Tatsuichiro Hashimoto

The mechanism of macrophage activation induced by Ca2+ ionophore.

The mechanism of macrophage activation by Ca2+ ionophore was studied. Peritoneal exudate macrophages from normal guinea pigs exposed continuously to or pulse treated for 1 hr with the ionophore, A23187, were activated, manifesting increased glucose consumption and inhibition of migration. Highly purified macrophages were also activated as effectively as crude macrophage preparations, and the culture supernatant of spleen lymphocytes treated with A23187 lacked a macrophage activating effect, showing that the macrophage activation resulted from the direct effect of A23187 on macrophages, not via lymphokines produced by lymphocytes. The macrophage activation by A23187 was suppressed in the presence of EGTA, but the suppressive effect was overcome by the addition of Ca2+, but not of Mg2+. A dilution experiment with Ca2+ and Mg2+ during the pulse treatment of cells with A23187 revealed that the activating effect of A23187 was more dependent on Ca2+ content than Mg2+. In addition, the Ca2+ antagonist, nicardipine, was found to suppress the activating effect of A23187. The Ca2+ uptake into macrophages was increased by treatment with A23187. These results indicate that Ca2+ influx into cells is primarily important in the macrophage activating effect of A23187. Trifluoperazine (TFP: a specific inhibitor of calmodulin that is an intracellular Ca2+ receptor protein) was found to inhibit the activating effect of A23187. Further, the cyclic nucleotides, dibutyryl-cAMP and -cGMP, did not activate macrophages. Therefore, macrophage activation was presumed not to be directly mediated by cyclic nucleotides. All these findings show that macrophage activation with the ionophore proceeds by the following scheme: Ca2+ influx leads to activation of Ca2+ receptor protein, calmodulin leads to activation of calmodulin-regulated enzymes leads to metabolic changes, activation. TFP was found to suppress the macrophage activation with highly purified guinea pig macrophage activation factor/macrophage migration inhibitory factor (MIF/MAF) or lipopolysaccharide (LPS), suggesting that calmodulin also played an important role in macrophage activation with MIF/MAF or LPS.

Production of an antibody against guinea pig MIF: III. Biological activity of MIF recovered from immunoadsorbent column chromatography

Abstract In this study, MIF-active substance recovered from the immunoadsorbent column of an anti-MIF antibody was examined for possession of activities of other lymphokines. As a result, this MIF-active substance showed no activities of macrophage chemotactic factor (MCF), neutrophil chemotactic factor (NCF), skin-reactive factor (SRF), and vascular permeability factor (VPF). On the other hand, macrophage activation factor (MAF) activity assessed by “stimulation of glucose consumption” and “stimulation of [ 3 H]glucosamine incorporation” was associated with the recovered MIF. These results indicated that MIF could be separated from four other lymphokines, MCF, NCF, SRF, and VPF, which were thought to induce the delayed-type skin reaction. In addition, our results strongly support the possibility that MIF is the same molecule as MAF.

Production of an antibody against guinea pig MIF: I. Specificity of the anti-MIF antibody

Abstract An antibody was produced in rabbits against partially purified MIF which was released from the specifically stimulated lymphocytes of tuberculin-hypersensitive guinea pigs. The MIF used as an antigen was fractionated by polyacrylamide gel electrophoresis. The antibody thus prepared was then examined for its specificity for several lymphokines by affinity column chromatography. It was observed that the antibody column adsorbed MIF, but not the other three lymphokines, MCF, NCF, and SRF, indicating a keen specificity of the antibody against MIF.

Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells

Experiments were conducted to purify the differentiation-inducing factor (D-factor), which induces differentiation of mouse myeloid leukemic cell line, Ml, into macrophage-like cells, in a conditioned medium of guinea pig peritoneal macrophages stimulated with lipopolysaccharide. On gel filtration under high performance liquid column chromatography (HPLC), D-factor eluted at the position of 45-15 KD. By the subsequent separation on DEAE HPLC the D-factor activity disappeared. However, in the presence of recombinant human IL 1 alpha the D-factor activity appeared at a position where tumor necrosis factor (TNF) eluted. Even after fractionation on hydroxyapatite HPLC the IL 1-dependent D-factor was co-chromatographed with TNF. Recombinant human TNF as well as the partially purified guinea pig TNF induced differentiation of Ml cells in conjunction with either the partially purified guinea pig IL 1 or recombinant human IL 1 alpha, although these factors by themselves did not induce differentiation. These findings suggest that a part of D-factor activity in the conditioned medium resulted from the cooperative effects between TNF and IL 1.

Differences between macrophage migration inhibitions by lymphokines and muramyl dipeptide (MDP) or lipopolysaccharide (LPS): Migration enhancement by lymphokines

Abstract The mode of macrophage migration inhibition by lymphokines or a purified macrophage migration inhibitory factor (MIF) was found to differ from that by muramyl dipeptide (MDP) or lipopolysaccharide (LPS). First, a number of spreading macrophages were observed on the border of an aggregated mass of macrophages, the migration of which was inhibited by lymphokines or MIF, whereas few macrophages were spread in the case of MDP or LPS. Second, macrophage migration enhancement occurred at Day 3 in the case of lymphokines or MIF, whereas neither MDP nor LPS caused such an enhancement. These results suggest that macrophage activation by MIF differs from that by MDP or LPS. Also, the enhancement of macrophage migration by MIF at Day 3 is a hitherto unreported property of MIF.

The mechanism of cell surface changes of guinea pig macrophages activated with purified migration inhibitory factor/macrophage activation factor

Abstract Guinea pig MIF (MIF/MAF), which was purified by immunoadsorbent column chromatography using an antibody against MIF/MAF, was observed to induce characteristic cell surface changes in macrophages under scanning electron microscopy (SEM). MIF/MAF induced enlarged petal-like ruffles in both rounded and spreading macrophages. The changes were observed as early as 2 hr after stimulation with MIF/MAF and continued for 24 hr. These morphological changes appeared to be a good indicator of macrophage activation and migration inhibition in the early phase. The mechanism of the characteristic ruffle formation was studied using metabolic inhibitors and reagents known to affect microfilaments and microtubules. When macrophages were treated with MIF/MAF in the presence of mitomycin C, actinomycin D, or puromycin, formation of the petal-like ruffles was not affected. However, vinblastine and cytochalasin B inhibited the induction of these ruffles. These results indicate that microtubule and microfilament assembly, but not synthesis of DNA, RNA, and protein, are required for the formation of the petal-like ruffles. In addition, treatment with a Ca 2+ ionophore induced the same petal-like ruffles in macrophages, while treatment with dibutyryl-cyclic AMP or-cyclic GMP did not. These findings suggest that Ca 2+ plays an important role in macrophage activation by MIF/MAF, especially in the early phase.

Production of an antibody against guinea pig MIF. II. Analysis of the antibody-reacting material using radiolabeled lymphokines.

Abstract An antibody rasied in a rabbit against guinea pig macrophage migration inhibitory factor (MIF) was proved to bind specifically to MIF, but not to three other lymphokines, macrophage chemotactic factor (MCF), neutrophil chemotactic factor (NCF), and skin reactive factor (SRF). In this study, the material which reacted with the antibody was analyzed using radiolabeled lymphokines. First, the postlabeled lymphokine or control preparation with tritiated N-succinimidyl propionate was applied onto the immunoadsorbent columns. The materials recovered from the columns were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By this method, the antibody appeared to react with a homogeneous protein in terms of its molecular weight (MW), 35,000. Second, the double-labeled lymphocyte culture supernatants were used, in which supernatants of stimulated and unstimulated cultures were labeled with either [ 3 H]- or [ 14 C]leucine, respectively. By this method, also a single polypeptide with a MW of 35,000 and an isoelectric point of p I 5.0–5.5, was detected. These results indicate that the protein with a MW of 35,000 and an isoelectric point of p I 5.0–5.5 may be MIF.

Different Mechanisms of Macrophage Activation with Guinea Pig Macrophage Activation Factor, Lipopolysaccharide and Muramyl Dipeptide

The mechanism of macrophage activation was studied using three activating substances, guinea pig macrophage activation factor (MAF), lipopolysaccharide (LPS) and muramyl dipeptide (MDP). Guinea pig peritoneal exudate macrophages were activated to exhibit the accelerated glucose consumption in response to these activating substances. Calmodulin-specific inhibitors, trifluoperazine and No. 233, inhibited macrophage activation with MAF and LPS, while these inhibitors did not affect the activation with MDP. Ca2+ uptake into macrophages was enhanced in MAF-treated macrophages, but LPS and MDP did not affect the Ca2+ uptake. Methylamine and ethylamine, inhibitors of transglutaminase-dependent protein internalization into cells and/or of lysosomal enzymes, effectively inhibited the activating effect of LPS, but not those of MAF and MDP. These results suggest that Ca2+ and calmodulin play a role in macrophage activation with MAF, and neither transglutaminase-dependent internalization nor lysosomal enzymes participate in the activation process. In case of LPS, internalization into cells would be necessary for its activating effect. The processing of the contrary, since the activating effect of MDP was not affected by any of these inhibitors, the mechanism of activation with MDP remains obscure. Thus, the mechanisms of macrophage activation with MAF, LPS and MDP appear to be different from each other.

Role of lymphokines in regulation of macrophage differentiation

The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages.

Different Effect of (L)-Fucose Binding Lectin on Macrophage Migration Inhibition Caused by Guinea Pig Migration Inhibitory Factor and Synthetic Muramyl Dipeptide

(L)-Fucose binding lectin of Ulex europeus (UEA-I) was applied for the study of receptors for macrophage migration inhibitory factor (MIF) and synthetic muramyl dipeptide (MDP). The lectin was found to reduce the responsiveness of macrophages towards MIF when the cells were pretreated with the lectin. The suppressive effect of the lectin on MIF was reversed by (L)-fucose, but not by (D)-galactose, indicating that the effect of lectin is a sugar-specific phenomenon. By immunofluorescence technique the lectin appeared to be on the cell surface of the macrophages. These results suggest that the receptor for MIF and UEA-I lectin was identical or at least partially common. The lectin, however, did not prevent the responsiveness of cells toward MDP, which is known to inhibit macrophage migration just as MIF. These findings suggest that the mechanisms of the inhibitory effect on macrophage migration caused by the two substances (MIF and MDP) may be different, at least at the level of the receptors.