COMPLEMENT ACTIVATION BY C-REACTIVE PROTEIN ON THE HEP-2 CELL SUBSTRATE

The complement (C) activation by C-reactive protein (CRP) in acute-pha se sera is routinely tested in our laboratory by means of an indirect immunofluorescence method (C3-IFT) on rat kidney sections. This C3-IFT assay is based on the binding of CRP to the renal tissue followed by the fixation of C4 and C3 components to distinct vessel-associated med ullary structures as a result of CRP-mediated C activation in vitro. W hile the activation cascade leading to the deposition of C4 and C3 cou ld previously be deduced experimentally, we were unable as yet to visu alize CRP and the components of the Cl complex on kidney sections when testing patients' sera by indirect immunofluorescence. In an attempt to analyze the mechanisms of unexpectedly negative C3-IFT results (e. g. bacterial endocarditis) we employed monolayers of fixed HEp-2 cells which have previously been shown to be a suitable substrate for CRP b inding. By incubating purified native CRP supplemented with normal hum an serum as a source of C we detected the C components Clq, Clr, Cls, C4 and C3 in the same speckled immunofluorescent pattern on HEp-2 cell nuclei as described characteristically for CRP binding. The serial ac tivation steps from CRP up to C3 could also be followed on HEp-2 cells using C3-IFT-positive acute-phase sera. However, certain C3-IFT-negat ive acute-phase sera showed an arrest between Cls and C4 of the CRP-me diated C activation cascade. HEp-2 cells can thus be used to monitor t he process of autologous C activation initiated by endogeneous CRP in patients' sera. In contrast to native CRP, urea-modified CRP (mCRP) di d not bind to HEp-2 cell nuclei, but was detected in association with distinct filamentous cytoplasmic structures. Unlike its native counter part, binding of mCRP was not followed by a deposition of C components .