Studies on Antibody Synthesis Initiated in Vitro

Abstract

Ten µg. of the lipopolysaccharide endotoxin of Salmonella typhosa was given to rabbits intravenously to enhance the subsequent antibody response to an unrelated substance. The spleens were removed 24 hours later, diced, and incubated 1 hour with the antigen, bovine-γ-globulin (BGG), in a protein-free medium. After washing, the tissues either were extracted at once or planted and the fluids and tissues harvested 1 to 3 days later. Antibody was determined by a modification of the Boyden hemagglutination technique. Small amounts of antibody were synthesized as early as 1 hour after the addition of antigen. The antibody formed could be specifically inhibited with BGG, was not dialyzable, and did not sediment at 105,000 g for 2 hours. Dose-response studies revealed no antibody formation when the BGG concentration was 0.005 or 0.05 mg./ml. The best responses were obtained at concentrations of 0.5 to 5.0 mg./ml. These results were found irrespective of whether the animal had previously received BGG in vivo. Forty per cent autologous serum increased antibody formation about 9-fold over that secured with protein-free medium or with 40 per cent homologous serum. Antibody formed with this system could be detected by 50 per cent complement fixation test, although at much lower titer than found by hemagglutination. While spleens from rabbits previously given BGG did not produce more antibody than spleens from normal rabbits, they differed in that they produced antibody without the involvement of endotoxin. Under appropriate circumstances, endotoxin was effective in vitro in enabling spleen fragments to produce antibody to BGG. Cortisone acetate administered to rabbits prior to the removal of the spleen severely inhibited antibody production in vitro. Sodium prednisolone phosphate added in vitro showed a similar irreversible effect at concentrations as low as 2 x 10–5 M. Nitrogen mustard inhibited antibody formation at concentrations as low as 10–4 M.

Studies on Antibody Formation by Peritoneal Exudate Cells in Vitro

“…However, the drop in response at a concentration of 10.0 mg./ml, may be significant since this same pattern was found when diced spleens were incubated with 50.0 mg./ml, of BGG (2). The data suggest that, once a threshold concentration of antigen obtains, the cells respond with a maximal effort and are in substantial agreement with the earlier studies from this laboratory (2) in which diced rabbit spleens were exposed to BGG in vitro. Two differences between the two systems were noted, however.…”

“…Geometric mean titers (GMT) were calculated from the individual titers of each experiment including all titers greater than 8. The over-all mean hemaggluti- (2) 8 (2) 2 (2) nating titers in tissue cultures of cells from rabbits which received either primary or secondary injections of BGG were essentially the same, i.e. 30 and 32, respectively, and the antibody persisted in these two systems for essentially the same time; i.e., 21 and 23 days, respectively.…”

“…For the most part, the materials used were identical with those described earlier (2), and only variations will be recorded here.…”

“…Antibody a,~says were carded out on freshly harvested fluids with tanned formalinized sheep red ceils as previously described (2). The ceils were coated with BGG unless otherwise noted.…”

Possible modes of action of ACTH and glucocorticoids in allergic diseases

“…Alternatively, as indicated by the findings of Stevens & McKenna (1958), it is possible that the in vitro dose requirements for complete inhibition of antibody formation may be species-as well as antigen-dependent. Using diced rabbit spleen and bovine gamma-globulin as the antigen these workers observed irreversible inhibition of antibody production in vitro, with prednisolone added to the culture medium in concentrations as low as 2x 10~^ M.…”