The effect of human recombinant interleukin 4 (IL-4) on antibody production by normal peripheral blood mononuclear cells enriched for B cells was investigated.

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Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, we obtained a homologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-afflity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of '12I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells-i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the highaffinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. We therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the a and ( subunits of the GM-CSF receptor, respectively.

Cytokines play a vital role in coordinating immune and inflammatory responses. Unlike growth factor receptors with a tyrosine kinase, cytokine receptors have no intrinsic tyrosine kinase activity. Based on their structure, cytokine receptors are classified into several groups. High affinity receptors for IL-2, IL-3, IL-5, IL-6, and GM-CSF are composed of at least two distinct subunits, alpha and beta. The alpha subunits are primary cytokine binding proteins, and the beta subunits are required for formation of high affinity binding sites as well as for signal transduction. The GM-CSF, IL-3, and IL-5 receptors appear to share the same beta subunit in human, and therefore cross-talk among these cytokines may occur at the receptor level. High affinity receptors presumably are linked to various signal transduction pathways that lead to different cytokine functions. Differential expression of the cytokine receptors as well as reorganization of intracellular signalling pathways are critical for development of hemopoietic cells.

Interleukin 3 (IL‐3) and granulocyte‐macrophage colony stimulating factor (GM‐CSF) exert their biological functions through acting on a specific receptor which consists of a ligand‐specific alpha subunit and the shared common beta subunit. Inhibition by genistein of a subset of IL‐3/GM‐CSF‐mediated signals, including c‐myc induction, resulted in the abrogation of DNA synthesis, however, IL‐3 still protected cells from apoptotic cell death. Conversely, a C‐terminal truncated form of the GM‐CSF receptor, which is missing a critical cytoplasmic region required for activation of the Ras/Raf‐1/MAP kinase pathway, induced DNA synthesis, but failed to prevent cell death in response to GM‐CSF. Consequently, cells died by apoptosis in the presence of GM‐CSF, despite displaying a transient mitogenic response. However, expression of activated Ras protein complemented defective signalling through the mutant receptor and supported long‐term proliferation in concert with GM‐CSF. These results indicate that IL‐3 and GM‐CSF prevent apoptosis of hematopoietic cells by activating a signalling pathway distinct from the induction of DNA synthesis and that long‐term cell proliferation requires the activation of both pathways.

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The high‐affinity receptors for granulocyte‐macrophage colony stimulating factor (GM‐CSF), interleukin 3 (IL‐3) and IL‐5 consist of two subunits, alpha and beta. The alpha subunits are specific to each cytokine and the same beta subunit (beta c) is shared by these three receptors. Although none of these receptor subunits has intrinsic kinase activity, these cytokines induce protein tyrosine phosphorylation, activation of Ras, Raf‐1 and MAP kinase, and transcriptional activation of nuclear proto‐oncogenes such as c‐myc, c‐fos and c‐jun. In this paper, we describe a detailed analysis of the signaling potential of the beta c subunit by using a series of cytoplasmic deletion mutants. The human beta c consists of 881 amino acid residues. A C‐terminal deletion mutant of beta c at amino acid 763 (beta 763) induced phosphorylation of Shc and activation of Ras, Raf‐1, MAP kinase and p70 S6 kinase, whereas a deletion at amino acid 626 (beta 626) induced none of these effects. The beta 763 mutant, as well as the full‐length beta c, induced transcription of c‐myc, c‐fos and c‐jun. Deletions at amino acid 517 (beta 517) and 626 (beta 626) induced c‐myc and pim‐1, but no induction of c‐fos and c‐jun was observed. GM‐CSF increased phosphatidylinositol 3 kinase (PI3‐K) activity in anti‐phosphotyrosine immunoprecipitates from cells expressing beta 763 as well as beta c, whereas it was only marginally increased from cells expressing beta 517 or beta 626. Thus, there are at least two distinct regions within the cytoplasmic domain of beta c that are responsible for different signals, i.e. a membrane proximal region of approximately 60 amino acid residues upstream of Glu517 is essential for induction of c‐myc and pim‐1, and a distal region of approximately 140 amino acid residues (between Leu626 and Ser763) is required for activation of Ras, Raf‐1, MAP kinase and p70 S6 kinase, as well as induction of c‐fos and c‐jun.

HTLV‐I transformed T cells not only express a large number of interleukin‐2 receptors [IL‐2R/p55(Tac)], but also produce a factor named ATL‐derived factor (ADF) that augments the expression of IL‐2R/p55(Tac). Based on a partial N‐terminal amino acid sequence, complementary DNA (cDNA) clones for human and mouse ADF were isolated and sequenced. Recombinant ADF produced by COS‐7 monkey kidney cells showed IL‐2R/Tac inducing activity on YT cells, which are sensitive for ADF. ADF mRNA was strongly expressed in HTLV‐I(+) T cells lines, but not in inactivated cells (THP‐1, unstimulated PBMC). Furthermore, in normal human peripheral blood mononuclear cells, the expression of ADF mRNA was enhanced by mitogens or phorbol myristate acetate, suggesting a possible involvement of ADF in the lymphocyte activation. Homology analysis revealed an unexpected relationship between ADF and dithiol‐reducing enzyme, thioredoxin, involved in many important biological reactions such as the conversion of ribonucleotides into deoxyribonucleotides, or the stabilization of glucocorticoid receptors. The biological significance of the generation of a redox potential in lymphocyte activation, and the possible involvement of dithiol reduction in the induction of IL‐2R/Tac are discussed.