Biochemical properties that accompany the production of homogeneous antibody response: a general mechanism hypothesis [proceedings].

Micrococcus lysodeikticus (A.T.C.C. 4698), a Gram-positive bacterium, stimulates the production of a high titre of antibodies of restricted heterogeneity, as seen by electrophoresis and sequence analysis (Van Hoegaerden et al., 1975; Van Hoegaerden & Strosberg, 1976). Simultaneous vaccination of rabbits with Micrococcus and another antigen, such as lysozyme methyl ester or tobacco-mosaic virus, yields antibodies of restricted heterogeneity against that second antigen, suggesting a similar immunoregulation, monitored by the anti-Micrococcus immunity. In this communication, we have investigated some specific features of the Micrococcus immunity that might be responsible for the restricted heterogeneity of the immune response. Preimmune sera taken from Balb/c mice or C5, black/6J mice seldom showed agglutination patterns when tested with a suspension of Micrococcus (2mg/ml) by using a standard procedure at 37°C. Preimmune sera from randomly bred rabbits often contained natural Micrococcus-binding agglutinins of low titre. Using a haemolytic assay for complement titration, we repeatedly observed that hyperimmune rabbits, injected intravenously for several months with Micrococcus suspension, had lowered complement titres. This phenomenon was not associated with a homogeneous antibody response. In several cases, complement titres returned to normal values after a resting period of 1 month. In a comparative haemolytic trial, with a quantitatively standardized procedure for complement fixation, we found that Micrococcus cells, Micrococcus cell walls, chitin, zymosan and dextran sulphate all could activate and deplete serum complement of guinea pig in a dose-dependent fashion, whereas dextrine and poly(ethy1ene glycol) were ineffective. It appears that Micrococcus may interact both in vitro and in vivo with the complement system, by antibodymediated activation of the classical pathway or by a bacterium-induced activation of the alternative pathway. It remains as yet unclear, however, whether the last property initiates macrophage activity or triggers or suppresses T-cell-independent B-cell responses, as some other complement-activating polysaccharides do. A standard method was worked out to test the rabbit anti-Micrococcus sera for agglutination with erythrocytes of different species. At physiological temperatures, no anti-Micrococcus serum agglutinated sheep or rabbit erythrocytes, and agglutination of autologous erythrocytes was never observed. Immunosorbent-purified anti-Micrococcus antibodies were incapable of agglutinating rabbit or sheep erythrocytes or of lysing them in the presence of guinea-pig complement, suggesting that no significant binding occurred at the subagglutinating concentration. However, when several blood samples of a series of Micrococcus-injected rabbits were tested, we found that some antisera showed positive agglutination patterns with sheep erythrocytes specifically coated with rabbit immunoglobulin G (isolated on DEAE-cellulose) or rabbit immunoglobulin M (purified on Sephadex G-200). Agglutination was unrelated to anti-Micrococcus antibody titre. Possibly this anti-immunoglobulin factor can be identified with an allotypic rheumatoid factor described by Hamers et a/. (1975). In a final set of experiments, we investigated the relationship between antibody specificity and the appearance, existence or persistence of a homogeneous antibody response. Antibodies were specifically eluted from a Sepharose affinity column coupled with bacterial cell wall. Anti-Micrococcus antibodies were directed against either the bacterium-specific Perkins’ antigen (containing polymers of glucose and mannosaminuronic acid) or the bacterial peptidoglycan (built up of repeating units of N-acetyl-Dglucosamine, N-acetyl-D-muramic acid and the cross-linking peptide; Wikler, 1975). Anti-carbohydrate antibodies were eluted from the immunoadsorbent with 1 M* This paper was not presented at the meeting.