Enhancement of murine bone marrow colony formation and L-transformation by Brucella antigen.

Brucella melitensis and Pseudomonas aeruginos antigens, in the form of heat-killed cells, enhanced serum stimulation of colony formation by mouse bone marrow progenitor cells. The antigens also enhanced colony formation in unstimulated medium. Prior sensitization of the C57BL mice by recent infection or by immunization six months earlier increased sensitivity of bone marrow cells to brucella antigen enhancement of colony formation. The immunized mice provided marrow cells more primed to colony formation than infected mice. Evidence is presented that antigen also leads to greater recovery of live brucellae from marrow cells of infected animals. This may be due both to stimulation of the marrow cells and to direct stimulation of the brucellae in those cells. L-forms of brucellae from stimulated marrow cell cultures were isolated. Some degree of stabilization of the L-form was accomplished through incorporation into the marrow culture medium of MgSO4, sucrose and penicillin G. The place in the infection process of L-forms is discussed in terms of the hypothesis that the L-form is a product of immune reactions that involve a step-wise degradation of the brucella cell wall during which the various cell-mediated immune reactions become operative and are themselves the reflection of a general stimulation by the brucellae of the hemopoietic and lymphopoietic systems.

Sensitization and recall of anti-Brucella immunity in Guinea pig macrophages by attenuated and virulent Brucella.

Peritoneal macrophages from guinea pigs vaccinated with strain Rev I of Brucella melitensis were only moderately activated thereby to limit, in an in vitro system, the intracellular growth of Rev I bacilli. Nevertheless, the appropriate memory cells had been primed, as demonstrated by the observation that reinfection of animals with virulent B. melitensis followed by intraperitoneal inoculation of mineral oil called forth macrophages in immunized guinea pigs which inhibited strongly the intracellular growth of brucellae. These macrophages slowed the growth of brucellae in the absence of immune serum. The intensity of the recall response was related to the challenge route and to the virulence of the challenge strain. After equal doses of attenuated or virulent brucellae, resistance was highest in macrophages recalled by the virulent strain. An important basis for the attenuation of the Rev I strain may lie in its initially low degree of macrophage activation during primary infection, although still retaining the capacity to prime stem cells. This property is associated with a protein found in fraction I, because 600 mug/ml in Freund's adjuvant primed guinea pigs so that challenge by strain 6015 evoked activated macrophages. This was seen microscopically as a reduced spread of infection in and amongst the macrophage population. Immune serum further reduced this spread and limited the number of viable intracellular brucellae.

Medium-dependent activity of immune serum on Brucella-infected macrophages.

Medium constituents affect the inhibitory action of anti-Brucella melitensis serum during in vitro infection of oil-induced peritoneal macrophages of the guinea pig with B. melitensis.

Serum-mediated immune cellular responses to Brucella melitensis. IV. Infection of macrophages under anaerobic conditions.

Immune mechanisms active against Brucella were studied under conditions of oxygen deficiency. B. melitensis grew in rabbit serum-Tyrode medium flooded with N(2) and CO(2) gas mixtures. Immune sera from rabbits injected with B. melitensis strain Rev I possessed growth-inhibitory activity that operated in anaerobic environments against Rev I and virulent strain 6015. When mixed with macrophages, immune sera mediated even greater inhibition of bacterial growth and slowed the spread of infection throughout the tissue culture. Although under anaerobic conditions the rate of phagocytosis was reduced, the macrophages in immune serum killed significant percentages of Brucella, suggesting that an antibacterial mechanism had been activated. Sonic extracts of macrophages prepared and tested under anaerobic conditions depressed the growth rate of strain Rev I. The extracts, however, exhibited no immediate killing capacity when tested in Tyrode solution. A factor from serum was required for depression of the growth rate.

Serum-mediated immune cellular responses to Brucella melitensis. I. Role of a macrophage-stimulating factor in promoting ingestion of Brucella by streptomycin-protected cells.

Injection of rabbits with living Brucella melitensis Rev I induced the appearance of a macrophage-stimulating-factor (MSF) in the sera of these animals. MSF was involved in ingestion of bacilli, hastening the formation of protected loci as measured by the addition of lethal amounts of dihydrostreptomycin. When sufficient time had been allowed for effective ingestion, streptomycin had little effect. This in turn allowed for multiplication of bacilli intracellularly in the presence of 5 to 250 mug of drug per ml. MSF mediated more effective ingestion by both immune and normal macrophages. Under such conditions, there was little, if any, intracellular growth restriction by macrophages from immune animals. The activity appeared within the first 5 days after injection with 10(9) organisms and was present for several months. Three weeks after injection, the activity of serum was partially heat-labile. All activity was removed by absorption with heat-killed or living Rev I cells, suggesting that a specific globulin is concerned.