Two dimensional gel electrophoresis of cellular and secreted proteins from rat alveolar macrophages after lipopolysaccharide treatment

Abstract

Two dimensional gel electrophoresis (2-D PAGE) and automated image analysis were used to study the effects of bacterial lipopolysaccharide (LPS) activation on secreted and cellular rat alveolar macrophage proteins. Primary alveolar macrophages were cultured and exposed to LPS in the presence of [35S]-methionine for 24 h. Image analysis of 2-D PAGE revealed that LPS treatment primarily modulated the proportions of several alveolar macrophage cellular proteins versus control in addition to limited protein induction and repression. The differential effect of LPS was more pronounced on secreted proteins where qualitative and quantitative differences from control cells were found. Immunoblots of secreted proteins with anti-tumor necrosis factor alpha (TNF alpha) and anti-interleukin-1 alpha(IL-1 alpha) antibodies identified these monokines from protein fluorographic patterns. IL-1 alpha was detected as a single polypeptide of 17 kD at pI = 5. Use of recombinant TNF alpha and monoclonal and polyclonal antibodies for immunodetection revealed the 17 kD form of TNF alpha as well as higher molecular weight species at 18 kD and 22 kD. Thus, analysis of radiolabeled rat macrophages treated with LPS reveals quantitative modulation of several cellular proteins as well as a distinctive pattern of secreted proteins which contain multiple monokine forms resolvable by 2-D PAGE