Research in the Immunopathology Section focuses on the biological mediators and signal transduction pathways involved in the modulation of human monocyte and lymphocyte functions that may contribute to the immunopathology associated with various inflammatory lesions. Monocytes/macrophages are prominent in many inflammatory diseases, such as periodontal disease, rheumatiod arthritis, atherosclerosis, and cancer. The pathology associated with these diseases involves alterations in the integrity of the connective tissue framework implicating a role for matrix metalloproteinases (MMPs). MMPs are comprised of a family of zinc dependent endopeptidases divided into major subgroups that include the interstitial collagenases, the gelatinases, stromelysins, membrane type MMPs and others. Collectively these enzymes are capable of degrading all the extracellular matrix components. Because 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/macrophage as well as in the reciprocal interaction between monocytes/macrophages and tumor cells. Cytokine-induced MMP production by human monocytes is negatively regulated by GSK-3βthrough p38 MAPK Studies initiated last year focused on the potential role of the PI3K/AKT/GSK-3 pathway in the regulation of human monocyte MMPs. We examined this pathway, particularly GSK-3, in the induction of monocyte MMPs by LPS and cytokines. Because many inflammatory lesions are cytokine mediated and are not related to sepsis we first focused on GSK-3 regulation of cytokine induced MMPs. We previously showed that while either TNF-αor GM-CSF induced MMP-9 both cytokines were required for the stimulation of MMP-1. However, preincubation of monocytes with SB216763, a specific inhibitor of GSK-3, resulted in the induction of MMP-1 following the addition of TNF-αor GM-CSF and enhancement of MMP-1 stimulated by TNF-αplus GM-CSF. SB216763 also enhanced MMP-9 production by individual cytokines and their combination. Treatment of monocytes with SB216763 alone failed to stimulate MMP production. Examination of the mechanism involved in GSK-3 regulation of MMPs revealed that inhibition of GSK-3βresulted in decreased phosphorylation of p38 MAPK. Further evidence for the central role of p38 and its interaction with GSK-3βin MMP production was the ability of SB23580 to induce MMP-1 in monocytes stimulated with either TNF-αor GM-CSF. Moreover, inhibition of p38 or GSK-3 resulted in an increased phosphorylation of ERK1/2 which is required for cytokine-induced MMP production. Because β-catenin has been shown to regulate certain MMPs current siRNA studies are addressing the extent to which β-catenin is involved in regulation of MMP-1 and -9. Our findings demonstrate that cytokine-induced MMP production by monocytes is negatively regulated by GSK-3βthrough p38 MAPK. Endotoxin tolerance impairs IL-1 receptor-associated kinase (IRAK) 4 and TGF-beta-activated kinase 1 activation, K63-linked polyubiquitination and assembly of IRAK1, TNF receptor-associated factor 6, and IkappaB kinase gamma and increases A20 expression. In an ongoing collaboration with investigators at the University of Maryland we have been examining the role of TLRs in endotoxin signaling which leads to endotoxin tolerance. TLRs are primary sensors of microbial pathogens that activate innate immune cells, as well as initiate and orchestrate adaptive immune responses. Our recent studies shows that endotoxin tolerization of THP1 cells and human monocytes impairs LPS-mediated receptor recruitment and activation of IRAK4, ablates K63-linked polyubiquitination of IRAK1 and TRAF6, compromises assembly of IRAK1-TRAF6 and IRAK1-IKKγplatforms, and inhibits TAK1 activation. Deficiencies in these signaling events in LPS-tolerant cells coincided with increased expression of A20, an essential deubiquitination enzyme, and sustained A20-IRAK1 associations. Overexpression of A20 inhibited LPS-induced activation of NF-κB and ablated NF-κB reporter activation driven by ectopic expression of MyD88, IRAK1, IRAK2, TRAF6, and TAK1/TAB1, while not affecting the responses induced by IKKβand p65. A20 shRNA knockdown abolished LPS tolerization of THP1 cells, mechanistically linking A20 and endotoxin tolerance. Thus, deficient LPS-induced activation of IRAK4 and TAK1, K63-linked polyubiquitination of IRAK1 and TRAF6, and disrupted IRAK1-TRAF6 and IRAK1-IKKγassembly associated with increased A20 expression and A20-IRAK1 interactions are new determinants of endotoxin tolerance.

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National Institute of Dental & Craniofacial Research
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