Kenmei Sakata

Eosinophil chemotactic factors from granuloma of Kimura's disease, with special reference to T lymphocyte derived factors

The granuloma of patients with Kimuras disease characterized by tissue and peripheral blood eosinophilia was reviewed with respect to eosinophil infiltration. An infiltrate of inflammatory cells with histiocytes and a sprinkling of eosinophils were observed in the fibrous stroma surrounding the newly formed vessels. Mast cells were rarely seen in the areas where eosinophils were grouped together. Three different eosinophil chemotactic factors (ECF) were isolated from the granulomas of Kimuras disease. They were termed as low molecular weight (LMW), intermediate molecular weight (IMW), and high molecular weight (HMW)‐ECF according to the profile on gel filtration (LMW‐ECF, about 500; IMW‐ECF, about 12,500; HMW‐ECF, 45,000–70,000). In terms of their activity when extracted from the granuloma, LMW‐ECF and HMW‐ECF seemed to be major natural mediators for the tissue eosinophilia, whereas IMW‐ECF was a minor one. In an in vitro system, it was shown that granuloma lymphoid cells produce spontaneously at least two ECF having similar properties to LMW‐ and HMW‐ECF, respectively. By analysis with monoclonal antibodies, granuloma T cells, probably OKT4‐positive cells, were shown to be responsible for the production of those two ECF. It was thus suggested that prolonged synthesis of LMW‐ and HMW‐ECF by OKT4‐positive T cells plays a crucial role in the local eosinophilia of Kimuras disease.

Production of an eosinophil chemotactic lymphokine by a monocyte-derived factor from patients with hypereosinophilia.

Peripheral OKT4‐positive T lymphocytes from patients with hypereosinophilia spontaneously and selectively produced an eosinophil chemotactic factor (ECF) with chemokinetic activity. The molecular weight of the ECF was about 45,000 to 70,000. A possible mechanism of its spontaneous production by T lymphocytes was analyzed. Culture supernatants of blood monocytes from the patients showed little or no ECF activity, but they had a potency to induce the ECF production from T lymphocytes from normal donors when the cells were stimulated by the supernatants, which suggests that a monocyte‐derived soluble factor (MDF) stimulated T lymphocytes to produce an ECF resembling this spontaneously produced ECF from the patients. MDF seemed to be a synthesized protein by the cells. Gel filtration indicated that molecular weight of MDF ranged between 70,000 and 100,000. MDF activity was stable at 56° for 30 min but labile at 80° for 30 min. MDF failed to show detectable IL‐1 and IL‐2 activity. Furthermore, supernatants of stimulated monocytes by lipopolysacchride or silica particles failed to show ECF‐producing activity, whereas they showed evident lymphocyte‐activation activity. Neither recombinant IL‐1 nor IL‐2 had ECF and ECF‐producing activity. From the present experiments, it was suggested that MDF was at least partly involved in the induction of ECF production by OKT4‐positive T lymphocytes in patients with hypereosinophilia.

Production of fibroblast proliferative cytokines from T lymphocytes stimulated by a B cell lymphoma line and their functional heterogeneity

Human mononuclear leukocytes (MNL) produced several factors with fibroblast proliferation activity (FPA) for HFL-1, a human lung fibroblast cell line, when MNL were cocultured with irradiated BALL-1, a B cell lymphoma line (BCLL), but not with other BCLL. The cellular source of BALL-1-induced FPA seemed to be CD4-positive T lymphocytes. On isoelectric electrophoresis, major activity of BALL-1-induced FPA was detected in the fractions around pH 4-5, and minor activity was present in the fractions around pH 6-7. Major BALL-1-induced FPA consisted of at least 4 different fibroblast proliferation factors (FPFs) according to their molecular weight; 320-600 kDa (P-I), 50-110 kDa (P-II), 22-38 kDa (P-III) and 4.6-11 kDa (P-IV). P-I had affinity to heparin though the rest had little or no affinity. FPA of P-I was suppressed by an antibody against acidic FGF, and FPA of P-III was suppressed by an antibody against IL-6. On the other hand, FPA of P-II and P-IV was suppressed by none of the antibodies against cytokines with FPA, such as FGF, IL-4, IL-6, IFN-gamma, TGF-beta and TNF-alpha. It was thus suggested that P-I was acidic FGF, that P-III was IL-6, and that P-II and P-IV were different cytokines from those described above. Furthermore, it was found that P-II and P-IV failed to exhibit proliferation activity for human umbilical vein endothelial cells (HUVEC).(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of tissue eosinophilia: V. Induction of lymphocyte-derived eosinophil chemotactic inhibitory factor production by a macrophage product from complete Freund's adjuvant-treated guinea pigs

Splenic T lymphocytes of complete Freunds adjuvant (CFA)-treated guinea pigs spontaneously produce a lymphokine, eosinophil-directed chemotactic inhibitory factor (ECIF), which selectively inhibits the chemotactic response of the treated eosinophils to a certain chemotactic lymphokine for eosinophils. In the present paper, induction of spontaneous ECIF production was examined in CFA-treated guinea pigs. Although short-time (3-hr) cultured supernatants of CFA-treated spleen cells exhibited little or no ECIF activity, the supernatants could stimulate normal T lymphocytes to produce and release distinct ECIF activity. The factor with ECIF-releasing activity (ECIF-RF) was produced by splenic adherent cells of CFA-treated animals. ECIF-RF activity was absorbed only by T lymphocytes, suggesting that target cells of ECIF are T lymphocytes. ECIF-RF activity was eluted near cytochrome c (MW 12,500) on a Sephadex G-100 column. It was further found that ECIF-RF was sensitive to heating at 56 degrees C, to enzyme treatment with trypsin and neuraminidase, and to acid and alkaline condition. We thus concluded that ECIF-RF derived from adherent cells of CFA-treated animals may be involved in the suppression of cell-mediated tissue eosinophilia by stimulation of T lymphocytes to produce and release ECIF activity.

The regulation of tissue eosinophilia: IV. Purification and properties of eosinophil-directed chemotactic inhibitory factors from complete Freund's adjuvant-treated guinea pigs

Eosinophil-directed chemotactic inhibitory factors (ECIF) from T lymphocytes of complete Freunds adjuvant-treated guinea pigs were recovered in two separate molecular weight (MW) fractions: the one is eluted near bovine serum albumin (MW about 70,000), and in pH range between 7.0 and 7.5 by chromatofocusing column, whereas the other is near cytochrome c (MW 12,500), and in pH range between 5.0 and 5.5. It was, however, found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoadsorption that the high molecular ECIF is probably an aggregated form of the low molecular ECIF. It was subsequently confirmed that both the ECIF had similar physicochemical properties: sensitive to heating at 56 or 80 degrees C sensitive to enzyme treatment with trypsin and neuraminidase, sensitive in acid but not alkaline condition, and bind to peanut agglutinin-or Limulus polyphemus agglutinin-coupled agarose beads. The inhibitory activity of ECIF was suppressed by previous treatment of eosinophils with D-galactose and sialic acid. ECIF activity was specifically absorbed by eosinophils; the absorption was inhibited by pretreatment of eosinophils with D-galactose and sialic acid. Previous treatment of eosinophils with beta-galactosidase and neuraminidase led the cells not to respond to both ECIF. It was thus suggested that the inhibition by ECIF is receptor-mediating phenomenon, and that ECIF and ECIF receptors on eosinophils contain galactose and sialic acid residues which may play an essential role for the biological activity.

Selective potentiation of lymphocyte-derived macrophage chemotactic factor release in complete Freund's adjuvant-treated guinea pigs.

Dinitrophenol (DNP)‐ovalbumin(OA)‐induced tissue macrophage reaction in sensitized guinea pigs is enhanced by treatment with complete Freunds adjuvant (CFA). The enhancement of the reaction may be due to the increased production of a T‐lymphocyte‐derived macrophage chemotactic factor (LDMCF) because treatment of animals with CFA potentiates antigen‐ and concanavalin A(ConA)‐induced release of LDMCF activity from spleen cells of the CFA‐treated animals in vitro. This potentiating effect by CFA seems to be ascribed to the release of an adherent‐cell‐derived soluble factor from the CFA‐treated animals. The adherent cell‐derived factor, LDMCF‐potentiating factor (LDMCF‐PF), preferentially potentiates the release of LDMCF activity but not of eosinophil chemotactic activity from antigen‐ or Con‐A‐stimulated T lymphocytes. Protein synthesis is required for release of LDMCF‐PF. Molecular weight of LDMCF‐PF activity is assumed to be about 10,000‐20,000. LDMCF‐PF activity is sensitive to trypsin, to neuraminidase, and also to alkalinity at pH 11, suggesting that LDMCF‐PF is a glycoprotein. The present study provides one explanation for the enhanced macrophage reaction in delayed‐type hypersensitivity reactions.

Aproach for the regulation of tissue eosinophilia using chemotaxis chamber.

The regulation of tissue eosinophilia in allergic skin lesions were examined using chemotactic chamber assay. Tissue eosinophilia manifested two phases: early phase peaking at 6hr and delayed phase at 24hr. More potent delayed phase was mediated by two different eosinophil chemotactic factors, delayed ECF-a and b. Delayed ECF-a was shown to be a lymphokine produced by T lymphocytes through antigenic or mitogenic stimulation, whereas delayed ECF-b was produced by reaction with anti-hapten IgG1 antibody. The Delayed eosinophilia was suppressed completly when animals were treated with complete Freunds adjuvant. This suppression was due to the production of eosinophil-directed chemotactic inhibitory factor (ECIF) which selectively inhibit the chemotactic response of eosinophils to delayed ECF-a but not to delayed ECF-b.On the other hands, tissue eosinophilia was markedly potentiated when animals were treated with alum hydroxy gel or Bordetella pertussis vaccine. This potentiation was ascribed to the potentiation of delayed ECF-a production by T lymphocytes. Macrophage products, named ECF-potentiating factors, could potentiate ECF-production but not macrophage chemotactic factor production. These results suggested that tissue eosinophilia, especially by a lymphokine (delayed ECF-a), is regulated by a lymphokine (ECIF) selectively, and that macrophage products are involved in the ECF production.