• View in gallery
    Figure 1—

    Photomicrograph of a cytocentrifuged preparation of cells isolated from a blood sample from a healthy dog showing highly pure, intact neutrophils. Gradient centrifugation followed by lysis of erythrocytes consistently yielded predominantly neutrophils (> 95%) and rare mononuclear cells against a background containing some cellular debris. The neutrophils were mostly seen individually, with rare clumping. Wright stain; bar = 20 μm.

  • View in gallery
    Figure 2—

    Dot plot of side light scatter (SSC-H) versus forward light scatter (FSC-H) obtained by fluorescence-activated cell-sorting analysis of neutrophils purified from blood samples collected from a healthy dog. Purified neutrophils have consistent light scatter and relative absence of other cells. Of the 500,000 events analyzed, 96.11% were identified as neutrophils on the basis of their characteristic forward and side light scatter (circle marked R1).

  • View in gallery
    Figure 3—

    Schematic representation of a sequencing strategy for the protein-encoding sequence of the canine PROCR gene (A), photograph of an electrophoretic gel of a canine neutrophil cDNA preparation (B), and graph depicting the results of flow cytometric detection of anti–human RCR-379 antibody bound to neutrophils from 1 healthy dog with phycoerythrin-conjugated anti–rat IgG antibody (C). A—The genomic sequence of Canis familiaris chromosome 24 encompassing PROCR was extracted from the latest canine genome assembly (CanFam3.1; 3,471 bp). All 4 predicted exons (thick areas in top diagram) were assembled to generate a consensus cDNA sequence (XM_542972.3) and used in multiple sequence alignment analysis (forward and reverse). A PCR strategy was developed for amplification of the entire PROCR complete cDNA (green color) from the start to the stop codon. B—An amplicon of 726 bp was detected by use of canine neutrophil cDNA preparations as template (lanes 1 and 2) but was absent when the template was omitted (−). M = DNA ladder (set of known DNA fragments varying in size from 100 bp to 12,000 bp. C—This histogram of counts versus flourecence intensity (FL2-H) is used to illustrate that, compared with cells not exposed to RCR-379 (solid blue area), increased fluorescence was detected in neutrophils incubated with RCR-379 (red line).

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    Figure 4—

    Representative photomicrograph of canine neutrophils labeled with DAPI (nuclei) and calcein AM (cytoplasm). Bar = 50 μm.

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    Figure 5—

    Mean Chx values for isolated neutrophils from healthy dogs incubated with CnPC or CnAPC (results combined) with (gray bars) or without (white bars) the anti–human EPCR antibody RCR-379 and then allowed to migrate across a porous membrane toward IL-8 (A; n = 3) or rhC5a (B; 3). The Chx value was defined as the fluorescence reading obtained from the sample well divided by the fluorescence reading obtained from the well containing untreated cells. All assays were performed in triplicate for each chemoattractant by use of neutrophils from different dogs (n = 6). The CnPC sample had been harvested from 6 plasma units from 6 different healthy dogs and used separately across 6 different experiments. Error bars indicate the 95% CI, and the dashed horizontal line indicates the Chx value for untreated cells (1.0). *†Value differs significantly (*P < 0.01; †P < 0.001) between cells treated with and without RCR-379 at this protein concentration.

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Endothelial protein C receptor–dependent antichemotactic effects of canine protein C

Valerie M. WongDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON NIG 2WI, Canada.

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Olivier CôtéDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON NIG 2WI, Canada.

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Dorothee BienzleDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON NIG 2WI, Canada.

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M. Anthony HayesDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON NIG 2WI, Canada.

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R. Darren WoodDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON NIG 2WI, Canada.

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Abstract

OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis.

SAMPLE Neutrophils isolated from blood samples from healthy dogs (n = 6) and sick dogs with (2) or without (3) an inflammatory leukogram.

PROCEDURES Neutrophils were analyzed by reverse transcriptase PCR assay and flow cytometry for detection of EPCR mRNA and protein expression, respectively. Neutrophils were incubated with CnPC zymogen or canine activated protein C (CnAPC), with or without RCR-379 (an anti–human EPCR antibody). Neutrophils were then allowed to migrate through a filter membrane toward a chemokine. Untreated neutrophils served as positive control samples. Migration was quantified by fluorescence measurement, and chemotaxis index (Chx) values (fluorescence of test sample/fluorescence of positive control sample) were computed.

RESULTS The cDNA for EPCR was amplified, and EPCR expression was detected on neutrophil surfaces. Obtained Chx values were significantly higher in cells treated with RCR-379 than in cells treated with CnPC or CnAPC alone. The Chx values for neutrophils treated with RCR-379 were not significantly different from 1, whereas those for neutrophils treated without RCR-379 were significantly less than 1. The effects of RCR-379 on neutrophil migration were independent of concentration or activation status of protein C.

CONCLUSIONS AND CLINICAL RELEVANCE Canine neutrophils expressed EPCR, and inhibition of neutrophil chemotaxis by CnPC and CnAPC depended on EPCR. Interventions with EPCR signaling may have therapeutic application in dogs.

Abstract

OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis.

SAMPLE Neutrophils isolated from blood samples from healthy dogs (n = 6) and sick dogs with (2) or without (3) an inflammatory leukogram.

PROCEDURES Neutrophils were analyzed by reverse transcriptase PCR assay and flow cytometry for detection of EPCR mRNA and protein expression, respectively. Neutrophils were incubated with CnPC zymogen or canine activated protein C (CnAPC), with or without RCR-379 (an anti–human EPCR antibody). Neutrophils were then allowed to migrate through a filter membrane toward a chemokine. Untreated neutrophils served as positive control samples. Migration was quantified by fluorescence measurement, and chemotaxis index (Chx) values (fluorescence of test sample/fluorescence of positive control sample) were computed.

RESULTS The cDNA for EPCR was amplified, and EPCR expression was detected on neutrophil surfaces. Obtained Chx values were significantly higher in cells treated with RCR-379 than in cells treated with CnPC or CnAPC alone. The Chx values for neutrophils treated with RCR-379 were not significantly different from 1, whereas those for neutrophils treated without RCR-379 were significantly less than 1. The effects of RCR-379 on neutrophil migration were independent of concentration or activation status of protein C.

CONCLUSIONS AND CLINICAL RELEVANCE Canine neutrophils expressed EPCR, and inhibition of neutrophil chemotaxis by CnPC and CnAPC depended on EPCR. Interventions with EPCR signaling may have therapeutic application in dogs.

Activated protein C is an endogenous anticoagulant found in plasma. It is derived from protein C zymogen, which is synthesized by hepatocytes and circulates in plasma at low concentrations. Upon activation by thrombin, APC inhibits the activities of factors V and VIII by irreversible proteolysis.1,2 Other than anticoagulant effects, rhAPC has anti-inflammatory3–5 and cytoprotective6,7 properties.

Previously, rhAPC was commercially available as drotrecogin alfa (activated) and approved by the US FDA and European Medicine Agency for treatment of sepsis in humans with a high risk of death on the basis of favorable interim results obtained in a multicenter clinical trial.8 However, subsequent studies9,10 failed to substantiate these results, and drotrecogin alfa (activated) was voluntarily withdrawn from the market worldwide by the manufacturer in 2011.11 Bleeding was the most common adverse reaction.9

Studies involving genetically engineered nonanticoagulant APC variants have shown retention of cytoprotective properties12,13 and improved survival rates in mice with experimentally induced sepsis.14 Some APC variants even confer a greater degree of cytoprotection than wild-type APC.15,16 Results from these studies suggest that the functional properties of APC that are unrelated to its anticoagulant properties may have therapeutic potential. Furthermore, a preclinical safety assessment trial17 involving administration of 3A3K-APC to mice and monkeys yielded favorable results.

Humans and dogs with sepsis share many clinicopathologic features, including a decrease in circulating protein C activity and the positive prognostic value of an increase in protein C activity early during hospitalization.18–21 Furthermore, the genomic and expression sequences of CnPC share a high degree of similarity with their human counterparts.22 Given these similarities and the success of treating rodents with experimentally induced sepsis14 or ischemic stroke23 with nonanticoagulant APC variants, we believed that nonanticoagulant canine APC would have therapeutic potential.

Knowledge of the biological functions of CnPC is limited, and therapeutic application of CnPC has not yet been reported. Inhibition of neutrophil chemotaxis has been suggested to confer therapeutic potential.24 The purpose of the study reported here was to determine whether CnPC had antichemotactic effects on canine neutrophils, whether EPCR was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis. The long-term goal was to provide data to assist in the generation of improved treatments for dogs with sepsis and other inflammatory diseases.

Materials and Methods

Animals

For evaluations of neutrophil chemotaxis, blood samples from 6 adult dogs (1 Beagle, 2 Great Danes, and 3 large-sized hound crosses) were used in 6 experiments over 6 days (3 dogs for each chemoattractant used and 1 dog/experiment). Four of these dogs were owned by the Ontario Veterinary College. Two were owned by members of the college, and owner consent was secured prior to inclusion. All dogs had no remarkable physical examination findings and no recent history of illness or medication (except for heartworm preventives). All procedures involving dogs were carried out in compliance with the University of Guelph's Animal Use Policy and Procedures (protocol Nos. 08E089 and 1704).

For detection of EPCR on canine neutrophils by flow cytometry, another 11 dogs were used. This group included 6 healthy dogs, 2 sick dogs with an inflammatory leukogram, and 3 sick dogs without an inflammatory leukogram.

Blood sample collection

For the experiments involving neutrophil chemotaxis, food was withheld from dogs for a minimum of 12 hours. Then, approximately 20 mL of blood was collected from a cephalic vein by use of a 21-gauge winged needle blood collection seta into multiple 6-mL plastic collection tubes containing 10.8 mg of potassium EDTA. Blood samples in the multiple collection tubes were pooled for each dog prior to neutrophil isolation.

Purification of protein C from plasma samples

Details on the purification of CnPC have been described elsewhere.25 Briefly, jugular venous blood samples were aseptically collected from dogs other than those in the present study, and plasma was harvested. Each of the 6 plasma units was then subjected to barium chloride and ammonium sulfate precipitation, followed by immunoaffinity chromatography by use of a matrix that is linked to HPC4,b an antibody that targets the heavy chain of human protein C. Plasma-derived CnPC was then activated by incubation with a specific activator of protein C as previously described.25 The purity of this protein preparation was confirmed by protein gel electrophoresis followed by silver staining and comparison with the migration pattern of previously purified protein samples confirmed by tandem mass spectrometry to contain CnPC. Protein concentration was determined with a Bradford microprotein assay.c

Isolation of neutrophils from healthy dogs for chemotaxis tests

For chemotaxis tests, neutrophils were isolated within 2 hours after blood sample collection. Pooled blood from each of the 6 healthy dogs was placed over 10 mL of prechilled polysucrose and sodium diatrizoate solution adjusted to a density of 1.077 g/mLd layered over 10 mL of a higher density solution (adjusted to 1.119 g/mL)e in a 50-mL polypropylene conical tube.f Tubes were centrifuged at 340 × g and 21°C for 30 minutes without braking. Cells located between the 2 solution layers were then removed and placed into a 15-mL polypropylene conical tubef that contained 5 mL of chilled PBS solution containing 5mM EDTA (pH, 7.4). The tube was centrifuged at 300 × g and 4°C for 10 minutes, and the supernatant was discarded.

To osmotically lyse the remaining erythrocytes, the harvested cells were resuspended in 1 mL of lysis buffer (8.99 g of NH4Cl/L, 1 g of KHCO3/L, and 37 mg of Na4EDTA/L; pH, 7.35) at room temperature (approx 22°C) for 10 minutes. The remaining intact cells were harvested by centrifugation at 300 × g and 4°C for 5 minutes. Another round of lysis was performed if the cell pellet had pink discoloration.

Cell pellets were washed once by resuspension in 1 mL of PBS solution, recovered by centrifugation at 300 × g and 4°C for 5 minutes, and resuspended in 1 mL of PBS solution. A slide was prepared by cytocentrifugation and stained with Wright stain for differential cell counting. Additionally, some cells were resuspended in PBS solution containing 5mM EDTA, 10% horse serum, and 1 mg of sodium azide/L (pH, 7.35) for assessment of the percentage of neutrophils within the sample by flow cytometry.g Half a million events were acquired, and neutrophils were identified by characteristic side versus forward light scatter.

Detection of mRNA for EPCR in isolated neutrophils from a healthy dog by reverse-transcriptase PCR assay

Approximately 1 × 106 isolated neutrophils from 1 healthy dog were prepared as previously described and resuspended in PBS solution. Total RNA was isolated by use of a commercial kith and oligo dT primersi in accordance with the manufacturer's protocol. First-strand cDNA was obtained by reverse-transcriptase PCR.j By use of the total cDNA as template and a commercial master mix kit,k 40 PCR cycles were performed in a 20-μL reaction mixture containing 0.4μM sense (5′-ATGTTGACAACATTGCTGCCA-3′) and antisense (5′-AACACCGCCGTCCACCT-3′) primer pairs with the following conditions: denaturing at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 90 seconds. Agarose gel analysis was applied to the resulting product, and the approximately 724-bp product was purified by use of a commercial gel extraction kitl on the basis of the manufacturer's protocol.

Sequencing of the PCR products was performed at Laboratory Services, University of Guelph. Approximately 3 to 20 ng of PCR product, the previously described primer pairs, and a commercial reaction kitm were used for the sequencing reactions in a thermocycler.n The sequencing reactions were set up and conducted as indicated in the protocol provided by the manufacturer of the reaction kit. The PCR reagents were removed from the cycle sequencing reactions by use of filter plateso loaded with a gel filtration medium.p The clean reactions were used for electrophoresis,q and a minimum read length of 700 bp was generated for each of the reactions. Resulting chromatograms were analyzed by use of computer softwarer to generate quality target sequences within the software's clear confidence range. The 2 resulting sequences were assembled with the aid of a commercial software packages and compared against predicted and confirmed gene sequences in the databases of the National Center for Biotechnology Information by use of a bioinformatic search tool.t

Detection of EPCR on neutrophils from dogs of various health statuses by flow cytometry

Approximately 200 μL of each EDTA-anticoagulated blood sample from each of the 11 dogs with various health statuses was incubated with 1,400 μL of lysis buffer at room temperature for 10 minutes and then washed in PBS solution containing 5mM EDTA, 10% horse serum, and 1 mg of sodium azide/L (pH, 7.35). The cell pellet was loosened and incubated at 4°C with or without (negative control substance) RCR-379,u which is a rat monoclonal antibody against the extracellular domain of human ECPR, at a dilution of 5 mg/L for 60 minutes. The cells were washed once and incubated at 4°C with 200 μL of goat serum for 60 minutes (blocking). The cells were washed again and then incubated at 4°C with goat anti-rat IgG antibody coupled to phycoerythrin at an optimized dilution (approx 5 mg/L).u Following another wash, the cells were analyzed by flow cytometry.g Neutrophils were identified on the basis of their characteristic position on side versus forward light scatter, and fluorescence was detected at 585/42 nm for all samples.

Chemotaxis assay using isolated neutrophils from healthy dogs

Isolated neutrophils from each of the 6 healthy dogs were labeled with the fluorochrome calcein AMv by combining 10 μL of the fluorochrome and 1 mL of cell suspension for 15 minutes in a water bath at 37°C with gentle agitation. To remove extracellular fluorochrome, the cell suspension was centrifuged at 300 × g and 4°C for 5 minutes and resuspended in fresh PBS solution. The cells were washed once more in a similar fashion. To confirm that all cells were labeled, the cells were examined by fluorescence microscopy. Briefly, a slide containing 500 to 2,000 calcein AM–labeled neutrophils was prepared by cytocentrifugation. A drop of liquid mounting medium containing 4′,6-diamidino-2-phenylindole nuclear stainw was applied, and the slide was read with 24 hours at excitation and emission wavelengths of 350 and 470 nm, respectively, for 4′,6-diamidino-2-phenylindole and 490 and 525 nm, respectively, for calcein AM.

Neutrophils were resuspended at a concentration of 106 cells/mL and treated with CnPC or CnAPC at protein concentrations of 0.1 ng/μL, 1 ng/μL, and 10 ng/μL or without these proteins. Samples were then incubated with PBS solution or antibody RCR-379 for 30 minutes at 4°C. Neutrophil chemotaxis was assessed in a 96-well microplate system.x Thirty microliters of the chemoattractant recombinant canine IL-8 (18 μgμL)y or rhC5a (10 ng/mL)y was deposited in each well (neutrophils from 3 healthy dogs/chemoattractant). For a negative control substance, PBS solution was used in place of a chemoattractant. A porous membrane was placed over the wells, and 50 μL of neutrophil suspension (106 cells/mL) was placed over the membrane.

All analyses were performed in triplicate. The assay was placed in an incubator at 37°C with 5% CO2 for 1 hour to allow the neutrophils to migrate. Cells that failed to migrate were gently scraped off the top of the membrane with a cell scraper. Fluorescence was measured at excitation and emission wavelengths of 485 and 528 nm, respectively, by use of a spectrophotometer and software.z Phosphate-buffered saline solution was used for blank measurement with an assigned value of 100 RFUs. The Chx values were calculated by dividing the fluorescence reading obtained from treated cells by that from untreated cells. Fluorescence measurements were also performed for 0.5 μg of CnPC, 0.5 μg of CnAPC, IL-8, and rhC5a placed under the membrane in the absence of neutrophils. The mean reading from the triplicate analyses was considered as 1 experimental replicate. In all, neutrophils from 6 dogs were used separately in 6 different chemotaxis assays, with each chemoattractant (IL-8 or rhC5a) used on 3 of these occasions.

Viability assay using isolated neutrophils from healthy dogs

Isolated neutrophils from 1 healthy dog were resuspended in PBS solution at 1 × 109 cells/L and incubated with CnPC or CnAPC at protein concentrations of 0.1 ng/μL, 1 ng/μL, and 10 ng/μL or without these proteins for 30 minutes at 4°C and then incubated at 37°C with 5% CO2 for 1 hour. The cell suspension was incubated with an equal volume of 0.4% trypan blue solution at room temperature for 2 minutes. The cells were then placed in the 2 chambers of a hemocytometer. For each sample, 500 cells from each chamber were examined and the mean percentage of unstained cells was used to assess cell viability of that particular sample.

Statistical analysis

For detection of EPCR by flow cytometry, a 2-factor split-plot in a completely randomized design was used to examine differences in MFI and MdFI among dogs by health status by means of ANOVA. The whole-plot factor was health status (healthy, sick with an inflammatory leukogram, or sick without an inflammatory leukogram), and the split-plot factor was treatment (with or without incubation with RCR-379). Dog nested within health status was treated as a random effect. The interaction between treatment and health status was included in the model.

For the chemotaxis assay, a 4-factor split plot in a completely randomized design with subsampling was used to compare fluorescence values between chemoattractants by means of ANOVA. The whole-plot factor was chemoattractant (IL-8 or rhC5a), and the split-plot factors were protein concentration (of CnPC or CnAPC), treatment (CnPC or CnAPC), and antibody (with or without RCR-379). Dog nested within chemoattractant was treated as a random effect. All main effects and all 2-way, 3-way, and 4-way interactions were fitted in the model. For comparisons with control values in the chemotaxis experiment, a 2-factor split plot was used in a completely randomized design. The whole-plot factor was chemoattractant, and the split-plot factor was control sample. Dog nested within chemoattractant was treated as a random effect. The interaction between chemoattractant and control samples was fitted in the model.

To assess the ANOVA assumptions, comprehensive residual analyses, including testing of the normality assumption by use of the Shapiro-Wilk, Kolmogorov-Smirnov, Cramer-von Mises, and Anderson-Darling tests, were performed. The residuals were plotted against predicted values as well as against the explanatory variables used in the model with the intent of revealing outliers, unequal variances, or the need for data transformation. Statistical softwareaa was used for all analyses. Values of P < 0.05 were considered significant.

Results

Isolation of neutrophils

The method described for neutrophil isolation from blood samples from healthy dogs consistently yielded > 95% intact neutrophils as assessed on cytocentrifuge preparations (Figure 1). Cell clumping was rarely observed. The remaining cells were predominantly lymphocytes. Flow cytometric analysis of 1 sample of isolated neutrophils revealed that 96.11% of cells in the sample were neutrophils (Figure 2). The number of cells recovered by this method ranged from 5.9 × 105 cells/mL to 1.4 × 106 cells/mL of blood.

Figure 1—
Figure 1—

Photomicrograph of a cytocentrifuged preparation of cells isolated from a blood sample from a healthy dog showing highly pure, intact neutrophils. Gradient centrifugation followed by lysis of erythrocytes consistently yielded predominantly neutrophils (> 95%) and rare mononuclear cells against a background containing some cellular debris. The neutrophils were mostly seen individually, with rare clumping. Wright stain; bar = 20 μm.

Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.186

Figure 2—
Figure 2—

Dot plot of side light scatter (SSC-H) versus forward light scatter (FSC-H) obtained by fluorescence-activated cell-sorting analysis of neutrophils purified from blood samples collected from a healthy dog. Purified neutrophils have consistent light scatter and relative absence of other cells. Of the 500,000 events analyzed, 96.11% were identified as neutrophils on the basis of their characteristic forward and side light scatter (circle marked R1).

Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.186

PCR detection of EPCR mRNA

Expression of EPCR was detected at the mRNA level. The described PCR strategy was used to detect a single product of approximately 726 bp in size. The product was sequenced in both sense and antisense directions, and the resulting sequences were assembled to generate a cDNA sequence. This sequence was 99.7% identical to the predicted expression sequence for gene PROCR given the latest canine genome assembly (CanFam3.1;3471 bp; Figure 3). Heterozygosity was identified in the sequence at 2 positions: T127C and A606G. For T127C, the predicted tyrosine residue was replaced by a histidine residue. The predicted serine residue remained unchanged with A606G. The sequence has been submitted to GenBank (accession number KF498853). Compared with the human EPCR mRNA transcript, 611 of 726 (84.2%) sites were identical. At the amino acid level, human and canine EPCR were predicted to be 76.4% identical.

Figure 3—
Figure 3—

Schematic representation of a sequencing strategy for the protein-encoding sequence of the canine PROCR gene (A), photograph of an electrophoretic gel of a canine neutrophil cDNA preparation (B), and graph depicting the results of flow cytometric detection of anti–human RCR-379 antibody bound to neutrophils from 1 healthy dog with phycoerythrin-conjugated anti–rat IgG antibody (C). A—The genomic sequence of Canis familiaris chromosome 24 encompassing PROCR was extracted from the latest canine genome assembly (CanFam3.1; 3,471 bp). All 4 predicted exons (thick areas in top diagram) were assembled to generate a consensus cDNA sequence (XM_542972.3) and used in multiple sequence alignment analysis (forward and reverse). A PCR strategy was developed for amplification of the entire PROCR complete cDNA (green color) from the start to the stop codon. B—An amplicon of 726 bp was detected by use of canine neutrophil cDNA preparations as template (lanes 1 and 2) but was absent when the template was omitted (−). M = DNA ladder (set of known DNA fragments varying in size from 100 bp to 12,000 bp. C—This histogram of counts versus flourecence intensity (FL2-H) is used to illustrate that, compared with cells not exposed to RCR-379 (solid blue area), increased fluorescence was detected in neutrophils incubated with RCR-379 (red line).

Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.186

Flow cytometric detection of EPCR

For both MFI and MdFI values obtained by flow cytometry, the ANOVA assumptions were adequately met except for 1 outlier that had no substantive impact on the results. The ANOVA models revealed that health status of dogs was significantly (P = 0.01) associated with MFI values. A significant (P = 0.003) association was also identified between neutrophil treatment with RCR-379 and MFI. No significant interaction was identified between neutrophil RCR-379 treatment and dog health status (ie, there was an effect of treatment on MFI regardless of health status).

The estimated difference in MFI between neutrophils incubated with and without RCR-379 was 12.14 (Table 1). The estimated difference in MFI between sick dogs with an inflammatory leukogram and healthy dogs was significant (P = 0.02), with an estimated difference of 21.14. The difference in MFI between isolated neutrophils from the 6 healthy dogs and those from the 3 sick dogs without an inflammatory leukogram was not significant (P = 0.14), with an estimated difference of 10.16. The difference in MFI between isolated neutrophils from sick dogs with an inflammatory leukogram and those from sick dogs without an inflammatory leukogram was significant (P = 0.005), with an estimated difference of 31.30. Results regarding MdFI values indicated similar differences.

Table 1—

Mean (95% CI) values for results of flow cytometric detection of EPCR expression* on neutrophils isolated from blood samples of dogs of various health statuses.

VariableMFIMdFI
Neutrophils incubated without RCR-379 (n = 6)32.44 (25.02 to 39.86)27.83 (21.88 to 33.78)
Neutrophils incubated with RCR-379 (n = 6)44.58 (37.16 to 52.00)37.83 (31.88 to 43.78)
Difference between neutrophil incubation with and without RCR-379 (n = 6)12.14 (5.25 to 19.02)10.00 (5.20 to 14.80)
Neutrophils from healthy dogs (n = 6)34.85 (26.62 to 43.19)28.03 (21.13 to 34.93)
Neutrophils from sick dogs with an inflammatory leukogram (n = 2)55.99 (41.56 to 70.42)50.44 (38.49 to 62.39)
Neutrophils from sick dogs without an inflammatory leukogram (n = 3)24.69 (12.91 to 36.47)20.02 (10.27 to 29.77)
Difference between healthy dogs and sick dogs with an inflammatory leukogram21.14 (4.47 to 37.80)22.41 (8.61 to 36.20)
Difference between healthy dogs and sick dogs without an inflammatory leukogram10.16 (−4.27 to 24.60)8.01 (−3.93 to 19.96)
Difference between sick dogs with an inflammatory leukogram and sick dogs without an inflammatory leukogram31.30 (12.67 to 49.93)30.42 (15.00 to 48.84)

Expression was evaluated by use of the anti–human EPCR antibody RCR-379.

Indicated difference is significant (P < 0.05).

Chemotaxis

Staining of neutrophils with calcein AM was confirmed by fluorescence microscopy (Figure 4). Significant (P < 0.001) differences in chemotaxis findings were identified between neutrophils stained with calcein AM and not incubated with CnPC, CnAPC, or RCR-379 (positive neutrophils) and negative control neutrophils (incubated with PBS solution). No significant (P = 0.08) difference was identified between the 2 chemoattractants in proportions of migrating cells. There was no significant interaction between the chemoattractant and the control samples. A significantly (P = 0.02) greater proportion of positive neutrophils than negative control neutrophils migrated toward the chemoattractants. In the absence of CnPC, CnAPC, or RCR-379, a significantly (P = 0.001) greater proportion of cells migrated toward both IL-8 and rhC5a (median, 614.59 RFUs; 95% CI, 370.26 to 1,020.15 RFUs) than toward PBS solution (median, 174.79 RFUs; 95% CI, 105.30 to 290.13 RFUs). Compared with the blank substance (PBS solution), neutrophils exposed to IL-8 (P = 0.49), rhC5a (P = 0.32), CnPC (P = 0.65), and CnAPC (P = 0.73) were statistically similar in fluorescence values.

Figure 4—
Figure 4—

Representative photomicrograph of canine neutrophils labeled with DAPI (nuclei) and calcein AM (cytoplasm). Bar = 50 μm.

Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.186

The chemotaxis data were not normally distributed despite logarithmic transformation. The final ANOVA model revealed no significant interactions involving treatment (CnPC or CnAPC), but treatment was retained as a main effect and was significant (P = 0.02). Two significant 2-way interactions were identified: protein concentration (of CnPC or CnAPC) by antibody (with or without RCR-379; P = 0.033) and chemoattractant by antibody (P = 0.002). However, because these 2 interactions had a variable in common (antibody), they could not be disentangled. Therefore, the 3-way interaction of chemoattractant, protein concentration, and antibody was retained in the final model despite lack of significance (P = 0.36).

The mean Chx value for cells treated with CnAPC in the absence of RCR-379 was 0.567 (95% CI, 0.409 to 0.790; P = 0.001), and the mean value for cells treated with CnPC in the absence of RCR-379 was 0.496 (95% CI, 0.355, 0.691; P < 0.001). Mean values for each evaluated concentration of CnPC or CnAPC were summarized by chemoattractant and antibody status (Table 2). The mean Chx value for cells treated with CnAPC was estimated to be 1.143 times higher than the value for cells treated with CnPC (95% CI, 1.021 to 1.280; P = 0.02). In the absence of RCR-379, Chx values for cells treated with either CnPC or CnAPC at all tested concentrations were all significantly less than 1 (Figure 5). In the presence of RCR-379, Chx values for cells treated with either CnPC or CnAPC at all but the lowest concentration tested (0.1 ng/μL), with IL-8 used as chemoattractant, were not significantly different from 1.

Table 1—

Mean (95% CI) Chx values for the effects of CnPC and CnAPC concentration and anti-human EPCR antibody (RCR-379) on IL-8- and rhC5a-induced chemotaxis of neutrophils isolated from blood samples of 6 healthy dogs.

 IL-8rhC5a
Protein concentration (ng/μL)No RCR-379RCR-379No RCR-379RCR-379
0.10.390 (0.234–0.649)0.466 (0.280–0.775)0.39* (0.235–0.651)0.636 (0.382–1.058)
10.415* (0.249–0.690)0.665 (0.400–1.107)0.360* (0.216–0.600)0.639 (0.384–1.064)
100.399* (0.240–0.663)0.664 (0.399–1.105)0.360* (0.217–0.600)0.909 (0.546–1.512)
1000.490* (0.294–0.815)0.755 (0.454–1.257)0.373* (0.224–0.620)1.126 (0.676–1.873)

Indicated value is significantly different from the value for no inhibition.

The Chx values were calculated as the fluorescence of test sample divided by the fluorescence of positive control sample.

Figure 5—
Figure 5—

Mean Chx values for isolated neutrophils from healthy dogs incubated with CnPC or CnAPC (results combined) with (gray bars) or without (white bars) the anti–human EPCR antibody RCR-379 and then allowed to migrate across a porous membrane toward IL-8 (A; n = 3) or rhC5a (B; 3). The Chx value was defined as the fluorescence reading obtained from the sample well divided by the fluorescence reading obtained from the well containing untreated cells. All assays were performed in triplicate for each chemoattractant by use of neutrophils from different dogs (n = 6). The CnPC sample had been harvested from 6 plasma units from 6 different healthy dogs and used separately across 6 different experiments. Error bars indicate the 95% CI, and the dashed horizontal line indicates the Chx value for untreated cells (1.0). *†Value differs significantly (*P < 0.01; †P < 0.001) between cells treated with and without RCR-379 at this protein concentration.

Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.186

To confirm that the protein preparations did not affect cell viability, thereby inhibiting chemotaxis, cells were incubated with CnPC and CnAPC at the concentrations used in the chemotaxis assay and then stained with trypan blue. Consistently, < 2% of the cells had positive results of this staining.

Discussion

Results of the study reported here indicated that plasma-derived CnPC zymogen purified from canine plasma and its Protac-activated form, CnAPC, inhibited canine neutrophil migration toward IL-8 and rhC5a. Protac is a fast-acting activator of protein C isolated from the venom of the copperhead snake Agkistrodon contortrix. Therefore, CnPC had antichemotactic effects similar to rhAPC26–28 This suggested that CnPC and CnAPC might have a therapeutic role in dogs with inflammatory conditions in which tissue injury caused by accumulation of neutrophils is a concern (eg, ischemia-reperfusion injury and immune-mediated polyarthritis).

The use of plasma-derived proteins has some advantages over the use of recombinant therapeutic proteins. The method we used to purify CnPC was less expensive and not as technically demanding as production of recombinant therapeutic proteins. However, our method required reliance on a supply of plasma from healthy dogs, providing a possible route for infectious disease transmission. On the other hand, production of recombinant CnPC or CnAPC would be more technically demanding and the glycosylation pattern that determines some of the functional properties of this protein would necessitate production in mammalian cells. However, genetic engineering could be presumed to allow for an infinite supply of therapeutic proteins. Furthermore, the option exists to express proteins in a cell line obtained from another animal species to minimize the risk of infectious disease transmission.

The antichemotactic effect of CnPC identified in the present study was likely independent of the activation status of CnAPC, given that both CnPC and CnAPC exposure reduced the Chx value of neutrophils to < 1. These findings are consistent with what is known about the structure of human and canine protein C.25 In another study,25 we showed that human and canine protein C are highly similar in structure, both consisting of a light chain linked to a heavy chain. Incubation with Protac resulted in removal of an 11-amino acid peptide from the N-terminus of CnPC zymogen, exposing the anticoagulant domain of the protein. Because both CnPC and CnAPC inhibited neutrophil chemotaxis, CnPC most likely interacted with canine neutrophils through a mechanism independent of its anticoagulant domain.

To explore this mechanism further, we tested whether EPCR was involved, given that EPCR has been found to play a role in inhibition of neutrophil chemotaxis by human protein C and rhAPC.26 We first confirmed the presence of EPCR transcripts in neutrophils. In preliminary research assessing surface expression of EPCR on canine neutrophils, we tested 2 monoclonal anti-human EPCR antibodies, RCR-252 and RCR-379. On flow cytometry, RCR-252 did not bind to canine neutrophils under various incubation conditions (data not shown). On the other hand, RCR-379 bound to the surface of canine neutrophils, suggesting that an EPCR-like receptor was expressed on canine neutrophils.

The degree of EPCR expression appeared low, as suggested by the slight shift in fluorescence values on flow cytometry, compared with results for negative control cells. This finding was similar to other reported findings when RCR-252 was used to detect EPCR expression on the surface of human neutrophils.26 In the presence of RCR-379, the antichemotactic effects of CnPC and CnAPC toward rhC5a were abolished at all protein concentrations, whereas they were abolished at all but the lowest concentration (0.1 ng/μL) for IL-8 induced chemotaxis. This suggested that the antichemotactic effect of CnPC and CnAPC was dependent on the EPCR we identified on canine neutrophils and that this receptor interacted with a domain in these proteins independent of the anticoagulant domain. Indeed, although not formally tested, it appeared that the blocking effects of RCR-379 might have been more pronounced when CnPC and CnAPC were at higher concentrations.

We observed that EPCR expression was significantly upregulated on neutrophils from sick dogs with an inflammatory leukogram relative to expression on neutrophils from healthy dogs. However, because the original intent of the present study did not include examining the degree of EPCR expression in neutrophils from sick dogs and the study design did not include consideration of the sample size needed to properly examine this, conclusions regarding these preliminary findings should be guarded. Expression of the EPCR is upregulated on monocytes during inflammation,29 but degree of its expression on neutrophils during inflammation is unknown. Given these preliminary findings, additional studies are needed to better characterize the expression and functional role of EPCR on neutrophils.

The present study had some limitations. First, this was an in vitro study that lacked the complex environment of a physiologic system. For example, the membrane used in the in vitro system did not reflect the reactive endothelium during inflammation. Only 1 cytokine was present in the assay system at a given time, which did not reflect the inflammatory milieu in vivo. Therefore, the biological importance of our results needs to be examined further in a more physiologically relevant model. Second, we examined the effects of CnPC and CnAPC on directional migration of neutrophils from only 6 healthy dogs (1 Beagle, 2 Great Danes, and 3 large-sized hound crosses). We observed that neutrophils isolated from different dogs had different degrees of readiness to migrate, even though the proportions of neutrophils in which migration was inhibited by CnPC or CnAPC did not vary greatly among dogs. These findings suggested that the neutrophils of some dogs were more reactive than those of other dogs. Also, results may have been biased by the sole use of mostly large-sized dogs. More studies are required to define this phenomenon and to confirm the results achieved in the present study are generalizable to other dogs, particularly diseased dogs, if CnPC were to be used as a therapeutic agent.

Results of the present study indicated that plasma-derived CnPC and Protac-activated CnAPC inhibited canine neutrophil chemotaxis in vitro, EPCR was expressed on canine neutrophils, and the antichemotactic effects of CnPC or CnAPC were dependent on EPCR and distinct from their anticoagulant properties. Future efforts should focus on better characterizing the roles of CnPC and canine EPCR regarding neutrophils from dogs in various disease states and the therapeutic potential of intervening with EPCR signaling.

Acknowledgments

This manuscript represents a portion of a thesis submitted by Dr. Wong to the University of Guelph as partial fulfillment of the requirements for a Doctor of Philosophy degree.

Supported in part by the Ontario Veterinary College Pet Trust and PhD Fellowship Program.

Presented in abstract form at the 48th Annual Meeting of the American Society for Veterinary Clinical Pathology, Montreal, November 2013.

The authors thank Mary Ellen Clark for technical assistance.

ABBREVIATIONS

APC

Activated protein C

Chx

Chemotaxis index

CnAPC

Canine activated protein C

CnPC

Canine protein C

EPCR

Endothelial protein C receptor

IL-8

Interleukin-8

MdFI

Median fluorescence index

MFI

Mean fluorescence index

RFU

Relative fluorescence unit

rhAPC

Recombinant human activated protein C

rhC5a

Recombinant human complement factor 5a

Footnotes

a.

BD Vacutainer safety-lok, Becton-Dickinson, Rutherford, NJ.

b.

Anti–protein C affinity matrix, Roche, Laval, QC, Canada.

c.

Bio-Rad protein assay kit, Bio-Rad, Mississauga, ON, Canada.

d.

Histopaque-1077, Sigma-Aldrich, Oakville, ON, Canada.

e.

Histopaque-1119, Sigma-Aldrich, Oakville, ON, Canada.

f.

BD Falcon polypropylene conical tube, BD Biosciences, Mississauga, ON, Canada.

g.

FACScan flow cytometer with CellQuest software, Becton-Dickinson, Franklin Lakes, NJ.

h.

RNeasy mini kit, Qiagen, Toronto, ON, Canada.

i.

Oligo(dT)20, Life Technologies, Burlington, ON, Canada.

j.

SuperScript III reverse transcriptase, Life Technologies, Burlington, ON, Canada.

k.

HotStart master mix kit, Qiagen, Toronto, ON, Canada.

l.

MiniElute gel extraction kit, Qiagen, Toronto, ON, Canada.

m.

ABI Prism BigDye terminator cycle sequencing ready reaction kit, version 3.1, Applied Biosystems, Foster City, Calif.

n.

GeneAmp PCR system 9700 or 2720 thermal cycler, Applied Biosystems, Foster City, Calif.

o.

ltiscreen-HV plates, Millipore, Mississauga, ON, Canada.

p.

Sephadex G-50 superfine, Sigma, Oakville, ON, Canada.

q.

Applied Biosystems 3730 DNA analyzer, Applied Biosystems, Foster City, Calif.

r.

ABI Prism DNA sequencing analysis software, version 3.7, Applied Biosystems, Foster City, Calif.

s.

Geneious, Biomatters Ltd, Auckland, New Zealand.

t.

BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/. Accessed July 30, 2013.

u.

Abcam, Toronto, ON, Canada.

v.

Invitrogen, Burlington, ON, Canada.

w.

DAPI, Life Technologies, Burlington, ON, Canada.

x.

ChemoTx, NeuroProbe Inc, Gaithersburg, Md.

y.

R&D Systems, Minneapolis, Minn.

z.

BioTek Synergy HT and Gen5 version 1.11, BioTek, Winooski, Vt.

aa.

SAS, version 9.1, SAS Institute, Cary, NC.

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Contributor Notes

Address correspondence to Dr. Wong (vwong@midwestern.edu).

Dr. Wong's present address is College of Veterinary Medicine, Midwestern University, Glendale, AZ 85308.