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ORIGINAL RESEARCH ARTICLE

Production of Modified C-Reactive Protein in U937-Derived Macrophages

Irina Ciubotaru^*, Lawrence A. Potempa and Rosemary C. Wander^*,1

^* Department of Nutrition, The University of North Carolina at Greensboro, Greensboro, North Carolina 27403; and Immtech International, Inc., 150 Fairway Drive, Suite 150, Vernon Hills, Illinois 60061-1860

To whom requests for reprints should be addressed at ¹ P.O. Box 26170, 201 Mossman Building, The University of North Carolina at Greensboro, Greensboro, NC 27402-6170. E-mail: rcwander{at}uncg.edu

Plasma C-reactive protein (CRP) has been proposed to be a strong independent predictor for cardiovascular disease. This circulating form of CRP (native CRP or nCRP) is well described. Recently, the existence of a conformationally distinct isoform of CRP (modified CRP or mCRP) has been reported. The relevance of each CRP isoform to atherosclerotic disease is unknown. The purpose of this study was to examine the natural expression of CRP in undifferentiated, differentiated, and stimulated macrophages, cells known to contribute to atherogenesis in vivo, and to determine whether transcribed CRP was expressed as nCRP or mCRP. Macrophages were generated from U937 monocytes using phorbol 12-myristate 13-acetate. Differentiated macrophages were further stimulated with lipopolysaccharides (LPS). In undifferentiated, differentiated, and stimulated cells, CRP expression was assessed by reverse transcription–polymerase chain reaction, and CRP protein production was measured by fluorescence microscopy and flow cytometry (cellular CRP) or high-sensitivity enzyme-linked immunosorbent assay (secreted CRP). CRP transcript was minimally expressed in undifferentiated cells. Expression increased markedly in macrophages during differentiation and was not affected by LPS at 24 hrs. Cellular CRP protein increased in a time-dependent manner after LPS stimulation, and this induction was mediated via interleukin (IL)-6 and IL-1ß. A small amount of secreted CRP was detected in the media of differentiated cells, but it was not significantly increased after LPS stimulation. Using specific monoclonal antibodies, our data indicate that cellular CRP is directly translated as the mCRP rather than the nCRP isomer. These results indicate that U937-derived macrophages are a good cell model to further study the production of mCRP under conditions relevant for the atherogenic process.

Key Words: native C-reactive protein (nCRP) • modified C-reactive protein (mCRP) • inflammation • interleukins • atherosclerosis

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