IntroductionAlthough IFN-γ plays a central role in controlling the host's response to bacterial and viral infection, the precise cellular mechanisms that contribute to this obligate role in host defense have not been fully elucidated (1). These IFN-γ-dependent events depend exclusively on signal transducer and activator of transcription factor 1 (STAT1) activation, since mice lacking this signal transducer are defective in their ability to resolve bacterial and viral infections (2-4). That IFN-γ is important in regulating host defense is emphasized by evidence of its effect on leukocyte recruitment. In this respect, IFN-γ can both enhance and suppress chemokine secretion in response to proinflammatory cytokines (IL-1β and TNF-α), modulate chemokine receptor expression, and affect cellular adhesion and transmigration (5-10). In vivo, however, it is unclear whether these actions on leukocyte trafficking are a direct result of IFN-γ or occur through its modulation of other mediator cascades during the inflammatory response. Recent data, however, suggest that the initial activation of resident CD4 T cells to produce IFN-γ (and IL-17) are important in directing neutrophil (PMN) recruitment and in resolving bacterial infection (11-13).During acute inflammation, PMN trafficking is tightly regulated to provide effective host defense without promoting tissue injury. Initially, PMN are recruited from the circulation toward sites of inflammatory insult or infection in response to local secretion of neutrophil-activating chemokines containing the ELR amino acid motif (ELR + ) (14). As inflammation proceeds, or infection subsides, PMN are removed in a controlled manner to prevent persistent tissue necrosis (15). The initial phase of PMN clearance results from the downregulation of local ELR + CXC chemokine expression and a switch to other chemokines selective for the attraction of mononuclear cells (MNC) ( 16). Central to the removal of PMN is the
These data demonstrate that HPMC synthesize IL-8, MCP-1 and RANTES in response to inflammatory cytokines. HPMC-derived C-x-C and C-C chemokines might contribute to the intra-peritoneal recruitment of leukocytes during peritoneal inflammation.
Leukocyte recruitment into the infected peritoneal cavity consists of an early, predominant polymorphonuclear leukocyte (PMN) influx and subsequent, prolonged mononuclear cell migration phase. Although chemokine secretion by resident peritoneal cells plays a primary role in mediating this migration, the mechanisms involved in controlling the switch in phenotype of cell infiltrate remain unclear. The present study investigates a potential role for the Th1-type cytokine IFN-γ in the process of leukocyte recruitment into the peritoneal cavity. Stimulation of cultured human peritoneal mesothelial cells with IFN-γ (1–100 U/ml) alone or in combination with IL-1β (100 pg/ml) or TNF-α (1000 pg/ml) resulted in significant up-regulation of monocyte chemoattractant protein-1 and RANTES protein secretion. In contrast, IFN-γ inhibited basal and IL-1β-, and TNF-α-induced production of IL-8. The modulating effects of IFN-γ on chemokine production occurred at the level of gene expression, and the degree of regulation observed was dependent on the doses of IL-1β and TNF-α used. Analysis of the functional effects of IFN-γ on IL-1β-induced transmesothelial PMN migration with an in vitro human transmigration system and an in vivo murine model of peritoneal inflammation demonstrated that IFN-γ was able to down-regulate PMN migration induced by optimal doses of IL-1β. These effects were mediated in vivo via down-regulation of CXC chemokine synthesis. These findings suggest that IFN-γ may play a role in controlling the phenotype of infiltrating leukocyte during the course of an inflammatory response, in part via regulation of resident cell chemokine synthesis.
We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic epithelial cell line HT‐29. Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin‐lα (IL‐lα), tumour necrosisfactor‐α (TNF‐α), interferon γ (IFN‐γ) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay. Unstimulated cells produced a small time‐dependent increase in nitrite generation of 50 ± 4, 75 ± 8, and 103 ±8 nM per 106 cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 μg ml−1) pretreatment and iNOS mRNA was not detected. None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL‐lα/IFN‐γ produced a highly significant (P< 0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective. Time course studies with IL‐lα/IFN‐γ combination revealed significant (P< 0.001) increases in nitrite at 24 h (153 ±7), 48 h (306 ±24), and 72 h (384 ±15) compared to basal values of 50 ±4, 75 ±8, and 103 ±8 nM per 106 cells respectively. Studies with IL‐lα/IFN‐γ combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL‐lα (0.3–10 ng ml−1) and IFN‐γ (10–300 u ml−1) in combination induced a concentration‐dependent expression of iNOS mRNA at 24 h. Pretreatment with cycloheximide before IL‐lα/IFN‐γ stimulation reduced nitrite levels to basal values. These data suggest that the IL‐lα/IFN‐γ‐induced nitrite production by HT‐29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme. The addition of TNF‐α produced a significant (P< 0.001) 3 fold increase of IL‐lα/IFN‐γ‐induced nitrite generation. In marked contrast the presence of TNF‐α had no effect on IL‐lα/IFN‐γ‐induced iNOS mRNA expression by HT‐29 cells. These findings suggest that the up‐regulation by TNF‐α of IL‐lα/IFN‐γ‐induced nitrite generation is at the post‐transcriptional level. These data suggest that pro‐inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.
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