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Activation of macrophage CD8: pharmacological studies of TNF and IL-1 beta production
T J Lin1, N Hirji, G R Stenton
1Department of Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada, USA.
Abstract:
Previously, we demonstrated that rat macrophages express CD8 and that Ab to CD8 stimulates NO production. We confirm that CD8 is expressed by rat macrophages and extend understanding of its functional significance. Activation of CD8 alpha (OX8 Ab) on alveolar macrophages stimulated mRNA expression for TNF and IL-1 beta and promoted TNF and IL-1 beta secretion. Similarly, OX8 Ab (CD8 alpha) stimulated NR8383 cells to secrete TNF, IL-1 beta, and NO. Activation of CD8 beta (Ab 341) on alveolar macrophages increased mRNA expression for TNF and IL-1 beta and stimulated secretion of TNF, but not IL-1 beta. Interestingly, anti-CD8 Abs did not stimulate IFN-gamma or PGE2 production, or phagocytosis by macrophages. OX8 (CD8 alpha)-induced TNF and IL-1 beta production by macrophages was blocked by inhibitors of protein tyrosine kinase(s), PP1, and genistein, but not by phosphatidylinositol-3 kinase inhibitor, wortmannin. Moreover, OX8 stimulated protein tyrosine kinase activity in NR8383 cells. Further analysis of kinase dependence using antisense to Syk kinase demonstrated that TNF, but not IL-1 beta, stimulation by CD8 alpha is Syk dependent. By contrast, protein kinase C inhibitor Ro 31-8220 had no effect on OX8-induced TNF production, whereas OX8-induced IL-1 beta production was blocked by Ro 31-8220. Thus, there are distinct signaling mechanisms involved in CD8 alpha (OX8)-induced TNF and IL-1 beta production. In summary, macrophages express CD8 molecules that, when activated, stimulate TNF and IL-1 beta expression, probably through mechanisms that include activation of Src and Syk kinases and protein kinase C. These findings identify a previously unknown pathway of macrophage activation likely to be involved in host defense and inflammation.
Insights
Rat macrophages express CD8 molecules. Activating CD8 on these cells stimulates the production of tumor necrosis factor (TNF) and interleukin-1 beta (IL-1 beta), revealing a new pathway for macrophage activation.
Area of Science:
- Immunology
- Cell Biology
Background:
- Rat macrophages express the CD8 molecule.
- Antibodies targeting CD8 can stimulate nitric oxide (NO) production.
Purpose of the Study:
- To investigate the functional significance of CD8 expression on rat macrophages.
- To elucidate the signaling pathways involved in CD8-mediated macrophage activation.
Main Methods:
- Activation of rat alveolar macrophages and NR8383 cells using anti-CD8 antibodies (OX8 for CD8 alpha, Ab 341 for CD8 beta).
- Measurement of mRNA expression and protein secretion for TNF, IL-1 beta, NO, IFN-gamma, and PGE2.
- Inhibition studies using protein tyrosine kinase inhibitors (PP1, genistein), PI3K inhibitor (wortmannin), and PKC inhibitor (Ro 31-8220).
- Assessment of Syk kinase dependence using antisense oligonucleotides.
- Analysis of protein tyrosine kinase activity.
Main Results:
- Activation of CD8 alpha (OX8) stimulated TNF and IL-1 beta mRNA and secretion, as well as NO production in NR8383 cells.
- Activation of CD8 beta (Ab 341) increased TNF and IL-1 beta mRNA and TNF secretion, but not IL-1 beta secretion.
- CD8 activation did not affect IFN-gamma or PGE2 production, or phagocytosis.
- CD8 alpha-induced cytokine production was dependent on protein tyrosine kinases (Src, Syk) and protein kinase C (PKC) for IL-1 beta, but only Syk for TNF.
- Distinct signaling pathways mediate CD8 alpha-induced TNF and IL-1 beta production.
Conclusions:
- Macrophages express functional CD8 molecules that, upon activation, induce the expression and secretion of pro-inflammatory cytokines TNF and IL-1 beta.
- CD8-mediated macrophage activation involves distinct signaling cascades, including Src, Syk kinases, and PKC.
- This pathway represents a novel mechanism of macrophage activation potentially involved in host defense and inflammatory responses.