In vitro reactivity of cryoglobulin IgM and IgG in hepatitis C virus-associated mixed cryoglobulinemia

Background/Aims: Mixed cryoglobulinemia is frequently associated with chronic hepatitis C virus infection. We aimed to clarify the mechanism, kinetics and participating proteins in cryoprecipitate formation, whcih are still being debated. Methods: Eighteen patients with cryoglobulinemia were studied. Isolated serum cryoprecipitates and purified cryoglobulin IgM and IgG fractions were analyzed in vitro by turbidimetry for temperature-dependent complex formation. Immunoglobulin reactivity, i.e., in cryoprecipitates and in cryoglobulin-free sera, was studied using immunoblot and enzyme immunoassays. HCV RNA was detected by reverse transcriptase/polymerase chain reaction. Results: By turbidimetry, purified cryo-IgM precipitated (in the absence of HCV RNA) with cryo-IgG as well as with non-cryoglobulin IgG and with IgG Fc or F(ab′)2 fragments. In contrast, purified cryo-IgG did not precipitate with non-cryoglobulin IgM. Anti-HCV IgG reactivity was found in cryoglobulin-free sera, in cryoprecipitates and in purified cryoglobulin IgG fractions. The respective titers were similar. Purified cryo-IgM did not react to HCV-encoded proteins. Binding of cryo-IgM to heterologous IgG was inhibited by intact IgG (up to a mean of about 52%) as well as by IgG Fc (33%) and F(ab′)2 fragments (17%). Binding of cryo-IgM to IgG was enhanced at low temperature (4°C vs. 37°C), particularly for type III cryoglobulin IgM. Conclusions: In hepatitis C virus-associated cryoglobulinemia the vitro precipitate formation depended on cryo-IgM, while IgG appeared to act as an unspecific antigenic partner. Hepatitis C viral particles were probably not required. Cryo-IgM binding occurred primarily to intact IgG. Anti-HCV reactivity of either cryo-IgM or cryo-IgG was not necessary for precipitate formation. Regarding the pathogenesis, a direct hepatitis C virus protein-dependent stimulation of B-cells producing cryo-IgM seems to be unlikely.