Colony-stimulating Factor Receptor Alpha Research Articles

Tu1747 - Reduced Neutrophil Granulocyte-Macrophage Colony Stimulating Factor Signaling is Associated with Granulocyte-Macrophage Colony Stimulating Factor Receptor Alpha Chain Gene Mutations and Stricturing Disease Complications in Pediatric Crohn Disease

Tu1747 - Reduced Neutrophil Granulocyte-Macrophage Colony Stimulating Factor Signaling is Associated with Granulocyte-Macrophage Colony Stimulating Factor Receptor Alpha Chain Gene Mutations and Stricturing Disease Complications in Pediatric Crohn Disease

EPA-0763 – IL3R gene RS6603272 variant and CSFR2A gene RS4129148 variant in patients with schizophrenia

Schizophrenia has genetic, epigenetic and environmental components. Recent reports showed that the IL3 gene, colony stimulating factor 2 receptor alpha (CSF2RA), beta (CSF2RB) and IL-3 receptor alpha (IL3RA), the IL-specific receptor subunits for CSF2 and IL3, respectively, are associated with schizophrenia. Association analyses of rs6603272 variant of the IL3R gene and rs4129148 variant of CSFR2A gene with schizophrenia have been studied. We had 166 schizophrenic patients and 246 healthy controls. In both case control studies, a PCR-RFLP method was used. The genoype frequencies of the IL3R gene rs6603272 variant were TT, 54.8, TG, 40.4, GG, 4.8 % in cases, TT, 57.7, TG, 36.2 and GG, 6.1% in controls. The frequency of T allele in controls and cases was 76% and 75% respectively. Similarly, the genotype frequencies of the CSFR2A gene rs4129148 variant were GG, 44.6; GC,39.8; CC,15.7 % in cases and GG,43.9; GC,43.1; and CC, 13.0% in controls. The G allele frequency was 64% in cases and 65% in controls. In our study, we did not find an association between the rs66033272 variant of IL3RA gene and schizophrenia (χ2=0.896; P=0.639). Nor did we find an association between the CSFR2A rs4129148 variant and schizophrenia (χ2= 0.770; P=0.681)

De novo complex X chromosome rearrangement unmasking maternally inherited CSF2RA deletion in a girl with pulmonary alveolar proteinosis

We report on a 3-year-old girl with a de novo complex X chromosome rearrangement associated with congenital pulmonary alveolar proteinosis (PAP) and short stature. Array comparative genome hybridization and FISH analyses contributed to characterize the complex rearrangement consisting of a 7.37 Mb terminal deletion of Xp22.33p22.2, a 17.3 Mb interstitial inverted duplication of Xp22.2p21.3, and a 10.14 Mb duplication of Xq27.3q28. PCR analysis of microsatellite markers supported a paternal origin of the X chromosome rearrangement. A pre-meiotic two-step mechanism may explain the occurrence of this complex X rearrangement: an inverted duplication deletion event on Xp, and duplication of the Xq27.3qter region through a telomere capture event stabilizing the broken chromosome Xp end. The girl has also inherited from her healthy mother an X chromosome with a colony stimulating factor 2 receptor, alpha (CSF2RA) gene deletion. Consistent with the recessive mode of inheritance, the de novo paternal Xp22.33p22.2 deletion combined to the maternally inherited CSF2RA gene deletion led to homozygous deletion of CSF2RA and PAP diagnosis in the girl. The Xp deletion encompasses the pseudoautosomal region 1 (PAR1) which contains genes that escape X inactivation. Short stature homeobox (SHOX) haploinsufficiency explains growth retardation. Absence of other symptoms in relation to the X deletion/amplification is most probably due to skewed X inactivation. Finally, inherited deletions may unmask rare pathogenic genomic rearrangement and contribute to clinical phenotypes by a recessive mode of gene action.

A family-based study of the IL3RA gene on susceptibility to schizophrenia in a Chinese Han population

Schizophrenia has been observed to be associated with various abnormalities in cytokines and cytokine receptors that have been one of the recent focal points of immunological research in schizophrenia. Recent reports have showed that IL-3 gene, colony stimulating factor 2 receptor alpha (CSF2RA) and IL-3 receptor alpha (IL3RA) are associated with schizophrenia. The aim of this study was to investigate IL3RA gene variants in schizophrenia among a Chinese population by using a family-based association approach. Our sample included 101 Chinese parent–offspring trios of Han descent. All subjects were genotype for IL3RA-rs6603272 and -rs6645249 using PCR techniques. Single marker analysis showed a significant association for rs6603272 (X2=5.15, df=1, P=0.023), but not for the rs6645249. However, there was a significant genotypic association of both the polymorphisms with schizophrenia (for rs6603272, X2=6.15, df=2, P=0.046; for rs6645249, X2=21.79, df=2, P=1.85e-005). Haplotype TDT was statistically significant (X2=5.14, df=1, P=0.023), with the rs6603272(T)–rs6645249(G) haplotype significantly associated with schizophrenia (OR=1.66; 95% CI=1.08–2.55). In conclusion, our family-based association study also revealed a small but significant contribution of the IL3RA variants to susceptibility to schizophrenia in a Chinese population.

Association between interleukin-3 receptor alpha polymorphism and schizophrenia in the Chinese population

Schizophrenia has been observed to be associated with various abnormalities in cytokines and cytokine receptors. Three very recent reports showed the evidence that the IL3 gene, colony stimulating factor 2 receptor alpha (CSF2RA), beta (CSF2RB) and IL-3 receptor alpha (IL3RA), the IL-specific receptor subunits for CSF2 and IL3, respectively, are associated with schizophrenia. To examine the association of the IL3RA polymorphism (rs6603272) with schizophrenia in a Chinese population, 310 physically healthy patients with schizophrenia were compared with 330 age-, sex- matched normal controls. Statistically significant differences were observed in both allelic and genotypic frequencies of the rs6603272 polymorphism (Allele, chi2=6.24, d.f.=1, p=0.013, odds ratio (OR)=1.35, 95% CI 1.07-1.71; Genotype, chi2=6.85, d.f.=2, p=0.033). Our results indicate a small but significant contribution of the IL3RA polymorphism to susceptibility to schizophrenia, suggesting that the IL3 pathway may be involved in schizophrenia.

Gene expression profiling of peripheral mononuclear cells in lame dairy cows with foot lesions

Lameness is a major health issue and likely the single most common cause of pain and discomfort in dairy cattle. Appropriate treatment is delayed or neglected due, in part, to lack of reliable detection. Assessment of cows with lameness is currently limited to subjective visual scoring systems based on locomotion and posture abnormalities. These systems are unreliable to detect lameness, and therefore, a large number of cows remain undiagnosed. The objective of this research was to search for potential biomarkers for lameness-associated painful inflammatory foot lesions in dairy cattle using microarray-based gene expression profiling of peripheral blood mononuclear cells (PBMC). BOTL5 microarrays spotted in duplicate with cDNA representing bovine immune response genes were interrogated with cDNA samples in an eight-array, balanced complete block design with dye swap. Samples from eight lame cows with inflammatory foot lesions and from eight sound cows were pair-matched by age, weight, days in lactation, and pregnancy status at time of PBMC collection and directly compared with each other on individual arrays. Statistical analysis of resulting fluorescence intensity data revealed 31 genes that were putatively differentially expressed in lame versus sound cows ( P < 0.05). Of these, BLASTn analysis and gene ontology information showed that 28 genes had high similarity or homology to known human and/or rodent genes. Validation of 15 of these genes known to be important in inflammation and pain was carried out using relative quantitative real-time RT-PCR, which confirmed the up-regulation of interleukin (IL)-2 (12.68 ± 1.47-fold increase) and IL-10 (2.39 ± 0.55-fold increase), matrix metalloproteinase-13 (MMP-13) (10.44 ± 1.14-fold increase), and chemokine C–C motif receptor-5 (CCR5) (5.26 ± 1.05-fold increase), in lame relative to sound cows ( P ≤ 0.05). Similarly, granulocyte-macrophage colony-stimulating factor receptor alpha chain precursor (GM-CSF-R-alpha) (2.30 ± 0.63-fold increase) and IL-4 (2.06 ± 0.59-fold increase) showed a tendency ( P = 0.10) for up-regulation in lame compared to sound cows. PBMC co-expression of IL-2, MMP-13, CCR5 and IL-10, and potentially IL-4 and GM-CSF-R-alpha appears to be a promising, objective sign of lameness-related inflammatory foot lesions in dairy cattle. In conclusion, this study revealed potential biomarkers of the presence of foot lesions that could boost diagnostic accuracy of lameness and, ultimately, help identify animals in need of pain relief.

Detection of differentiation‐ and activation‐linked cell surface antigens on cultured mast cell progenitors

Mast cells (MC) are multifunctional effector cells of the immune system. They derive from uncommitted CD34(+) hemopoietic progenitor cells (HPC). Depending on the stage of maturation and the environment, MC variably express differentiation- and activation-linked antigens. Little is known, however, about the regulation of expression of such antigens in immature human MC. We analyzed expression of CD antigens on human MC grown from cord blood-derived CD34(+) HPC. The HPC were isolated by magnetic cell sorting (MACS) and FACS to >97% purity, and were cultured in stem cell factor (SCF) and interleukin (IL)-6 with or without additional cytokines (IL-4 or IL-10) in serum-free medium. The cell surface phenotype of MC was determined by monoclonal antibodies and flow cytometry. Cultured MC progenitors were found to react with antibodies against various CD antigens including CD58, CD63, CD117, CD147, CD151, CD203c, and CD172a, independent of the growth factors used and time-point investigated (days 14-42). CD116 [granulocyte-macrophage colony-stimulating factor receptor alpha (GM-CSFRalpha)] and CD123 (IL-3Ralpha) were expressed on MC precursors on day 14, but disappeared thereafter. Cultured MC did not express CD2, CD3, CD5, CD10, CD19, or CD25. Addition of IL-10 to MC cultures showed no effect on expression of CD antigens. However, IL-4 was found to promote expression of CD35 and CD88 on cultured MC without changing expression of other CD antigens. Most MC antigens may already be expressed at an early stage of mastopoiesis. Whereas IL-3R and GM-CSFRs are lost during differentiation of MC, these cells may acquire complement receptors (CD35, CD88) under the influence of distinct cytokines.

Physical mapping of CSF2RA, ANT3 and STS on the pseudoautosomal region of bovine chromosome X.

The pseudoautosomal region (PAR) of bovine chromosome X (BTA X) has a particularly low representation of genes and markers, making comparative gene mapping in this region difficult. We describe the localization of three genes, colony-stimulating factor 2 receptor alpha (CSF2RA), ADP/ATP translocase 3 (ANT3) and steroid sulphatase (STS) on PAR of BTA X using a 5000 rad whole-genome radiation hybrid panel. The relationship of these genes to a number of previously mapped simple sequence repeat (microsatellite) markers is determined by physical and radiation hybrid mapping methods. The resulting radiation hybrid map resolves a discrepancy between the two major bovine linkage maps in the PAR of BTA X.

Myeloid differentiation antigen and cytokine receptor expression on acute myelocytic leukaemia cells with t(16;21)(p11;q22): frequent expression of CD56 and interleukin-2 receptor alpha chain.

We report the cellular characteristics of cells from three patients with de novo acute myelocytic leukaemia (AML) with t(16;21)(p11;q22), two M4 and one M5a according to the FAB classification, and two permanent cell lines with t(16;21)(p11;q22), TSU1621MT and YNH-1. The FUS/ERG fusion mRNA was demonstrated in all cases by reverse transcriptase-polymerase chain reaction (RT-PCR). The immunophenotypes of the AML cells, and YNH-1 and TSU1621MT cell lines with t(16;21) were characterized as CD34+CD33+CD13+CD11b+CD18+CD56+ HLA-DR-/+. Cells from all samples strongly expressed c-kit, granulocyte colony-stimulating factor receptor (G-CSFR), c-fms (macrophage colony-stimulating factor receptor), interleukin-3 receptor alpha chain (IL-3Ralpha), and granulocyte macrophage colony-stimulating factor receptor alpha chain (GM-CSFRalpha), and these data corresponded well to the growth responsiveness to the cytokines. IL-2Ralpha expression was also found in all t(16;21) samples, but IL-2 did not act on the proliferation of the leukaemic cells in in vitro cultures. G-CSF distinctly promoted the proliferation of leukaemic cells of t(16;21) AML, but did not enhance the expression of MPO and neutrophil differentiation of these cells. Our findings indicate that AML cells with t(16;21) preserve stem cell properties such as CD34 and c-kit expression, and suggest that they have the potential to differentiate into a monocytic lineage. The relationship between the unique cellular characteristics (especially CD56 and IL-2Ralpha expression) and FUS/ERG protein remains undetermined.

Xp pseudoautosomal gene haploinsufficiency and linear growth deficiency in three girls with chromosome Xp22;Yq11 translocation.

Colony stimulating factor-2 receptor alpha (CSF2RA) and interleukin-3 receptor alpha (IL3RA), two genes from the chromosome Xp and Yp pseudoautosomal region (PAR), have been suggested as candidate genes for short...

Functional Replacement of Cytokine Receptor Extracellular Domains by Leucine Zippers

Granulocyte-macrophage colony-stimulating factor receptor signals by a complex which includes the ligand and two different receptor subunits: a low affinity alpha receptor binding chain (granulocyte-macrophage colony-stimulating factor receptor alpha subunit (GM-Ralpha)) and a signal-transducing beta chain (GM-Rbeta). To investigate two unresolved issues in the initiation of signaling, the role of receptor extracellular domains and receptor stoichiometry, we replaced the mouse GM-Ralpha and GM-Rbeta extracellular domains with the leucine zipper domain of either the Fos or Jun molecule. We co-transfected combinations of chimeric receptors into Ba/F3 cells and found that both simple heterodimers of the GM-Ralpha and GM-Rbeta intracellular domains and homodimers of the GM-Rbeta intracellular domain signaled for proliferation. Surprisingly, homodimers of the GM-Ralpha intracellular domain also signaled for prevention of apoptosis in transfected cells. We similarly engineered dimers of the intracellular domain of the human interferon gamma receptor beta subunit and found that homodimers of the intracellular domain signaled for proliferation. When Fos peptide was added to Ba/F3 cells expressing the human interferon gamma receptor beta subunit construct, thereby preventing homodimer formation, the cells no longer proliferated in the absence of mouse interleukin 3.

Investigation of newly synthesized peptides by capillary zone electrophoresis/electrospray mass spectrometry.

A series of newly synthesized peptides (M(r) = 1600-2250 Da), corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor alpha subunit have been examined by capillary zone electrophoresis/electrospray-mass spectrometry (CZE/ES-MS). The separation efficiency of CZE combined with the specificity of mass spectrometry allowed rapid and reliable identification of the target peptides and a number of associated side products. In solid-phase synthesis of peptides and small proteins such side products are inevitable, therefore the use of CZE and/or high-performance liquid chromatography combined with mass spectrometry is gaining a central role in such synthesis procedures.

Binding of human GM-CSF to synthetic peptides of the alpha subunit of its receptor.

We have synthesized a series of peptides corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor alpha subunit (hGM-CSFR alpha). The sequences were chosen according to the homology between hGM-CSFR alpha and the growth hormone receptor (GHR) and correspond to the regions reported to form the binding site of the latter receptor. The peptides were examined for their binding activity to hGM-CSF by affinity chromatography on resin-immobilized hGM-CSF and by a solid phase binding assay. For peptides endowed with hGM-CSF binding activity were identified and the postulated homology between the binding sites of hGM-CSFR alpha GHR was confirmed.

PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene.

Growth factor receptors play an important role in hematopoiesis. In order to further understand the mechanisms directing the expression of these key regulators of hematopoiesis, we initiated a study investigating the transcription factors activating the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha gene. Here, we demonstrate that the human GM-CSF receptor alpha promoter directs reporter gene activity in a tissue-specific fashion in myelomonocytic cells, which correlates with its expression pattern as analyzed by reverse transcription PCR. The GM-CSF receptor alpha promoter contains an important functional site between positions -53 and -41 as identified by deletion analysis of reporter constructs. We show that the myeloid and B cell transcription factor PU.1 binds specifically to this site. Furthermore, we demonstrate that a CCAAT site located upstream of the PU.1 site between positions -70 and -54 is involved in positive-negative regulation of the GM-CSF receptor alpha promoter activity. C/EBP alpha is the major CCAAT/enhancer-binding protein (C/EBP) form binding to this site in nuclear extracts of U937 cells. Point mutations of either the PU.1 site or the C/EBP site that abolish the binding of the respective factors result in a significant decrease of GM-CSF receptor alpha promoter activity in myelomonocytic cells only. Furthermore, we demonstrate that in myeloid and B cell extracts, PU.1 forms a novel, specific, more slowly migrating complex (PU-SF) when binding the GM-CSF receptor alpha promoter PU.1 site. This is the first demonstration of a specific interaction with PU.1 on a myeloid PU.1 binding site. The novel complex is distinct from that described previously as binding to B cell enhancer sites and can be formed by addition of PU.1 to extracts from certain nonmyeloid cell types which do not express PU.1, including T cells and epithelial cells, but not from erythroid cells. Furthermore, we demonstrate that the PU-SF complex binds to PU.1 sites found on a number of myeloid promoters, and its formation requires an intact PU.1 site adjacent to a single-stranded region. Expression of PU.1 in nonmyeloid cells can activate the GM-CSF receptor alpha promoter. Deletion of the amino-terminal region of PU.1 results in a failure to form the PU-SF complex and in a concomitant loss of transactivation, suggesting that formation of the PU-SF complex is of functional importance for the activity of the GM-CSF receptor alpha promoter. Finally, we demonstrate that C/EBP alpha can also active the GM-CSF receptor alpha promoter in nonmyeloid cells. These results suggest that PU.1 and C/EBP alpha direct the cell-type-specific expression of GM-CSF receptor alpha, further establish the role of PU.1 as a key regulator of hematopoiesis, and point to C/EBP alpha as an additional important factor in this process.

CD34-positive early human thymocytes: T cell receptor and cytokine receptor gene expression.

CD34, a stem cell marker, has been shown to be expressed on human CD3-CD4-CD8- (triple-negative; TN) thymocytes. Phenotypic and functional analyses suggest the following differentiation sequence: CD34+1-3-4-8(-)--> CD34+1+3-4 +/- 8(-)-->CD34-1+3-4+8(+/-)-->CD34-1++3-4+8+. In this report, we examined cytokine receptor gene expression on these subsets by reverse transcription-polymerase chain reaction analysis (RT-PCR). We were able to detect interleukin-7 receptor (IL-7R), c-kit and IL-2R gamma in all CD34+ thymocyte subsets, consistent with previous functional studies. We found IL-1R, granulocyte/macrophage colony-stimulating factor receptor-alpha and IL-4R transcripts in CD3- and CD34+ subsets. Secondly, we investigated T cell receptor (TCR)-delta and -beta gene rearrangement and transcription in CD34+ thymocytes. Our results show that a full-length TCR-delta transcript and the recombination activating genes RAG-1 and RAG-2 mRNA were already expressed in the CD34+1- subset. Mature V beta-containing TCR transcripts were also detected in the CD34+1+ subset, but not in the CD1- fraction. Furthermore, PCR analysis of D-J beta gene rearrangements showed that > or = 70% of CD34+1- cells are in a TCR beta germ-line configuration, although D-J beta recombination had already started in this population.

The Murine Interleukin-3 Receptor α Subunit Gene: Chromosomal Localization, Genomic Structure, and Promoter Function

The interleukin-3 receptor (IL-3R) is composed of alpha and beta subunits, members of the class I cytokine receptor family. Here we describe isolation and characterization of the chromosomal gene for the mouse IL-3R alpha subunit (mIL-3R alpha). Whereas the human IL-3R alpha gene is tightly linked with the granulocyte-macrophage colony-stimulating factor receptor alpha subunit (GM-CSFR alpha) gene in the pseudoautosomal region of the X and Y chromosomes, the mIL-3R alpha gene (II3ra) is located in the proximal region of mouse chromosome 14, separated from the mouse GM-CSFR alpha gene, which is on chromosome 19. The mIL-3R alpha gene spans about 10 kb and is divided into 12 exons. All the exon-intron boundaries possess the splicing junction consensus sequences (5'GT-AG3'), and the whole genomic structure is similar to those of the previously reported class I cytokine receptor genes. There are two major transcription initiation sites that are located at 215 and 188 nucleotides upstream of the initiator codon. The promoter region is GC-rich and contains potential binding sites for GATA, Ets, c-myb,, Sp1, Ap-2, and G-C boxes, but not a typical TATA or CAAT sequence. A fusion gene containing 0.8 kb of the 5' noncoding sequence linked to the firefly luciferase gene directed the transcription in mouse mast cells but not in fibroblasts or T cells, suggesting that this promoter functions in a cell type-specific manner. Further sequential deletion of the 5' region suggests two potential regulatory regions for transcription of the mIL-3R alpha gene.

Identification of conserved amino acids in the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit critical for function. Evidence for formation of a heterodimeric receptor complex prior to ligand binding.

A superfamily of growth factor and cytokine receptors has recently been identified, which is characterized by four spatially conserved cysteine residues, a tryptophan-serine motif (WSXWS) in the extracellular domain, and a proline-rich cytoplasmic domain. The high affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (hGM-CSFR) consists of two subunits, alpha (hGM-CSFR alpha) and beta (hGM-CSFR beta), both of which are members of the receptor superfamily. In this study, we prepared mutations in conserved amino acids of the receptor subunit necessary for GM-CSF binding (hGM-CSFR alpha) and analyzed mutant receptors for low affinity binding, internalization, and high affinity binding when complexed with the beta subunit. Mutations in the cytoplasmic domain did not affect GM-CSF binding or receptor internalization. Mutation of a single conserved serine residue within the WSXWS motif diminishes cell surface receptor expression but not ligand binding. Mutation of either the second or third conserved cysteine residue of hGM-CSFR alpha resulted in complete loss of low affinity binding; however, co-expression of the cysteine 2 mutant with hGM-CSFR beta yielded a high affinity receptor complex. Since neither the cysteine 2 mutant nor the beta subunit can bind ligand alone, this result suggests that hGM-CSFR alpha and hGM-CSFR beta exist in a preformed heterodimeric protein complex on the plasma membrane.

The human pseudoautosomal GM-CSF receptor alpha subunit gene is autosomal in mouse.

The gene encoding the granulocyte macrophage colony stimulating factor receptor alpha subunit (CSF2RA) has previously been mapped to the pseudoautosomal region of the human sex chromosomes. In contrast, we report that the murine locus, Csf2ra, maps to an autosome in the laboratory mouse. By in situ hybridization and genetic mapping, Csf2ra maps at telomeric band D2 of mouse chromosome 19. This first instance of a pseudoautosomal locus in human being autosomal in mouse, indicates incomplete conservation between the human and mouse X chromosomes and suggests that the genetic content of the pseudoautosomal region may differ between species of eutherian mammals due to chromosomal rearrangements.