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Results for “Interleukin-3 Receptor alpha Subunit”

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PubMed1996

Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Animals↗

PubMed1998

Identification of a Cys motif in the common beta chain of the interleukin 3, granulocyte-macrophage colony-stimulating factor, and interleukin 5 receptors essential for disulfide-linked receptor heterodimerization and activation of all three receptors.

The human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors undergo covalent dimerization of the respective specific alpha chains with the common beta subunit (betac) in the presence of the cognate ligand. We have now performed alanine substitutions of individual Cys residues in betac to identify the Cys residues involved and their contribution to activation of the IL-3, GM-CSF, and IL-5 receptors. We found that substitution of Cys-86, Cys-91, and Cys-96 in betac but not of Cys-100 or Cys-234 abrogated disulfide-linked IL-3 receptor dimerization. However, although Cys-86 and Cys-91 betac mutants retained their ability to form non-disulfide-linked dimers with IL-3Ralpha, substitution of Cys-96 eliminated this interaction. Binding studies demonstrated that all betac mutants with the exception of C96A supported high affinity binding of IL-3 and GM-CSF. In receptor activation experiments, we found that betac mutants C86A, C91A, and C96A but not C100A or C234A abolished phosphorylation of betac in response to IL-3, GM-CSF, or IL-5. These data show that although Cys-96 is important for the structural integrity of betac, Cys-86 and Cys-91 participate in disulfide-linked receptor heterodimerization and that this linkage is essential for tyrosine phosphorylation of betac. Sequence alignment of betac with other cytokine receptor signaling subunits in light of these data shows that Cys-86 and Cys-91 represent a motif restricted to human and mouse beta chains, suggesting a unique mechanism of activation utilized by the IL-3, GM-CSF, and IL-5 receptors.

Amino Acid Sequence↗

PubMed1995

Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively.

The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony-stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice that express the mouse IL-5R alpha constitutively by phosphoglycerate kinase promoter. The transgenic mouse expressed IL-5R alpha ubiquitously, and the bone marrow cells formed various types of colonies, including multi-lineage colonies, in response to IL-5. IL-5 also supported formation of both multi-lineage and blast cell colonies from dormant progenitors of the 5-fluorouracil-treated transgenic mice. The cells composing the blast cell colony gave rise to many colonies including multi-lineage colonies when they were replated in secondary culture containing either Il-5 or IL-3. There was no significant difference in replating efficiency or in types of secondary colonies between IL-5- and IL-3-stimulated cultures. Conversely, the cells from the IL-3-induced blast cell colonies of the transgenic mice proliferated in response to either IL-3 or IL-5. Thus, the development of the progenitors can be equally supported by either IL-5 or IL-3, suggesting that intracellular signals from the IL-3R can be replaced by those from IL-5. These results strongly suggest that the lineage specificity of IL-5 is mainly due to the restricted expression of IL-5R alpha.

Animals↗

PubMed1994

Regulation of IL-3 receptor expression: evidence for a post-transcriptional mechanism that dominantly suppresses the expression of beta subunits.

The IL-3 receptor (IL-3R) is composed of alpha and beta subunits. Two homologous beta subunits of IL-3R are present in the mouse: AIC2A is the IL-3 specific beta subunit, and AIC2B is the common beta subunit shared by IL-3, IL-5 and granulocyte macrophage colony stimulating (GM-CSF) factor receptors. Both beta subunits form functionally indistinguishable high-affinity IL-3Rs with the same IL-3R specific alpha subunit (IL-3R alpha or SUT-1). Cell surface expression of the alpha and beta subunits of IL-3R was found to be diminished in an IL-3 non-responsive variant (MC/9.IL-4) derived from an IL-3-dependent mast cell line, MC/9. This IL-3R-defective phenotype was dominant based on cell fusion experiments. Moreover, regulatory mechanisms of the alpha and beta subunits are distinct since cell hybrids between MC/9.IL-4 and a CTLL-2 transfectant (CTLL/AS) expressing AIC2A and IL-3R alpha showed a significantly reduced expression of the AIC2A mRNA, while the IL-3R alpha expression was unchanged. Since transcription of both AIC2A and IL-3R alpha cDNAs in the CTLL/AS was driven by an artificial promoter, SR alpha, and nuclear run-off assays showed similar transcriptional rates of the AIC2A gene in both CTLL/AS and the cell hybrids between MC/9.IL-4 and CTLL/AS, the dominant suppression of the beta subunits is post-transcriptional and sequence-specific. A target sequence of the negative regulator must be present within 2756 bases of AIC2A mRNA, which is transcribed from the transfected cDNA in CTLL/AS cells. Similar dominant suppression of the beta subunit expression was also found in a B cell line WEHI231. As the negative regulator suppresses expression of the beta subunits of IL-3, IL-5 and GM-CSF receptors, it has the potential to eliminate all three high-affinity receptors simultaneously.

Animals↗

PubMed1993

Dynamic modulation of the cell surface expression of the granulocyte-macrophage colony-stimulating factor receptor.

The GM-CSF receptor (GM-CSFR) is composed of alpha and beta subunits. Surface expression of the alpha chain alone leads to low affinity GM-CSF binding and of both subunits to high affinity binding; the beta chain is required for transducing a proliferative signal. Studies of GM-CSFR expression have concentrated largely on static events occurring under conditions of binding equilibrium. We have examined the dynamic regulation of high and low affinity GM-CSFR expression in neutrophils (1100 +/- 200 R/cell, KD 50 +/- 15 pM) and a GM-CSF dependent human leukaemic cell line, TF-1 (2000 +/- 450 R/cell KD 15 +/- 5 pM) and 8600 +/- 1150 R/cell KD 1.8 +/- 0.3 nM). The addition of GM-CSF to TF-1 cells (350 pM, 4 h at 37 degrees C) caused a reduction in subsequent binding of 125I-GM-CSF at low ligand concentration (100 pM) (following a low pH wash to remove surface bound ligand) to 16 +/- 4% and a reduction in binding at high ligand concentration (2 nM 125I-GM-CSF) to 36 +/- 9% of control. Scatchard analysis showed complete down-regulation of high affinity GM-CSFR and a significant reduction in low affinity GM-CSFR. In neutrophils, concentration-response curves of ligand induced receptor down-regulation at 37 degrees C showed that observed down-modulation was more than 10-fold greater than predicted by static equilibrium binding data and correlated closely with GM-CSF priming of the neutrophil respiratory burst. The addition of IL-3 to TF-1 cells at 37 degrees C reduced 100 pM 125I-GM-CSF binding to 18 +/- 4% and 2 nM 125I-GM-CSF binding to 46 +/- 5% of control. TF-1 cells, but not neutrophils, were able to re-express GM-CSFR following removal of GM-CSF from medium. TF-1 proliferation assays showed that pulsed GM-CSF (0.35-3.5 nM) for up to 4 h did not cause a significant increase in 3H-thymidine incorporation which required the continued presence of GM-CSF (control 2875 +/- 208 cpm, pulsed GM-CSF 5 ng/ml 4972 +/- 1344, continuous GM-CSF 5 ng/ml 17249 +/- 2982). Therefore, proliferation of TF-1 cells required the continued presence of GM-CSF at a time when there was no detectable surface high affinity GM-CSFR.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗

PubMed1993

Interleukin 3 stimulates proliferation and triggers endothelial-leukocyte adhesion molecule 1 gene activation of human endothelial cells.

Proliferation and functional activation of endothelial cells within a tissue site of inflammation are regulated by humoral factors released by cells, such as T lymphocytes and monocytes, infiltrating the perivascular space. In the present study we investigated the effects of interleukin 3 (IL-3), an activated T lymphocyte-derived cytokine, on cultured human umbilical vein endothelial cells (HUVEC). Proliferative activity, evaluated both by estimation of the fraction of cells in the S phase and by direct cell count demonstrated that IL-3, at the dose of 25 ng/ml, enhances more than threefold both DNA synthesis and cell proliferation above baseline control conditions. Binding studies with radioiodinated ligand demonstrated that HUVEC constitutively express a smaller number of IL-3 binding sites (approximately 99 binding sites per cell, with an apparent Kd of 149 pM). Accordingly, molecular analysis showed the presence of transcripts for both alpha and beta subunits of the IL-3 receptor. Functional activation of endothelial cells was evaluated by the expression of the endothelial-leukocyte adhesion molecule 1 (ELAM-1) transcript and by leukocyte adhesion. The ELAM-1 gene transcript was clearly detectable 4 h after IL-3 addition and started to decrease after 12 h. Moreover, IL-3-induced ELAM-1 transcription was followed by enhanced adhesion of neutrophils and CD4+ T cells to HUVEC. The findings that IL-3 can stimulate both proliferation and functional activation of endothelial cells suggest that this cytokine can be involved in sustaining the process of chronic inflammation.

CD4-Positive T-Lymphocytes↗

PubMed1994

Interleukin-11.

Interleukin-11 (IL-11), a stromal cell-derived cytokine, has been known to act widely in hematopoietic and non-hematopoietic systems. IL-11 supports the growth of certain types of plasmacytoma and hybridoma cells, acts with interleukin-3 (IL-3) in shortening the Go period of early progenitors. IL-11 supports megakaryocyte colony formation and maturation, and acts as an autocrine growth factor in megakaryoblastic cell lines. In addition, IL-11 stimulates erythrocytopoiesis, enhances antigen-specific antibody responses, induces the synthesis of acute phase proteins, inhibits lipoprotein lipase activity and adipocyte differentiation, and promotes neuronal development. Administration of rhIL-11 to mice resulted in an increase of neutrophils and platelets. The human IL-11 gene is localized at 19q13.3-13.4, and codes 199 amino acids and 23 kDa with no N glycosylation. Its receptor and signal transduction share partially those of interleukin-6 (IL-6). Further analysis of its role in normal and pathological state is necessary to determine the exact function and its application for clinical uses.

Acute-Phase Proteins↗

PubMed1992

[IL-3 gene, receptor and signal transduction].

Interleukin 3 (IL-3) plays a critical role in growth and differentiation of myeloid cells. The human IL-3 gene, located on chromosome 5 contains several cis-acting DNA sequences, i.e. CLE (conserved lymphokine element) and a GC rich region, similar to the GM-CSF gene. The expression of the GM-CSF and IL-3 genes in activated T cells may be regulated coordinately through these cis-regulatory elements. Recently, reconstitution experiments using isolated cDNA clones for receptor subunits have revealed that two distinct proteins are required for the high affinity receptors for GM-CSF, IL-3 and IL-5; the alpha subunits bind with the corresponding ligands with low affinity, and the beta subunit is shared in common. Tyrosine kinase activity appears to be involved in the signal transduction of the IL-3 and GM-CSF receptors.

Animals↗

PubMed1991

Connective tissue mast cells in contact with fibroblasts express IL-3 mRNA. Analysis of single cells by polymerase chain reaction.

Mast cell-fibroblast interactions have been extensively investigated in the last few years. Fibroblasts support the in vitro survival but not proliferation of mouse connective-tissue type mast cells. However, the factor(s) that allow their survival on fibroblast monolayers has not been identified. We have investigated the presence of mRNA for IL-3 and granulocyte-macrophage-CSF in single mouse mast cells, before and after co-culture with 3T3 fibroblasts, using the polymerase chain reaction technique. The system was calibrated first by using in vitro generated population of mouse bone-marrow derived mast cells (BMMC). Significant differences in the amplification of IL-3 cDNA were observed in each of the BMMC cells examined, whereas the amplification of cDNA for the alpha-subunit of the Fc epsilon RI were similar. Inasmuch as murine cultured IL-3-dependent mast cells differentiate into connective tissue-like mast cells when co-cultured with 3T3 fibroblasts without any exogenous supply of growth factors, it was of interest to determine whether these connective tissue-like mast cells produce IL-3 message. Separation of the differentiated BMMC from the fibroblast monolayer, by either trypsinization or by single cell manipulation revealed the synthesis of a detectable amount of IL-3 mRNA in these mast cells. Whether this IL-3 mRNA was induced by fibroblasts was further investigated using connective tissue mast cells freshly purified from the mouse peritoneal cavity. Only about 20% of these connective tissue mast cells produced detectable amount of granulocyte-macrophage-CSF mRNA whereas in less than 10% of the cells IL-3 mRNA was detected. However, when these connective tissue mast cells were co-cultured with 3T3 fibroblasts for 18 hours and then separated, IL-3 mRNA were detected in most of the cells whereas no such mRNA was detected in tissue mast cells incubated for 18 h with medium derived from 3T3 fibroblasts. Therefore we conclude that fibroblasts induce the accumulation of IL-3 mRNA in connective tissue mast cells. The production of IL-3 may play a role in the survival of this type of mast cells on the fibroblast monolayer.

Animals↗

PubMed1991

Leukemia inhibitory factor can potentiate murine megakaryocyte production in vitro.

Receptors for murine leukemia inhibitory factor (LIF) were demonstrated on immature and mature murine megakaryocytes. LIF alone had no effects in culture on the survival or proliferation of normal murine megakaryocytes or their precursors. However, combination of LIF with multipotential-colony-stimulating factor (Multi-CSF) (interleukin-3) enhanced the megakaryocyte colony formation able to be stimulated by Multi-CSF; the enhancement involved all types of megakaryocyte colony and resulted in the formation of increased numbers of megakaryocytes. These observations provide a possible basis for the observation that, when LIF is injected in vivo, elevations are observed in megakaryocyte numbers and platelet levels.

Animals↗

PubMed1994

Acute phase response factor and additional members of the interferon-stimulated gene factor 3 family integrate diverse signals from cytokines, interferons, and growth factors.

Cytokines and growth factors elicit responses in target cells through induction of gene expression. Signaling mechanisms leading to gene transcription from cell surface receptors often require tyrosine phosphorylation. A family of transcription factors comprising the interferon (IFN)-stimulated gene factor 3 (ISGF3) multimeric complex are phosphorylated and activated in response to interferon. We describe a protein 50% identical to the 91-kDa subunit of ISGF3 that constitutes the acute phase response factor (APRF). This protein was rapidly activated by interleukin-6 to bind an enhancer element common to genes activated in liver cells during the acute phase response to inflammation. Remarkably, APRF was also activated by IFN alpha, IFN gamma, epidermal growth factor, platelet-derived growth factor, colony stimulating factor-1, and the cytokines leukemia inhibitory factor and oncostatin M. The growth factors also activated a third, distinct but related, DNA-binding protein in addition to APRF and p91. This novel factor or a closely related one, but neither APRF nor p91, was also activated in lymphoid cells by interleukin-2, erythropoietin, and interleukin-3. Activation of APRF, p91, and additional members of the ISGF3 family is thus a general feature of a wide variety of signaling pathways, integrating diverse signals through common transcriptional regulators.

Animals↗

PubMed1995

Induction of differentiation of WEHI-3B D+ leukemic cells transfected with differentiation-stimulating factor/leukemia inhibitory factor receptor cDNA.

Differentiation-stimulating factor (D-factor)/leukemia inhibitory factor can induce the differentiation of mouse myeloid leukemia M1 cells and also stimulate proliferation of the interleukin-3 (IL-3)-dependent cell line, DA-1a. To determine whether D-factor can induce the differentiation of leukemia cells other than M1 cells, WEHI-3B D+ mouse myelomonocytic leukemia cells were transfected with a plasmid containing mouse D-factor receptor cDNA. Expression of D-factor receptor in transfected cells was determined by binding of [125]D-factor and analyzed by Scatchard's method. The transfected cells had high-affinity D-factor receptors with a dissociation constant of 100 to 200 pmol/L and binding sites per cell varied from 67 to 1,500 among several clones. The cells expressing a high level of D-factor receptor were induced to differentiate by D-factor; about 60% of the cells exhibited the ability to reduce nitroblue tetrazolium and expression of the differentiation antigen Mac-1 (CD11b) on the cell surface increased. The effect of cytokines, which induce the differentiation of M1 cells, on the transfected WEHI-3B cells was examined. The sensitivity to oncostatin M was identical to that against D-factor in the cells of each clone. Expression of D-factor receptor in WEHI-3B cells promoted sensitivity to IL-6 and granulocyte colony-stimulating factor (G-CSF). Induction of differentiation of the cells accompanied the suppression of proliferation. Treatment of the cells with D-factor for longer than 5 days resulted in 50% inhibition of growth. These results indicate that the stimulating effect of D-factor on the differentiation of malignant myeloid cells is not unique to M1 cells.

Animals↗

PubMed1995

Human IL-3 receptor signaling: rapid induction of phosphatidylcholine hydrolysis is independent of protein kinase C but dependent on tyrosine phosphorylation in transfected NIH 3T3 cells.

Although tyrosine kinases are clearly activated after ligand engagement of the human IL-3R in both hematopoietic and nonhematopoietic cytoplasmic environments, a role for phospholipid hydrolysis and protein kinase C in IL-3R signal transduction is emerging. We have used NIH 3T3 cells transiently transfected with human IL-3R alpha- and beta-subunits to study phosphatidylcholine hydrolysis in response to human IL-3. We have found that NIH 3T3 cells expressing the complete human IL-3R respond to human IL-3 with a rapid and sustained increase in sn-1'2'-diacylglycerol. Accompanying this was a rapid increase in intracellular levels of phosphorylcholine. The protein kinase C inhibitor H-7, however, was not effective in inhibiting phosphatidylcholine hydrolysis in response to human IL-3 in NIH 3T3 cells expressing the human receptor. Thus the human IL-3R induces a rapid protein-kinase-C-independent hydrolysis induced by the murine receptor. Simultaneous with the increase in phosphatidylcholine hydrolysis induced by the murine receptor. Simultaneous with the increase in diacylglycerol levels was an increase in membrane-bound protein kinase C enzyme activity. Immunoblotting with isoform-specific Abs against protein kinase C showed that, whereas the zeta-isoform is constitutively membrane bound, the alpha-isoform of protein kinase C is translocated to the membrane in response to IL-3. Activation of phosphatidylcholine hydrolysis and protein kinase C activation required both alpha- and beta-receptor subunits. To determine the relationship of tyrosine phosphorylation to the activation of phosphatidylcholine hydrolysis and protein kinase C translocation, we used the specific and structurally unrelated tyrosine kinase inhibitors genistein and herbimycin. Both inhibitors effectively blocked human IL-3-induced tyrosine phosphorylation. In addition, both inhibitors blocked phosphatidylcholine hydrolysis and protein kinase C translocation. These data, combined with our previous report showing that c-jun induction by IL-3 is dependent on protein kinase C, suggest that, in hematopoietic and nonhematopoietic cells expressing the human IL-3R, phosphatidylcholine hydrolysis and protein kinase C are downstream effectors of tyrosine phosphorylation in the IL-3 signal transduction cascade resulting in immediate early response gene induction.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Overexpression of Shc proteins potentiates the proliferative response to the granulocyte-macrophage colony-stimulating factor and recruitment of Grb2/SoS and Grb2/p140 complexes to the beta receptor subunit.

The high affinity receptor for GM-CSF consists of a unique alpha subunit and a beta subunit that is shared with receptors for IL-3 and IL-5. Activation of GM-CSF receptor (GMR) triggers two distinct cytoplasmic signalling pathways, JAK2 and Ras, and is sufficient to maintain proliferation of growth factor-dependent cell lines. Shc proteins are phosphorylated upon activation of GMR and may be involved in the transmission of GM-CSF signals to Ras. To define the role of Shc proteins in cells stimulated with GM-CSF, we investigated both the network of interactions that involve Shc after GM-CSF stimulation and the effects of overexpressing Shc proteins on the proliferative response to GM-CSF. Two cytoplasmic complexes, Grb2/Sos and Grb2/p140 bind through the Grb2 SH2 domain to phosphorylated Shc, and are thereby recruited to the beta subunit. Both complexes are stable, even in the absence of ligand, and depend on the direct association of p140 and Sos respectively with the SH3 domains of Grb2. p140 is an uncharacterized protein constitutively phosphorylated on tyrosine and, in its Grb2-bound form, expressed only in hematopoietic cells, the oligomeric complex formed by phosphorylated beta subunit-phosphorylated Shc-Grb2-SoS-p140 is also induced by IL-3 and L-5 stimulation of growth-factor dependent cell lines. Overexpression of wild-type Shc proteins in growth factor-dependent cells increases both MAP kinase activation and proliferation in response to GM-CSF. These effects require the association of Shc with Grb2. Taken together these results indicate that phosphorylation of Shc proteins is a crucial step in the transmission of GM-CSF proliferative stimuli, since it creates a high affinity binding site for the Grb2/SoS complex, whose function is to activate Ras and, for the Grb2/p140 complex, whose function remains unknown.

Adaptor Proteins, Signal Transducing↗

PubMed1993

Transcription of genes encoding granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 6 receptors and lack of proliferative response to exogenous cytokines in nonhematopoietic human malignant cell lines.

Studies in recent years have suggested that human tumor cell lines are capable of responding in vitro to hematopoietic growth factors. In the present study, we investigate the transcription of the alpha and beta subunits of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the alpha and beta subunits of interleukin 3 (IL-3) receptor, and the single subunit of interleukin 6 (IL-6) receptor and its associated gp130 transduction protein by PCR amplification of reverse-transcribed cellular mRNA in 34 malignant cell lines derived from a variety of histological cell types. mRNA for only a single subunit polypeptide was found in a significant minority of cell lines (23%), while in 20% both the alpha and beta subunits of either the GM-CSF receptor or the IL-3 receptor were detected among a number of different histological cell types. Transcription of the gene encoding the IL-6 receptor was found in 38% of cell lines, and all lines transcribed the gp130 transduction protein, consistent with previous observations on the ubiquity of that polypeptide. In order to test the in vitro effect of exogenously added growth factors on those malignant cell lines transcribing complete cytokine receptor, either GM-CSF, IL-3, or IL-6 was added in therapeutic concentrations (20-500 ng/ml) and cellular proliferation was measured by incorporation of [3H]thymidine. No stimulation was seen at either 3 and 6 days of culture. Production of cytokine by these cell lines was investigated at the level of transcription and by assay of peptide product. None transcribed mRNA for either GM-CSF or IL-3, while 5 of 6 (STD, DOZ, ADE, Hep-2, and Detroit) expressed IL-6 mRNA. Of these latter, 2 cell lines (ADE and Hep-2) produced IL-6 as determined by bioassay, while none produced GM-CSF or IL-3 by enzyme-linked immunosorbent assay. This suggests that in the case of GM-CSF and IL-3, failure to proliferate on addition of cytokine is not due to the prior presence of endogenous production. In contrast, at least a subset of malignant cell lines may involve a closed IL-6 autocrine loop saturating cell surface sites. These findings suggest that the ability to transcribe the genes encoding cytokine receptor is by itself insufficient to render cells cytokine responsive and that malignant cells may lack the cellular machinery for cytokine-induced proliferation. This in turn suggests that therapeutic administration of either GM-CSF, IL-3, or IL-6 may involve no additional risk of tumor regrowth in vivo.

Base Sequence↗

PubMed1993

Granulocyte-macrophage colony-stimulating factor receptors alter their binding characteristics during myeloid maturation through up-regulation of the affinity converting beta subunit (KH97).

Acute myeloid leukemia blasts express dual affinity (high and low) granulocyte-macrophage colony-stimulating factor (GM-CSF) binding, and the high affinity GM-CSF binding is counteracted by excess interleukin-3 (IL-3). Neutrophils express a single class of GM-CSF-R with intermediate affinity that lack IL-3 cross-reactivity. Here we demonstrate the differentiation associated changes of GM-CSF binding characteristics in three models representative of different stages of myeloid maturation. We find that high affinity GM-CSF binding is converted into intermediate affinity binding, which still cross-reacts with IL-3, beyond the stage of promyelocytes. During terminal maturation towards neutrophils, IL-3 cross-reactivity is gradually lost. We sought to determine the mechanism underlying the affinity conversion of the GM-CSF-R. Northern and reverse transcriptase-polymerase chain reaction analysis of GM-CSF-R alpha and -beta c (KH97) transcripts did not provide indications for the involvement of GM-CSF-R splice variants in the formation of the intermediate affinity GM-CSFR complex. In COS-cell transfectants with increasing amounts of beta c in the presence of a fixed number of GM-CSF-R alpha chains, the high affinity GM-CSF binding converted into intermediate affinity GM-CSF binding. These results are discussed in view of the concept that increasing expression of beta c subunits may cause alternative oligomerization of the GM-CSF-R alpha and -beta c subunits resulting in the formation of intermediate rather than high affinity GM-CSFR alpha.beta c complexes.

Acute Disease↗

PubMed1996

IL-13 released by and localized in human basophils.

We and others have shown that human basophils can synthesize and release IL-4. However, IL-13, a cytokine that closely resembles IL-4, has not hitherto been described as a basophil product. The production of IL-13 by basophils was demonstrated by immunocytochemistry. Approximately 70% of basophils stimulated with anti-FcepsilonRIalpha (antibody to the alpha subunit of IgE receptor type I) stained for IL-13. Under similar experimental conditions, mononuclear cells failed to stain for IL-13. The cytokine was localized to basophilic granules by electron microscopic examination of immunogold staining. The secretion of IL-13 into the culture supernatant was assayed by ELISA. Kinetic studies showed detectable IL-13 release at 3 h, which steadily increased up to 24 h. This is significantly different from the kinetics of basophil histamine and IL-4 release. IL-13 production was also observed upon stimulation with anti-IgE, anti-FcepsilonRIalpha, IL-3, and A23187 in a dose-dependent manner. PBMC, neutrophils, and eosinophils isolated from the same donors did not release IL-13 after anti-IgE stimulation. The anti-IgE-induced basophil IL-13 synthesis could be enhanced by IL-3 preincubation (with and without IL-3 preincubation, anti-IgE-induced IL-13 production was 227 +/- 99 and 42 +/- 13 pg/10(6) basophils, respectively). PBMC produced a significant amount of IL-1 3 upon stimulation with PHA, but a low level of IL-13 in response to A23187 and/or PMA. Eosinophils and neutrophils did not produce IL-13 when cultured with A23187, IL-5, and anti-FcepsilonRIalpha. This is the first demonstration of IL-1 3 production by basophils. Our data suggest that basophils, in addition to secreting mediators, can represent an important source of proallergic cytokines.

Basophils↗

PubMed1996

The production of macrophage inflammatory protein-1 alpha by human basophils.

Macrophage inflammatory protein-1alpha (MIP-1alpha) has previously been shown to be produced by mononuclear cells, eosinophils, and neutrophils. Its production by basophils has not been investigated. The objective of this study was to investigate the production of MIP-1alpha by basophils. Peripheral blood basophils were separated by Percoll gradient centrifugation, cultured overnight, and processed for double immunocytochemistry using Abs against MIP-1alpha and FcepsilonRIalpha (alpha subunit of IgE receptor type 1). We demonstrated that basophils expressed immunoreactive MIP-1alpha upon stimulation with anti-IgE. Less than 5% of the basophils stained for MIP-1alpha without stimulation. The secretion of MIP-1alpha by basophils was studied by ELISA. In these experiments, basophils were further enriched to 65 to 99% (median, 86%) by a negative selection method. Basophils released MIP-1alpha when stimulated by Abs against IgE and FCepsilonRIalpha as well as IL-3 and the calcium ionophore, A23187. In parallel experiments, PBMC, eosinophils, and neutrophils did not produce MIP-1alpha in response to anti-IgE, but they did so in response to A23187. No MIP-1alpha release was detected in platelet preparations. Preincubation with IL-3 (15 min or 18 h) augmented anti-IgE-included basophil MIP-1alpha production. The secretion of MIP-1alpha by basophils was detectable shortly after stimulation and gradually increased over 24 h. Since MIP-1alpha has potent inflammatory and histamine-releasing activities, its production by basophils may indicate a positive feedback mechanism for allergic inflammation.

Antibodies, Anti-Idiotypic↗