We have previously reported on the induction, in mice, of a systemic (splenic) immune response with IgA as the dominant antibody, as a result of a short (4 day) intragastric immunization course with foreign erythrocytes. This response was followed by a prolonged period of hyporesponsiveness to similarly administered antigen. Here it is shown that this hyporesponsiveness is also manifested towards antigen given intraperitoneally, and that one is therefore dealing with tolerance, not with failure to absorb antigen from the gut. In contrast, mice primed parenterally and then challenged intragastrically behaved as if never having any previous contact with the antigen, i.e., with a primary-type splenic response of predominant IgA character. This agrees with our former conclusion that splenic responses to enterically absorbed antigen reflect colonization of the spleen by cells sensitized locally in the gut wall, a site not readily primed by the parenteral route. Serum from intragastrically immunized mice contained a very active tolerogen. In vivo, it was capable of conferring tolerance to nonimmune recipient mice. In vitro, it paralyzed the activity of antibody-producing cells. Inhibitory sera has weak antibody activity, restricted to the IgA class, and contained immune complexes reacting with rheumatoid factor but not with C1q. Elimination of these complexes by means by insolubilized rheumatoid factor abolished the tolerogenic effect. In conclusion, the enterically induced tolerogen seems to consist of immune complexes with IgA as the antibody.

The capacity for fixation and activation of hemolytic complement by polyclonal IgM rheumatoid factors (RF) isolated from sera of patients with rheumatoid arthritis and monoclonal IgM-RF isolated from the cryoprecipitates of patients with IgM-IgG mixed cryoglobulinemia was examined. RF mixed with aggregated, reduced, and alkylated human IgG (Agg-R/A-IgG) in the fluid phase failed to significantly reduce the level of total hemolytic complement, CH50, or of individual complement components, C1, C2, C3, and C5. However, sheep erythrocytes (SRC) coated with Agg-R/A-IgG or with reduced and alkylated rabbit IgG anti-SRC antibody were hemolyzed by complement in the presence of polyclonal IgM-RF. Human and guinea pig complement worked equally well. The degree of hemolysis was in direct proportion to the hemagglutination titer of the RF against the same coated cells. Monoclonal IgM-RF, normal human IgM, and purified Waldenström macroglobulins without antiglobulin activity were all inert. Hemolysis of coated SRC by RF and complement was inhibited by prior treatment of the complement source with chelating agents, hydrazine, cobra venom factor, specific antisera to C1q, CR, C5, C6, or C8, or by heating at 56 degrees C for 30 min. Purified radiolabeled C4, C3, and C8 included in the complement source were bound to hemolysed SRC in direct proportion to the degree of hemolysis. These data indicate that polyclonal IgM-RF fix and activate complement via the classic pathway. The system described for assessing complement fixation by isolated RF is readily adaptable to use with whole human serum.

The agglutination of Ig-coated particles by human RF or Clq can be inhibited by Ig aggregates or AgAb complexes. The effect of Ig class was studied by means of agarose-linked human monoclonal Igs. RF was inhibited by all subclasses of IgG and IgA but not by IgM, whereas Clq reacted with IgM, IgG3 and IgG1. Heat-aggregated IgG3 was fractionated by gel-filtration on Ultrogel. Inhibition was restricted to certain fractions of aggregates, viz (IgG3) approximately 7 and (IgG3) approximately 21 for RF, and (IgG3) approximately 10,(IgG3) approximately 14 and (IgG3) approximately 27 for Clq. In a precipitin curve experiment, it was found that RF was inhibited by soluble complexes over an extended range of AgAb ratios, the inactivation of Clq being limited to complexes with 2-5 times antigen excess. Inhibiting factors were found in patients with various diseases and, at low titres, in 22% of healthy people. In 27% of patients' sera, the inhibitors were demonstrable by Clq only after removal of endogenous RF by adsorption on insolubilized IgG. In several patients endogenous agglutinating activity and direct inhibitory activity tended to alternate during the course of the disease. Sera from various patients were also filtrated on Ultrogel and the elution was monitored by immunoassay of IgA, IgM and IgG, as well as by the two inhibition tests. The inhibiting factors were distributed over several peaks which only partially coincided with the elution profiles of IgG and IgM.