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Bovine CD14 gene characterization and relationship between polymorphisms and surface expression on monocytes and polymorphonuclear neutrophils.

Ibeagha-Awemu EM, Lee JW, Ibeagha AE, Zhao X - BMC Genet. (2008)

Bottom Line: The study has provided information on sequence variations within the CD14 gene and proteins of cattle.Further observations involving a larger sample size are required to validate our findings.The computational analysis on the promoter and comparative analysis with other species has revealed regions of regulatory element motifs that may indicate important regulatory effects on the gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Science, McGill University, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada. eveline.ibeagha-awemu@mcgill.ca

ABSTRACT

Background: CD14 is an important player in host innate immunity in that it confers lipopolysaccharide sensitivity to cell types like neutrophils, monocytes and macrophages. The study was aimed at characterizing the CD14 gene of cattle for sequence variations and to determine the effect of variations on the expression of the protein on the surfaces of monocytes and neutrophils in healthy dairy cows.

Results: Five SNPs were identified: two within the coding regions (g.A1908G and g.A2318G, numbering is according to GenBank No. EU148609), one in the 5' (g.C1291T) and two in the 3' (g.A2601G and g.G2621T) untranslated regions. SNP 1908 changes amino acid 175 of the protein (p.Asn175Asp, numbering is according to GenBank No. ABV68569), while SNP 2318 involves a synonymous codon change. Coding region SNPs characterized three gene alleles A (GenBank No. EU148609), A1 (GenBank No. EU148610) and B (GenBank No. EU148611) and two deduced protein variants A (ABV68569 and ABV68570) and B (ABV68571). Protein variant A is more common in the breeds analyzed. All SNPs gave rise to 3 haplotypes for the breeds. SNP genotype 1908AG was significantly (P<0.01) associated with a higher percentage of neutrophils expressing more CD14 molecules on their surfaces. The promoter region contains several transcription factor binding sites, including multiple AP-1 and SP1 sites and there is a high conservation of amino acid residues between the proteins of closely related species.

Conclusion: The study has provided information on sequence variations within the CD14 gene and proteins of cattle. The SNP responsible for an amino acid exchange may play an important role in the expression of CD14 on the surfaces of neutrophils. Further observations involving a larger sample size are required to validate our findings. Our SNP and association analyses have provided baseline information that may be used at defining the role of CD14 in mediating bacterial infections. The computational analysis on the promoter and comparative analysis with other species has revealed regions of regulatory element motifs that may indicate important regulatory effects on the gene.

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Flow cytometyric analyses of relationship of surface expression of CD14 on monocytes and neutrophils with CD14 genotypes. Cells were stained with fluorescein isothiocyanate (FITC)-labelled mouse anti human CD14 antibody and 20,000 events were gated and analyzed with Windows Multiple Document Interface for flow cytometry (WinMDI) software version 2.8. (a) Histogram showing a control sample that was not stained with antibody and occupies the Log100 to 101 region and known as the control zone. M1 (Log101 to 102) is the zone of low expression or lower fluorescence zone indicating lower number of CD14 antigens on cells, M2 (Log102 and higher) is the zone of higher fluorescence emitted by a higher rate of absorption by more CD14 antigens on cells and M3 is the total area of expression. (b) Histogram showing stained monocytes with a higher percentage of cells in M2 and a higher overall MCF intensity of 201.44 as compared to 34.76 for polymorphonuclear neutrophils; (c) Histogram showing stained polymorphonuclear neutrophils with a higher percentage of cells in M1.
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Figure 1: Flow cytometyric analyses of relationship of surface expression of CD14 on monocytes and neutrophils with CD14 genotypes. Cells were stained with fluorescein isothiocyanate (FITC)-labelled mouse anti human CD14 antibody and 20,000 events were gated and analyzed with Windows Multiple Document Interface for flow cytometry (WinMDI) software version 2.8. (a) Histogram showing a control sample that was not stained with antibody and occupies the Log100 to 101 region and known as the control zone. M1 (Log101 to 102) is the zone of low expression or lower fluorescence zone indicating lower number of CD14 antigens on cells, M2 (Log102 and higher) is the zone of higher fluorescence emitted by a higher rate of absorption by more CD14 antigens on cells and M3 is the total area of expression. (b) Histogram showing stained monocytes with a higher percentage of cells in M2 and a higher overall MCF intensity of 201.44 as compared to 34.76 for polymorphonuclear neutrophils; (c) Histogram showing stained polymorphonuclear neutrophils with a higher percentage of cells in M1.

Mentions: Whole blood from healthy Holstein cows (animals showing no outward symptoms of infection and farm record indicating milk somatic cell counts below 200,000 cells/ml) with different CD14 genotypes were incubated with fluorescein isothiocyanate (FITC)-labeled mouse anti-human CD14 antibody to determine the effects of genotypes on the expression of CD14 antigens on the surfaces of monocytes and neutrophils. The results are presented in Figure 1 and Table 2. In Figure 1, a higher percentage of gated monocyte cells were found in the LogFITC region labeled M2 (log102 and above) which indicates a higher fluorescence intensity coming from cells with the most CD14 antigens on their surfaces and termed the high expression region. On the other hand, more neutrophils were in the M1 or low expression region (Log101 to 102). The mean channel fluorescence (MCF) intensities (for all cows) observed for the gated regions were M1 = 32.04 (range 16.67 – 65.18), M2 = 288.97 (149.85 – 566.47) and M3 = 201.44 (73.57 – 437.56) for monocytes and M1 = 22.76 (16.20 – 35.52), M2 = 267.33 (148.26 – 536.87) and M3 = 34.76 (19.49–60.83) for neutrophils. As presented, the M3 (M1 + M2) MCF intensity for monocyte was higher than for neutrophils. SNP A1908G that changes amino acid 175Asn to Asp, and thus protein A to B, is significantly (P < 0.01) associated with a higher number of neutrophils in the M2 or higher expression zone. 7.26% of gated neutrophils from cows of genotype 1908AG were found in M2 as compared to 4.36% from cows of genotype 1908AA (Table 2). In the M1 zone, the percentage of neutrophils from cows of both genotypes was the same. For monocytes, a higher number of total cells stained was observed for 1291CT (P < 0.05) and the other genotypes (2318AG, 2601AG and 2621GT) in perfect linkage disequilibrium with C1291T but this difference disappeared when Scheffe's adjustments was applied to means. A similar result was recorded for haplotypes.


Bovine CD14 gene characterization and relationship between polymorphisms and surface expression on monocytes and polymorphonuclear neutrophils.

Ibeagha-Awemu EM, Lee JW, Ibeagha AE, Zhao X - BMC Genet. (2008)

Flow cytometyric analyses of relationship of surface expression of CD14 on monocytes and neutrophils with CD14 genotypes. Cells were stained with fluorescein isothiocyanate (FITC)-labelled mouse anti human CD14 antibody and 20,000 events were gated and analyzed with Windows Multiple Document Interface for flow cytometry (WinMDI) software version 2.8. (a) Histogram showing a control sample that was not stained with antibody and occupies the Log100 to 101 region and known as the control zone. M1 (Log101 to 102) is the zone of low expression or lower fluorescence zone indicating lower number of CD14 antigens on cells, M2 (Log102 and higher) is the zone of higher fluorescence emitted by a higher rate of absorption by more CD14 antigens on cells and M3 is the total area of expression. (b) Histogram showing stained monocytes with a higher percentage of cells in M2 and a higher overall MCF intensity of 201.44 as compared to 34.76 for polymorphonuclear neutrophils; (c) Histogram showing stained polymorphonuclear neutrophils with a higher percentage of cells in M1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2536669&req=5

Figure 1: Flow cytometyric analyses of relationship of surface expression of CD14 on monocytes and neutrophils with CD14 genotypes. Cells were stained with fluorescein isothiocyanate (FITC)-labelled mouse anti human CD14 antibody and 20,000 events were gated and analyzed with Windows Multiple Document Interface for flow cytometry (WinMDI) software version 2.8. (a) Histogram showing a control sample that was not stained with antibody and occupies the Log100 to 101 region and known as the control zone. M1 (Log101 to 102) is the zone of low expression or lower fluorescence zone indicating lower number of CD14 antigens on cells, M2 (Log102 and higher) is the zone of higher fluorescence emitted by a higher rate of absorption by more CD14 antigens on cells and M3 is the total area of expression. (b) Histogram showing stained monocytes with a higher percentage of cells in M2 and a higher overall MCF intensity of 201.44 as compared to 34.76 for polymorphonuclear neutrophils; (c) Histogram showing stained polymorphonuclear neutrophils with a higher percentage of cells in M1.
Mentions: Whole blood from healthy Holstein cows (animals showing no outward symptoms of infection and farm record indicating milk somatic cell counts below 200,000 cells/ml) with different CD14 genotypes were incubated with fluorescein isothiocyanate (FITC)-labeled mouse anti-human CD14 antibody to determine the effects of genotypes on the expression of CD14 antigens on the surfaces of monocytes and neutrophils. The results are presented in Figure 1 and Table 2. In Figure 1, a higher percentage of gated monocyte cells were found in the LogFITC region labeled M2 (log102 and above) which indicates a higher fluorescence intensity coming from cells with the most CD14 antigens on their surfaces and termed the high expression region. On the other hand, more neutrophils were in the M1 or low expression region (Log101 to 102). The mean channel fluorescence (MCF) intensities (for all cows) observed for the gated regions were M1 = 32.04 (range 16.67 – 65.18), M2 = 288.97 (149.85 – 566.47) and M3 = 201.44 (73.57 – 437.56) for monocytes and M1 = 22.76 (16.20 – 35.52), M2 = 267.33 (148.26 – 536.87) and M3 = 34.76 (19.49–60.83) for neutrophils. As presented, the M3 (M1 + M2) MCF intensity for monocyte was higher than for neutrophils. SNP A1908G that changes amino acid 175Asn to Asp, and thus protein A to B, is significantly (P < 0.01) associated with a higher number of neutrophils in the M2 or higher expression zone. 7.26% of gated neutrophils from cows of genotype 1908AG were found in M2 as compared to 4.36% from cows of genotype 1908AA (Table 2). In the M1 zone, the percentage of neutrophils from cows of both genotypes was the same. For monocytes, a higher number of total cells stained was observed for 1291CT (P < 0.05) and the other genotypes (2318AG, 2601AG and 2621GT) in perfect linkage disequilibrium with C1291T but this difference disappeared when Scheffe's adjustments was applied to means. A similar result was recorded for haplotypes.

Bottom Line: The study has provided information on sequence variations within the CD14 gene and proteins of cattle.Further observations involving a larger sample size are required to validate our findings.The computational analysis on the promoter and comparative analysis with other species has revealed regions of regulatory element motifs that may indicate important regulatory effects on the gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Science, McGill University, Ste-Anne-de-Bellevue, Quebec H9X 3V9, Canada. eveline.ibeagha-awemu@mcgill.ca

ABSTRACT

Background: CD14 is an important player in host innate immunity in that it confers lipopolysaccharide sensitivity to cell types like neutrophils, monocytes and macrophages. The study was aimed at characterizing the CD14 gene of cattle for sequence variations and to determine the effect of variations on the expression of the protein on the surfaces of monocytes and neutrophils in healthy dairy cows.

Results: Five SNPs were identified: two within the coding regions (g.A1908G and g.A2318G, numbering is according to GenBank No. EU148609), one in the 5' (g.C1291T) and two in the 3' (g.A2601G and g.G2621T) untranslated regions. SNP 1908 changes amino acid 175 of the protein (p.Asn175Asp, numbering is according to GenBank No. ABV68569), while SNP 2318 involves a synonymous codon change. Coding region SNPs characterized three gene alleles A (GenBank No. EU148609), A1 (GenBank No. EU148610) and B (GenBank No. EU148611) and two deduced protein variants A (ABV68569 and ABV68570) and B (ABV68571). Protein variant A is more common in the breeds analyzed. All SNPs gave rise to 3 haplotypes for the breeds. SNP genotype 1908AG was significantly (P<0.01) associated with a higher percentage of neutrophils expressing more CD14 molecules on their surfaces. The promoter region contains several transcription factor binding sites, including multiple AP-1 and SP1 sites and there is a high conservation of amino acid residues between the proteins of closely related species.

Conclusion: The study has provided information on sequence variations within the CD14 gene and proteins of cattle. The SNP responsible for an amino acid exchange may play an important role in the expression of CD14 on the surfaces of neutrophils. Further observations involving a larger sample size are required to validate our findings. Our SNP and association analyses have provided baseline information that may be used at defining the role of CD14 in mediating bacterial infections. The computational analysis on the promoter and comparative analysis with other species has revealed regions of regulatory element motifs that may indicate important regulatory effects on the gene.

Show MeSH
Related in: MedlinePlus