Antibody Formation in Vitro

Abstract

The successful initiation of a primary antibody response in tissue culture has been reported by several workers (1--4). I n a previous report from this laboratory (3) it has been suggested that the sequential action of at least two different types of cells was responsible for the formation of antibody in tissue culture. One type was a mononucleated cell with phagocytic activity, referred to as a macrophage, and the other was a non-phagocytic cell belonging to the lymphocytic series. The macrophage, presumably a non-antibody--producing cell, was thought to interact with the antigen yielding a product which in some manner stimulated the production of specific antibody in the lymphocytic cells. The role of the macrophage, and the nature of the active substance(s) that result from the interaction of antigen and macrophages, were further investigated. The results of these experiments in which tissue culture and chick embryo techniques were used are presented in this paper. Materials and MahodsLymph Node Cdls.--Young Wistar strain rats (60 gin) obtained from Blue Spruce Farms, Altamount, New York, were used as the source of lymph node cells. The animals were sacrificed by a blow on the head and the two lumbar nodes, the renal node, and an intestinal node (5) were excised. The nodes from 10 rats were teased manually in 6 ml of a Special Buffer Solution (SBS) of the following composition: NaC10.8 per cent, KC1 0.04 per cent, Na~HPO4 0.0568 per cent, KHsPO4 0.0095 per cent, MgSO4 0.0060 per cent, phenol red 0.002 per cent, and CAC12.2 H20 0.014 per cent. The suspension of teased cells was then filtered through sterile gauze to remove tissue fragments as well as clumps of cells. Mter centrifuging the cells at 200 X g for 10 minutes in the cold, the cells were washed once with SBS and then counted. Approximately 20 X 106 cells were obtained per rat; not more than 5 per cent of these cells were macrophages.Rabbit lymph node cells were obtained by teasing the mesenteric and popllteal nodes in a similar manner. A much higher yield of lymph node cells (480 X 106) per rabbit was obtained; approximately 15 per cent of the cells were macrophages.lffacrophages.--Rat macrophages were obtained by injecting 5 ml of a beef heart infusion broth fortified with 10 per cent proteose-peptone No. 3 (6) intraperitoneally into 200 to 250 gin Wistar rats obtained from Blue Spruce Farms. The rats were sacrificed by a blow on

Cells Involved in the Immune Response

“…The evidence favoring an eminent role for the macrophage in the induction of the immune response has been well documented and has accrued from both in vitro and in vivo investigations (36)(37)(38)(39)(40)(41)(42). However, the exact stage in the immune response where the macrophage acts has hitherto not been precisely stated or defined.…”

Gamma Globulin and Antibody Formation in Vitro

“…These latter studies were performed with a bacterial antigen (Shigella) and still remain to be confirmed and repeated with protein antigens. The difference in effectiveness of X-ray on tissue receiving antigen immediately as compared to tissue receiving antigen 12-24 hr later, could possibly be explained on the basis of an inhibitory effect of irradiation on a unique antigen-processing function of macrophages (29,30). If this were the case, however, it is clear that the irradiated macrophages must lose this special property over a period of 12-24 hr after irradiation.…”

Cells as Antigen Carriers and as Immunoglobulin Producers

“…The size may affect the period within the body during which an antigen may retain its configuration and may also affect the rate at which it is phagocytized. Whether such phagocytosis is itself an active step in the initiation of the immune response (37,38) it simply serves to reduce the ambient antigen concentration in the immediate surroundings of antibody-forming cells must remain open. Promoting capacity of cells does not solely depend on size of the antigen: the promoting capacity of red blood corpuscles and nucleated cells shows considerable differences.…”