Masao J. Tanabe

Polyclonal B Cell Activation by Cell Wall Preparations of Gram‐Positive Bacteria

The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram‐positive bacteria were studied using the anti‐sheep red blood cell (SRBC) or anti‐trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS‐responder), C3H/HeJ (LPS‐non‐responder), (CBA/N × Balb/c) F1 male with an X‐linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA‐ability of synthetic peptidoglycan, muramyl dipeptide (N‐acetylmuramyl‐L‐alanyl‐D‐isoglutamine, MDP), was also examined, and a similar activity was observed in MDP.

Immunological Properties of Vibrio cholerae Lipopolysaccharides

Immunological effects of wall lipopolysaccharide (LPS) preparations obtained from Vibrio cholerae Inaba 569B, Ogawa NIH 41 and NAG 4715 strains by the hot phenol‐water procedure were examined in mice. Although these LPS lack KDO, which are basic components of the core region of most gram‐negative LPS, they still have potencies as B‐cell mitogens, adjuvants, immunosuppressants, polyclonal B‐cell activators and phagocytic stimulants for macrophages. The activities of these V. cholerae LPS on murine immune system seemed to be weaker than those of Salmonella typhimurium LT2‐LPS. Among these V. cholerae LPS, NAG 4715‐LPS showed the strongest mitogenic activity and phagocytic stimulation, while the potencies of this NAG 4715‐LPS for the induction of polyclonal B cell activation, adjuvant effects and immunosuppression did not seem to be greater to those of the other LPS.

An endotoxin induced serum factor that causes enhancement of antibody response to heterologous erythrocytes.

The sera obtained from blood of the mice, which had been intravenously injected with LPS several hours in advance, contained some active substance capable of enhancing anti‐sheep red blood cell (SRBC) antibody responses in mice. Activity of the sera was still retained after passage through a rabbit anti‐LPS antibody‐coated Sepharose 4B column, but greatly reduced by passage through a rabbit anti‐mouse thymocyte antibody‐coated Sepharose 4B column. The active substance in the sera was eluted through a Sephadex G‐200 column at the same position as the serum albumin.

Lipopolysaccharide-Induced Mediators Assisting the Proliferative Response of C3H/HeJ Thymocytes to Concanavalin A

3H‐Thymidine uptake of thymocytes from LPS‐responder Balb/c mice in the presence of a submitogenic dose (0.5 μg/ml) of con A in vitro was significantly enhanced by adding LPS (0.1 to 2.5 μg/ml), while the uptake of thymocytes from LPS‐nonresponder C3H/HeJ was not enhanced by LPS. However, “endotoxin soups,” which were prepared from the supernatants of LPS‐responder murine spleen cell cultures in the presence of LPS, clearly increased the incorporation of 3H‐thymidine into C3H/HeJ thymocytes in the presence of this small amount of con A. The soup prepared from C3H/HeJ spleen cell cultures did not show any synergistic effect with con A. Even if the major histocompatibility between soup‐producer cells and responder cells to con A was different, the soups were still effective. The active substance in the “endotoxin soups” was eluted through a Sepharose CL‐4B column, and its molecular size was estimated to be about 20,000 daltons. The activity of the soups was destroyed by heating at 70 C for 30 min or at 80 C for 10 min. Digestion with trypsin destroyed the activity of the soups, but digestion with DNase or RNase did not. The role of the active substance in the soups in synergy with con A and its relation to the synergistic effect of con A and LPS are discussed.

Effect of Anti‐Ia Serum In Vitro on Lipopolysaccharide‐Induced Proliferative Responses of Murine Bone Marrow and Spleen Cells

The in vitro proliferative response of murine bone marrow cells and spleen cells to bacterial lipopolysaccharide (LPS) and the effect of anti‐Ia serum on the response were studied. The incorporation of [3H]thymidine into cells prepared from bone marrow increased in the presence of LPS, but the addition of anti‐Ia serum to the cultures reduced the incorporation. Pretreatment of bone marrow cells with anti‐Ia serum and complement did not abolish the ability of the cells to respond to LPS, while the same pretreatment destroyed this ability in spleen cells. These results suggest that cultures of Ia‐negative bone marrow cells generate Ia‐positive cells during the culture period, and the Ia‐positive cells are responsive cells to LPS.

Protective Effect of Passively Transferred Immune Peritoneal Exudate Cells in Mice Infected with Salmonella typhimurium

Protective immunity to salmonella infection can be passively transferred into recipients with immune lymphoid cells (1 ). Transfer of either purified populations of bone marrow-derived (B ) lymphocytes or thymus-derived (T ) lymphocytes obtained from immune donors increases the protective capacity of the recipients against the infecting organisms (6 ). However, the transfer of protective activity seems not to be limited to these lymphocyte populations of the immune donors. It was previously demonstrated that cultured peritoneal macrophages collected from mice which had been injected intravenously with macrophages of mice im munized with live vaccine exerted an inhibitory action against intracellular virulent Salmonella enteritidis; this inhibitory action was attributed to the protective capacity of the recipients own macrophages (8 ). There was no precise technique for separating lymphocytes from macrophages in a peritoneal cell population at that time. Because of the advance in immunological methods, purified populations of macro -

Proliferative response of bone marrow cells to concanavalin A does not require Ia antigen-positive cells.

The in vitro proliferative response of murine bone marrow cells to concanavalin A (Con A) and the effect of anti‐Ia serum on the response were studied. The incorporation of [3H]thymidine into cells prepared from the bone marrow of C3H/He, ATL, ATH, and C57BL/6 mice increased in the presence of certain doses of Con A. The bone marrow cells of athymic nude mice were also capable of responding to Con A, but cells prepared from the spleens of such mice were not. The addition of anti‐Ia serum to the cultures of bone marrow cells did not affect the responses of these cells to Con A, though their proliferative response to bacterial lipopolysaccharide was greatly reduced in the presence of the serum. Moreover, pretreatment of the bone marrow cells with anti‐Ia serum or anti‐Thy. 1.2 serum and rabbit complement did not abolish the ability of these cells to respond to Con A. These results indicate that there are some Ia negative and Thy. 1.2 negative cell populations in the marrow capable of responding to Con A. Furthermore, the effect of anti‐Ia serum on the Con A‐induced proliferative response of the spleen cells which had been obtained from γ‐irradiated and syngeneic bone marrow cell‐reconstituted mice was examined. The ability of these cells to respond to Con A increased gradually week by week after the reconstitution. The suppressive effect of anti‐Ia serum on the response of these cells gradually became much more pronounced after the reconstitution.

Proliferative and Polyclonal Plaque Forming Cell Responses of CBA/N-Defective Spleen Cells by Staphylococcal Protein A or Killed Organisms of Staphylococcus aureus Cowan I Strain

Staphylococcal protein A (SpA) is known to bind to the Fe region of IgG of mammals (1,2,5) and to be a T cell independent polyclonal B cell mitogen for peripheral human lymphocytes (3, 6) via specific interaction with surface IgG molecules (3). Staphylococcal organisms are capable. of inducing polyclonal B cell activation in mice characterized by the spontaneous appearance of plaque forming cells (PFC) which produce antibodies specific for antigens unrelated to the organisms (6, 7). Moller and Landwall (6) reported that SpA in the cell wall of the staphylococcal organisms was responsible for the B cell activation since the SpA (0.1-1.0 mgfmlfculture) and Cowan I strain organisms containing SpA had the ability to induce the polyclonal PFC response in spleen cell cultures, but some strains which lacked SpA did not. However, we demonstrated that purified SpA (~O.l mgfmlfculture) was unable to induce polyclonal antibody production in the spleen cell cultures and SpA-deficient mutant organisms from Cowan I strain which lack SpA in their cell walls were capable of inducing polyclonal PFC responses in the cultures (7). To clarify the triggering mechanisms by SpA and the organisms, we examined the in vitro proliferative and polyclonal PFC responses of spleen cells obtained from CBAfN-defective mice. The CBAfN subline shows a number of immunologic defects, all of which are apparently related to an X-chromosome linked abnormality in the function of B lymphocytes to respond to thymus-independent antigens (11) and cell wall preparations of gram-positive organisms (9, 10). Since the CBAfNdefect is X-chromosome linked, CBAfN females mated to normal males (Balbjc in this series of experiments) produced Fl males which showed some defect in the function of B lymphocytes and female littermates which, although heterozygous for the defective X-chromosome, were phenotypically normal. The spleen cells of hybrid Fl male and female mice (18-20 wk of age) in RPMI-1640 medium (Flow Laboratories, Rockville, Md., U.S.A.) containing 10 mM HEPES, 2 mM L-glutamine, penicillin (100 Ufml) , streptomycin (100 p,gf

Dysfunction of Thymocytes of C3H/HeJ Mice in Blastogenic Response to Anti‐Thymocyte Serum In Vitro and Its Repair with Lipopolysaccharide Induced Mediator

In vitro mitogenic responses of thymocytes to rabbit anti‐mouse thymocyte serum (ATS) have been compared in several mouse strains. The response of thymocytes of C3H/HeJ mice is about one‐third of those of thymocytes of C3H/He, ATL or ATH strains. Phenol‐extracted bacterial lipopolysaccharide (LPS) does not induce mitogenic response in cultured C3H/HeJ spleen cells, but the spleen cells of all other strains used are capable of responding to LPS. The low response of C3H/HeJ thymocytes to ATS is restored by adding “endotoxin soup” prepared from spleen cell cultures of LPS‐responder mice in the presence of LPS. Neither soup prepared from C3H/HeJ spleen cell cultures without the addition of LPS nor soup prepared from cell cultures with LPS show such restoration of the response of C3H/HeJ thymocytes to ATS. The molecular size of the active factor in “endotoxin soup” was estimated on a Sepharose CL‐4B column and determined to be about 20,000 daltons. The activity of “endotoxin soup” is destroyed by heating at 70 C for 30 min or 80 C for 10 min and diminished by digestion with trypsin. The mechanisms of restoration of low response of C3H/HeJ thymocytes to ATS by “endotoxin soup” are discussed.