Kaoru Onoue

Inhibition of macrophage migration by synthetic muramyl dipeptide.

Abstract N-acetylmuramyl-L-alanyl-D-isoglutamine, a synthetic compound which is known to have a minimal effective structure for an adjuvant activity of cell wall peptidoglycans, was found to inhibit the migration of normal macrophages. It was shown that the inhibition was neither due to cytotoxic or agglutinating effect of the muramyl dipeptide on macrophages nor due to lymphokine production uopn stimulation of lymphocytes by the muramyl dipeptide.

Functional activation of immune lymphocytes by antigenic stimulation in cell mediated immunity: I. Requirement for macrophages in antigen-induced MIF production by guinea pig immune lymphocytes in vitro

Abstract The role of macrophages in the process of antigen-induced production of mediators in cellular immune response was studied, using the antigen-induced production of migration inhibitory factor (MIF) as a measure of the activation of immune lymphocytes. The production of MIF by guinea pig immune lymph node cells in response to the stimulation with PPD was abolished when the lymph node cells were depleted of adherent cell population by passing the cells through a Tetron fiber column and incubating the effluent cells in plastic dishes. These purified immune lymphocytes did not respond to particle-bound PPD, either. However, the response was obviously restored by the addition of a small number of the purified peritoneal adherent cells (macrophages) which had been pulse-treated with PPD. The PPD-pulsed macrophages produced no MIF by themselves. Thus, the results clearly indicated the requirement for macrophages in the process of antigen-induced MIF production by immune lymphocytes. Destruction of PPD-pulsed macrophages by freezing and thawing or by homogenization abrogated their ability to stimulate immune lymphocytes. Attempts to restore the response of the purified immune lymphocytes to PPD by adding 2-mercaptoethanol or the culture supernatant of macrophages to the medium have so far been unsuccessful.

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. II. Analysis of the macrophage-replacing activity of the LPS-stimulated macrophage culture supernatant in the antigenic activation of purified immune lymphocytes.

Abstract A requirement for the cooperation of macrophages (adherent cell population) in the process of the antigenic activation of immune lymphocytes for the production of migration inhibitory factor (MIF) has been demonstrated previously. It was found, in the present study, that the culture supernatant of peritoneal macrophages, which had been pulse-stimulated with a bacterial lipopolysaccharide (LPS), could be substituted for live macrophages. Fractionation of the supernatant by gel filtration revealed its activity in the fraction of 15,000–100,000 MW and the activity was completely abolished by heat treatment at 85°C for 30 min. These results distinguished the nature of the active component from that of LPS which was found to be present in a trace amount in the supernatant and strongly suggested the presence of a factor(s) derived from macrophages which effects, in some way, the process of the antigenic activation of immune lymphocytes. Our experiment showed that the cooperating function of macrophages was inhibited by the treatment of macrophages with vinblastine. It may be that vinblastine affects the release of an active factor(s) from macrophages although other possibilities remain that the inhibition resulted from its effects on other functioning processes of macrophages.

Superoxide Anion‐Generating Activities of Macrophages as Studied by Using Cytochalasin E and Lectins as Synergistic Stimulants for Superoxide Release

Treatment of macrophages with cytochalasin E in combination with a lectin was found to stimulate the generation of superoxide anions (O2‐) very efficiently. The macrophages stimulated with concanavalin A, phytohemagglutinin or wheat germ agglutinin released superoxide, but cells pretreated with cytochalasin E released much greater amounts of superoxide, without notable lag time, upon stimulation with the lectin. Wheat germ agglutinin was found to be the most efficient stimulant among the lectins tested. Superoxide generation in guinea pig macrophages was shown to be dependent largely on cytoplasmic glucose metabolism and to some extent on mitochondrial respiration, since the superoxide release was largely but not totally inhibited by 2‐deoxyglucose and to a lesser extent by antimycin A or KCN. The method presented is sensitive and allows rapid assay of the superoxide‐generating activity with only 1–5 × 105 macrophages for a single determination. In application of this technique, elevation of the superoxide‐generating activity was shown with macrophages elicited by chemical inflammation or those obtained from mice after treatment with tubercle bacilli.

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity: III. A soluble factor released from LPS-stimulated peritoneal adherent cells effective in antigenic activation of sensitized lymphocytes☆

Antigen-induced production of migration inhibitory factor (MIF) by sensitized lymphocytes requires macrophages to effectively stimulate lymphocytes with soluble antigen in vitro. The present study showed that macrophage-depleted lymphocytes of sensitized guinea pigs could be activated with antigens when the culture supernatant of peritoneal adherent cells pulse-stimulated with a macromolecular fraction of bacterial lipopolysaccharide (LPS) was added to the lymphocyte culture. The apparent macrophage-replacing activity was found in the fraction which emerged slightly ahead of serum albumin upon gel filtration of the culture supernatant, and the activity was shown to be destroyed by heating at 65 °C for 30 min or by trypsin digestion. These results appeared to show that the activity was due to a protein component, most probably released from macrophages. Two-step culture experiments revealed that the soluble factor should be present in the early stage of the culture to activate the macrophage-depleted immune lymphocytes with antigen, as well as in the later stage when the presence of antigen in the medium is no longer required. Furthermore, the factor was shown to act in the activation of a T-cell-enriched fraction of immune lymphocytes. The factor appeared to be playing some essential role in making an antigenic stimulus effective for the activation of immune lymphocytes.

Induction of Delayed Type Hypersensitivity-Like Skin Reaction by Peptidoglycans of Bacterial Cell Walls

Water‐soluble glycopeptides isolated from Lactobacillus plantarum and Staphylococcus epidermidis cell walls elicited a delayed type hypersensitivity (DTH)‐like skin reaction in rats previously immunized with Mycobacterium tuberculosis cell walls, but not in unimmunized rats. Histological examination of the skin reaction sites in immunized animals revealed a close similarity of this skin reaction to a typical DTH reaction with respect to the time course of development and the types of cells that infiltrated into the skin reaction sites, which were characterized by a predominant infiltration of mononuclear cells at 48 hr. This DTH‐like reaction was also demonstrated by immunizing the rats with the cell wall peptidoglycans of L. plantarum or S. epidermidis and skin testing them with homologous as well as heterologous peptidoglycans. The DTH‐like reaction appeared to be caused by peptidoglycans that exist in common in the cell walls of phylogenetically distant bacterial species. Furthermore, it was also suggested that the putative antigenic determinants) might include both the glycan chain and part of the peptide moieties of the cell wall peptidoglycan rather than either of the single moieties.

A Sensitive Solid Phase Radioimmunoassay for Secretory IgA

A sensitive solid phase radioimmunoassay method was established for the specific quantitative determination of secretory IgA (sIgA) by taking advantage of the dual antigenicities of sIgA, one specific for α‐chain and the other for secretory component (SC). The sIgA and IgA in the sample were first bound by anti‐IgA antibodies coated on the polystyrene tube, then the amount of bound sIgA was quantified by the use of 125I‐labeled anti‐SC antibodies. This method is quite sensitive and allows us to distinguish sIgA from IgA and free SC which usually coexist in exocrine secretions. Linear relationship was observed between the bound radioactivity of radioiodinated anti‐SC and the amount of sIgA in the range of 5 to 60 ng of sIgA.