Katsuo Kumagai

Pretreatment of plastic petri dishes with fetal calf serum. A simple method for macrophage isolation.

We have developed a simple method which can recover the highly purified macrophages or monocytes in suspension from mouse peritoneal exudate cells and human perpheral blood mononuclear cells. Plastic Petri dishes coated overnight with heat-inactivated fetal calf serum (FCS) selectively bind macrophages and monocytes. The adherent macrophages and monocytes are easily removed by incubation in phosphate-buffered saline containing 0.2% ethylenediamine tetraacetate (EDTA) and 5% FCS, and recovered as a cell suspension with greater than 95% purity. A small number of isolated cells can restore the mitogenic response to phytohemagglutinin (PHA-P) of macrophages-depleted lymphocytes and can lyse 51Cr-labeled target cells in an antibody-dependent cell-mediated cytotoxicity system. Thus, the method should be valuable for studies of various functions of macrophages and monocytes from different immune tissues of man and animals.

Neutrophil Proteinase 3-Mediated Induction of Bioactive IL-18 Secretion by Human Oral Epithelial Cells

IL-18, a potent IFN-γ-inducing cytokine, is expressed by various nonimmune cells as well as macrophages, suggesting that it has important physiological and immunological roles. The present study focused on the mechanism of active IL-18 induction from human oral epithelial cells. The epithelial cells and the cell lines constitutively express IL-18 mRNA and the 24-kDa precursor form of IL-18. Bioactive IL-18 exhibiting IFN-γ-inducing activity was detected in the supernatant of the cells on costimulation with neutrophil proteinase 3 (PR3) and LPS for 24 h after IFN-γ-priming for 3 days. An active 18-kDa form of IL-18 was detected in lysate and supernatant of the cells only after the above treatment and the induction was inhibited by cycloheximide and by serine proteinase inhibitors. After the treatment, lactate dehydrogenase activity was not detected in the cell culture supernatant, and PR3 was detected only in the membrane and not in cytoplasm fractions of the cells. Caspase-1 was not detected in the cells even after the treatment and the IL-18 induction was not inhibited by a caspase-1 inhibitor. These results suggest that the PR3-mediated induction of bioactive IL-18 secretion from oral epithelial cells in combination with LPS after IFN-γ-priming occurred via a caspase-1-independent pathway, and provide new insight into the possible involvement of a neutrophil proteinase in the induction of bioactive IL-18 in oral inflammation such as periodontitis.

Immunologic aspects of the nonobese diabetic (nod) mouse. Abnormalities of cellular immunity.

Experiments were performed on 12-wk-old nonobese diabetic (NOD) mice to investigate the immunologic background of the condition, using ICR mice as controls. The results indicate the following: (1) absolute decreases in number of T lymphocytes, (2) depression of natural killer activity, (3) normal responsiveness in delayed type hypersensitivity and functional depression of killer T cells against allogeneic tumors, (4) diminished resistance to herpes virus infection, and (5) enhanced production of polyclonal antibodies to T cell-dependent antigens. These features are similar to those noted in other autoimmune diseases of man and in their experimental models in laboratory animals. Elucidation of the pathogenetic mechanism of autoimmune diabetes mellitus in NOD mice, therefore, may contribute to the diagnosis, treatment, and prevention of a wide variety of autoimmune diseases.

A simple method to eliminate the antigenicity of surface class I MHC molecules from the membrane of viable cells by acid treatment at pH 3

We describe here a simple, reproducible method which specifically eliminates the antigenicity of surface class I major histocompatibility complex (MHC) molecules by acid treatment at pH 3 from the membrane of viable cells. When fresh mononuclear cells (MNC) or established cultured cell lines were treated at 4 degrees C for 2 min with citric acid buffer at pH 3 containing 1% bovine serum albumin, the antigenicity of class I MHC molecules, but not those of class II MHC and the other non-MHC antigens, was eliminated from the surface membrane without significant cell death. This method was effective for both human and murine cells with various origins. Monoclonal and polyclonal antibodies were used to identify the expression of surface antigens in conjunction with immunofluorescence tests. The eliminated antigenicity of human class I MHC antigens (i.e., HLA-A,B,C) on MNC regenerated when cells were incubated in the medium at 37 degrees C for 10 h. The pretreatment of cells with emetine (10(-4) M), a protein synthesis inhibitor, was found to be effective in inhibiting this regeneration. The acid treatment method might be useful for future studies on the functional characterization of surface class I MHC antigens.

Aminoalkylbisphosphonates, potent inhibitors of bone resorption, induce a prolonged stimulation of histamine synthesis and increase macrophages, granulocytes, and osteoclasts in vivo

SummaryAminoalkyl derivatives of bisphosphonates are potent inhibitors of bone resorption. A single I.P. injection of 4-amino-1-hydroxybutylidene-1,1-bis-phosphonate (AHBuBP) induced a prolonged enhancement of histidine decarboxylase (HDC) activity in the bone marrow, spleen, lung, and liver of mice and resulted in an increase in histamine. The induction of HDC by the agent was dose dependent (16–80 μmol/kg) and peaked 3–4 days after its injection (40 μmol/kg). Repeated S.C. injections of smaller doses of AHBuBP (0.32 or 1.6 μmol/kg/day) for 4 days also enhanced HDC activity. However, the minimum dose capable of inhibiting bone resorption (0.064 μmol/kg/day) was lower than that inducing HDC. Unexpectedly, AHBuBP, at the doses inducing HDC, increased macrophages, granulocytes, and even osteoclasts. The size of osteoclasts was also enlarged by the agent. Another aminobisphosphonate, 3-amino-1-hydroxypropylidene-1,1-bisphosphonate, but none of nonamino derivatives, also exhibited essentially the same effects as those of AHBuBP. These results indicate that in spite of increase in osteoclasts and their enlargement, bone resorption is still inhibited by amino bisphosphonates. As granulocyte and granulocyte-macrophage colony-stimulating factors and interleukin-3 induce HDC in hematopoietic organs, and histamine has a hematopoietic activity, the HDC induction by aminobisphosphonates may be relevant to the proliferation of progenitor cells of macrophages, granulocytes, and osteoclasts.

Contrast between effects of aminobisphosphonates and non-aminobisphosphonates on collagen-induced arthritis in mice.

1 Bisphosphonates (BPs) are inhibitors of bone resorption, and many derivatives have been developed for the treatment of enhanced bone resorption. Aminobisphosphonates (aminoBPs) are particularly potent in this respect. We have shown previously that aminoBPs, such as 4‐amino‐1‐hydroxybutylidene‐1,1‐bisphosphonic acid (AHBuBP), induce histidine decarboxylase, the enzyme forming histamine, and increase macrophages, granulocytes and osteoclast numbers. Non‐aminoBPs do not show this activity. 2 In the present study, an additional aminoBP, cycloheptyl‐aminomethylene bisphosphonate (CHAMBP), was shown to have similar properties to AHBuBP suggesting that these actions are common among aminoBPs. 3 In experiments carried out to determine if aminoBPs affect immune responses, we found that CHAMBP and AHBuBP each exacerbated the arthritis induced in mice by the co‐injection of type II collagen and an adjuvant, a model for rheumatoid arthritis. In contrast, dichloromethylene bisphosphonate (C12MBP), a typical non‐aminoBP, did suppress the arthritis. 4 On the basis of these results, and those obtained previously, we propose that the exacerbating effects of CHAMBP and AHBuBP may be related to their ability to stimulate the synthesis of histamine and to increase macrophages and granulocytes. Conversely, we propose that the suppressive effect of C12MBP on arthritis is related to its cytotoxic action on macrophages or granulocytes.

Effects of macrophage depletion on the induction of histidine decarboxylase by lipopolysaccharide, interleukin 1 and tumour necrosis factor

1 Our previous work has shown that injection into mice of lipopolysaccharide (LPS) and the cytokines interleukin 1 (IL‐1) and tumour necrosis factor (TNF) induces histidine decarboxylase (HDC), the enzyme forming histamine, in various tissues such as liver, lung, spleen and bone marrow, but not in the blood. The induction of HDC also occurs in nude mice and mast cell‐deficient mice. On the other hand, haematopoietic cytokines such as IL‐3, granulocyte colony‐stimulating factor (G‐CSF) and granulocyte – macrophage CSF (GM‐CSF) only induce HDC in the haematopoietic organs, i.e. bone marrow and spleen. In the present study, the effect of macrophage depletion on the induction of HDC was examined. 2 On day 1 after a single intravenous injection of a macrophage depletor (liposomes encapsulating dichloromethylene diphosphonate, which is toxic when ingested into macrophages), macrophages were almost completely depleted in the liver and reduced by about 50% in the spleen and bone marrow, but not significantly affected in the lung. On day 3, the degrees of the depletion were similar to those of day 1. In the spleen, macrophages were depleted in the red pulp, and there was a structural destruction. 3 In macrophage‐depleted mice, the induction of HDC by LPS, IL‐1α or TNF‐α was not impaired in the liver, and was potentiated in the lung and bone marrow. The induction of HDC was decreased only in the spleen at day 3. 4 HDC was not induced by LPS in the spleen of the adult rat, which is correspondingly inactive in haematopoiesis. 5 These results indicate that the major cells in which HDC activity is induced in response to LPS, IL‐1 and TNF are not circulating granulocytes, circulating monocytes, T cells derived from thymus, mast cells or phagocytic macrophages. Based on these results, we discuss the possibility that the major cells in which HDC was induced in non‐haematopoietic and haematopoietic organs were endothelial cells and haematopoietic precursor cells respectively.

The potent activity of fresh water fish kidney cells in cell-killing I. Characterization and species-distribution of cytotoxicity

Abstract Kidney cells of fresh water fishes, Cyprinus carpio (carp), Carassius cuvieri (crucian carp), Ctenopharyngodon idella (grass carp), Misgrunus anguillicandatus (orienta) weather fish) and Channa argus (northern snakehead) were markedly cytotoxic against certain established mammalian cell lines when incubated at 25°C in the absence of specific antibody. Cytotoxicity occurred immediately after incubation with targets, increased exponentially and was accomplished within 6 hrs. The cytotoxicity was mediated by the intact cells but not by either their extracts or culture supernatants. The cytotoxic activity was directed against established cell lines but not normal resting cells. Under similar conditions, kidney cells of salt water fishes showed no cytotoxicity against any targets tested. Leukocytes obtained from various lymphoid organs of other normal higher vertebrates also showed no such potent cytotoxicity. These results indicate that a population(s) of kidney cells in fresh water fishes of stenohaline type carrys the killing activity with a spectrum of target cells similar to that of activated macrophages or natural killer cells in mammals.

Variation in CD4+ T and CD8+ T lymphocyte subpopulations in bovine mammary gland secretions during lactating and non-lactating periods.

Mammary gland secretions (MGS) of dairy cows at different stages of lactation were studied by immunofluorescence cytometry for T lymphocyte subpopulations using monoclonal antibodies. During early and late lactation, the mean ratio of CD4+/CD8+ T lymphocytes in the MGS was 0.5 and 0.8, respectively. A large proportion of the CD8+ cells coexpressed the activation molecule, ACT2. These results indicate that CD8+ ACT2+ cells constituted the major phenotype in the T lymphocytes throughout lactation. In the mammary gland of cows in which drying off was induced, however, the proportion of CD8+ ACT2+ cells decreased, resulting in the increase of the CD4+/ CD8+ ratio in the MGS. At the late non-lactation stage, the ratio reached a maximal level of 2.5-4.0, which was similar to or higher than that found in the peripheral blood. This selective increase of CD4+/CD8+ cell ratio correlated with an increase in the concentrations of total cells in the MGS. This high CD4+/CD8+ cell ratio during the drying off stage rapidly decreased just before parturition, correlating with the decrease in concentrations of total cells in the MGS, reaching the lowest level at early lactation. The cells isolated at the non-lactation stage produced the cytokines IL-2 and IL-4 at a level much higher than those of cells isolated at lactation stages, and the increases were correlated with the CD4+ T lymphocyte proportions.

GM-CSF and G-CSF stimulate the synthesis of histamine and putrescine in the hematopoietic organs in vivo

Histamine and putrescine (a precursor of polyamines) are formed by histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), respectively. Within a few hours after injection of a lipopolysaccharide (LPS) into mice, HDC is induced in the liver, spleen, lung and bone marrow, and ODC is induced in the liver, spleen and bone marrow. Since LPS is known to stimulate the production of various cytokines, the abilities of various cytokines to induce HDC and ODC in the tissues of mice were examined. IL-2, IL-6, IL-8, IFN gamma and M-CSF were ineffective. IL-1 alpha, IL-1 beta, TNF alpha and TNF beta induced HDC and ODC, as does LPS. On the other hand, GM-CSF and G-CSF induced HDC and ODC only in the spleen and bone marrow within a few hours after their injection. These results suggest that, in addition to their roles in inflammation or immune responses, HDC and ODC are also involved in an early stage of hematopoiesis.