[show abstract][hide abstract] ABSTRACT: We and others have reported that rheumatoid arthritis (RA) synovial T cells can activate human monocytes/macrophages in a contact-dependent manner to induce the expression of inflammatory cytokines, including tumour necrosis factor alpha (TNFalpha). In the present study we demonstrate that RA synovial T cells without further activation can also induce monocyte CC and CXC chemokine production in a contact-dependent manner. The transcription factor NFkappaB is differentially involved in this process as CXC chemokines but not CC chemokines are inhibited after overexpression of IkappaBalpha, the natural inhibitor of NFkappaB. This effector function of RA synovial T cells is also shared by T cells activated with a cytokine cocktail containing IL-2, IL-6 and TNFalpha, but not T cells activated by anti-CD3 cross-linking that mimics TCR engagement. This study demonstrates for the first time that RA synovial T cells as well as cytokine-activated T cells are able to induce monocyte chemokine production in a contact-dependent manner and through NFkappaB-dependent and NFkappaB-independent mechanisms, in a process influenced by the phosphatidyl-inositol-3-kinase pathway. Moreover, this study provides further evidence that cytokine-activated T cells share aspects of their effector function with RA synovial T cells and that their targeting in the clinic has therapeutic potential.
Arthritis research & therapy 02/2006; 8(6):R168. · 4.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: TNF-alpha is a key factor in a variety of inflammatory diseases. This study examines the role of p38 MAPK in the regulation of TNF-alpha 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-alpha 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-alpha transcription. This is mediated through p38 MAPK regulation of NF-kappaB. 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-alpha 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 Journal of Immunology 01/2005; 173(11):6928-37. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Interleukin 10 knockout (IL-10-/-) mice spontaneously develop a Th1 T cell mediated colitis with many similarities to Crohn's disease. Daily injections of IL-10 are unable to induce remission in mice with established disease. In contrast, we have shown previously that intravenous administration of adenoviral vectors encoding IL-10 (AdvmuIL-10) induces hepatic IL-10 release and leads to long term disease suppression with profound systemic immunoregulatory changes.
To determine whether rectal delivery of AdvmuIL-10 induces localised colonic IL-10 expression without systemic immune suppression, and assess its therapeutic efficacy in IL-10-/- mice with established colitis.
A single rectal infusion of 5 x 10(8) PFU AdvmuIL-10 to 10 week IL- 10-/- mice resulted in a median level of 27.3 pg/mg IL-10 in colonic homogenates harvested one week later. IL-10-/- mice with established colitis treated with an enema of 5 x 10(8) PFU AdvmuIL-10 entered clinical and histological remission whereas empty cassette adenovirus (Adv0) or phosphate buffered saline (PBS) treated mice developed progressive disease. After four weeks, the histological score of AdvmuIL-10 treated mice (4.4 (1.5)) was significantly lower than that of Adv0 (11.1 (1.1); p<0.001) and PBS (10.9 (1.0); p<0.01) treated controls. In addition, the stool concentration of IL-1beta over the four week experiment was significantly higher in mice treated with saline or Adv0 than in those treated with AdvmuIL-10 (p<0.01).
Local AdvmuIL-10 therapy reverses colitis in IL-10-/- mice without the systemic effects seen after intravenous administration. Gene therapy strategies using adenoviral vectors encoding immunoregulatory cytokines may prove to be a potent approach to the treatment of chronic inflammatory diseases such as Crohn's disease.
[show abstract][hide abstract] ABSTRACT: The transcription factor NF-kappaB 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-kappaB plays in TNFalpha- or lipopolysaccharides (LPS)-induced chemokine secretion by human monocyte-derived macrophages was examined. Secretion of the CXC chemokines IL-8, GROalpha and ENA-78, induced by TNFalpha, was significantly suppressed by inhibiting NF-kappaB, using overexpression of IkappaBalpha. However, when induced by LPS the expression of these chemokines was unaffected. In contrast, expression of the CC chemokines MIP-1alpha, MCP-1 and RANTES inducedby TNFalpha or LPS was significantly inhibited by the overexpression of IkappaBalpha. Therefore, there appear to be different mechanisms regulating CC and CXC chemokine secretion by macrophages, depending on the stimulus and that TNFalpha and LPS can use different signaling mechanisms in macrophages to regulate chemokine synthesis.
European Journal of Immunology 08/2002; 32(7):2037-45. · 4.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 TNF- or lipopolysaccharides (LPS)-induced chemokine secretion by human monocyte-derived macrophages was examined. Secretion of the CXC chemokines IL-8, GRO and ENA-78, induced by TNF, was significantly suppressed by inhibiting NF-κB, using overexpression of IκB. However, when induced by LPS the expression of these chemokines was unaffected. In contrast, expression of the CC chemokines MIP-1, MCP-1 and RANTES inducedby TNF or LPS was significantly inhibited by the overexpression of IκB. Therefore, there appear to be different mechanisms regulating CC and CXC chemokine secretion by macrophages, depending on the stimulus and that TNF and LPS can use different signaling mechanisms in macrophages to regulate chemokine synthesis.
European Journal of Immunology 06/2002; 32(7):2037 - 2045. · 4.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: IL-10 is a potent anti-inflammatory cytokine and inhibitor of TNF-alpha production. The molecular pathways by which IL-10 inhibits TNF-alpha 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-alpha 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-alpha gene. When added, either with or after the stimulus, IL-10 required the 3' untranslated region of the TNF-alpha 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-alpha production addressed the effect of IL-10 on the activation of p38 mitogen-activated protein kinase and NF-kappaB. However, these studies could demonstrate no requirement for the inhibition of p38 mitogen-activated protein kinase or NF-kappaB activation as potential mechanisms. Overall, these results may explain the diversity previously ascribed to the complex mechanisms of IL-10 anti-inflammatory activity.
The Journal of Immunology 06/2002; 168(10):4837-45. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate the mechanism that leads to the spontaneous production of tumor necrosis factor alpha (TNFalpha) in rheumatoid arthritis (RA) synovial tissue.
Normal blood monocytes were cocultured either with fixed activated T cells generated from normal blood or RA synovial T cells purified from synovium. TNFalpha production was measured in supernatants from these cocultures following blockade of the transcription factor nuclear factor kappaB (NF-kappaB) using adenoviral transfer of the inhibitor of NF-kappaB kinase alpha into the responding monocytes, or blockade of phosphatidylinositol 3-kinase (PI 3-kinase) using the inhibitory drugs wortmannin or LY294002. TNFalpha production was measured by enzyme-linked immunosorbent assay.
TNFalpha production in synovial tissue from patients with RA but not osteoarthritis was found to be T cell dependent. The RA synovial joint T cells resembled normal T cells that had been activated for 8 days using a cocktail of cytokines. These T cells, designated Tck (cytokine-activated T cells), and RA synovial T cells both induced TNFalpha production in resting monocytes in a cell-contact-dependent manner, which was abrogated by blockage of the transcription factor NF-kappaB but augmented if PI 3-kinase was inhibited. Normal blood T cells activated conventionally via the T cell receptor with crosslinked anti-CD3 antibody resulted in TNFalpha production from monocytes; this was unaffected by NF-kappaB blockade, but was inhibited in the presence of PI 3-kinase-blocking drugs.
These data provide strong evidence for the importance of T cells in inducing TNFalpha in chronic inflammatory rheumatoid tissue, and give insight into the mechanism whereby these T cells are activated in vivo. Furthermore, they indicate that production of TNFalpha in pathologic tissue is regulated differently from physiologic antigen-dependent TNFalpha production, which raises the possibility that selective inhibitors of TNFalpha in disease may be developed.
[show abstract][hide abstract] ABSTRACT: 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-1beta 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-alpha, IFN-gamma, and RANTES release from stimulated splenocyte cultures, and also by a change in the proportion of CD45RB(high/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.
The Journal of Immunology 07/2001; 166(12):7625-33. · 5.52 Impact Factor