Identification of macrophage activation associated proteins by two-dimensional gel electrophoresis and microsequencing

To understand activation in monocytes and macrophages we have studied changes in protein synthesis using the human monocytoid U937 cell line and two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and protein sequencing. U937 cells that had been metabolically labeled during treatment with PMA, LPS, or IFN-γ showed appreciable increases or decreases the in synthesis of 14 proteins when analyzed by 2D PAGE. Although some 20 proteins are reported to be affected by these agents in U937 cells, none of them correspond with the 14 proteins studied here. Of the 14 observed changes, four spots (p41/65, p35/65, p26/44, p20/53) were upregulated by PMA only, one (p16/44) by LPS only, five spots (p29/47, p26/45, p26/48, p12/47, p10/45) by both LPS and PMA, and, finally, one (p29/45) by all three agents. Two spots (p20/59 and p20/61) were down-regulated by IFN-γ and one of these spots (p20/59) was up-regulated by LPS. Only one spot (p20/48) was up-regulated by IFN-γ. eleven spots with matching mobilities (both M(r) and pI) to those identified in U937 were observed on 2D PAGE gels from human culture derived macrophages. Ten spots from U937 were sequenced by Edman degradation. Two were not identified from information contained in the available DNA and protein databases and thus represent novel proteins, whereas a further six of the proteins were N-terminally blocked. The remaining two (29/47 and 12/47, respectively) were identified from existing protein databases as translationally controlled tumor protein (TCTP) and cytokeratin. This is the first report of the presence of TCTP in hemopoietic cells and its modulation by PMA or LPS in any cell type. We believe that 2D PAGE and sequencing is a powerful approach for identifying key proteins in macrophage cellular activation.