| The role of macrophages in the defense against tumors has been investigated extensively over the last decades. Compelling evidence has accumulated that activated macrophages have the capacity to recognize and destroy tumor cells. Also, the mechanisms by which macrophages kill tumor cells have been studied in a large number of in vitro experiments. One major mechanism through which macrophages effectively kill tumor cells requires cell to cell contact; indicating that certain molecules expressed on cell surface of activated macrophages may mediate the tumoricidal capability. Tumor necrosis factor (TNF) and Nitric oxide (NO) produced by activated macrophages are the two classical mediators of tumor cell death.However; evidence of discrepancy is accumulating indicating these two mediators do not appear to account for the broad, potent tumoricidal activity of activated macrophages. These conflicting observations may be reasoned by the pleiotropic nature and ill-defined tumoricidal mechanisms of macrophages.1. Membrane bound TNF-αindependent tumoricidal capacity of BCG activated macrophagesIn order to focus on the contact dependent tumoricidal function and exclude any secretion of cytotoxic factors, the cytotoxicity assays were carried out using paraformaldehyde fixed macrophages as effectors. The result indicates macrophages activated by BCG in vivo possess a potent MCA207 cells killing ability at E: T ratio of 50:1. In contrast, inflammatory macrophages induced by thioglycolate did not show cytotoxicity in these assays. Previous studies demonstrated the expression of membrane form of TNF-αmay explain why chemically fixed macrophages remain cytotoxic to some types of tumor cells. Therefore, we analyzed the expression level of TNF-αon macrophage. To our surprise, BCG activation did not increase the expression level of TNF-αcompared with thioglycolate elicited macrophages. As a positive control, LPS stimulation in vitro can strongly enhance the expression of membrane bound TNF-α.2. High-throughput proteomics analysis of macrophage membrane proteomesWe applied cutting edge proteomics techniques to obtain valuable information about membrane protein expression patterns in macrophages under different conditions. Purified membrane proteins were isolated from BCG-activated and thioglycolate elicited mouse peritoneal macrophages and separated by one dimensional SDS-PAGE. The proteins present in each gel slice were subjected to trypsinolysis, and the resulting peptide fragments were detected using the LCQ Deca XP system. Using the criteria mentioned above, we obtained a list of over 1000 proteins identified in each of the two populations of macrophages respectively. Analyzing the available information about the identified membrane proteins manually, many of the identified proteins were found to be known markers of peritoneal macrophages. These included CD11b, F4/80, CD14, CD86, CD18, CD16, CD44, Toll-like receptor-1. Other CD antigens detected were CD243, CD98, CD107a, CD107b, CD191, CD36, CD97, CD205, CD206, CD177, CD180, CD300, CD45, and CD29. Several members of the integrins, integrin alphaM, integrin beta-2, integrin alpha5 and integrin beta-1 were also detected. In addition, a significant number of the identified proteins were"hypothetical proteins"that have not yet been reported to be translated into proteins. Further analyses of these proteins using bioinformatics tools, gene expression and specific antibodies will likely yield new scientific insights into macrophage biology.To assess the sensitivity of the method, we selected CD11b and TNF-α, two classical proteins expressed on macrophages, for analysis by flow cytometry. The expression levels of the two molecules were 93.6% and 18.2%, respectively. Both molecules were identified using the method described here and the peptide numbers identified were in accordance with their abundance. Thus, all membrane proteins from high to moderate abundance can be identified by the method.3. Proteins uniquely expressed by BCG-activated tumoricidal macrophagesProteins identified from each gel piece at the same size position were compared manually between the two samples. Only the proteins detected in BCG-activated macrophages were selected. Complication of the proteins from all the eight comparisons resulted in a list of 454 proteins which were identified from activated microphages only. Of these 454 proteins, 168 were identified with at least two peptides. This set of proteins represents an extremely high confidence profile of membrane protein specifically expressed in activated macrophages. Investigating the available information about the differentially expressed proteins manually, the classical marker of macrophage tumoricidal activation, iNOS, was detected with 33 peptides, indicating a very high expression level of this protein. Another mouse macrophage activation-associated marker protein, cyclooxygenase, was also detected. To our surprise, the recently discovered macrophage alternative activation marker, Ym1, was detected too. In addition to those known macrophage activation-associated proteins, several novel proteins were identified specifically on activated macrophages which are potential markers for macrophage tumoricidal activation. As the firt step to investigate the functions of these target proteins, we have cloned one of the novel proteins and constructed pET expression vector of it, which has settled the foundation for further studies.
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