Biomedical subjects

A Ishiguro

Publications and source records attributed to A Ishiguro.

At least 55 records · Page 3Linked to original sources

PubMed1996

Id2 expression increases with differentiation of human myeloid cells.

Id proteins are helix-loop-helix (HLH) transcriptional factors that lack the basic DNA binding domain. The Id proteins have been reported generally to function as inhibitors of cell differentiation, and their gene expression is often downregulated during cell differentiation. We examined the expression of human Id mRNAs by Northern hybridization in 11 human myeloid cell lines, several myeloid cell lines induced to differentiate, fresh myeloid leukemia samples, and normal human myeloid cells. Id2 mRNA was expressed in myelomonoblastic and monoblastic leukemic cells (PLB-985, THP-1, and U-937) but was weakly expressed in myeloblastic leukemic cells (KG-1 and HL-60). Id2 mRNA levels markedly increased with induction of differentiation of myeloid blasts (HL-60, PLB-985, THP-1, and U-937) toward either granulocytes or macrophages. Examination of fresh acute myeloid leukemic samples from 22 individuals also showed prominent Id2 mRNA expression in those samples having more differentiated blasts. Using the French-American-British classification, only 2 of 8 M0/M1 samples expressed Id2 mRNA; however, 10 of 13 M2/M3/M4 samples expressed it. In normal human myeloid cells, Id2 mRNA was expressed in cultured macrophages from bone marrow and in mature granulocytes and monocytes from peripheral blood. The half-life of Id2 mRNA was short (1 hour), and its expression was inducible by cessation of protein synthesis. Id3 mRNA was moderately expressed in monoblastic cell lines (THP-1 and U-937), and levels decreased with their differentiation. Almost no Id3 expression was detectable in either other myeloid leukemia lines, fresh leukemic samples, or normal human myeloid cells by Northern analyses. Id1 mRNA was not detected by polymerase chain reaction in either leukemic or normal myeloid cells except in K562 myeloid/erythroid cells. These results showed that Id2 mRNA was constitutively expressed in more mature myeloid blast cells and level markedly increased with terminal myeloid differentiation, suggesting that Id2 protein may inhibit an HLH transcriptional complex that normally represses myeloid differentiation.

Acute Disease↗

PubMed1996

Reference intervals for serum granulocyte colony-stimulating factor levels in children.

OBJECTIVE: To determine age-adjusted reference intervals for children for serum granulocyte colony-stimulating factor (G-CSF) levels. DESIGN: We determined the serum G-CSF levels of 168 disease-free children who were term neonates to 15 years of age with a highly sensitive chemiluminescent enzyme immunoassay. RESULTS: The lowest values (5 ng/L) exceeded the detectable limit (1 ng/L) of this assay technique. The G-CSF levels were highest on the day of birth (mean +/- SD 147 +/- 146 ng/L); thereafter values decreased to 46 +/- 33 ng/L in the early neonatal period and to 23 +/- 10 ng/L in the late neonatal period. The G-CSF levels both on the day of birth and in the early neonatal period were significantly higher than in all older age groups. No significant differences were found among any of the age groups after 4 weeks of age. The G-CSF values were correlated with both blood leukocyte counts (r = 0.496, p < 0.0001) and neutrophil counts (r = 0.547, p < 0.0001). In children older than 4 weeks of age (n = 128), the 95% reference interval for G-CSF values was 5 to 42 ng/L. CONCLUSIONS: Our study in disease-free children revealed that change in serum G-CSF levels are age dependent and are correlated with neutrophil counts. Determination of reference intervals for the neonatal period requires further study.

Adolescent↗

PubMed1996

Evidence for the presence of immunoglobulin E antibodies specific to the cell wall phosphomannoproteins of Candida albicans in patients with allergies.

To determine the major antigenic component of Candida albicans against immunoglobulin E (IgE) antibodies in the sera of patients with allergies who were positive for IgE antibodies to C. albicans crude antigen in a CAP system, phosphomannoproteins (CAMP/A or CAMP/B for serotype A or B strain, respectively) and their acid-stable portions (CAMP-S/A or CAMP-S/B) were isolated from beta-mercaptoethanol (2-ME) extracts of C. albicans cells of serotypes A and B, and IgE antibodies against these components were compared with those against protein complex and enolase (CAE) fractions isolated from C. albicans cells. The dot blot test, which was used to detect IgE antibodies to the C. albicans antigens, showed that IgE antibodies to the 2-ME extract and phosphomannoprotein fractions were present in the sera of 98.0% (2-ME extract), 96.8% (CAMP/A), 93.2% (CAMP-S/A), 97.2% (CAMP/B), and 81.5% (CAMP-S/B) of the patients, whereas IgE antibodies to the protein complex and CAE fractions were found in the sera of 73.6 and 48.8% of the patients, respectively. The extent of IgE binding to the 2-ME extract and phosphomannoproteins was well correlated with the fluorescence intensities estimated with the CAP system. Furthermore, the results obtained from the inhibition experiment with the CAP system indicated that the binding of IgE antibodies to Candida antigens is strongly inhibited by the phosphomannoprotein fraction and is an indication that the serum of the patients contained IgE antibodies specific to the cell wall phosphomannoproteins of C. albicans. Finally, an initial chemical analysis indicated that the epitopes for IgE antibodies on the phosphomannoproteins is a carbohydrate portion, since the ability of CAMP/A to inhibit the binding of IgE antibodies to the homologous CAMP/A was destroyed after oxidation by sodium periodate but not after digestion with proteinase K.

Antibodies, Fungal↗

PubMed1996

Production of interleukin-10 in the cerebrospinal fluid in aseptic meningitis of children.

IL-10 is a cytokine that has antiinflammatory properties. We investigated IL-10 using ELISA and a reverse-transcribed polymerase chain reaction in the cerebrospinal fluid (CSF) of children with or without aseptic meningitis. When the patients with aseptic meningitis had meningeal symptoms, IL-10 in the CSF was detectable in 14 of 22 patients (88 +/- 146 ng/L, n = 31). The IL-10 levels decreased as meningeal symptoms disappeared. In 20 of 21 control children without meningitis, CSF samples had no detectable levels of IL-10 (< 10 ng/L). Serum IL-10 levels were lower than the corresponding levels in the CSF from the same individuals with aseptic meningitis. Significant correlations were found between IL-10 levels and mononuclear cell counts in the CSF of the affected patients (r = 0.644, p < 0.001). The IL-10 mRNA was detected by reverse-transcribed polymerase chain reaction-assisted amplification in the CSF cells in four of seven patients with the disease. The culture of CSF mononuclear cells produced high levels of IL-10 (152-485 ng/L) in all of five patients. Cytokine kinetics in the CSF showed that mean IL-10 levels reached the peak on the 2nd to 3rd d of the illness, although all mean levels of IL-6, IL-8, and granulocyte colony-stimulating factor were the highest on the 1st d of the illness. In summary, IL-10 is produced in the CSF in aseptic meningitis, and may increase relatively late compared with the proinflammatory cytokines. IL-10 may play an immunoregulatory role in the meningeal inflammatory network.

Adolescent↗

PubMed1995

Expression of Id2 and Id3 mRNA in human lymphocytes.

Helix-loop-helix (HLH) transcription factors are involved in cellular growth and differentiation. The Id (inhibitor of DNA binding and differentiation) HLH proteins, in a dominantly negative fashion, regulate transcriptional activities of basic HLH proteins. We examined by northern hybridization the expression of Id2 and Id3 mRNA in human leukemia/lymphoma lines and patient samples, as well as resting and activated normal human lymphocytes from peripheral blood (PBL). The Id2 mRNA was abundantly expressed in 5/12 T-cell and 3/4 B-cell lines, and Id3 mRNA was detected in 4/12 T-cell and 3/4 B-cell lines. Interestingly, Id2, but not Id3, mRNA was strongly expressed in 4/5 T-cell lines infected with human T-cell leukemia virus type I (HTLV-I) (ATL-1k, MT-2, S-LB1) and type II (Mo). Another unexpected finding was that T-cell leukemias and T-cell lines often expressed either Id2 or Id3 mRNA. In addition, resting PBL constitutively expressed prominent levels of Id2 mRNA, but not Id3 mRNA. Upon PHA-stimulation, Id2 expression decreased and Id3 levels increased with biphasic kinetics. Taken together, our studies revealed three unexpected findings which require further analysis: (1) expression of Id2 mRNA is often associated with lymphocytic transformation by HTLV-I or -II; (2) T-cells usually express either Id2 or Id3 mRNA, but B-cells often express both simultaneously; (3) non-dividing, normal PBL express high levels of Id2 and no Id3 mRNA; and with the onset of cellular proliferation, levels of Id2 mRNA decrease while levels of Id3 mRNA increase, suggesting that regulation of expression of these closely related genes is disparate.

B-Lymphocytes↗

PubMed1995

Detection of IgE antibody against Candida albicans enolase and its crossreactivity to Saccharomyces cerevisiae enolase.

Candida albicans 46 kDa protein, a glycolytic enolase enzyme, is an important allergen of the yeast. The purpose of the study was to detect circulating IgE and IgG antibodies against C. albicans enolase (CAE). We isolated CAE using sequential DEAE Sephacel and P11 column chromatography from spheroplasts of C. albicans, and detected IgE and IgG antibody against CAE by immunoblotting. Crossreactivity of enolase of C. albicans and Saccharomyces cerevisiae was also examined by immunoblotting and immunoblot inhibition test. Among 54 sera with positive IgE RAST to C. albicans, IgE antibody against CAE was detected in 20 sera (37%) and IgG antibody in 27 sera (50%). The allergenic potency of CAE was confirmed using a skin-prick test in three patients. Simultaneous IgE binding to S. cerevisiae enolase was only observed in four out of 20 sera reacting to CAE. Pre-treatment of sera with CAE completely inhibited IgE binding to S. cerevisiae enolase. Whereas the latter only partially inhibited IgE binding to CAE. These results suggest that CAE shares some crossreacting epitopes with S. cerevisiae enolase, representing minor components of CAE but dominant segments of S. cerevisiae enolase.

Antibodies, Fungal↗

PubMed1995

Characterization of IgE-binding epitopes on Candida albicans enolase.

Candida albicans enolase is one of the important allergens in Candida allergy. We isolated and purified 46kDa C. albicans enolase (CAE) from C. albicans and characterized epitopes for IgE antibody by lectin-blotting and enzymatic digestion followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Lectin blotting and deglycozilation indicated that this protein did not contain polysaccharide side chains. The purified CAE and recombinant fusion protein produced from CAE gene possessed common epitopes for IgE antibody. We estimated IgE binding epitopes on the basis of reported amino acid sequences from the analysis of cDNA encoding CAE. V8 protease digestion of CAE gave six polypeptide fragments (A-F). The N-termini of each fragment were confirmed by amino acid sequence and the C-termini were estimated by molecular weights of each fragment and the specific cutting site of V8 protease. Fragment C (25.0 kDa; F-171-I-399) reacted to 90% IgE antibodies examined, whereas fragments D (21.0 kDa; F-171-I-360), E (16.2 kDa; F-171-D-317) and F (13.0 kDa; A-47-E-170) showed no IgE binding. Our results suggest that epitopes for IgE antibodies exist near the C-terminal of the protein.

Amino Acid Sequence↗

PubMed1995

Transient elevation of granulocyte colony-stimulating factor levels in cerebrospinal fluid at the initial stage of aseptic meningitis in children.

At the early stage of aseptic meningitis, there is a transient increase in neutrophil counts in the cerebrospinal fluid. Some factors in the cerebrospinal fluid might induce migration of neutrophils into the cerebrospinal fluid. Granulocyte colony-stimulating factor (G-CSF) plays an important role, not only as a hemopoietic factor but also as a regulating factor for a biologic defense system by neutrophils in the foci of infection. To analyze the role of G-CSF on accumulating neutrophils in the cerebrospinal fluid, we have measured G-CSF levels in the cerebrospinal fluid of children with aseptic meningitis, paying particular attention to the phasal transition. Within the first 2 d from the onset, G-CSF levels in the cerebrospinal fluid were 223 +/- 97 ng/L, significantly higher than those of the patients without meningitis (p < 0.01). Beyond the second day after the onset, the G-CSF levels rapidly decreased to below the detectable level, even though the patients manifested meningeal signs and symptoms. There was a direct relationship between G-CSF levels and neutrophil counts in the cerebrospinal fluid (r = 0.763, p < 0.01). During the first 2 d after the onset, the G-CSF level in the cerebrospinal fluid in each case was remarkably higher than that in the serum. This finding suggests that the G-CSF in the cerebrospinal fluid was produced in the spinal cavity. From our results, the transient elevation of G-CSF levels might lead to the transient increase in neutrophil counts in the cerebrospinal fluid by recruiting them from the peripheral blood at the initial stage of aseptic meningitis.

Adolescent↗

PubMed1993

Improvement of neutropenia and neutrophil dysfunction by granulocyte colony-stimulating factor in a patient with glycogen storage disease type Ib.

Patients with glycogen storage disease type Ib (GSD Ib) suffer from recurrent bacterial infections due to neutropenia and neutrophil dysfunction. To improve the quality of life in a 9-year-old boy with GSD Ib, we subcutaneously administered recombinant human granulocyte colony-stimulating factor (G-CSF). Daily injections of 100 micrograms/m2 of G-CSF significantly increased absolute neutrophil counts and augmented neutrophil mobility. The patient was then treated with 70 and 100 micrograms/m2 of G-CSF daily and twice-weekly. The treatment maintained absolute neutrophil counts at significantly higher levels than those without treatment for 22 months and markedly decreased the frequency of infections and the necessity for hospitalisation. Weekly injections of 70 micrograms/m2 of G-CSF were less efficient. No adverse effects were observed during treatment. These findings indicate that daily and twice-weekly treatment with G-CSF of long duration are safe and effective for patients with GSD Ib. G-CSF may be a useful therapeutic agent in patients with neutrophilic impairment as a consequence of a metabolic disorder.

Cell Movement↗

PubMed1992

Immunoblotting analysis of sera from patients with candidal vaginitis and healthy females.

Antigenic components of Candida albicans were extracted from whole cells with a buffer containing SDS and 2-mercaptoethanol, and separated by SDS-polyacrylamide gel electrophoresis. The components reactive with IgG, IgA, IgM and IgE antibodies in sera from patients with (14 subjects) and without (15 subjects) C. albicans in the vagina, and from healthy females (34 subjects), were investigated by immunoblotting using immunoglobulin class-specific antibodies. Many components reacted with IgG and IgA in all sera tested; the major antigens that reacted strongly with the sera were 67, 62, 29 and 25 kDa components. Several components were observed which reacted with IgM in 63% of the sera; the 67, 62 and 25 kDa components that reacted with IgG and IgA also reacted with IgM. No components reacting with IgE were detected in any of the sera. No striking differences in antibody binding profiles to whole cell antigens were detected among the C. albicans positive and negative patients or the healthy subjects. On the other hand, IgG against extracellular proteinase was more frequently detected in the C. albicans positive patients than in the C. albicans negative group or the healthy subjects. This may suggest that vaginal infection with C. albicans contributes to a rise in anti-proteinase antibody levels.

Adult↗

PubMed1992

Identification of Candida albicans antigens reactive with immunoglobulin E antibody of human sera.

Candida albicans antigens which reacted with immunoglobulin E (IgE) antibodies of 57 allergic patients were detected by immunoblotting. Of the various antigens, the 175-, 125-, 46-, 43-, and 37-kDa antigenic components reacted most frequently with the patient sera. To purify the major antigens, C. albicans cells were fractionated. The 46-, 43-, and 37-kDa antigens were recovered in cytoplasmic fractions, but the 175- and 125-kDa antigens were not recovered in any fraction. The 46-, 43-, and 37-kDa antigens were purified from cytoplasmic fractions by DEAE and P11 ion-exchange chromatography. Antigens were isolated by cutting bands out of sodium dodecyl sulfate-polyacrylamide gels. The purified components confirmed by immunoblotting were next processed for amino acid sequencing. Parts of the sequences of the 46-, 43-, and 37-kDa antigens had significant levels of homology with Saccharomyces cerevisiae glycolytic enzyme enolase, phosphoglycerate kinase, and aldolase, respectively. Rabbit IgG antibodies prepared against the 46- and 43-kDa antigens strongly cross-reacted with the homologous proteins of S. cerevisiae. However, S. cerevisiae enolase and phosphoglycerate kinase did not cross-react with IgE of patient sera. This result suggests that IgE antibodies against only small parts of their epitopes are elevated in the allergic patients. Since enolase is reported to be a major antigen for systemic candidiasis, this enzyme may be the immunodominant protein in both allergies and fungal infections.

Adolescent↗

PubMed1992

[Elevated production of GM-CSF by peripheral blood mononuclear cells stimulated with mite antigens in children with bronchial asthma].

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine which can stimulate the proliferation and functions of eosinophils and neutrophils. We have studied whether peripheral blood mononuclear cells (PBMC), obtained from children with stable asthma, can produce GM-CSF through stimulation with crude Dermatophagoides farinae (Df) antigen in vitro. The patients had strongly positive RAST results for house dust mite Df. Levels of GM-CSF in PBMC culture supernatant were assayed by an enzyme immunoassay. When stimulated with Df antigen, patient's PBMC released significantly increased levels of GM-CSF compared with unstimulated ones. PBMC of non-allergic controls, however, did not. The addition of polymyxin B, an inactivator of endotoxin, did not suppress the Df-antigen-induced production of GM-CSF. Levels of GM-CSF produced by PBMC stimulated with Df antigens were significantly higher in the patients than in the controls. These results indicate that PBMC of children with Df-allergic asthma, unlike those of non-allergic children, can produce elevated levels of GM-CSF by stimulation with Df antigens.

Animals↗

PubMed1992

[Suppression of in vitro hemopoiesis by the monocytes in a patient with aplastic anemia associated with hepatitis].

The cause of aplastic anemia associated with hepatitis (AAH) is as yet still unknown. There is a supposed relation to the immune mechanisms, however few reports have shown the effects of monocytes on the pathogenesis in the patients with AAH. We have reported a case of a 12-year-old boy with AAH related to cytomegaloviruses, and studied the hemopoietic progenitors. He showed pancytopenia and hypoplasia of the bone marrow on admission to our hospital. The culture studies showed that mononuclear cells (MNC) of the bone marrow produced few hemopoietic colonies in all cell lineages. However, the depletion of adherent cells from the MNC increased numbers of erythroid, neutrophil-macrophage and megakaryocyte colonies. Furthermore, the addition of adherent cells of the peripheral blood suppressed the colony formation in the aforementioned cell lineages by marrow MNC from which adherent cells, phagocytic cells and T-cells were abrogated. The results way suggest that monocytes play some soles in the pathogenesis of aplasia through inhibitor of hemopoiesis.

Anemia, Aplastic↗

PubMed1991

Induction of granulocyte and granulocyte-macrophage colony-stimulating factors from human monocytes stimulated by Fc fragments of human IgG.

The effect of human IgG on human haemopoiesis has been studied in vitro. Dialysed purified IgG stimulated haemopoietic colony growth by bone marrow mononuclear cells (MNC) but not by monocyte-depleted MNC. Culture media, conditioned by IgG-stimulated peripheral blood MNC, augmented formation of neutrophil-macrophage, eosinophil, and megakaryocyte colonies by monocyte-depleted marrow MNC. Serum-free IgG-conditioned media also contained colony-stimulating activity (CSA). IgG Fc fragments and heat-aggregated IgG promoted the secretion of CSA, but F(ab')2 fragments, Fab fragments or ultracentrifuged IgG did not. In the cell-selection studies, CSA was produced by highly enriched monocytes following stimulation with Fc fragments. The antiserum against human granulocyte colony-stimulating factor (G-CSF) and/or granulocyte-macrophage CSF (GM-CSF) neutralized the CSA produced by Fc fragment-activated monocytes. Enzyme immunoassays showed G-CSF and GM-CSF in media conditioned by monocytes stimulated with the Fc fragments, heat-aggregated IgG and anti-D-sensitized red blood cells (RBC). Northern hybridization analysis showed mRNA encoding G-CSF and GM-CSF in RNA extracted from MNC and monocytes cultured with the Fc fragments, but not in the RNA from unstimulated cells or monocyte-depleted MNC. These results indicate that IgG Fc fragments, aggregated IgG and antigen-antibody complexes induce monocytes to produce G-CSF and GM-CSF in vitro. The CSFs release induced by IgG may be involved in the in vivo regulatory network in haemopoiesis.

Cell Division↗

PubMed1991

The relationship of the proliferating cell nuclear antigen protein to cis-diamminedichloroplatinum (II) resistance of a murine leukemia cell line P388/CDDP.

We investigated whether the proliferating cell nuclear antigen (PCNA) protein takes part in cis-diamminedichloroplatinum (II) (CDDP) resistance, using a murine leukemia cell line P388 and its CDDP resistant cell line. P388/CDDP was 4 times more resistant to CDDP than P388. The cell lines were maintained in the DBA/2 female mouse peritoneal space. In total cells, the amount of the PCNA protein decreased to 90% in P388 after 1 h CDDP treatment, but that of P388/CDDP increased to 127%. The difference was statistically significant (p = 0.012, n = 5). As for G2/M phase cells, the difference was also significant at 1 h (p = 0.016, n = 5) and at 2 h (p = 0.036, n = 5) after treatment. In P388 the amount of the PCNA protein decreased in accordance with the inhibited cell proliferation, whereas in P388/CDDP, the amount of the PCNA protein increased in spite of the inhibited cell proliferation. This increase of the PCNA protein suggests that the PCNA protein is involved in CDDP resistance of P388/CDDP through enhanced DNA repair synthesis.

Animals↗

PubMed1990

Interleukin-6 enhances murine megakaryocytopoiesis in serum-free culture.

We investigated the effect of interleukin-6 (IL-6) on murine megakaryocytopoiesis in a serum-free culture system. The addition of IL-6 to a culture containing interleukin-3 (IL-3) resulted in a significant increase in the number of megakaryocyte colonies by bone marrow cells of normal mice. The megakaryocytic progenitors that survive exposure to 5-fluorouracil (5-FU) exhibited a more significant response to IL-6 and IL-3. Polyclonal anti-IL-6 antibody neutralized the stimulatory effect of IL-6 on megakaryocyte colony growth supported by IL-3. Delayed addition experiments and replating experiments of blast cell colonies showed that megakaryocytic progenitors are supported by IL-3 in the early stage of the development but require IL-6 for their subsequent proliferation and differentiation. In addition, IL-6 increased the size of megakaryocytes in granulocyte-macrophage-megakaryocyte colonies. The combination of granulocyte colony-stimulating factor or granulocyte-macrophage colony stimulating factor with IL-3 resulted in an increase in the granulocyte-macrophage colony growth of bone marrow cells of 5-FU-treated mice or normal mice, respectively, but had little effect on the enhancement of pure and mixed megakaryocyte colony growth. These results suggest that IL-6 plays an important role in murine megakaryocytopoiesis.

Animals↗

PubMed1990

Effects of interleukin-3 and interleukin-4 on the development of "connective tissue-type" mast cells: interleukin-3 supports their survival and interleukin-4 triggers and supports their proliferation synergistically with interleukin-3.

We examined the effects of interleukin-3 (IL-3) and interleukin-4 (IL-4) on connective tissue-type mast cells (CTMC) purified from murine peritoneal cells. Although both factors failed to induce extensive proliferation of CTMC, they stimulated CTMC proliferation synergistically in a dose-dependent manner. Pretreatment of CTMC with IL-3 and/or IL-4 indicated that the sustained presence of both factors was required for the development of type 1 mast cell colonies. The delayed addition of IL-3 to cultures of purified CTMC with IL-4 induced no colony formation, while the delayed addition of IL-4 to cultures with IL-3, even on day 28 of culture, induced type 1 colony formation. In replating type 1 colonies induced by IL-3 and IL-4 to secondary cultures with IL-3 alone, few secondary colonies developed. However, the delayed addition of IL-4 to the secondary culture induced many type 1 colonies. The purified CTMC cultured with IL-3 retained the morphological and cytochemical characteristics of CTMC, as well as proliferative ability. These observations indicate that IL-3 supports the survival of CTMC in methylcellulose culture and that IL-4 triggers and supports CTMC proliferation synergistically with IL-3. The serum-free culture of purified CTMC and the culture of single CTMC demonstrated that the synergistic effect of IL-3 and IL-4 on colony growth and the surviving effect of IL-3 on CTMC require no influence from accessory cells or other humoral factors.

Animals↗

PubMed1990

Synergistic action of phorbol ester and IL-3 in the induction of "connective tissue-type" mast cell proliferation.

12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting phorbol ester, induced the proliferation of connective tissue-type mast cells (CTMC) synergistically with IL-3 in a methylcellulose culture, as well as with IL-4. The culture of single CTMC and the serum-free culture of CTMC fractionated by Percoll density gradient centrifugation showed that this synergistic action of IL-3 and TPA required no effects of accessory cells or other humoral factors. Although the populations of CTMC acted on by TPA and IL-4 seemed to be close to each other, the velocity of colony growth induced by the simultaneous stimulation of the combination of TPA and IL-4 was faster than that induced by either TPA or IL-4 in the presence of IL-3. In addition, the addition of anti-IL-4 antibody did not neutralize the effect of TPA on the proliferation of CTMC. These results suggest that TPA and IL-4 act on the proliferation of CTMC synergistically with IL-3 via a different pathway. Beside TPA, other phorbol derivatives capable of activating protein kinase C (PKC) induced the proliferation of CTMC synergistically with IL-3, but phorbol derivatives which were unable to activate PKC did not. These results indicate that the activation of PKC is involved in the process of TPA action on the proliferation of CTMC. Furthermore, the facts that 1-oleoyl-2-acetylglycerol, which activated membrane PKC transiently, and staurosporine, which has been reported to inhibit PKC, did not induce the proliferation of CTMC in the presence of IL-3 and that the effect of TPA was exhibited by the sustained stimulation suggest that the action of TPA on the proliferation of CTMC requires at least two steps. The first one is the primary activation of membrane PKC and the second one is the disappearance of PKC from the cells, "down-regulation."

Alkaloids↗