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, which 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 degrees C vs. 37 degrees C), particularly for type III cryoglobulin IgM. Conclusions: In hepatitis C virus-associated cryoglobulinemia the in 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.