- Research article
- Open Access
Bovine CD14 gene characterization and relationship between polymorphisms and surface expression on monocytes and polymorphonuclear neutrophils
© Ibeagha-Awemu et al; licensee BioMed Central Ltd. 2008
- Received: 27 February 2008
- Accepted: 08 August 2008
- Published: 08 August 2008
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.
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.
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.
- CD14 Gene
- CD14 SNPs
- CD14 Promoter
- Host Innate Immunity
- CD14 Genotype
The bovine cluster of differentiation (CD) 14 is an important player in host innate immunity in that it mediates host defense against Gram-negative bacterial infections and also confers immunity against viral infections [1, 2]. It is abundant (about 99,500 to 134,600) on the cell membrane of monocytes and to a lesser extent (about 1,900 to 4,400) on neutrophils (polymorphonuclear neutrophil leukocytes) [3, 4]. Two forms exist, a membrane bound form (mCD14) and a soluble (sCD14) form . sCD14 is known to confer lipopolysaccharide (LPS) sensitivity to cells lacking mCD14, including epithelial cells and endothelial cells [6, 7]. Also, recombinant bovine sCD14 can sensitize mammary epithelial cells to low concentrations of LPS in vivo and in vitro thus indicating an important role of sCD14 in initiating host responses to Gram-negative bacterial infections [8, 9]. During the periparturient period, individual variations have been noticed in the response of cows to Gram-negative and Gram-positive bacteria infections . Therefore, sequence variations of the CD14 gene may play important roles in the presentation of CD14 molecules and thus, LPS sensitivity.
The CD14 gene of cattle was initially cloned and sequenced by Ikeda et al.  and recently by the bovine genome project. It is mapped to BTA 7. A SNP of human CD14 promoter has been shown to influence the activities of the gene . Baldini et al.  reported a SNP in the human CD14 gene promoter involving a C to T transition at position -159 (or -260 by ), and associated TT homozygotes with significantly higher levels of sCD14 and lower levels of IgE in children as compared to carriers of TC or CC. Reports of association of the -159 CD14 polymorphism with several human disease conditions have emerged [15–17]. Other authors however did not find any association between this polymorphism and several diseases [18, 19]. Despite the importance of this protein and the effect of the promoter polymorphism in humans, there is no report of sequence variations of this gene in cattle and their possible effects on circulating CD14 levels and disease susceptibility.
The objectives of this study were therefore to: (1) investigate the CD14 gene of cattle for sequence variations; (2) determine the effect of variations on CD14 expression on the surfaces of monocytes and neutrophils; and (3) use bioinformatics tools to computationally characterize the promoter region. In this study we present information on sequence variations within the CD14 gene of Canadian Holstein and Jersey cows and a possible role of one SNP in influencing the surface expression of the antigen on the surfaces of neutrophils. Furthermore, identified conserved regions of regulatory element motifs may have important regulatory effects on the gene. The results may provide baseline information that may be used in candidate gene studies aimed at defining the role of CD14 in mediating bacterial infections.
SNPs in the CD14 gene of Canadian Holsteins and Jersey cows
Comparison of the CD14 sequences of 106 Canadian Holsteins and 46 Jersey cows with published sequences (GenBank Nos. NW_001495367 and D84509) revealed a total of five SNPs including one in the 5' untranslated region (UTR) (g.C1291T, numbering is according to GenBank No. EU148609), two in the coding regions (g.A1908G and g.A2318G) and two in the 3' UTR (g.A2601G and g.G2621T) (Table 1). Four of the SNPs are transitional mutations while SNP 2621 involves the transversion of guanine to thymine. SNP 1908 is responsible for a non-synonymous codon change in amino acid 175 of the protein, from Asn (a ac) to Asp (g ac), while SNP 2318 results in a synonymous codon change without a change in amino acid 311 (Pro, cca vs ccg) of the protein. The coding region SNPs characterizes three gene alleles A (A1908A2318) (GenBank No. EU148609), A1 (A1908G2318) (GenBank No. EU148610) and B (G1908G2318) (GenBank No. EU148611) and two deduced protein variants A (Asn175) (A1908A2318, GenBank No. ABV68569 or A1908G2318, GenBank No. ABV68570) and B (Asp175) (G1908G2318, GenBank No. ABV68571). Protein variant A is more common in the breeds analyzed with a frequency of 88.6% in Holsteins and fixed in Jerseys (Table 1). Within protein variant A, gene allele A1 occurred at a very high frequency (80.2%) in Jerseys. The non-coding SNPs occurred at about equal magnitudes in Holsteins while the frequencies of T1291, G2601 and T2621 were above 80% in Jerseys.
SNPs, gene alleles, protein variants, haplotypes and their frequencies in the analyzed breeds
Holstein (n = 106)
Jersey (n = 43)
Gene alleles (haplotypes)
A or **175Asn
B or 175Asp
Haplotye structure of the breeds
Considering the five SNPs identified and the genotype information of all individuals sequenced, the program PHASE V2.2.1 determined a total of four potential haplotype combinations (CAAAG-C1291A1908A2318A2601G2621, CGAAG, TGGGT, TAGGT) in the analyzed breeds. TGGGT was absent in Jersey while CGAAG was absent in both populations. The gene alleles A1 and B were observed from the sequencing data to be associated with SNPs T1291, G2601 and T2621 while allele A was associated with C1291, A2601 and G2621 therefore giving rise to three actual haplotypes (TAGGT, TGGGT and CAAAG) in the analyzed populations (Table 1). The frequency of the haplotype associated with allele A (CAAAG) was highest (65.8%) in Holsteins while the haplotype associated with allele A1 (TAGGT) was highest in Jerseys (80.2%) (Table 1).
Effects of CD14 genotypes on the expression of CD14 on the surfaces of monocytes and neutrophils
Effects of CD14 genotypes on its expression (in %) on the surfaces of monocytes and neutrophils in Holstein cows (n = 64)
29.57 ± 1.54
57.00 ± 1.81
86.57a ± 0.96
73.25 ± 2.52
4.48 ± 0.62
77.74 ± 2.73
28.25 ± 1.48
61.26 ± 1.75
89.51b ± 0.93
76.21 ± 2.43
5.54 ± 0.60
81.75 ± 2.64
32.02 ± 3.32
58.94 ± 3.91
90.97b ± 2.07
81.57 ± 5.43
4.11 ± 1.34
85.68 ± 5.91
29.10 ± 1.14
59.12 ± 1.36
88.22 ± 0.74
75.25 ± 1.88
4.36A ± 0.43
79.60 ± 2.03
29.49 ± 2.26
59.39 ± 2.70
88.88 ± 1.47
76.09 ± 3.72
7.26B ± 0.86
83.35 ± 4.03
29.57 ± 1.56
57.00 ± 1.82
86.57a ± 0.97
73.25 ± 2.55
4.48a ± 0.59
77.74 ± 2.78
27.75 ± 1.84
62.46 ± 2.16
90.21b ± 1.14
77.36 ± 3.01
4.45a ± 0.70
81.81 ± 3.28
29.24 ± 2.60
58.85 ± 3.05
88.09a ± 1.61
73.90 ± 4.26
7.73bc ± 0.98
81.63 ± 4.65
30.33 ± 4.75
61.18 ± 5.56
91.51a ± 2.95
83.41 ± 7.78
5.68ab ± 1.80
89.08 ± 8.48
33.72 ± 4.75
56.72 ± 5.56
90.42a ± 2.95
79.73 ± 7.78
2.55a ± 1.80
82.28 ± 8.48
Characterization of CD14 promoter and comparative analysis ofCD14 proteins
We report here sequence variations of the bovine CD14 gene of Canadian Holstein and Jersey cows, through genomic DNA sequencing and computational analysis of the promoter. A gene is made up of both coding and non-coding regions which are all important in its expression and functionality. The complete description of a gene must therefore contain necessary information about the protein coding regions  and non-coding regions. The CD14 gene is an important component in host immunity [1, 8] and detailed information on its structure and sequence variations as shown in this study may provide further insight into its mode of action.
The coding region SNPs in our study and comparative analysis of our sequences with published sequences show that the CD14 gene of cattle codes for three putative CD14 proteins-A (GenBank No. ABV68569 and ABV68570), B (GenBank No. ABV68571) and C (UniProt AAD32215 and UniProt NP_776433), with A and B described herein. The A variant, fixed in Jerseys and with a high frequency of 88.6% in Holsteins, may be the original wild type allele for the gene. Furthermore, the sequence of UniProt BAA21517 is similar to variant A and the haplotyes that contain the A variant SNPs are at the highest frequencies in the studied breeds. The other variants may therefore be the result of recent mutational events. Further three SNPs described in the 5' and 3'UTRs and one synonymous SNP in the coding region of the gene indicates a higher sequence variation for the gene in Canadian Holsteins than Jerseys.
The variations, both in the coding and non-coding regions of the gene may affect the surface expression of CD14 molecules on monocytes and neutrophils. Interestingly, the coding region SNP that gave rise to the B variant of the protein (g.A1908G or p.Asn175Asp) had the greatest effect by being associated with the highest number of neutrophils expressing more CD14 molecules on their surfaces. It is well known that, monocytes express more CD14 receptors on their surfaces, about 99,500 to 134,600 as compared to 1,900 to 4,400 for neutrophils (3). This difference was clearly shown by the pattern of expression depicted in Figure 1 whereby, more monocytes were recorded in the M2 gated zone (region of higher expression) and more neutrophils in the M1 zone (lower expression). These results suggest that, the characteristic B protein SNP or g.A1908G may play a role in cell surface expression of CD14 on neutrophils. Also, the SNP in the 5'UTR region could be important in influencing the expression of this receptor on monocytes. Our data was however based on a small sample size necessitating further verifications on a larger scale. Neutrophils form a major line of defense against bacterial infections and their effectiveness depends on their availability at the site of infections. For Gram-negative bacterial infections, the CD14 molecule confers LPS sensitivity to neutrophils , which is necessary to initiate host immune responses. The complex of TLR4, CD14 and myeloid differentiation protein 2, enhanced by the presence of LPS binding protein is crucial in LPS signaling; leading to the release of cytokines [21–23].
Even though no promoter polymorphism was detected in this study, a promoter polymorphism of the gene in human is a risk factor in several diseases [15–17]. The promoter region in bovine may probably be under strong purifying selection which may explain the lack of SNPs in this region. This is a positive factor considering the important role of the gene in mediating Gram-negative bacteria attack and the possible effect of the 1908 SNP on the abundance of the molecule on the surfaces of neutrophils. Determination of the roles of the individual SNPs and haplotypes on the activities of the gene under disease conditions will further shed more light on their biological significance.
Our analysis on promoter characterization indicates that part of the exon 1 reported by Ikeda et al.  constitutes the promoter. Since evolutionary pressures lead to the conservation of important non-protein-coding regulatory regions, including transcription factor binding sites (TFBs) across closely related species, identification of TFBs described in the CD14 genes of human  and rat  in the present study was expected. In particular, the perfect conservation of the TATA box, 9 motifs of 6 REs motifs across cattle, human, rat, mouse and pig and 4 other motifs across at least 4 of these species including cattle shows common regions in the core promoter that act together in the same biological context to control the expression of gene products and functions. In our study, up to 5 SP1 and 8 AP1 sites (with no bp mismatch) were identified which may indicate possible roles in controlling the expression of the gene as demonstrated in the rat and humans [24, 25]. In the rat, Lui et al.  through mobility shift assays demonstrated that the SP1 and AP1 elements located respectively at positions -836 and -270 were required for basal promoter activity in liver cells. Also, Zhang et al.  showed that the SP1 transcription factor bound to three different regions of the human CD14 promoter and that a mutation of the major SP1 binding site decreased tissue specific promoter activity. One of the AP1 sites in our study, also shared by c-Fos and c-Jun (position 960–967, Figure 2), is similar to an AP1 site in rat promoter were JunD and Fra-2 proteins have been shown to bind . This site is also thought to transactivate the basal expression of the gene . This site in the mouse also plays a major role in the expression of the CD14 gene in macrophages . Furthermore, three motifs of PEA3 interestingly were conserved in the promoters of all species studied. PEA3 belongs to the ETS transcription factor super family and is known to appear on the promoters of many cellular genes, including HER-2/neu  and CD226 antigen . Conservation of C/EBP or CCAAT/enhancer binding proteins motifs in the studied species may be explained by their involvement in many aspects of cell growth. The high conservation of the amino acids of the proteins of bovine, buffalo, sheep and goat proteins was reflected in the tree of their phylogenetic relationships and is in line with other studies that found a high rate of conservation of genes and protein coding nucleotide positions between bovine, sheep and goat genes [29, 30]. This further supports the fact that information from the sequencing of the bovine genome will greatly enhance studies in other very closely related species.
Overall, this study provides information on sequence variations of the CD14 gene of Canadian Holstein and Jersey cows. The identified variations and association data have provided information that may shade more light on cell surface expression of CD14 by neutrophils, which are needed to control bacterial infections. Further data on the biological significance of the mutations is however necessary. Our computational analysis highlighted on the regulatory element motifs present in the promoter region of the gene. The comparative analysis with other species revealed conserved regions of regulatory element motifs that may have important regulatory effects on the gene.
Animals and genomic DNA extraction
Genomic DNA was extracted from the blood of 106 Canadian Holstein cows kept at the Howard Webster Centre-Macdonald Teaching Farm, McGill University and the milks of 46 Jersey cows enrolled in the Quebec Dairy Production Centre of Expertise program http://www.valacta.com using Nucleospin Blood Mini Kit (Macherey-Nagel Inc. Easton, PA) as described by the manufacturer. In the case of milk DNA isolation, the manufacturer's protocol was slightly modified. Milk samples were initially centrifuged at 13000 rpm for 15 min at 4°C to remove excess fat before proceeding with the manufacturer's protocol.
PCR amplification and sequencing
Primers used in the amplification of the whole bovine CD14 gene and other PCR conditions
Amplicon size (bp)
5'ATT ACC TTC TTC TGC ACC TCC A 3'
5' GGC AGC CTC TGA GAG TTT ATG T 3'
5' CTT CCT GTT ATA GCC CCT TTC C 3'
5' CAC GAT ACG TTA CGG AGA CTG A 3'
5' GGG TAC TCT CGT CTC AAG GAA C 3'
5' CTG AGC CAA TTC ATT CCT CTT C 3'
5' ACC TGA CTC TGG ACG GAA ATC 3'
5' TAC AGG AGA GCA ACC CTG AAA 3'
PCR reactions with all primer pairs were each carried out in a total volume of 45 μL containing 50 ng DNA, 0.25 mM dNTPs, 2.0 to 2.5 mM MgCl2 (Table 3), 10 μM of each primer, 2 units Tag DNA polymerase (Fermentas Life Sciences, Burlington, ON, Canada) and 1× Taq buffer. The cycling conditions, with PTC-100™ thermal cycler (MJ Research, Inc., Watertown, MA, USA) included an initial denaturation for 2 min at 94°C followed by 30 cycles comprising 30 sec at 94°C, 30 sec at 60°C, 50 sec at 72°C, and a final elongation for 5 min at 72°C. Both directions of amplified PCR products were sequenced by McGill University/Genome Quebec Innovation Centre using the big dye termination technique and an ABI 3700 sequencer.
Sequences were processed with Chromas, version 1.45 http://www.technelysium.com.au/chromas14x.html) and comparison with other published sequences was done with the multiple sequence alignment program with hierarchical clustering, Multalign http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html. CD14 protein sequences of different species (cattle, buffalo, goat, sheep, pig, man, mouse and rat) were aligned or processed with MEGA3.1 software  and phylogenetic relationships also constructed with the same software.
Computational characterization of the bovine CD14 promoter
The promoter region was analyzed for the presence of putative transcription factor-binding sites using the combined search query against the TRANSFAC database with a maximum allowable string mismatch of 10% [; http://www.cbil.upenn.edu/cgi-bin/tess/tess]. The combined search query option was used to take advantage of the full power of combined string and weight matrix searching, pre-filtering of factors, significance p-values, and new information in new databases. Particular attention was paid to binding sites already proven to be of significance in regulating the CD14 gene of other species and other common binding sites in mouse and human.
Identification of conserved motifs of regulatory elements in the non-coding regions of the CD14 gene of cattle and other species
Since regions of conserved non-coding sequences between closely related or divergent species are likely to have common functional roles, we searched the region, about 500 bps of the core promoters of cattle (this study or GenBank No. EU148609), human (GenBank No. U00699), mouse (GenBank No. X13987), rat (GenBank No. AF087944) and pig (GenBank No. DQ079063) for described conserved regulatory element (RE) motifs against the NSITEM data base http://linux1.softberry.com/berry.phtml?topic=nsitem&group=programs&subgroup=promoter. These species were chosen because of the availability of complete or partial promoter sequence information.
Flow cytometry was used to study the effects of identified CD14 SNPs on the expression of CD14 on the surfaces of neutrophils and monocytes in healthy cows. Blood was collected from the caudal vein of 64 Holstein cows with known CD14 genotypes by venipuncture into vacutainer tubes coated with heparin anticoagulant (BD Biosciences, Franklin Lakes, NJ, USA). After collection, samples were stored on ice and analyzed within four hours. One hundred microlitre of heparinized whole blood was placed in a 12 × 75 mm flow cytometric (FCM) tube and incubated with 10 μL of fluorescein isothiocyanate (FITC)-labeled mouse anti human CD14 antibody (ABD Serotec Inc., Raleigh, NC, USA). This was mixed (Barnstead Thermolyne, Dubuque, IOWA, USA) thoroughly and incubated at room temperature on an orbitron rotator (Boekel Ind. Inc., PA, USA) for 30 minutes. Lysis and fixation of erythrocytes was done by adding 2 mL of lysing solution (PHAGOTEST® Kit, Orpegen Pharma, Heidelberg, Germany) to the mixture. This was mixed gently, incubated for 20 minutes on an orbitron rotator at room temperature and centrifuged at 250 g for 5 minutes at 4°C. The supernatant was aspirated leaving approximately 400 μL of cells in the FCM tube. This was washed with 3 mL of Dulbecco's phosphate buffered saline (DPBS) pH 7.2 (Life Technologies) by centrifuging at 250 g for 5 minutes at 4°C. The supernatant was aspirated as described above and the cells resuspended in 1 mL of DPBS and analyzed by flow cytometry (Becton Dickinson Immunocytochemistry Systems, San José, CA, USA) within 30 minutes. Excitation of samples was at 488 nm; with FITC fluorescence measured at 525 nm ± 10 nm. Acquisition was stopped when 20,000 gated events were collected in the fluorescence cell count histogram. Gating of monocytes and polymorphonuclear leukocytes was based on forward scatter and side scatter dot plots by encircling the populations with amorphous regions. All parameters were recorded with logarithmic amplifications. List mode flow cytometric data from 20,000 events were stored and processed with the Windows Multiple Document Interface for flow cytometry (WinMDI) software version 2.8 (Joseph Trotter, The Sripps Research Institute, http://facs.scripps.edu/software.html) on a personal computer.
The viability of neutrophils and monocytes in whole blood was determined by propidium iodide (PI) exclusion (50 μg/mL, final concentration) using flow cytometry after cells were incubated for 10 minutes in the dark at room temperature. The cells showed 99% viability.
Allele frequencies were estimated with GENEPOP program  while haplotypes and their frequencies were determined with the program PHASE V2.1.1 [34, 35]. PHASE implements a Bayesian method of haplotype reconstruction based on genealogies reconstructed from coalescent theory under a Markov Chain Monte Carlo framework and has been shown to outperform other strategies such as the maximum likelihood expectation maximization algorithm in most cases .
Flow cytometric data were analyzed as a one-way ANOVA using the MIXED procedure SAS . Treatment means were separated using the least square means option of SAS. Differences between treatment means were tested using Scheffe's Multiple Comparison test and statistical significance was declared at P < 0.05.
Statistical model used: Yij = μ + genotypei + eij
This research was financed by NSERC, Alberta Milk, Dairy Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait Inc., Dairy Farmers of Canada, Canadian Dairy Network, AAFC, PHAC, Technology PEI Inc., Université de Montréal and University of Prince Edward Island through the Canadian Bovine Mastitis Research Network. We thank Jaime Sanchez-Dardon for technical support in flow cytometry.
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