiol cathepsin activities were increased selectively in PEC. Latex bead-exposed PEC were not more potent APC, and their thiol cathepsin activities were unchanged, indicating that phagocytosis and nonspecific irritation were not responsible. The phenotype of PEC directly exposed to GaAs mirrored cytokine-activated macrophages, in contrast to splenic macrophages from a distant site. Therefore, GaAs exposure differentially modulated cathepsin activities in splenic macrophages and PEC, which correlated with their Ag-processing efficiency. Perhaps such distinct alterations may contribute to the local inflammation and systemic immunotoxicity caused by chemical exposure.