Impaired Neutrophil Migration Associated with Specific Bovine CXCR2 Genotypes
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感染与免疫杂志 2005年第8期
Department of Animal Science, The University of Tennessee, Knoxville, Tennessee 37996
ABSTRACT
Bovine mastitis continues to be the most detrimental factor for profitable dairying. Recent research conducted within our laboratory has identified a genetic marker in the CXCR2 gene associated with mastitis susceptibility. The objective of the present study was to evaluate the migratory ability of neutrophils from cows with different CXCR2 +777 genotypes. Neutrophils isolated from peripheral blood of 30 Holstein cows were tested for in vitro migration and adhesion molecule expression. Cows with the CC or GC genotype at CXCR2 +777 showed significantly lower neutrophil migration to recombinant human interleukin-8 (rhIL-8) than cows with the GG genotype (P < 0.05). Cows with the CC genotype at CXCR2 +777 also showed decreased neutrophil migration to zymosan-activated serum compared to these same cows (P < 0.05). Decreased upregulation of CD18 expression was observed after stimulation with rhIL-8 in cows expressing the CXCR2 +777 CC genotype compared to cows expressing the GG genotype (P < 0.05). A similar trend was observed for CD11b (P < 0.10). However, no difference in CD62 downregulation was observed with respect to genotype. These results provide initial evidence for a phenotypic association between a single nucleotide polymorphism and neutrophil function in dairy cows, as well as potential insight into specific mechanisms affected in cows more susceptible to mastitis.
INTRODUCTION
Infectious diseases caused by bacteria negatively impact every animal industry. Effective elimination of bacterial infections, such as mastitis in dairy cattle, requires four basic steps: bacterial recognition, inflammatory mediator release, leukocyte recruitment from the bloodstream, and bacteria removal. When infection occurs, resident macrophages and mammary epithelial cells release inflammatory mediators that signal the body for help. These chemical messengers include chemotactic complement fractions, prostaglandins, leukotrienes, and acute-phase cytokines (interleukin-1 [IL-1], tumor necrosis factor, and IL-8) and are responsible for massive neutrophil migration from the circulation to the site of infection (10, 28, 31, 32).
The ability of neutrophils to migrate into infected tissues is dependent upon recognition of inflammatory mediators by cytokine, chemokine, and complement receptors. Two chemokine receptors present on neutrophil surfaces, CXCR1 and CXCR2, are required for maximum neutrophil function during infection (12, 16). Interleukin-8 is a high-affinity ligand for both receptors but shares CXCR1 with the lower-affinity ligand granulocyte chemotactic protein 2. Besides IL-8, CXCR2 also can bind growth-related oncogene alpha (GRO-), GRO-, GRO-, neutrophil-activating peptide 2, and epithelial-derived neutrophil attractant 78 (2, 38). In neutrophils, recognition of chemokines by CXCR1 and CXCR2 induces 2 integrin upregulation and chemotaxis, as well as enhancement of reactive oxygen species generation and phagocytosis of pathogens (2, 9, 21, 22, 24). The CXC receptors are members of the large family of serpentine receptors with seven transmembrane domains (7TMD) that couple to Bordetella pertussis toxin-sensitive heterotrimeric G-proteins for signal transduction. G-protein coupling to the receptor results in activation of phospholipase C, formation of the second messenger inositol 1,4,5-trisphosphate, and the subsequent increase in cytosolic free calcium concentration (4, 37), as well as activation of phosphatidylinositol 3'-kinase and consequent generation of phosphatidylinositol 3-phosphate (25). All these mechanisms mediate many of the events necessary for proper neutrophil activation and migration to eliminate invading pathogens.
Effective neutrophil recruitment to the site of infection also requires adhesion molecules from the selectin and 2 integrin families. Neutrophil selectin CD62L slows down cells upon contact with its specific ligands (7) and allows neutrophils to roll along vascular endothelial cells. Neutrophils activated by cytokines or chemoattractants subsequently shed CD62L (20) as a prerequisite for 2 integrin-mediated tight adhesion. The most important 2 integrin involved in neutrophil recruitment into inflamed tissue is CD11b/CD18 (3), which is predominantly stored in cytoplasmic granules. Upon cellular activation by inflammatory mediators, these cytoplasmic granules translocate to the cell surface and CD11b/CD18 expression increases (23), causing neutrophils to bind firmly to endothelial cells through interaction with its specific ligand, intercellular adhesion molecule 1.
Influx of neutrophils into the mammary gland is critical for the resolution of mastitis; without quick and efficient neutrophil migration, infections can persist and lead to acute clinical mastitis, chronic infections, severe tissue damage, and potentially death of the cow (15, 19). Interleukin-8 is one of the ligands that binds CXCR2 with high affinity (2), and it is at least partly responsible for the massive influx of neutrophils to the mammary gland during bacterial infection (29, 31, 35). Upon arrival at the site of infection, neutrophils phagocytose and kill intramammary bacteria through reactive oxygen species generation and antibacterial granule proteins (33). Once an infection is cleared, neutrophils undergo apoptosis and are phagocytosed by macrophages (34). This rapid clearance is important because neutrophil lysosomal enzymes and respiratory burst products can damage mammary gland tissue (8, 14, 27).
Single nucleotide polymorphisms (SNPs) within the CXCR2 gene have been identified and evaluated for their potential association with disease in humans (18, 30). Of these, homozygosity at position +785 CC and at position +1208 TT was found in high frequency in individuals with systemic sclerosis compared to healthy controls (30). It is possible that polymorphisms in bovine CXCR2 are also associated with disease susceptibility or progression and may potentially be used to select cows that are more resistant to disease. Recent research conducted in our laboratory has identified five SNPs in a 311-bp segment of the CXCR2 gene of dairy cows (39). Polymorphisms at positions +612, +777, and +861 showed a significant association with subclinical mastitis (40). One of these, the SNP located at position +777 (GC), results in amino acid 245 glutaminehistidine replacement. This may have direct implications in the functionality of the receptor, since amino acid 245 is located in the receptor's third intracellular loop, a critical site for G-protein coupling and activation (11). Holstein cows expressing the CC genotype at position +777 had increased incidence of subclinical mastitis (37%) compared to Holstein cows that expressed the CG (21%) or GG (22%) genotype (40). Based on this phenotypic observation, and since neutrophil recruitment to the site of infection is critical for the resolution of mastitis, we hypothesized that neutrophil migration and adhesion molecule expression would differ among cows of different CXCR2 +777 genotypes.
MATERIALS AND METHODS
Animal selection and sample collection. Thirty Holstein cows (six cows/day; two cows from each genotype) from the Knoxville Experiment Station in mid- to late lactation (at least 100 days in milk) were used in this study. Cows were selected based on their CXCR2 +777 genotype (GG, GC, or CC; n = 10 each), which was determined by matrix-assisted laser desorption ionization-time of flight mass spectroscopy at a commercial facility (Geneseek, Lincoln, NE). All cows were free from clinical mastitis, and milk samples were collected aseptically immediately prior to blood collection to determine the presence or absence of subclinical intramammary infection. Blood (60 ml) was collected by jugular venipuncture in syringes containing 2x acid citrate dextrose anticoagulant (10%, vol/vol) and processed immediately for neutrophil isolation. Prior to isolation, an aliquot was removed for determination of red and white blood cell counts using an automated cell counter (VetCount IIIB; Mallinckrodt, Phillipsburg, NJ). In addition, smears were obtained to determine leukocyte differential counts.
Neutrophil isolation. Neutrophils were isolated from blood as described previously (1) with some modifications. Blood was centrifuged at 860 x g for 30 min at 4°C, and the plasma and buffy coat were discarded. After repeating this step, neutrophils were isolated from the remaining erythrocytes by adding double-distilled water for 30 seconds, and then a 3x concentration of RPMI 1640 medium (Sigma, St. Louis, MO) was added to regain isotonicity of the solution. After a second lysis of red blood cells, remaining neutrophils (96% average purity) were washed with Hanks' balanced salt solution (HBSS; pH 7.2; Cellgro, Herndon, VA) and resuspended in the appropriate medium and cell concentration for the different functional assays as described below. Viability was assessed by trypan blue dye exclusion and always exceeded 97%. Average white blood cell count among cows was 7.5 x 103 cells/ml.
ZAS preparation. Zymosan-activated serum (ZAS) was obtained by incubating bovine serum with yeast cell wall particles (zymosan A; Sigma, St. Louis, MO) at a concentration of 10 mg/ml for 30 min at 37°C in a shaking water bath. After incubation, zymosan particles were pelleted by centrifugation, and serum was collected and stored at –20°C until use.
Adhesion molecule expression. Indirect immunofluorescent analysis of CD11b, CD18, and CD62L expression on isolated neutrophils was performed using murine monoclonal antibodies and fluorescein-conjugated antibody to mouse immunoglobulin G (IgG). Neutrophils (4 x 106 cells/ml in HBSS) were preincubated with HBSS (unstimulated control), 400 ng/ml recombinant human IL-8 (rhIL-8; R&D Systems, Minneapolis, MN), 10% ZAS, or 10 nM phorbol myristate acetate (PMA; Sigma, St. Louis, MO) at 39°C for 30 min while mixing horizontally. After preincubation, 50-μl aliquots of neutrophil suspension were added to 96-well round-bottom microtiter plates containing 50 μl IgG as a negative control (10 μg/ml; Caltag Laboratories, Burlingame, CA), monoclonal antibodies to bovine CD11b (MM10A; 5 μg/ml; VMRD Inc., Pullman, WA), bovine CD18 (BAQ30A; 10 μg/ml; VMRD Inc.), or bovine CD62 (BAQ92A; 5 μg/ml; VMRD Inc.). Plates were incubated at 4°C for 30 min and washed twice with HBSS, and 100 μl of fluorescently labeled goat anti-mouse IgG (Calbiochem, La Jolla, CA) was added to each well. Samples were incubated at 4°C for 30 min, washed as before, and resuspended in 2% formaldehyde in 0.15 M phosphate-buffered saline (PBS). Samples were stored at 4°C in the dark until analyzed by flow cytometry. Data are expressed as percentage of median fluorescence intensity increase over the HBSS-stimulated (negative) control.
Flow cytometry. For adhesion molecule expression, neutrophils were analyzed using a Beckman Coulter Epics XL flow cytometer (Beckman Coulter, Fullerton, CA). Dot plots were gated for neutrophils based on forward and side scatter characteristics, and the median fluorescence intensity was calculated after plotting fluorescence of histograms. Fluorescence associated with neutrophils incubated with nonspecific mouse IgG instead of anti-CD11b, -CD18, or -CD62L was considered the control for nonspecific fluorescence.
Neutrophil migration. The abilities of neutrophils to migrate towards rhIL-8 and ZAS were evaluated using a 48-well chemotaxis chamber (Neuroprobe, Gaithersburg, MD) as described previously (5). Briefly, PBS-0.1% bovine serum albumin (BSA) as negative control, 100 ng/ml rhIL-8, or 5% ZAS was seeded in the lower compartment of the chemotaxis chamber, whereas neutrophils in PBS-0.1% BSA (4 x 105 total cells/well) were added to the upper chamber. The chamber was incubated at 39°C, 5% CO2 for 30 min, and the polycarbonate filter separating the compartments was removed, washed with PBS and scraped three times, fixed with methanol, and stained with DiffQuik for counting. A total of 10 fields at 40x amplification were counted per well. Samples were assayed in triplicate; results were expressed as mean total number of neutrophils migrated per well.
Statistical analysis. Analysis of variance was done with mixed models using SAS software (SAS 8.2; SAS Institute Inc., Cary, NC). A randomized block design with replication was used to determine the effect of CXCR2 +777 genotype on different neutrophil functions. The statistical model was yijk = μ + Di + Gj + DGij + C(DG)ijk, where μ = overall mean, D = day, G = genotype, and C = cow. Data are presented as least square means with associated standard error. Statistical significance was declared at a P level of <0.05, and a trend toward significance was declared at a P level of <0.10.
RESULTS AND DISCUSSION
During mastitis and other inflammatory diseases, potent cytokines (interleukin-1, tumor necrosis factor, IL-8, etc.) and other inflammatory mediators (complement fractions such as C3a and C5a, prostaglandins, leukotrienes, etc.) guide neutrophils towards the site of infection (13, 29, 31, 32, 35, 36). This recruitment and the functional ability of neutrophils once there often determine whether a bacterial infection is cleared or becomes chronic (15, 19). A slow or ineffective neutrophil attack allows bacteria to continue their assault and generate a longer-lasting and potentially more damaging infection. Since neutrophils are key players in the resolution of mastitis and a polymorphism in CXCR2 +777 (GC) had been associated with mastitis susceptibility, we hypothesized that different CXCR2 +777 genotypes would be associated with efficiency of neutrophil migratory ability in vitro.
As a prerequisite for neutrophil migration from blood into tissues, CD62L must be shed and 2 integrin expression and/or affinity must be increased by neutrophils (6). Therefore, we evaluated adhesion molecule expression following stimulation with IL-8, complement fractions C3a and C5a through stimulation with ZAS, and PMA. No differences in CD62L downregulation in response to rhIL-8, ZAS, or PMA were observed among CXCR2 genotypes (P > 0.05) (data not shown). However, following incubation with rhIL-8, but not PMA or ZAS, the percent increase in CD18 expression by neutrophils from cattle with a CC genotype was approximately half that of neutrophils from cattle with a GG genotype and resulted in a significant difference between the two (P < 0.05) (Fig. 1; stimulation with PMA or ZAS not shown). Similarly, the relative upregulation of CD11b after stimulation with IL-8 was reduced by approximately a third in neutrophils from cattle with a CC genotype compared to those with a GG genotype (P < 0.10) (Fig. 1; stimulation with PMA or ZAS not shown). As both CD11b and CD18 are stored predominately in cytoplasmic granules (3), the decreased expression in neutrophils from cattle with the CC genotype may be due to lower baseline expression of these proteins in the cytoplasmic granules or reduced signaling from CXCR1 and/or CXCR2, which impairs granule translocation to the cell surface (3, 23). However, no differences were observed in CD11b or CD18 expression increase after stimulation with PMA or ZAS, which act independently of CXCR activation; this finding would suggest that overall CD11b or CD18 protein levels do not differ among genotypes. Regardless of the mechanism, altered upregulation of CD11b/CD18 may interfere with firm adherence of the neutrophil to the endothelium and the promptness of migration to the inflammatory site. Studies are currently under way to determine if firm adhesion is impacted.
Once firm adhesion occurs by binding of the 2 integrins to the endothelial surface, transmigration through the blood vessel to the site of infection takes place, and potent chemoattractants such as IL-8, C5a, and bacterial products direct neutrophils towards the site of infection. The effectiveness of this response upon bacterial invasion plays a critical role in the resolution of mastitis (15, 19). As such, we evaluated the ability of neutrophils from cattle with different CXCR2 genotypes to migrate in vitro (Fig. 2). Cows expressing the CC genotype for CXCR2 +777 had significantly fewer neutrophils migrate in response to both rhIL-8 and ZAS compared to cattle with the GG genotype (P < 0.05). Interestingly, neutrophils from cattle with a heterozygous genotype responded differently depending upon the stimulus. When neutrophils were stimulated with rhIL-8, those from heterozygous cows responded similarly to neutrophils from cattle with the CC genotype. In contrast, when stimulated with ZAS, neutrophils from heterozygous cows migrated at levels comparable to those cells from cows with the GG genotype.
The reduction in neutrophil migration, as well as CD11b/CD18 upregulation in cattle with a CC genotype, suggests that neutrophil migration also may be impaired in vivo in cattle with the same genotype and may partially explain the greater incidence of subclinical intramammary infections, as determined by bacteriological culture, observed in these cows (40). With the presence of subclinical intramammary infections, it would be expected that higher numbers of neutrophils would be recruited to the mammary gland in order to fight off infection. However, this expected increase in neutrophil recruitment, as measured by the average somatic cell count for each lactation, was not observed in cows with a CC genotype (40) and supports the hypothesis of impaired neutrophil migration in vivo in these animals. The combination of the in vitro and in vivo data also indicates that the defect(s) does not result in an all-or-none response but is a subtle reduction in the number and/or timing of neutrophils migrating. A "slight" delay in either of these factors may be sufficient to allow initial proliferation and invasion of bacteria and subsequent establishment of chronic subclinical infections.
Both neutrophil adhesion molecule (CD11b/CD18) upregulation and migration in response to rhIL-8 were lower in cattle with a CC genotype. One possible explanation for this response may be directly related to the identified polymorphism in CXCR2 (GC), which a causes glutaminehistidine replacement. This amino acid lies within the third intracellular loop of the receptor and has the potential to affect neutrophil activity during infection. This hypothesis is supported by work reported by Damaj et al. (11), which identified amino acid residues within the third intracellular loop of both human IL-8 receptors, CXCR1 and CXCR2, that were involved in mediating neutrophil calcium signaling and mobilization during IL-8 stimulation. Thus, the amino acid substitution at residue 245 may affect CXCR2 signaling and consequent neutrophil function in dairy cattle. Alternatively, the CXCR2 +777 polymorphism may serve as a marker for SNPs located within other segments of the CXCR2 coding sequence that more directly affect receptor binding and function.
Neutrophil migration was impaired in response to not only IL-8 but also ZAS in cattle with the CC genotype in comparison to the GG genotype. As ZAS does not activate CXCR2, this would suggest that polymorphisms within that gene may not be the cause of reduced neutrophil migration and altered adhesion molecule expression in cattle with a CC genotype. Zymosan-activated serum contains the complement fraction C5a. Similar to IL-8, C5a acts on neutrophils through a specific 7TMD receptor (17). Since the C5a receptor is also a G-protein-coupled receptor, it is possible that intracellular pathways that lead to neutrophil migration and are common to both receptors may be altered in cows expressing the CC genotype. Further supporting this idea, CXCR2 and C5a both activate phospholipase C in a pertussis toxin-sensitive manner (26, 37).
In conclusion, this study provides a potential mechanism to explain the genetic differences in mastitis susceptibility observed in cattle with different CXCR2 +777 genotypes. Cows expressing the CXCR2 +777 CC genotype, which have previously shown a higher prevalence of intramammary infections, exhibited impaired neutrophil migration and adhesion molecule upregulation compared to cows of the GG genotype. This information may offer insight into specific mechanisms that are affected in cows more susceptible to mastitis and provide possible targets for generating new strategies to either prevent or treat mastitis in dairy cattle.
ACKNOWLEDGMENTS
The aid of John Hodges, Mark Campbell, Charlie Young, and Dean Jenkins for each of their roles with respect to this project is appreciated greatly.
The research reported within the manuscript was supported by The University of Tennessee Food Safety Center of Excellence, The College of Veterinary Medicine Center of Excellence, and The University of Tennessee Agricultural Experiment Station.
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Bovine mastitis continues to be the most detrimental factor for profitable dairying. Recent research conducted within our laboratory has identified a genetic marker in the CXCR2 gene associated with mastitis susceptibility. The objective of the present study was to evaluate the migratory ability of neutrophils from cows with different CXCR2 +777 genotypes. Neutrophils isolated from peripheral blood of 30 Holstein cows were tested for in vitro migration and adhesion molecule expression. Cows with the CC or GC genotype at CXCR2 +777 showed significantly lower neutrophil migration to recombinant human interleukin-8 (rhIL-8) than cows with the GG genotype (P < 0.05). Cows with the CC genotype at CXCR2 +777 also showed decreased neutrophil migration to zymosan-activated serum compared to these same cows (P < 0.05). Decreased upregulation of CD18 expression was observed after stimulation with rhIL-8 in cows expressing the CXCR2 +777 CC genotype compared to cows expressing the GG genotype (P < 0.05). A similar trend was observed for CD11b (P < 0.10). However, no difference in CD62 downregulation was observed with respect to genotype. These results provide initial evidence for a phenotypic association between a single nucleotide polymorphism and neutrophil function in dairy cows, as well as potential insight into specific mechanisms affected in cows more susceptible to mastitis.
INTRODUCTION
Infectious diseases caused by bacteria negatively impact every animal industry. Effective elimination of bacterial infections, such as mastitis in dairy cattle, requires four basic steps: bacterial recognition, inflammatory mediator release, leukocyte recruitment from the bloodstream, and bacteria removal. When infection occurs, resident macrophages and mammary epithelial cells release inflammatory mediators that signal the body for help. These chemical messengers include chemotactic complement fractions, prostaglandins, leukotrienes, and acute-phase cytokines (interleukin-1 [IL-1], tumor necrosis factor, and IL-8) and are responsible for massive neutrophil migration from the circulation to the site of infection (10, 28, 31, 32).
The ability of neutrophils to migrate into infected tissues is dependent upon recognition of inflammatory mediators by cytokine, chemokine, and complement receptors. Two chemokine receptors present on neutrophil surfaces, CXCR1 and CXCR2, are required for maximum neutrophil function during infection (12, 16). Interleukin-8 is a high-affinity ligand for both receptors but shares CXCR1 with the lower-affinity ligand granulocyte chemotactic protein 2. Besides IL-8, CXCR2 also can bind growth-related oncogene alpha (GRO-), GRO-, GRO-, neutrophil-activating peptide 2, and epithelial-derived neutrophil attractant 78 (2, 38). In neutrophils, recognition of chemokines by CXCR1 and CXCR2 induces 2 integrin upregulation and chemotaxis, as well as enhancement of reactive oxygen species generation and phagocytosis of pathogens (2, 9, 21, 22, 24). The CXC receptors are members of the large family of serpentine receptors with seven transmembrane domains (7TMD) that couple to Bordetella pertussis toxin-sensitive heterotrimeric G-proteins for signal transduction. G-protein coupling to the receptor results in activation of phospholipase C, formation of the second messenger inositol 1,4,5-trisphosphate, and the subsequent increase in cytosolic free calcium concentration (4, 37), as well as activation of phosphatidylinositol 3'-kinase and consequent generation of phosphatidylinositol 3-phosphate (25). All these mechanisms mediate many of the events necessary for proper neutrophil activation and migration to eliminate invading pathogens.
Effective neutrophil recruitment to the site of infection also requires adhesion molecules from the selectin and 2 integrin families. Neutrophil selectin CD62L slows down cells upon contact with its specific ligands (7) and allows neutrophils to roll along vascular endothelial cells. Neutrophils activated by cytokines or chemoattractants subsequently shed CD62L (20) as a prerequisite for 2 integrin-mediated tight adhesion. The most important 2 integrin involved in neutrophil recruitment into inflamed tissue is CD11b/CD18 (3), which is predominantly stored in cytoplasmic granules. Upon cellular activation by inflammatory mediators, these cytoplasmic granules translocate to the cell surface and CD11b/CD18 expression increases (23), causing neutrophils to bind firmly to endothelial cells through interaction with its specific ligand, intercellular adhesion molecule 1.
Influx of neutrophils into the mammary gland is critical for the resolution of mastitis; without quick and efficient neutrophil migration, infections can persist and lead to acute clinical mastitis, chronic infections, severe tissue damage, and potentially death of the cow (15, 19). Interleukin-8 is one of the ligands that binds CXCR2 with high affinity (2), and it is at least partly responsible for the massive influx of neutrophils to the mammary gland during bacterial infection (29, 31, 35). Upon arrival at the site of infection, neutrophils phagocytose and kill intramammary bacteria through reactive oxygen species generation and antibacterial granule proteins (33). Once an infection is cleared, neutrophils undergo apoptosis and are phagocytosed by macrophages (34). This rapid clearance is important because neutrophil lysosomal enzymes and respiratory burst products can damage mammary gland tissue (8, 14, 27).
Single nucleotide polymorphisms (SNPs) within the CXCR2 gene have been identified and evaluated for their potential association with disease in humans (18, 30). Of these, homozygosity at position +785 CC and at position +1208 TT was found in high frequency in individuals with systemic sclerosis compared to healthy controls (30). It is possible that polymorphisms in bovine CXCR2 are also associated with disease susceptibility or progression and may potentially be used to select cows that are more resistant to disease. Recent research conducted in our laboratory has identified five SNPs in a 311-bp segment of the CXCR2 gene of dairy cows (39). Polymorphisms at positions +612, +777, and +861 showed a significant association with subclinical mastitis (40). One of these, the SNP located at position +777 (GC), results in amino acid 245 glutaminehistidine replacement. This may have direct implications in the functionality of the receptor, since amino acid 245 is located in the receptor's third intracellular loop, a critical site for G-protein coupling and activation (11). Holstein cows expressing the CC genotype at position +777 had increased incidence of subclinical mastitis (37%) compared to Holstein cows that expressed the CG (21%) or GG (22%) genotype (40). Based on this phenotypic observation, and since neutrophil recruitment to the site of infection is critical for the resolution of mastitis, we hypothesized that neutrophil migration and adhesion molecule expression would differ among cows of different CXCR2 +777 genotypes.
MATERIALS AND METHODS
Animal selection and sample collection. Thirty Holstein cows (six cows/day; two cows from each genotype) from the Knoxville Experiment Station in mid- to late lactation (at least 100 days in milk) were used in this study. Cows were selected based on their CXCR2 +777 genotype (GG, GC, or CC; n = 10 each), which was determined by matrix-assisted laser desorption ionization-time of flight mass spectroscopy at a commercial facility (Geneseek, Lincoln, NE). All cows were free from clinical mastitis, and milk samples were collected aseptically immediately prior to blood collection to determine the presence or absence of subclinical intramammary infection. Blood (60 ml) was collected by jugular venipuncture in syringes containing 2x acid citrate dextrose anticoagulant (10%, vol/vol) and processed immediately for neutrophil isolation. Prior to isolation, an aliquot was removed for determination of red and white blood cell counts using an automated cell counter (VetCount IIIB; Mallinckrodt, Phillipsburg, NJ). In addition, smears were obtained to determine leukocyte differential counts.
Neutrophil isolation. Neutrophils were isolated from blood as described previously (1) with some modifications. Blood was centrifuged at 860 x g for 30 min at 4°C, and the plasma and buffy coat were discarded. After repeating this step, neutrophils were isolated from the remaining erythrocytes by adding double-distilled water for 30 seconds, and then a 3x concentration of RPMI 1640 medium (Sigma, St. Louis, MO) was added to regain isotonicity of the solution. After a second lysis of red blood cells, remaining neutrophils (96% average purity) were washed with Hanks' balanced salt solution (HBSS; pH 7.2; Cellgro, Herndon, VA) and resuspended in the appropriate medium and cell concentration for the different functional assays as described below. Viability was assessed by trypan blue dye exclusion and always exceeded 97%. Average white blood cell count among cows was 7.5 x 103 cells/ml.
ZAS preparation. Zymosan-activated serum (ZAS) was obtained by incubating bovine serum with yeast cell wall particles (zymosan A; Sigma, St. Louis, MO) at a concentration of 10 mg/ml for 30 min at 37°C in a shaking water bath. After incubation, zymosan particles were pelleted by centrifugation, and serum was collected and stored at –20°C until use.
Adhesion molecule expression. Indirect immunofluorescent analysis of CD11b, CD18, and CD62L expression on isolated neutrophils was performed using murine monoclonal antibodies and fluorescein-conjugated antibody to mouse immunoglobulin G (IgG). Neutrophils (4 x 106 cells/ml in HBSS) were preincubated with HBSS (unstimulated control), 400 ng/ml recombinant human IL-8 (rhIL-8; R&D Systems, Minneapolis, MN), 10% ZAS, or 10 nM phorbol myristate acetate (PMA; Sigma, St. Louis, MO) at 39°C for 30 min while mixing horizontally. After preincubation, 50-μl aliquots of neutrophil suspension were added to 96-well round-bottom microtiter plates containing 50 μl IgG as a negative control (10 μg/ml; Caltag Laboratories, Burlingame, CA), monoclonal antibodies to bovine CD11b (MM10A; 5 μg/ml; VMRD Inc., Pullman, WA), bovine CD18 (BAQ30A; 10 μg/ml; VMRD Inc.), or bovine CD62 (BAQ92A; 5 μg/ml; VMRD Inc.). Plates were incubated at 4°C for 30 min and washed twice with HBSS, and 100 μl of fluorescently labeled goat anti-mouse IgG (Calbiochem, La Jolla, CA) was added to each well. Samples were incubated at 4°C for 30 min, washed as before, and resuspended in 2% formaldehyde in 0.15 M phosphate-buffered saline (PBS). Samples were stored at 4°C in the dark until analyzed by flow cytometry. Data are expressed as percentage of median fluorescence intensity increase over the HBSS-stimulated (negative) control.
Flow cytometry. For adhesion molecule expression, neutrophils were analyzed using a Beckman Coulter Epics XL flow cytometer (Beckman Coulter, Fullerton, CA). Dot plots were gated for neutrophils based on forward and side scatter characteristics, and the median fluorescence intensity was calculated after plotting fluorescence of histograms. Fluorescence associated with neutrophils incubated with nonspecific mouse IgG instead of anti-CD11b, -CD18, or -CD62L was considered the control for nonspecific fluorescence.
Neutrophil migration. The abilities of neutrophils to migrate towards rhIL-8 and ZAS were evaluated using a 48-well chemotaxis chamber (Neuroprobe, Gaithersburg, MD) as described previously (5). Briefly, PBS-0.1% bovine serum albumin (BSA) as negative control, 100 ng/ml rhIL-8, or 5% ZAS was seeded in the lower compartment of the chemotaxis chamber, whereas neutrophils in PBS-0.1% BSA (4 x 105 total cells/well) were added to the upper chamber. The chamber was incubated at 39°C, 5% CO2 for 30 min, and the polycarbonate filter separating the compartments was removed, washed with PBS and scraped three times, fixed with methanol, and stained with DiffQuik for counting. A total of 10 fields at 40x amplification were counted per well. Samples were assayed in triplicate; results were expressed as mean total number of neutrophils migrated per well.
Statistical analysis. Analysis of variance was done with mixed models using SAS software (SAS 8.2; SAS Institute Inc., Cary, NC). A randomized block design with replication was used to determine the effect of CXCR2 +777 genotype on different neutrophil functions. The statistical model was yijk = μ + Di + Gj + DGij + C(DG)ijk, where μ = overall mean, D = day, G = genotype, and C = cow. Data are presented as least square means with associated standard error. Statistical significance was declared at a P level of <0.05, and a trend toward significance was declared at a P level of <0.10.
RESULTS AND DISCUSSION
During mastitis and other inflammatory diseases, potent cytokines (interleukin-1, tumor necrosis factor, IL-8, etc.) and other inflammatory mediators (complement fractions such as C3a and C5a, prostaglandins, leukotrienes, etc.) guide neutrophils towards the site of infection (13, 29, 31, 32, 35, 36). This recruitment and the functional ability of neutrophils once there often determine whether a bacterial infection is cleared or becomes chronic (15, 19). A slow or ineffective neutrophil attack allows bacteria to continue their assault and generate a longer-lasting and potentially more damaging infection. Since neutrophils are key players in the resolution of mastitis and a polymorphism in CXCR2 +777 (GC) had been associated with mastitis susceptibility, we hypothesized that different CXCR2 +777 genotypes would be associated with efficiency of neutrophil migratory ability in vitro.
As a prerequisite for neutrophil migration from blood into tissues, CD62L must be shed and 2 integrin expression and/or affinity must be increased by neutrophils (6). Therefore, we evaluated adhesion molecule expression following stimulation with IL-8, complement fractions C3a and C5a through stimulation with ZAS, and PMA. No differences in CD62L downregulation in response to rhIL-8, ZAS, or PMA were observed among CXCR2 genotypes (P > 0.05) (data not shown). However, following incubation with rhIL-8, but not PMA or ZAS, the percent increase in CD18 expression by neutrophils from cattle with a CC genotype was approximately half that of neutrophils from cattle with a GG genotype and resulted in a significant difference between the two (P < 0.05) (Fig. 1; stimulation with PMA or ZAS not shown). Similarly, the relative upregulation of CD11b after stimulation with IL-8 was reduced by approximately a third in neutrophils from cattle with a CC genotype compared to those with a GG genotype (P < 0.10) (Fig. 1; stimulation with PMA or ZAS not shown). As both CD11b and CD18 are stored predominately in cytoplasmic granules (3), the decreased expression in neutrophils from cattle with the CC genotype may be due to lower baseline expression of these proteins in the cytoplasmic granules or reduced signaling from CXCR1 and/or CXCR2, which impairs granule translocation to the cell surface (3, 23). However, no differences were observed in CD11b or CD18 expression increase after stimulation with PMA or ZAS, which act independently of CXCR activation; this finding would suggest that overall CD11b or CD18 protein levels do not differ among genotypes. Regardless of the mechanism, altered upregulation of CD11b/CD18 may interfere with firm adherence of the neutrophil to the endothelium and the promptness of migration to the inflammatory site. Studies are currently under way to determine if firm adhesion is impacted.
Once firm adhesion occurs by binding of the 2 integrins to the endothelial surface, transmigration through the blood vessel to the site of infection takes place, and potent chemoattractants such as IL-8, C5a, and bacterial products direct neutrophils towards the site of infection. The effectiveness of this response upon bacterial invasion plays a critical role in the resolution of mastitis (15, 19). As such, we evaluated the ability of neutrophils from cattle with different CXCR2 genotypes to migrate in vitro (Fig. 2). Cows expressing the CC genotype for CXCR2 +777 had significantly fewer neutrophils migrate in response to both rhIL-8 and ZAS compared to cattle with the GG genotype (P < 0.05). Interestingly, neutrophils from cattle with a heterozygous genotype responded differently depending upon the stimulus. When neutrophils were stimulated with rhIL-8, those from heterozygous cows responded similarly to neutrophils from cattle with the CC genotype. In contrast, when stimulated with ZAS, neutrophils from heterozygous cows migrated at levels comparable to those cells from cows with the GG genotype.
The reduction in neutrophil migration, as well as CD11b/CD18 upregulation in cattle with a CC genotype, suggests that neutrophil migration also may be impaired in vivo in cattle with the same genotype and may partially explain the greater incidence of subclinical intramammary infections, as determined by bacteriological culture, observed in these cows (40). With the presence of subclinical intramammary infections, it would be expected that higher numbers of neutrophils would be recruited to the mammary gland in order to fight off infection. However, this expected increase in neutrophil recruitment, as measured by the average somatic cell count for each lactation, was not observed in cows with a CC genotype (40) and supports the hypothesis of impaired neutrophil migration in vivo in these animals. The combination of the in vitro and in vivo data also indicates that the defect(s) does not result in an all-or-none response but is a subtle reduction in the number and/or timing of neutrophils migrating. A "slight" delay in either of these factors may be sufficient to allow initial proliferation and invasion of bacteria and subsequent establishment of chronic subclinical infections.
Both neutrophil adhesion molecule (CD11b/CD18) upregulation and migration in response to rhIL-8 were lower in cattle with a CC genotype. One possible explanation for this response may be directly related to the identified polymorphism in CXCR2 (GC), which a causes glutaminehistidine replacement. This amino acid lies within the third intracellular loop of the receptor and has the potential to affect neutrophil activity during infection. This hypothesis is supported by work reported by Damaj et al. (11), which identified amino acid residues within the third intracellular loop of both human IL-8 receptors, CXCR1 and CXCR2, that were involved in mediating neutrophil calcium signaling and mobilization during IL-8 stimulation. Thus, the amino acid substitution at residue 245 may affect CXCR2 signaling and consequent neutrophil function in dairy cattle. Alternatively, the CXCR2 +777 polymorphism may serve as a marker for SNPs located within other segments of the CXCR2 coding sequence that more directly affect receptor binding and function.
Neutrophil migration was impaired in response to not only IL-8 but also ZAS in cattle with the CC genotype in comparison to the GG genotype. As ZAS does not activate CXCR2, this would suggest that polymorphisms within that gene may not be the cause of reduced neutrophil migration and altered adhesion molecule expression in cattle with a CC genotype. Zymosan-activated serum contains the complement fraction C5a. Similar to IL-8, C5a acts on neutrophils through a specific 7TMD receptor (17). Since the C5a receptor is also a G-protein-coupled receptor, it is possible that intracellular pathways that lead to neutrophil migration and are common to both receptors may be altered in cows expressing the CC genotype. Further supporting this idea, CXCR2 and C5a both activate phospholipase C in a pertussis toxin-sensitive manner (26, 37).
In conclusion, this study provides a potential mechanism to explain the genetic differences in mastitis susceptibility observed in cattle with different CXCR2 +777 genotypes. Cows expressing the CXCR2 +777 CC genotype, which have previously shown a higher prevalence of intramammary infections, exhibited impaired neutrophil migration and adhesion molecule upregulation compared to cows of the GG genotype. This information may offer insight into specific mechanisms that are affected in cows more susceptible to mastitis and provide possible targets for generating new strategies to either prevent or treat mastitis in dairy cattle.
ACKNOWLEDGMENTS
The aid of John Hodges, Mark Campbell, Charlie Young, and Dean Jenkins for each of their roles with respect to this project is appreciated greatly.
The research reported within the manuscript was supported by The University of Tennessee Food Safety Center of Excellence, The College of Veterinary Medicine Center of Excellence, and The University of Tennessee Agricultural Experiment Station.
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