No abstract

IL-10 is a potent anti-inflammatory cytokine and inhibitor of TNF-α production. The molecular pathways by which IL-10 inhibits TNF-α production are obscure, with diverse mechanisms having been published. In this study, a new approach has been taken for the study of human cells. Adenovirus was used to deliver TNF-α promoter-based luciferase reporter genes to primary human monocytic cells. The reporter genes were highly responsive to macrophage activation and appeared to mirror the behavior of the endogenous TNF-α gene. When added, either with or after the stimulus, IL-10 required the 3′ untranslated region of the TNF-α gene to inhibit luciferase mRNA and protein expression, indicating a posttranscriptional mechanism. However, if macrophages were incubated with IL-10 before activation, inhibition of gene expression was also mediated by the 5′ promoter, suggesting a transcriptional mechanism. To our knowledge, this is the first time that a dual mechanism for IL-10 function has been demonstrated. Studies to elucidate the mechanisms underlying the inhibition of TNF-α production addressed the effect of IL-10 on the activation of p38 mitogen-activated protein kinase and NF-κB. However, these studies could demonstrate no requirement for the inhibition of p38 mitogen-activated protein kinase or NF-κB activation as potential mechanisms. Overall, these results may explain the diversity previously ascribed to the complex mechanisms of IL-10 anti-inflammatory activity.

TNF-α is a key factor in a variety of inflammatory diseases. This study examines the role of p38 MAPK in the regulation of TNF-α in primary human cells relevant to inflammation, e.g., macrophages and rheumatoid synovial cells. Using a dominant negative variant (D168A) of p38 MAPK and a kinase inhibitor, SB203580, we confirm in primary human macrophages that p38 MAPK regulates TNF-α production using a posttranscriptional mechanism requiring the 3′ untranslated region of the gene. However, in LPS-activated primary human macrophages we also detect a second previously unidentified mechanism, the p38 MAPK modulation of TNF-α transcription. This is mediated through p38 MAPK regulation of NF-κB. Interestingly this mechanism was not observed in rheumatoid synovial cells. Importantly however, the dominant negative mutant of p38 MAPK, but not SB203580 was effective at inhibiting spontaneous TNF-α production in these ex vivo rheumatoid synovial cell cultures. These data indicate there are potential major differences in the role of p38 MAPK in inflammatory signaling that have a bearing on the use of this kinase as a target for therapy. These results indicate despite disappointing results with p38 MAPK inhibitors in the clinic, this kinase is a valid target in rheumatoid disease.

The transcription factor NF‐κB is a pivotal intracellular regulator of many inflammatory responses and it has been proposed that it represents a potential therapeutic target. As chemokines are important for the progress of an inflammatory response by the recruitment of immuno‐competent cells, the role NF‐κB plays in TNFα‐ or lipopolysaccharides (LPS)‐induced chemokine secretion by human monocyte‐derived macrophages was examined. Secretion of the CXC chemokines IL‐8, GROα and ENA‐78, induced by TNFα, was significantly suppressed by inhibiting NF‐κB, using overexpression of IκBα. However, when induced by LPS the expression of these chemokines was unaffected. In contrast, expression of the CC chemokines MIP‐1α, MCP‐1 and RANTES inducedby TNFα or LPS was significantly inhibited by the overexpression of IκBα. Therefore, there appear to be different mechanisms regulating CC and CXC chemokine secretion by macrophages, depending on the stimulus and that TNFα and LPS can use different signaling mechanisms in macrophages to regulate chemokine synthesis.

IL-10-deficient (IL-10−/−) mice develop colitis with many similarities to Crohn’s disease. Daily IL-10 injections have a short systemic half-life and are unable to induce complete remission in IL-10−/− mice with established disease. In this paper, we investigate the duration, potency, and immunogenicity of gene therapy using an adenoviral vector encoding murine IL-10 (AdvmuIL-10). A single systemic injection of AdvmuIL-10 was sufficient not only to prevent the onset of colitis for at least 10 wk but also to induce clinical and histological remission in mice with established disease. In addition, AdvmuIL-10 diminished the systemic manifestations of disease, including elevated acute-phase proteins, as well as the local consequences of inflammation such as raised stool IL-1β concentrations. Both IL-10 protein and the effects of secreted IL-10 were detectable for 10 wk after AdvmuIL-10 injection. Furthermore, the immunoregulatory effect of a single AdvmuIL-10 injection was manifest both by a reduction in TNF-α, IFN-γ, and RANTES release from stimulated splenocyte cultures, and also by a change in the proportion of CD45RBhigh/low lymphocytes in the spleen compared with control mice. The delivery of AdvmuIL-10 resulted in a significantly diminished host antiadenoviral response compared with control adenoviral vectors. Thus, gene therapy strategies using adenoviral vectors encoding immunoregulatory and antiinflammatory cytokines may prove to be a potent approach for the treatment of chronic inflammatory disease. Antiinflammatory cytokine expression protects against immune responses directed at gene vectors.