Tatsuo Saito-Taki

Phagocytosis and bactericidal action of mouse peritoneal macrophages treated with leukotriene B4

The effects of exogenous leukotriene B4 (LTB4) on the resistance of mouse peritoneal macrophages against Salmonella (S.) typhimurium and Pseudomonas (P.) aeruginosa infections were studied. In vitro, LTB4 added to macrophage monolayers at final concentrations of 10(-12)-10(-8) M, enhanced their phagocytosis of S. typhimurium to 2.3 times the control level and that of P. aeruginosa to 1.8 times the control level. The intracellular killing rates were also elevated by the addition of LTB4: for S. typhimurium, 83.3% (LTB4) vs 59.1% (control) and for P. aeruginosa, 46.5% (LTB4) vs 9.2% (control). In vivo, intraperitoneally injected LTB4 (5 ng) enhanced the clearance at 24 h of intraperitoneally injected S. typhimurium from the mouse peritoneal cavity (2.38 x 10(3) +/- 0.94 x 10(3) cells [LTB4] vs 5.73 x 10(5) +/- 1.90 x 10(5) [control]) and spleen (5.00 x 10(2) +/- 0.94 x 10(2) [LTB4] vs 2.47 x 10(4) +/- 0.84 x 10(4) [control]), but this effect disappeared by 48 h. In contrast, in beige mice, an experimental model of the Chédiak-Higashi syndrome that is characterized by susceptibility to bacterial infection, there was no induction of the eliminating effect by intraperitoneal injection of LTB4. Activation of macrophages by exogenous LTB4 seemed to have contributed to such an augmented resistance of macrophages to bacterial infection. This study suggested a possible use of LTB4 in bacterial infectious diseases whereby phagocytes are able to play a key role in host defense.

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.

Toxicity of Microcystis aeruginosa K‐139 Strain

Toxicity of the cells of a newly established axenic Microcystis aeruginosa K‐139 strain to mice was studied. LD50 of the cells harvested in the mid‐log phase was 7.3 mg/kg. The organs of acute dead mice were examined histopathologically. The blood congestion and necrosis of the parenchymal cells around the central veins in the liver were observed, but other organs seemed to be normal. The liver damage was confirmed by the tests of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities in the sera of the mice after the injection with the K‐139 cells. Furthermore, the K‐139 cells were capable of inducing interleukin 1 (IL‐1) production by peritoneal macrophages in vitro.

Effect of Muramyl Dipeptide Analog on Salmonella enteritidis Infection in Beige Mice with Chediak-Higashi Syndrome

Beige mutant (bg/bg) mice with Chediak‐Higashi syndrome (CHS) were much more sensitive to virulent Salmonella enteritidis No. 11 strain than parental C57 BL/6 (+/+) or heterozygous (bg/ +) mice, and they had weaker bactericidal activity against the organisms. Muramyl dipeptide (MDP) and Nα‐(N‐acetyl‐muramyl‐L‐alanyl‐D‐isoglutamyl)‐Nε‐stearoyl‐L‐lysine [MDP‐Lys(L18)], a synthetic derivative of MDP, failed to confer any protection against the infection, but the MDPs showed some ability to stimulate the bactericidal activity in the peritoneal cavities and spleens of these mice. The bactericidal effect of MDP‐Lys(L18) was dose‐dependent, and the greatest effect was seen when it had been injected 24 hr before the infection. Multiple injections of MDP were much more beneficial than a single injection.

Delayed Hypersensitivity in Murine Salmonellosis: Specificity of Footpad Reaction in Mice Infected with Rough Mutants of Salmonella typhimurium

Delayed type (footpad) hypersensitivity (DTH) in BALB/c mice immunized with rough mutant strains of Salmonella typhimurium LT2 was examined. Injection of live organisms of an Rb mutant TV148 strain induced DTH in mice, while injection of the heat‐killed organisms did not. The mice immunized with live organisms of the Ra, Rb, Rc, Rd, and Re mutant strains showed positive footpad reactions to the heat‐killed cell antigen of LT2 (wild type) strain. The mice immunized with the Rb mutant strain also showed positive footpad swellings in response to heat‐killed cell antigens of S. paratyphi A, S. paratyphi B, S. typhi, S. enteritidis, and S. cholerae‐suis. Furthermore, positive reactions to antigens of Escherichia coli and Shigella flexneri were seen in the TV148‐immunized mice, but the mice did not respond to heat‐killed organisms of Pseudomonas aeruginosa or Staphylococcus aureus. The cross‐reactive footpad reaction to E. coli could be transferred adoptively with T cells prepared from the spleens of TV148‐immunized mice into syngeneic recipients. These results suggest that the cross‐reactive DTH antigen(s) is widely distributed among related organisms such as Shigella and Escherichia.

Comparison of Murine B Cell Clonal Expansions by Synthetic Lipid A and Muramyl Dipeptide Analogs

Direct stimulations of murine B lymphocytes with synthetic lipid A analogs and synthetic muramyl dipeptide (MDP) derivatives were studied using a limiting dilution assay system. Synthetic lipid A analogs, GLA‐27 and GLA‐40, when conjugated with bovine serum albumin (BSA) had the ability to induce B cell clonal expansion of a single B cell from the spleen or bone‐marrow. Their activities were almost the same as those of naturally obtained lipid A, but were lower than that of bacterial lipopolysaccharide (LPS). Addition of dextran sulfate (DXS) enhanced the effect of lipid A analogs. In contrast, synthetic MDP and its derivatives, although they had many biological and immunological activities in experimental animals, could not stimulate a single B cell to induce clonal expansion regardless of the presence or absence of DXS. These results suggested that lipid A analogs can directly cause the proliferation of B cells, but MDPs can not.

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 -

IgG2b-dependent down regulation of the LPS-induced PFC-response and its blockade by Fcγ2bR protein

Fc gamma receptor (Fc gamma R)-dependent immunoregulation by murine heat-aggregated (HAgg) IgG subclasses on the bacterial lipopolysaccharide (LPS)-induced plaque forming cell (PFC) response to trinitrophenylated sheep red blood cell (TNP-SRBC) antigen and the competitive effect by Fc gamma 2bR-protein on the down regulation by HAgg-IgG2b were studied in murine T-cell-deprived spleen cell cultures. HAgg-IgG1 and HAgg-IgG3 enhanced the PFC response, but HAgg-IgG2b strongly suppressed the LPS-induced PFC response. HAgg-IgG1 could not compete with the suppressive effect of HAgg-IgG2b. The HAgg-IgG2b seemed to act on both macrophages (M phi) and B-cells, because the cell cultures that had been reconstituted with HAgg-IgG2b-pretreated M phi and untreated B-cells and vice versa showed poor PFC responses. The suppression induced by HAgg-IgG2b on the LPS-induced PFC response in the T-cell-deprived cultures was abolished by the addition of phospholipase C (PLC)-treated Fc gamma 2bR protein at the early stage of the culture. The mechanisms by which HAgg-IgG2b suppress the LPS-induced PFC response and PLC-treated Fc gamma 2bR protein restores this response were discussed.

The adjuvant effect of a muramyl dipeptide (MDP) analog on temperature-sensitive Salmonella mutant vaccine

Adjuvant effect of a synthetic muramyl dipeptide analog, MDP-Lys (L18), MurNAc-L-Ala-D-glu[Lys(CO-(CH2)16-CH3)-OH]NH2 on a live Salmonella enteritidis vaccine, a temperature-sensitive mutant Ts-O strain that was obtained from the virulent S. enteritidis No.11 strain and could not grow at 37 degrees C, but could multiply at 25 degrees C as fast as the parent strain, was examined. Although the Ts-O organisms were avirulent and could hardly multiply in vivo when the organisms were injected into C57BL/6 mice and its mutant beige strain, which has a malfunction of phagocytic cells, injection of these mice with Ts-O endowed them with some protective immunity against infection by the virulent No.11 strain. When MDP-Lys(L18) was injected with Ts-O vaccine, the protection and the bactericidal capacity in the peritoneal cavities and spleens of these mice were augmented. MDP-Lys(L18) was still effective when it was injected at 48 h before or after the inoculation of Ts-O vaccine. Its effect was also observed against infection by the virulent S. cholerae-suis Hokkaido strain, a strain that does not share common O-antigenic determinants with the S. enteritidis No.11 or Ts-O strain. In addition, the mice that were inoculated simultaneously with Ts-O organisms and MDP-Lys(L18) were examined 10 days later for their footpad delayed type hypersensitivity reactions against Ts-O antigen. Mice inoculated with MDP-Lys(L18) and Ts-O showed augmented footpad swelling in comparison with the controls. These findings indicate that MDP-Lys(L18) is capable of augmenting the cellular immunity by live vaccine.

In vitro proliferative activity of spleen cells in mice infected with attenuated Salmonella typhimurium.

Proliferative activity of cultured spleen cells obtained from mice 1 to 5 weeks after infection with attenuated strains of Salmonella typhimurium was examined in the presence or absence of lipopolysaccharide (LPS) or concanavalin A (Con A). Spontaneous uptake of 3H‐thymidine (TdR) by cells taken from infected mice at the 2nd and 3rd weeks was obviously lower than that by cells from uninfected, control mice. Cells from infected mice at the 4th and 5th weeks also showed a lower proliferative response to LPS than that of the controls. However, the responses of the cells to Con A remained virtually unchanged during the entire period. Furthermore, the reduction of spontaneous 3H‐TdR uptake by the cells could be achieved also by the injection of heat‐killed instead of living organisms.