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 in which the monocyte/macrophage is a prominent cell. Examples of these chronic inflammatory lesions include periodontal disease and rheumatoid arthritis where destruction of connective tissue leads to loss of structural integrity and atherosclerosis where degradation of connective tissue in vulnerable plaques leads to rupture and subsequent ischemic events. Additionally, the interaction of monocytes/macrophages with cancer cells has the potential for regulating or contributing to metastasis through the induction of connective tissue degrading enzymes. 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 in the human monocyte and how the monocyte may regulate the function of other cell types. ? ? Determining the inflammatory agents and signal transduction pathways that lead to the production of MMPs by monocytes is important to potential therapeutic intervention. ? Reactive oxygen species such as hydrogen peroxide (H2O2) have been associated with the initiation or aggravation of diverse pathological states. In a manuscript published this fiscal year we report that addition of H2O2 to LPS activated monocytes, but not alone, caused a significant enhancement of the LPS-induced production of MMP-1, cyclooxygenase-2 (COX-2), and PGE2. The mechanism by which H2O2 increased these mediators was through the enhancement of IkappaB degradation, the inhibitor of the activation of the transcription factor NF-kappaB. The subsequent increase in NF-kappaB activation by H2O2 is responsible for the increase in MMP-1 production. The effect of H2O2 on IkB degradation and NF-kappaB activation was demonstrated through studies of co-immunprecipitation of IkB with p50 and ELISA assay of NF-kappaB p65 activity. The key role for NF-kappaB in this process was demonstrated by the ability of MG-132 or lactacystin (NF-kappaB inhibitors) to block the enhanced production of MMP-1, COX-2 and PGE2. In contrast, indomethacin, which inhibited PGE2 production, partially blocked the enhanced MMP-1 production. Moreover, while PGE2 restored MMP-1 production in indomethacin treated monocyte cultures it failed to significantly restore MMP-1 production in NF-kappaB inhibitor treated cultures. Thus in the presence of LPS and H2O2, NF-kappaB plays a dominate role in the regulation of production of MMP-1, COX-2 and PGE2 as well as in the PGE2 induction of MMP-1.? ? A number of inflammation factors serve as biomarkers for chronic inflammatory diseases. We have investigated how some of these biomarkers interact in the regulation of monocyte MMP production and the potential relationship of these affects on the pathology associated with inflammatory lesions. Our findings have demonstrated that in many cases in order for biomarkers to affect monocyte MMP production there is a requirement that more than one of these inflammatory agents must be present. An example of this requirement is demonstrated in one of our studies published this last year on C reactive protein. We have demonstrated that C reactive protein (CRP), a biomarker of inflammation and cardiovascular disease, enhances MMP-1 production by monocytes only in the presence of activators such as inflammatory cytokines. Augmentation of MMP-1 by CRP occurs, in part, through the induction of an intermediate, monocyte chemotactic protein-1 (MCP-1). The role of MCP-1 in the enhancement by MMP-1 by CRP was demonstrated by suppression of MMP-1 by neutralizing antibodies against MCP-1 and the ability of exogenous MCP-1 to increase MMP-1 production by cytokine stimulated monocytes. In addition to CRP, oxidized LDL (ox-LDL) is another biomarker of atherosclerosis and potentially other diseases. When cytokine activated monocytes were exposed to CRP and ox-LDL there was a synergistic enhancement of MMP-1. The synergistic increase in MMP-1 by CRP and ox-LDL resulted from a differential regulation through MCP-1 and prostaglandin E2, respectively. These findings indicate that multiple biomarkers or risk factors may be involved in the initiation of an inflammatory disease. ? ? In a collaborative study with investigators at The Johns Hopkins University School of Medicine we have a manuscript in press that provides evidence for the association of MCP-1 gene polymorphism with increased occult ischemia in individuals when additional risk factors are present. A single nucleotide polymorphism in the MCP-1 gene promoter (-2578A>G) results in greater production of MCP-1 protein. We genotyped 679 apparently healthy siblings of people with premature coronary artery disease (CAD), tested for occult ischemia with exercise treadmill tests and thallium-201 single photon emission computed tomography, and assessed CAD risk factors to calculate the Framingham risk score (FRS). For subjects in the first three quartiles of FRS, the prevalence of occult ischemia did not differ significantly by genotype, but in the highest quartile of FRS, individuals with the -2578A>G MCP-1 polymorphism had a significantly greater prevalence of ischemia (44.4% vs 26.1%, p=0.017). These findings provide further support for the interaction of multiple inflammatory mediators in the pathology associated with inflammatory events.
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