Research in the Immunopathology Section focuses on the biological mediators and signal transduction pathways involved in the modulation of human monocyte functions that may contribute to the immunopathology associated with various inflammatory lesions. Connective tissue destruction is associated with many diseases, such as periodontal disease, atherosclerosis and rheumatoid arthritis, in which the monocyte/macrophage is a prominent cell. Since matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are believed to play a major role in the destruction and remodeling of connective tissue, a major emphasis has been placed on how these enzymes and inhibitors are regulated. Research is also conducted on the role of MMPs and TIMPs in the regulation of cellular functions that are not directly related to connective tissue metabolism. Interaction between cytokines is believed to play a major role in the severity of the pathology associated with inflammatory lesions. Findings that we have published this year demonstrate that interferon (IFN)-gamma modulates the ability of monocytes to produce MMPs when these cells are exposed to other cytokines. Tumor necrosis factor (TNF)- alpha or granulocyte monocyte-colony-stimulating factor (GM-CSF) enhance the production of MMP-9 by monocytes, however exposure to both cytokines is required for the induction of MMP-1 production by monocytes. While IFN-gamma alone fails to induce MMP production by monocytes, in the presence of GM-CSF it stimulates the production of TNF-alpha that results in the synthesis of MMP-1. In contrast, IFN-gamma suppresses the TNF-alpha-induced production of MMP-9. The mechanism of this inhibition involves IFN-gamma mediated activation of caspase 8 that in turn activates p38 mitogen activated protein kinase (MAPK) that phosphorylates STAT1. Activated STAT1 induces IRF-1 that binds to IFN-gamma stimulated responsive elements, and as shown by other investigators, that overlap NF-kB binding sites in the MMP-9 promoter thus inhibiting MMP-9. Therefore, the NF-kB induced by TNF-alpha is blocked by IRF-1 from binding to the MMP-9 promoter. Thus, IFN-gamma in the presence of GM-CSF and/or TNF-alpha differentially regulates monocyte MMPs through induction of TNF-alpha and a novel mechanism involving caspase 8. The regulation of monocyte MMP production involves multiple signal transduction components. We have shown that lipopolysaccharide (LPS) induction of MMP-9 occurs, in part, through a PI-3 kinase/protein kinase B (Akt)/NF-kB pathway. This was demonstrated by through the use of specific inhibitors and immunoprecipitation studies. Inhibitors of PI3-K suppressed LPS-induced Akt activity and MMP-9 production. Evidence for the participation of Akt in monocyte MMP-9 synthesis was demonstrated by the inhibition of MMP-9 by SH-5, a specific inhibitor of Akt. The mechanism by which Akt regulates MMP-9 is through the activation of NF-kB as shown by co-immunoprecipitation of the phosphorylated form of Ik-B kinase alpha and Akt as well as the suppression of the dissociation of Ik-B from NF-kB by SH-5. The role of NF-kB in regulation of MMP-9 was further demonstrated by the inhibition of MMP-9 production by proteasome inhibitors, lactacystin and MG-132, that prevented the ubiquitination and dissociation of IkB from NF-kB. Monocytes/macrophages are intimately involved with and regulated by infectious disease agents. Chlamydia pneumoniae (Cp) has been associated with inflammatory diseases such as atherosclerosis where the monocyte/macrophage may, through the production of MMPs, play a critical role in the rupture of vulnerable plaques in the vessel wall. Our studies with Cp have demonstrated that this infectious agent can prime monocytes to produce significantly higher levels of MMPs when exposed to TNF-alpha and GM-CSF, cytokines associated with inflammation. Moreover, the mechanism by which Cp enhhances MMP production is through the induction of higher levels cyclooxygenase-2 leading to increased PGE2 production that regulates MMP synthesis. HIV-1 is another infectious agent that interacts with monocytes/macrophages in the progression of infection by this virus. In collaborative studies with investigators from the FDA we have elucidated a mechanism by which macrophages induce activation of latently HIV infected cells to produce virions. Co-culture of macrophages with HIV-1 latently infected U1 or ACH2 cells resulted in rapid nuclear localization of NF-kB p50/p65 dimers with a concomitant increase in the expression of pro-inflammatory cytokines and HIV-1 replication. The cytokines produced by macrophages when co-cultured with the HIV-1-latently infected cells included IL-1B, IL-6, IL-8, TNF-alpha, and TGF-beta. Pretreatment of macrophage-U1 or macrophage-ACH2 co-cultures with neutralizing anti-TNF-alpha down-regulated the replication of HIV-1. Moreover, treatment of the co-cultures with a NF-kB inhibitor prevented the induction of cytokine expression, indicating a pivotal role of NF-kB-mediated signaling in the re-activation of HIV-1 in latently infected cells by macrophages.
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