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. Interaction between monocytes/macrophages and tumor cells in the production and activation of MMPs Subsequent to entering an inflammation site monocytes differentiate into macrophages and exposure to stimulants such as IFN-alpha;or IL-4 result in their polarization into M1 (classically activated) or M2 (alternatively activated) macrophages, respectively. The reciprocal interaction between tumor cells and monocytes and monocyte-derived macrophages may play a significant role in the production of MMPs involved in the alteration of connective tissue leading to tumor progression. To assess this possibility and identify MMPs that are potentially involved in this process we are carrying out research in the following areas: (1) determine by real time PCR the MMP family members and TIMPs expressed by human monocytes, resting macrophages, M1 and M2 macrophages;(2) determine by real time PCR the MMP family members and TIMPs expressed by normal keratinocytes and two oral squamous carcinoma cell lines, HN-12 and HN-30;(3) examine the reciprocal interaction between monocytes/macrophages and tumor cells on MMP and TIMP expression;(4) study the mechanism of activation of pro-MMPs in the interaction between monocytes/ macrophages and cancer cells;and (5) determine the signal signal transduction pathways involved in the regulation of MMP production by squamous cell carcinomas. 1) Expression of MMPs and TIMPs in monocytes, macrophages, M1 macrophages and M2 macrophages Expression of MMPs was determined from RNA obtained from donor matched monocytes and macrophages. Major findings include: monocytes express significantly more MMP-1, -10, and -14 when activated than macrophages whereas monocytes express low levels of MMP-2 and TIMP-3 compared to macrophages;monocyte MMP expression is more responsive to LPS and TNF-alpha than macrophages;LPS induced higher levels of MMP-1, -7, and -10 in monocytes than TNF-alpha;and GM-CSF whereas the converse was the case for MMP-9;interestingly, LPS caused a significant decrease in TIMP-2 and TIMP-4 expression by monocytes; with the exception of MMP-1, -7, and -14, macrophage expression of MMPs was relatively unaffected by LPS or TNF-alpha;and GM-CSF; comparison of resting macrophages with M1 and M2 macrophages revealed that these subsets had lower levels of MMP-1 as well as MMP-2 with the greatest decrease occurring in MMP-2 expression in M2 macrophages. In contrast M2 macrophages had higher levels of MMP-12. 2) Real Time PCR analysis of MMPs and TIMPs produced by keratinocytes, HN12 and HN30 cell lines. We are currently analyzing 23 MMP family members and TIMPs in keratinocytes, HN12 and HN30 cell lines by qPCR. Western analysis has shown that there is a difference between HN12 and HN30 cells in MMP-1 production with HN12 cells producing low levels of MMP-1 whereas HN30 produce large amounts of MMP-1. It is anticipated that other differences in MMPs as well as TIMPs will be found which may affect the degree of invasiveness of these cell lines. 3) Effect of mediators from normal keratinocytes, HN12 and HN30 cells on MMP and TIMP production by monocytes and macrophages. In the first stage of the study on the reciprocal interaction of monocytes/macrophage with tumor cells we are testing the mediators found in the culture supernatants of normal keratinocytes and HN12 and HN30 cells on the expression of monocyte and macrophage MMPs and TIMPs by qPCR. The MMPs selected for analysis are those based on the qPCR analysis results described in item 1. 4) Conversion of HN30 pro-MMP-1 to active MMP-1 by monocytes. Interaction of stromal cells, such as monocytes/macrophages, with tumor cells is believed to be involved in the induction and activation of MMPs. We have previous found that MMP-1 in monocyte cultures is primarily in the active form as compared to macrophage cultures in which only pro-MMP-1 is detected. In our recent studies we have shown the squamous carcinoma cell line HN30 produces significant amounts of pro-MMP-1 which can be converted to active MMP-1 by exposure to cell membranes from monocytes but not macrophages. The ability of monocytes to activate tumor cell pro-MMP-1 appears to be due to cell surface serine protease activity. Plasminogen activator inhibitor-1 (PAI-1) or alpha 2-antiplasmin block this conversion indicating the role of cell surface associated plasminogen on monocytes in this process. PCR analysis has revealed low levels of plasminogen mRNA in monocytes. Current studies are determining if plasminogen synthesis is lost as monocytes mature into macrophages. These results indicate that one of the affects of monocytes on tumor progression is the activation of MMP-1. 5) Signaling pathways involved in the regulation of MMP production by squamous carcinoma cells. Examination of the regulatory pathways involved in the endogenous production of MMP-1 by HN30 cells revealed that the ERK1/2 MAPK is a positive regulator of MMP-1. In contrast, p38 MAPK functions as a negative regulator of MMP-1 as evidenced by a significant increase in MMP-1 production by HN30 cells when the action of phosphorylated p38 is inhibited. Of interest, NH12, which do not produce significant levels of MMP-1, have high levels of phosphorylated ERK1/2 and p38 in contrast to HN30 cells which have lower levels of phosphorylated ERK1/2 and p38. However, when HN12 cells are stimulated with plasmin they secrete detectable levels of MMP-1. Initial findings suggest that this may be due to a change to a higher ratio of phosphorylated ERK1/2 compared to p38.
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