Kinetic analysis of chemotactic factor generation in human serum via activation of the classical and alternate complement pathways

John I. Gallin, Robert A. Clark, Michael M. Frank

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The kinetics of complement-related chemotactic factor generation in human serum were evaluated. Addition of immune complexes to serum resulted in formation of chemotactic activity within 5 min of incubation while activation with S. typhosa endotoxin of serum from normal subjects resulted in a prolonged (15-20 min) latent period prior to generation of chemotactic activity. In contrast to S. typhosa, endotoxin incubation of serum with E. coli endotoxin resulted in rapid formation of chemotactic activity with a kinetic pattern similar to that noted with immune complexes. In these sera antibody to E. coli but not S. typhosa endotoxin was demonstrable; analysis of sera from immunized individuals with demonstrable S. typhosa antibody showed a pattern of chemotactic factor formation similar to that of immune complexes and E. coli endotoxin. Activation of human serum deficient in C2 (<0.05% normal C2) with either immune complexes or E. coli endotoxin resulted in a prolonged latent period before formation of chemotactic activity although total chemotactic activity at 60 min was normal; addition of purified C2 restored normal kinetics. The addition of Mg2+-EGTA to normal serum activated with immune complexes or E. coli endotoxin changed the pattern from rapid to delayed formation of chemotactic activity. EDTA completely inhibited generation of chemotactic factor with immune complexes or endotoxin. Activation with immune complexes of serum heated for 20 min at 50°C resulted in the rapid formation of chemotactic activity, while activation with S. typhosa endotoxin failed to generate chemotactic activity. These results indicate that in human serum generation of chemotactic factors proceeds rapidly when mediated by antibody and components of the classical pathway, but only after a latent period when mediated by the alternate pathway. Further studies demonstrated that activation of either complement pathway in human serum results in the generation of a chemotactic factor, which is heat stable (30 min at 56°C), has an estimated molecular weight of 15,500 daltons, is inhibited by goat antibody to human C5 but not C3 and, therefore, represents the C5 cleavage product C5a. Thus, the classical and alternate complement pathways in human serum can be easily distinguished by kinetic analysis. Since normal levels of chemotactic factor may be present after 60 min of incubation, despite complete block of one complement pathway, kinetic analysis may be required to detect clinically important abnormalities.

Original languageEnglish (US)
Pages (from-to)334-346
Number of pages13
JournalClinical Immunology and Immunopathology
Issue number3
StatePublished - Jan 1975


ASJC Scopus subject areas

  • Immunology and Allergy
  • Pathology and Forensic Medicine
  • Immunology

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