Human Granulocyte-macrophage Colony-stimulating Factor Receptor Research Articles

Characterization of the Role of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor α Subunit in the Activation of JAK2 and STAT5

The high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) consists of an alpha (GMRα) and a common beta (βc) subunit. The intracellular domain of βc has been extensively characterized and has been shown to be critical for the activation of both the JAK/STAT and MAP kinase pathways. The function of the intracellular domain of GMRα, however, is not as well characterized. To determine the role of this domain in GMR signaling, an extensive structure-function analysis was performed. Truncation mutants α362, α371, and α375 were generated, as well as the site-directed mutants αVQVQ and αVVVV. Although α375β, αVQNQβ, and αVVVVβ stimulated proliferation in response to human GM-CSF, the truncation mutants α362β and α371β were incapable of transducing a proliferative signal. In addition, both α371 and αVVVV were expressed at markedly reduced levels, indicating the importance of residues 372 to 374 for proper protein expression. More importantly, we show that GMRα plays a direct role in the activation of the JAK/STAT pathway, and electrophoretic mobility shift assays (EMSA) indicate that both GMRα and βc play a role in determining the STAT5 DNA binding complex activated by the GMR. Thus, the intracellular domain of the human GMRα is important for activation of the JAK/STAT pathway and protein stabilization.© 1998 by The American Society of Hematology.

Characterization of the Role of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor α Subunit in the Activation of JAK2 and STAT5

AbstractThe high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) consists of an alpha (GMRα) and a common beta (βc) subunit. The intracellular domain of βc has been extensively characterized and has been shown to be critical for the activation of both the JAK/STAT and MAP kinase pathways. The function of the intracellular domain of GMRα, however, is not as well characterized. To determine the role of this domain in GMR signaling, an extensive structure-function analysis was performed. Truncation mutants α362, α371, and α375 were generated, as well as the site-directed mutants αVQVQ and αVVVV. Although α375β, αVQNQβ, and αVVVVβ stimulated proliferation in response to human GM-CSF, the truncation mutants α362β and α371β were incapable of transducing a proliferative signal. In addition, both α371 and αVVVV were expressed at markedly reduced levels, indicating the importance of residues 372 to 374 for proper protein expression. More importantly, we show that GMRα plays a direct role in the activation of the JAK/STAT pathway, and electrophoretic mobility shift assays (EMSA) indicate that both GMRα and βc play a role in determining the STAT5 DNA binding complex activated by the GMR. Thus, the intracellular domain of the human GMRα is important for activation of the JAK/STAT pathway and protein stabilization.© 1998 by The American Society of Hematology.

Cytoplasmic Domains of the Human Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Receptor β Chain (hβc) Responsible for Human GM-CSF-induced Myeloid Cell Differentiation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of myeloid progenitor cells. The biologic actions of GM-CSF are mediated by its binding to the alpha and beta subunits of the GM-CSF receptor (GM-CSFRalpha and betac, respectively). To determine whether identical regions of the betac protein mediate both cell growth and differentiation, we expressed cDNA constructs encoding the human wild-type (897 amino acids) and truncated betac (hbetac) subunits along with the wild-type human GM-CSFRalpha subunit in the murine WT19 cell line, an FDC-P1-derived cell line that differentiates toward the monocytic lineage in response to murine GM-CSF. Whereas the WT19 cell line carrying the C-terminal deleted hbetac subunit of 627 amino acids was still able to grow in human GM-CSF (hGM-CSF), 681 amino acids of the hbetac were necessary for cell differentiation. The addition of hGM-CSF to WT19 cell lines containing the hbetac627 subunit stimulated the phosphorylation of ERK (extracellular signal-regulated kinase) and induced the tyrosine-phosphorylation of SHP-2 and STAT5, suggesting that the activation of these molecules is insufficient to mediate the induction of differentiation. A point mutation of tyrosine 628 to phenylalanine (Y628F) within hbetac681 abolished the ability of hGM-CSF to induce differentiation. Our results indicate that the signals required for hGM-CSF-induced differentiation and cell growth are mediated by different regions of the hbetac subunit.

A Selective Switch-on System for Self-Renewal of Embryonic Stem Cells Using Chimeric Cytokine Receptors

Propagation of embryonic stem (ES) cells with an undifferentiated pluripotential phenotype depends on leukemia inhibitory factor (LIF). The LIF receptor complex is composed of a heterodimer of LIF receptor α (LIFRα) and gp130. To activate LIFR signaling pathways independently from endogenous ones, we constructed chimeric receptors by linking the extracellular domain of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α or β (hGMRα or β) to the transmembrane and cytoplasmic regions of either mouse LIFRα or gp130. hGMRα-mLIFR/ hGMRβ-mgp130 or hGMRα-mgp130/ hGMRβ-mgp130, but not hGMRα-mLIFR/ hGMRβ-mLIFR, preserved the self-renewal activity in A3 ES cells. All of these chimeric receptors were phosphorylated after hGM-CSF stimulation without phosphorylation of endogenous gp130. Phosphorylation of the signal transducer and activator of transcription 3 through chimeric receptors correlated with the undifferentiated phenotype. Therefore, these chimeric receptors prove useful to analyze mechanisms of the self-renewal of ES cells.

Stoichiometric structure-function analysis of the prolactin receptor signaling domain by receptor chimeras.

The intracellular domain of the prolactin (PRL) receptor (PRLr) is required for PRL-induced signaling and proliferation. To identify and test the functional stoichiometry of those PRLr motifs required for transduction and growth, chimeras consisting of the extracellular domain of either the alpha or beta subunit of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-CSFr) and the intracellular domain of the rat PRLr were synthesized. Because the high-affinity binding of GM-CSF results from the specific pairing of one alpha- and one beta-GM-CSFr, use of GM-CSFr/PRLr chimera enabled targeted dimerization of the PRLr intracellular domain. To that end, the extracellular domains of the alpha- and beta-GM-CSFr were conjugated to one of the following mutations: (i) PRLr C-terminal truncations, termed alpha278, alpha294, alpha300, alpha322, or beta322; (ii) PRLr tyrosine replacements, termed Y309F, Y382F, or Y309+382F; or, (iii) PRLr wild-type short, intermediate, or long isoforms. These chimeras were cotransfected into the cytokine-responsive Ba/F3 line, and their expression was confirmed by ligand binding and Northern and Western blot analyses. Data from these studies revealed that heterodimeric complexes of the wild type with C-terminal truncation mutants of the PRLr intracellular domain were incapable of ligand-induced signaling or proliferation. Replacement of any single tyrosine residue (Y309F or Y382F) in the dimerized PRLr complex resulted in a moderate reduction of receptor-associated Jak2 activation and proliferation. In contrast, trans replacement of these residues (i.e., alphaY309F and betaY382F) markedly reduced ligand-driven Jak2 activation and proliferation, while cis replacement of both tyrosine residues in a single intracellular domain (i.e., alphaY309+382F) produced an inactive signaling complex. Analysis of these GM-CSFr-PRLr complexes revealed equivalent levels of Jak2 in association with the mutant receptor chains, suggesting that the tyrosine residues at 309 and 382 do not contribute to Jak association, but instead to its activation. Heterodimeric pairings of the intracellular domains from the known PRLr receptor isoforms (short-intermediate, short-long, and intermediate-long) also yielded inactive receptor complexes. These data demonstrate that the tyrosine residues at 309 and 382, as well as additional residues within the C terminus of the dimerized PRLr complex, contribute to PRL-driven signaling and proliferation. Furthermore, these findings indicate a functional requirement for the pairing of Y309 and Y382 in trans within the dimerized receptor complex.

Definition of the role of tyrosine residues of the common beta subunit regulating multiple signaling pathways of granulocyte-macrophage colony-stimulating factor receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces various functions, including the proliferation and differentiation of a broad range of hematopoietic cells. We previously reported that at least two distinct pathways are involved in human GM-CSF receptor signaling; both require the box 1 region of the common beta subunit (beta c). This region is essential for the activation of JAK2, which is necessary for all the biological functions of GM-CSF. The activation of JAK2 by GM-CSF leads to rapid tyrosine phosphorylation of cellular proteins, including the beta c. However, the significance of beta c phosphorylation with regard to the regulation of signaling molecules and the expression of GM-CSF functions is less well understood. Here we investigated the role of the cytoplasmic tyrosine residues of the beta c by using a series of beta c mutants expressed in murine BA/F3 cells. A mutant beta c with all eight cytoplasmic tyrosines converted to phenylalanine (Fall) activated JAK2 but not SHP-2, MAPK cascades, STAT5, or the c-fos promoter in BA/F3 cells, and it did not effectively induce proliferation. Adding back each tyrosine to Fall revealed that Tyr577, Tyr612, and Tyr695 are involved in the activation of SHP-2, MAPK cascades, and c-fos transcription, while every tyrosine, particularly Tyr612, Tyr695, Tyr750, and Tyr806, facilitated STAT5 activation. Impaired growth was also restored, at least partly, by any of the tyrosines. These results provide evidence that beta c tyrosines possess distinct yet overlapping functions in activating multiple signaling pathways induced by GM-CSF.

Thymocyte proliferation and differentiation in human granulocyte-macrophage colony-stimulating factor receptor transgenic mice

Thymocytes display varying responses to cytokines depending on their stage of differentiation. Whether these responses are due to stage-specific cytokine receptor expression or to downstream signaling mechanisms is unknown. We examined the relationship between receptor expression and thymocyte proliferation or differentiation by using thymocytes from transgenic mice that constitutively expressed the human granulocyte-macrophage colony stimulating factor (hGM-CSF) receptor. Transgenic CD4-CD8-, CD4+CD8-, and CD4-CD8+ thymocyte populations expressing the hGM-CSF receptor proliferated when cultured with hGM-CSF, whereas CD4+CD8+ cells failed to proliferate despite expressing this receptor. We next examined the effect of hGM-CSF receptor signaling on thymocyte differentiation in fetal thymic organ culture supporting a full program of T cell development in vitro. Addition of hGM-CSF to the transgenic fetal thymic organ culture resulted in failure of CD4-CD8- cells to differentiate into CD4+CD8+ cells. To investigate this maturational inhibition more closely, we repopulated wild-type fetal lobes with sorted pro-T, pre-T or post pre-T precursor cells from hGM-CSF receptor transgenic mice. In these cultures hGM-CSF blocked both pro-T and pre-T cell differentiation, whereas the more mature post pre-T cells differentiated normally. These results suggest that hGM-CSF receptor signaling during thymocyte differentiation causes stage-specific inhibition of precursor cell maturation. In addition, repopulation studies of transgenic fetal lobes with sorted wild-type thymocyte precursors indicated that hGM-CSF inhibited proliferation and differentiation of the wild-type precursors, suggesting a secondary effect via transgenic stromal cells. (J Allergy Clin Immunol 1997;100:S87-96.)

The Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Exists as a Preformed Receptor Complex That Can Be Activated by GM-CSF, Interleukin-3, or Interleukin-5

AbstractThe granulocyte-macrophage colony-stimulating factor (GM-CSF ) receptor is expressed on normal and malignant hematopoietic cells as well as on cells from other organs in which it transduces a variety of functions. Despite the widespread expression and pleiotropic nature of the GM-CSF receptor, little is known about its assembly and activation mechanism. Using a combination of biochemical and functional approaches, we have found that the human GM-CSF receptor exists as an inducible complex, analogous to the interleukin-3 (IL-3) receptor, and also as a preformed complex, unlike the IL-3 receptor or indeed other members of the cytokine receptor superfamily. We found that monoclonal antibodies to the GM-CSF receptor α chain (GMRα) and to the common β chain of the GM-CSF, IL-3, and IL-5 receptors (βc ) immunoprecipitated both GMRα and βc from the surface of primary myeloid cells, myeloid cell lines, and transfected cells in the absence of GM-CSF. Further association of the two chains could be induced by the addition of GM-CSF. The preformed complex required only the extracellular regions of GMRα and βc , as shown by the ability of soluble βc to associate with membrane-anchored GMRα or soluble GMRα. Kinetic experiments on eosinophils and monocytes with radiolabeled GM-CSF, IL-3, and IL-5 showed association characteristics unique to GM-CSF. Significantly, receptor phosphorylation experiments showed that not only GM-CSF but also IL-3 and IL-5 stimulated the phosphorylation of GMRα-associated βc . These results indicate a pattern of assembly of the heterodimeric GM-CSF receptor that is unique among receptors of the cytokine receptor superfamily. These results also suggest that the preformed GM-CSF receptor complex mediates the instantaneous binding of GM-CSF and is a target of phosphorylation by IL-3 and IL-5, raising the possibility that some of the biologic activities of IL-3 and IL-5 are mediated through the GM-CSF receptor complex.

Expression and functional analysis of two isoforms of the human GM-CSF receptor alpha chain in myeloid development and leukaemia.

The human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha chain RNA is alternatively spliced to yield receptor isoforms. Two of these, alpha 1 and alpha 2, differ in their cytoplasmic domains. Because the GM-CSFR beta chain (beta c) is shared with the receptors for interleukins 3 and 5 it is possible that the alpha chain confers specificity on the GM-CSF response and that the different isoforms might refine this response further. Studies have been directed at determination of the respective biological roles of the alpha 1 and alpha 2 isoforms. Expression of the isoforms was examined by RNase protection analysis in normal granulocytes and a variety of cell lines of haemopoietic origin, at different stages of differentiation and activation. Expression was also analysed in cells from patients with a variety of leukaemic subtypes. Results demonstrated that the relative abundance of the isoforms was similar in all cell populations examined. The human GM-CSFR alpha 1 or alpha 2 receptors were independently expressed in the murine factor-dependent cell line FDC-P1, so that the properties of the receptors could be compared. Cell lines that expressed either receptor could be converted to growth in response to human GM-CSF and assumed a more differentiated phenotype when compared with the parental cell line. However, the morphology, expression of cell surface antigens and dose-growth response characteristics did not differ significantly between cells that expressed either the alpha 1 or alpha 2 receptor. These studies demonstrate that the alpha 1 and alpha 2 subunits of the GM-CSF receptor are co-ordinately regulated in both normal and malignant haemopoiesis. Furthermore, each receptor is able to deliver both proliferative and differentiative signals to myeloid cells.

Hematopoietic and Lymphopoietic Responses in Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Transgenic Mice Injected With Human GM-CSF

Using a clonal assay of bone marrow (BM) cells from transgenic mice (Tg-mice) expressing the human granulocyte-macrophage colony-stimulating factor receptor (hGM-CSFR), we found in earlier studies that hGM-CSF alone supported the development not only of granulocyte-macrophage colonies, but also of erythrocytes, megakaryocytes, mast cells, blast cells, and mixed hematopoietic colonies. In this report, we evaluated the in vivo effects of hGM-CSF on hematopoietic and lymphopoietic responses in the hGM-CSFR Tg-mice. Administration of this factor to Tg-mice resulted in dose-dependent increases in numbers of reticulocytes and white blood cells (WBCs) in the peripheral blood. Morphological analysis of WBCs showed that the numbers of all types of the cell, including neutrophils, eosinophils, monocytes, and lymphocytes increased; the most remarkable being in lymphocytes that contained a number of large granular lymphocytes (LGLs) in addition to mature T and B cells. However, total cellularity of the BM of the Tg-mice decreased in a dose-dependent manner when hGM-CSF was injected. In sharp contrast to the BM, spleens of the Tg-mice were grossly enlarged. Although all types of blood cells and hematopoietic progenitors increased in the spleen, erythroid cells and their progenitors showed the most significant increase. Increased numbers of megakaryocytes and LGLs were also observed in spleen and liver of the treated Tg-mice. Flow cytometric analysis showed that LGLs expanded in Tg-mice expressed Mac-1+ CD3- NK1.1+. The thymus of Tg-mice treated with hGM-CSF exhibited a dose-dependent shrinkage and a remarkable decrease in CD4+ CD8+ cells. Thus, hGM-CSF stimulated not only myelopoiesis but also erythropoiesis and megakaryopoiesis of hGM-CSFR Tg-mice in vivo, in accordance with our reported in vitro findings. In addition, hGM-CSF affected the development of lymphoid cells, including natural killer cells of these Tg-mice.

Hematopoietic and Lymphopoietic Responses in Human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF ) Receptor Transgenic Mice Injected With Human GM-CSF

AbstractUsing a clonal assay of bone marrow (BM) cells from transgenic mice (Tg-mice) expressing the human granulocyte-macrophage colony-stimulating factor receptor (hGM-CSFR), we found in earlier studies that hGM-CSF alone supported the development not only of granulocyte-macrophage colonies, but also of erythrocytes, megakaryocytes, mast cells, blast cells, and mixed hematopoietic colonies. In this report, we evaluated the in vivo effects of hGM-CSF on hematopoietic and lymphopoietic responses in the hGM-CSFR Tg-mice. Administration of this factor to Tg-mice resulted in dose-dependent increases in numbers of reticulocytes and white blood cells (WBCs) in the peripheral blood. Morphological analysis of WBCs showed that the numbers of all types of the cell, including neutrophils, eosinophils, monocytes, and lymphocytes increased; the most remarkable being in lymphocytes that contained a number of large granular lymphocytes (LGLs) in addition to mature T and B cells. However, total cellularity of the BM of the Tg-mice decreased in a dose-dependent manner when hGM-CSF was injected. In sharp contrast to the BM, spleens of the Tg-mice were grossly enlarged. Although all types of blood cells and hematopoietic progenitors increased in the spleen, erythroid cells and their progenitors showed the most significant increase. Increased numbers of megakaryocytes and LGLs were also observed in spleen and liver of the treated Tg-mice. Flow cytometric analysis showed that LGLs expanded in Tg-mice expressed Mac-1+CD3−NK1.1+. The thymus of Tg-mice treated with hGM-CSF exhibited a dose-dependent shrinkage and a remarkable decrease in CD4+CD8+ cells. Thus, hGM-CSF stimulated not only myelopoiesis but also erythropoiesis and megakaryopoiesis of hGM-CSFR Tg-mice in vivo, in accordance with our reported in vitro findings. In addition, hGM-CSF affected the development of lymphoid cells, including natural killer cells of these Tg-mice.

The cytoplasmic domain of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha subunit is essential for both GM-CSF-mediated growth and differentiation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage progenitor cells. The biologic actions of GM-CSF are mediated by binding to a specific receptor consisting of two chains designated as alpha and beta subunits. We have demonstrated that the murine FDC-P1-derived cell line WT-19 transfected with the human GM-CSF receptor alpha and beta subunits (GM-CSFRalpha and beta) can be induced to differentiate by the addition of human GM-CSF (hGM-CSF). By expressing a series of GM-CSFRalpha mutants in WT19 cells, we have determined the amino acid domains of the GM-CSFRalpha cytoplasmic domain that regulate cell differentiation, proliferation, and survival. We found that the membrane proximal proline-rich domain and adjacent 16 residues are essential for both hGM-CSF-dependent cell proliferation and differentiation. In contrast, the C-terminal region of the GM-CSFRalpha cytoplasmic domain was not necessary for cell differentiation mediated by hGM-CSF, but the removal of this region severely impaired the ability of hGM-CSF to support cell survival. While the activation of JAK2, Shc, Erk, and STAT5 proteins correlated with hGM-CSF-mediated cell growth, cellular differentiation occurred in the absence of activation of these signal transduction pathways.

Recombinant Toxins Containing Human Granulocyte-Macrophage Colony-Stimulating Factor and Either Pseudomonas Exotoxin or Diphtheria Toxin Kill Gastrointestinal Cancer and Leukemia Cells

The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is a potential target for toxin-directed therapy, because it is overexpressed on many leukemias and solid tumors and apparently not on stem cells. To investigate the potential therapeutic use of GM-CSF toxins, we fused human GM-CSF to truncated forms of either Pseudomonas exotoxin (PE) or diphtheria toxin (DT) and tested the cytotoxicity of the resulting GM-CSF–PE38KDEL and DT388–GM-CSF on human gastrointestinal (GI) carcinomas and leukemias. Toward gastric and colon cancer cell lines, GM-CSF–PE38KDEL was much more cytotoxic than DT388–GM-CSF, with IC50s (concentration resulting in 50% inhibition of protein synthesis) of 0.5 to 10 ng/mL compared with 4 to 400 ng/mL, respectively. In contrast, toward leukemia lines and fresh bone marrow cells DT388–GM-CSF was more cytotoxic than GM-CSF–PE38KDEL. The cytotoxicity of both GM-CSF–PE38KDEL and DT388–GM-CSF toward the human cells was specific, because it could be competed by an excess of GM-CSF. Binding studies indicated that human GM-CSF receptors were present on all of the human GI and leukemic cell lines tested, at levels of 540 to 3,700 sites per cell (kd = 0.2 to 2 nmol/L), and the number of sites per cell did not correlate with the cell type. A similar pattern of cytotoxicity was found with recombinant immunotoxins binding to the transferrin receptor, in that anti-TFR(Fv)–PE38KDEL was much more cytotoxic than DT388–anti-TFR(Fv) toward GI cells, but both were similar in their cytotoxic activity toward leukemia cells. The fact that PE is more effective than DT in killing GI but not leukemic tumor cells targeted by GM-CSF indicates a fundamental difference in the way PE or DT gains access to the cytosol in these cells. GM-CSF–PE38KDEL and DT388–GM-CSF deserve further evaluation as possible treatments for selected tumors.

Recombinant Toxins Containing Human Granulocyte-Macrophage Colony-Stimulating Factor and Either Pseudomonas Exotoxin or Diphtheria Toxin Kill Gastrointestinal Cancer and Leukemia Cells

AbstractThe granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is a potential target for toxin-directed therapy, because it is overexpressed on many leukemias and solid tumors and apparently not on stem cells. To investigate the potential therapeutic use of GM-CSF toxins, we fused human GM-CSF to truncated forms of either Pseudomonas exotoxin (PE) or diphtheria toxin (DT) and tested the cytotoxicity of the resulting GM-CSF–PE38KDEL and DT388–GM-CSF on human gastrointestinal (GI) carcinomas and leukemias. Toward gastric and colon cancer cell lines, GM-CSF–PE38KDEL was much more cytotoxic than DT388–GM-CSF, with IC50s (concentration resulting in 50% inhibition of protein synthesis) of 0.5 to 10 ng/mL compared with 4 to 400 ng/mL, respectively. In contrast, toward leukemia lines and fresh bone marrow cells DT388–GM-CSF was more cytotoxic than GM-CSF–PE38KDEL. The cytotoxicity of both GM-CSF–PE38KDEL and DT388–GM-CSF toward the human cells was specific, because it could be competed by an excess of GM-CSF. Binding studies indicated that human GM-CSF receptors were present on all of the human GI and leukemic cell lines tested, at levels of 540 to 3,700 sites per cell (kd = 0.2 to 2 nmol/L), and the number of sites per cell did not correlate with the cell type. A similar pattern of cytotoxicity was found with recombinant immunotoxins binding to the transferrin receptor, in that anti-TFR(Fv)–PE38KDEL was much more cytotoxic than DT388–anti-TFR(Fv) toward GI cells, but both were similar in their cytotoxic activity toward leukemia cells. The fact that PE is more effective than DT in killing GI but not leukemic tumor cells targeted by GM-CSF indicates a fundamental difference in the way PE or DT gains access to the cytosol in these cells. GM-CSF–PE38KDEL and DT388–GM-CSF deserve further evaluation as possible treatments for selected tumors.

Intrathymic T cell development in human GM-CSF receptor transgenic mice is blocked by human GM-CSF

Intrathymic T cell development in human GM-CSF receptor transgenic mice is blocked by human GM-CSF

Intrathymic T cell development in human GM-CSF receptor transgenic mice is blocked by human GM-CSF

Intrathymic T cell development in human GM-CSF receptor transgenic mice is blocked by human GM-CSF

OLIGOMERIZATION OF THE SOLUBLE GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR: IDENTIFICATION OF THE FUNCTIONAL LIGAND-BINDING SPECIES

The ligand-specific α subunit of the dimeric human GM-CSF receptor exists in both transmembrane anchored (tmGM-CSFRα) and soluble (sGM-CSFRα) isoforms. sGM-CSFRα binds to GM-CSF in solution and antagonizes GM-CSF biological activity in vitro. In an effort to better understand the biological properties of sGM-CSFRα the authors have attempted to define the exact stoichiometry of the interaction between GM-CSF and sGM-CSFRα. Size separation of sGM-CSFRα by polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions demonstrated that sGM-CSFRα can exist in solution not only in a monomeric state but also in higher order oligometers. FPLC analysis of ligand/sGM-CSFRα complexes suggested that only one of these sGM-CSFRα species could functionally bind GM-CSF. PAGE analysis of FPLC fractions demonstrated that the peak of GM-CSF binding activity corresponded to the presence of a monomeric form of sGM-CSFRα. The experiments demonstrate that while sGM-CSFRα can adopt oligomeric forms in solution, the binding of GM-CSF to sGM-CSFRα most likely occurs in a (GM-CSF)1(sGM-CSFRα)1configuration.

A chimeric serine/threonine kinase receptor system reveals the potential of multiple type II receptors to cooperate with transforming growth factor-beta type I receptor.

Receptor-type serine/threonine kinases (RSKs) have been organized into two distinct classes known as types I and II on the basis of sequence similarity. However, experiments have shown ligand specificities in the two classes and as a result type I and type II receptors can often bind to a common ligand. The transforming growth factor-beta- (TGF-beta) specific receptors represent such a case, where both type I and II receptors (T beta RI and T beta RII) are observed. Of additional interest is the observation that heteromeric associations of type I and II receptors can also enable signaling. To further elucidate the function of various RSKs, the extracellular domains of both alpha and beta chains from human granulocyte-macrophage colony-stimulating factor receptors were linked to transmembrane cytoplasmic domains of RSKs. Chimeric receptors of human granulocyte-macrophage receptor (hGMR) alpha with T beta RI and hGMR beta with T beta RII were expressed in murine pre-B cell-derived Ba/F3 cells. These chimeras formed heteromeric complexes, transmitted TGF-beta signals, and were down-modulated in response to human granulocyte-macrophage colony-stimulating factor. However, experiments utilizing these chimeric receptors in different combinations revealed that only heteromeric associations of transmembrane cytoplasmic domains mediated signaling and down-modulation. Chimeric receptors with transmembrane cytoplasmic domains of activin receptor type II and bone morphogenetic protein receptor type II also provided signals in conjunction with chimeric T beta RI. As a result, these type II receptors may share a common potential to signal via T beta RI. hGMR-RSK chimeric receptors may be useful tools for the identification and characterization of the divergent signals mediated by individual RSKs.

Human Granulocyte-Macrophage Colony-Stimulating Factor (hGM-CSF) Induces Inhibition of Intrathymic T-Cell Development in hGM-CSF Receptor Transgenic Mice

AbstractThymocytes show differential cytokine responses, depending on the stage of differentiation. Whether these responses are due to preferential cytokine receptor expression or due to downstream signaling mechanisms is unknown. In this study, we examined the relationship between receptor expression and T-cell proliferation or differentiation using thymocytes from transgenic mice constitutively expressing the human granulocyte-macrophage colony-stimulating factor (hGM-CSF ) receptor. Transgenic CD4−CD8−, CD4+CD8−, and CD4−CD8+ cells proliferated when cultured with hGM-CSF in vitro, whereas CD4+CD8+ cells failed to proliferate. To examine the effect of hGM-CSF receptor signaling on T-cell development, we used fetal thymic organ cultures. The addition of exogenous hGM-CSF resulted in the failure of CD4−CD8− cells to differentiate into CD4+CD8+ cells. To more closely identify this maturational inhibition, we reconstituted normal fetal lobes with sorted pro-T–, pre-T–, or post-pre-T–precursor cells from transgenic mice. The addition of hGM-CSF to these cultures led to a block in both pro-T– and pre-T–cell differentiation, whereas the more mature post-pre-T cells differentiated normally. We propose that hGM-CSF receptor signaling during T-cell development results in a stage-specific inhibition of thymic precursor maturation.

A Recombinant fusion toxin targeted to the granulocyte-Macrophage colony-Stimulating factor receptor

Human granulocyte-macrophage colony stimulating factor (GMCSF) and its high affinity receptor function to regulate the proliferation and differentiation of myeloid lineage hematopoietic cells, and may participate in the pathogenesis of many malignant myeloid diseases. We have used genetic engineering based on the elucidated molecular structures of human granulocyte-macrophage colony-stimulating factor and diphtheria toxin (DT) to pro-duce a recombinant fusion toxin, DTctGMCSF, that targets diphtheria toxin to high affinity GMCSF receptors expressed on the surface of blast cells from a large fraction of patients with acute myeloid leukemia (AML). DTctGMCSF was specifically immunoreactive with anti-diphtheria toxin and anti-GMCSF antiseras, and exhibited the characteristic catalytic activity of diphtheria toxin, catalyzing the in vitro ADP-ribosylation of purified elongation factor 2. The cytotoxic effects of DTctGMCSF were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium (MTT) bromide assay of cell viability and in vivo assays of protein synthesis inhibition. DTctGMCSF were specifically cytotoxic to human leukemia cell lines bearing high affinity receptors for human GMCSF with IC50 of 10−9 to 10−11M. It was not toxic to mammalian hematopoietic cell lines lacking human GMCSF (hGMCSF) receptors. In receptor positive cells, cytotoxicity can be specifically blocked by a large excess of hGMCSF, confirming that its cytotoxicity is mediated through the hGMCSF receptor. Though DTctGMCSF inhibited granulocyte-macrophage colony formation by committed myeloid progenitor cells (CFU-GM), it did not significantly affect erythroid burst formation by committed erythroid progenitor cells (BFU-E), or mixed granulocyte-erythroid-macrophage-megakaryocyte colony formation by pluripotent multilineage progenitor cells (CFU-GEMM). DTctGMCSF holds promise for the treatment of myeloid lineage malignancies, and is a useful reagent to study hematopoiesis.