• View in gallery

    Representative photomicrographs depicting qualitative histologic scoring of neutrophil infiltration (arrowheads) at local injection sites in equids after SC administration of ReqIL-4, ReqIL-8, or negative control solution, each mixed with the same biopolymer carrier. A 6-mm-diameter biopsy punch was used to collect cutaneous and subcutaneous tissues, including the biopolymer plug, at 1 injection site/treatment type/animal at selected time points, and samples were fixed in neutral-buffered 10% formalin solution prior to paraffin embedding and sectioning. Slides were excluded from analysis if biopolymer was not visible in the section. A—Absence of neutrophils (score of 0). B—Rare neutrophils (score of 1). C—Occasional neutrophils (score of 2). D—Moderate infiltration (score of 3). E—Strong infiltration (score of 4). F—Very dense infiltration (score of 5). The biopolymer (asterisk) is seen as a homogenous bright-pink substance surrounded by saffron-yellow to orange host tissue and collagen fibers. HPS stain; bar = 50 μm.

  • View in gallery

    Scatterplots of histologic scores for neutrophil (A) and mononuclear cell (B) infiltration into injection site tissue samples collected at various time points after SC injection of biopolymer (15 mg/mL) mixed with 100 ng of ReqIL-4 (squares), 100 ng of ReqIL-8 (positive control; triangles), or 10 μL of DPBSS (negative control; circles) in the lateral aspect of the neck in 6 equids (experiment 1). Infiltration scores for cells of each type were assigned from 0 (absent) to 5 (very dense) as depicted for neutrophils in Figure 1. The total injection volume for each treatment was 100 μL/site. Each point represents the cellular infiltration score for 1 sample/equid. Slides where no biopolymer was visible were excluded from the analysis. Horizontal lines depict the mean for all samples for a given treatment at the specified time point.

  • View in gallery

    Scatterplots of histologic scores for neutrophil infiltration into injection site tissue samples 3 and 12 hours after SC injection of biopolymer (8 mg/mL) mixed with 250 ng of ReqIL-4 (squares), 250 ng of ReqIL-8 (triangles), or 10 μL of the same culture medium used for dilution of cytokines (negative control; circles) in the lateral aspect of the neck in 6 equids (experiment 2). See Figure 2 for remainder of key.

  • View in gallery

    Scatterplots of histologic scores for neutrophil infiltration into injection site tissue samples 3 hours after SC injection of biopolymer (8 mg/mL) mixed with 500 ng of ReqIL-4, 1 μg of ReqIL-8, or 10 μL of the same culture medium used for dilution of cytokines (negative control) in the lateral aspect of the neck in 6 equids (experiment 3). Results for 1 animal were excluded from analysis because of poor-quality slides. The total injection volume for each treatment was 250 μL/site. The Cochran-Mantel-Haenszel test indicated a significant (P = 0.049) difference in scores among groups; however, the greatest difference between treatments did not meet significance criteria in pairwise comparisons (P = 0.059). NC = Negative control. See Figure 2 for remainder of key.

  • View in gallery

    Scatterplots of log-transformed neutrophil counts in injection site tissue samples as determined by flow cytometry 3 hours (A) and 6 hours (B) after SC injection of biopolymer mixed with ReqIL-4, ReqIL-8, or culture medium in experiment 3. Results of ANOVA revealed a significant (P = 0.025) difference in mean neutrophil counts among treatments; pairwise comparison with the Fisher least significant difference test indicated mean neutrophil counts (normalized to sample weight prior to enzymatic digestion for preparation of cell suspensions) were significantly greater for the positive control (ReqIL-8) treatment than for the negative control treatment 3 hours (P = 0.032), but not 6 hours (P = 0.054), after injection. See Figures 2 and 4 for key.

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Use of a biopolymer delivery system to investigate the influence of interleukin-4 on recruitment of neutrophils in equids

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  • 1 1Département des Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 2M2, Canada.
  • | 2 2Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 2M2, Canada.

Abstract

OBJECTIVE

To use a biopolymer delivery system to investigate the ability of interleukin (IL)-4 to recruit neutrophils into subcutaneous tissues of equids.

ANIMALS

16 horses and 2 ponies.

PROCEDURES

Animals were assigned to 3 experiments (6/experiment). Effects of recombinant equine (Req) IL-4 (100, 250, or 500 ng/site) versus a positive control (ReqIL-8; 100 ng, 250 ng, or 1 μg/site) and a negative control (Dulbecco PBSS or culture medium) on neutrophil chemotaxis were assessed after SC injection into the neck with an injectable biopolymer used as the vehicle. Tissue samples including the biopolymer plug were collected by biopsy at various time points from 3 hours to 7 days after injection. Neutrophil infiltration was evaluated by histologic scoring (experiments 1, 2, and 3) or flow cytometry (experiment 3).

RESULTS

Histologic neutrophil infiltration scores did not differ significantly among treatments at most evaluated time points. On flow cytometric analysis, log-transformed neutrophil counts in biopsy specimens were significantly greater for the ReqIL-8 treatment (1 μg/site) than the negative control treatment at 3 but not 6 hours after injection; results did not differ between ReqIL-4 and control treatments at either time point. Negative control treatments induced an inflammatory response in most equids in all experiments.

CONCLUSIONS AND CLINICAL RELEVANCE

Flow cytometry was a more reliable method to estimate neutrophil migration than histologic score analysis. The ReqIL-4 treatment did not induce a detectable neutrophil response, compared with the negative control treatment in this study. Evidence of inflammation in negative control samples suggested the biopolymer is not a suitable vehicle for use in equids.

Abstract

OBJECTIVE

To use a biopolymer delivery system to investigate the ability of interleukin (IL)-4 to recruit neutrophils into subcutaneous tissues of equids.

ANIMALS

16 horses and 2 ponies.

PROCEDURES

Animals were assigned to 3 experiments (6/experiment). Effects of recombinant equine (Req) IL-4 (100, 250, or 500 ng/site) versus a positive control (ReqIL-8; 100 ng, 250 ng, or 1 μg/site) and a negative control (Dulbecco PBSS or culture medium) on neutrophil chemotaxis were assessed after SC injection into the neck with an injectable biopolymer used as the vehicle. Tissue samples including the biopolymer plug were collected by biopsy at various time points from 3 hours to 7 days after injection. Neutrophil infiltration was evaluated by histologic scoring (experiments 1, 2, and 3) or flow cytometry (experiment 3).

RESULTS

Histologic neutrophil infiltration scores did not differ significantly among treatments at most evaluated time points. On flow cytometric analysis, log-transformed neutrophil counts in biopsy specimens were significantly greater for the ReqIL-8 treatment (1 μg/site) than the negative control treatment at 3 but not 6 hours after injection; results did not differ between ReqIL-4 and control treatments at either time point. Negative control treatments induced an inflammatory response in most equids in all experiments.

CONCLUSIONS AND CLINICAL RELEVANCE

Flow cytometry was a more reliable method to estimate neutrophil migration than histologic score analysis. The ReqIL-4 treatment did not induce a detectable neutrophil response, compared with the negative control treatment in this study. Evidence of inflammation in negative control samples suggested the biopolymer is not a suitable vehicle for use in equids.

A Th-2 cytokine profile is recognized as the main orchestrator in allergic inflammation.1 This response is classically associated with an inflammation profile largely mediated by IgE and a resulting eosinophilic infiltrate.2 However, noneosinophilic responses are also reported, with research largely focusing on respiratory diseases such as asthma and rhinitis. For example, it is accepted that asthmatic patients may have neutrophil-predominant phenotypes defined by specific cellular and molecular markers.3 Neutrophils also play a role in contact hypersensitivity and allergic contact dermatitis during the sensitization and elicitation phases when they can contribute to tissue damage.45 In horses with severe asthma (also described as heaves or recurrent airway obstruction), which is characterized by chronic airflow obstruction, bronchial hyperresponsiveness, and remodeling of the airways,6 neutrophils are also the predominant inflammatory cells that infiltrate the airway. Results of a previous study7 by our group revealed that neutrophils are closely associated with a Th-2 cytokine-predominant profile.

In other studies,7,8 in situ hybridization experiments showed that lymphocytes present in bronchoalveolar lavage fluid of horses with severe asthma had increased expression of Th-2-type cytokines (IL-4 and IL-5) and decreased expression of the Th-1-type cytokine interferon-γ, compared with results for control horses without signs of asthma. There is also evidence that pathways involving IL-4 and the IL-4 receptor are implicated in severe equine asthma.9,10 As IL-4 is one of the main effectors that influence activated cell differentiation marker 4-positive T cells (ie, CD4+ T cells) to acquire Th-2 characteristics,11 various researchers have investigated its role in neutrophil recruitment and activation. Equine pulmonary artery endothelial cells stimulated with IL-4 express IL-8, vascular endothelial growth factor, and E-selectin,12 which are factors known to contribute to neutrophil chemotaxis and migration into inflamed tissues.13,14 Similarly, IL-4 activates equine neutrophils in vitro by inducing morphological changes described as polarization and by activating signal transducer and activator of transcription-6.15 Interleukin-4 also appears to regulate the transcription factors p38, mitogen-activated protein kinase, and phosphoinositide-3 kinase in neutrophils, as their activity is correlated with an upregulation of mRNA for cell differentiation marker 23 (an IgE binding receptor).15 Results of investigations involving mice and rats revealed an indirect mechanism by which IL-4 may participate in neutrophil recruitment by increasing the expression of chemokine (C-C motif) ligand-2 (also called monocyte chemotactic protein-1),16 C-X-C motif chemokine ligand 1 (also called cytokine-induced neutrophil chemoattractant-1 in rats),17 neutrophil chemokines, and intracellular adhesion molecule 1,17 which is an adhesion molecule required for neutrophil transmigration processes. These findings support that IL-4 may contribute to the activation of neutrophils in allergic inflammation. Nonetheless, pathways leading to neutrophilia in chronic inflammatory diseases, neutrophil activation, and interactions between neutrophils and Th-2 cytokines remain incompletely understood.

Thus, the purpose of the study reported here was to develop a method for monitoring of neutrophil migration and assessment of neutrophil responses to IL-4 in equids. We chose an injectable biopolymer as a vehicle with cytokines added prior to SC injection to provide the stimulus for an inflammatory response. The biopolymer, an extract of a murine Engelbreth-Holm-Swarm tumor rich in basement membrane components (laminin, collagen IV, nidogen, and perlecan),18 has been widely used in vivo in studies involving mice,19,20 dogs,21 rabbits,22 and annelids.23 Use of the biopolymer has been investigated for drug-delivery applications24,25 and for recruitment of specific cell infiltrates,23 including neutrophils.26,27

Materials and Methods

Animals

Sixteen healthy Standardbred mares and 2 mixed-breed gelding ponies (mean ± SD age for the study sample, 7.8 ± 4.2 years) from the equine research and teaching herds of the Université de Montréal Faculté de Médecine Vétérinaire were included in the study. A complete physical examination was performed for each animal at the start of the study, and vaccination and deworming status of mares from the teaching herd were current (this information was not available for the 2 ponies). Equids were arbitrarily assigned to 1 of 3 experiments (6 equids/experiment). They were stabled with wood shavings for bedding and fed %%%good-quality hay and grain twice daily. The study was approved by the Animal Care and Use Committee of the Université de Montréal (protocol Rech-1324) and conducted in compliance with guidelines of the Canadian Council on Animal Care.

Reagents

Cytokines ReqIL-4a and ReqIL-8b (a potent neutrophil chemoattractant28,29 used as a positive control) were each reconstituted under aseptic conditions with filtered pure water and stored at −80°C until use. The biopolymerc was kept frozen at −20°C. Before use, it was thawed slowly on ice at 4°C for 48 hours. Because of its highly viscous properties at a concentration of 20 mg/mL, the biopolymer was diluted with sterile culture mediumd to facilitate manipulation, pipetting, and SC injection on the basis of manufacturer recommendations as detailed for each experiment. Cytokine-supplemented biopolymer aliquots were kept on ice until they were injected SC, while the mixtures were still fluid. The biopolymer rapidly gels at temperatures > 10°C, forming a polymerized pellet and sequestrating the added cytokines.

Experiment 1

Four horses and the 2 ponies were studied. Cytokine preparations were as follows: on ice and in aseptic conditions, the biopolymer was diluted with sterile culture mediumd to a concentration of 15 mg/mL and divided into nine 100-μL aliquots. The ReqIL-4 or ReqIL-8 was mixed into biopolymer aliquots to achieve a final cytokine concentration of 100 ng/sample, with a total of 3 aliquots for each preparation. Three aliquots of biopolymer were mixed with 10 μL of sterile DPBSSe (used as negative controls). The 9 prepared aliquots were loaded into 1-mL syringes with 21-gauge, 1-inch needlesf and kept on ice for 1 hour until injection.

Prior to injection, the left aspect of the neck was shaved of hair and cleaned with a chlorhexidine scrub solution to limit contamination. Each equid received 9 prepared biopolymer aliquots by SC injection in a pattern of 3 vertical columns of 3 injections with sites ≥ 3 cm apart. Each column comprised 1 injection of biopolymer mixed with ReqIL-4, 1 injection of biopolymer mixed with ReqIL-8, and 1 injection of biopolymer mixed with DPBSS (as a negative control).

To determine the general cellular kinetics in response to administered cytokines and to confirm the ability of ReqIL-4 and ReqIL-8 to influence mobilization of equine neutrophils in vivo, a broad time course was chosen for histologic analysis of the injection sites. Each column was arbitrarily assigned to a biopsy time (6 hours, 48 hours, or 7 days after injection). Inflammatory responses were monitored by measuring the degree of swelling at each injection site with a caliper every 2 hours for the first 12 hours and then every 24 hours until recovery. Prior to biopsy of the 3 injection sites in the selected column, equids were sedated with xylazineg (0.1 mg/kg, IV), and lidocaineh (1 mL/site, SC) was injected in an inverted L shape at a distance of approximately 0.5 cm around each biopolymer plug for local anesthesia. Cutaneous and subcutaneous tissue including the biopolymer plug at the 3 injection sites was collected with a 6-mm-diameter biopsy punch,i the biopsy sites were sutured closedj with a simple interrupted stitch, and tissues were fixed in neutral-buffered 10% formalin solution for histologic analysis.

Experiment 2

Cytokine preparations and protocols for injection, biopsy, and tissue sample fixation for the 6 horses used for experiment 2 were the same as described for experiment 1 with the following modifications: the biopolymer was diluted with the same culture medium to a concentration of 8 mg/mL, cytokine concentrations were increased to 250 ng/sample, and the same culture medium instead of DPBSS was mixed with the biopolymer as a negative control. Instead of 3 vertical columns of three 100-μL injections, 2 horizontal rows of three 100-μL injections were created and arbitrarily assigned to different biopsy times (3 or 12 hours after injection) for a total of 6 sites/horse.

Experiment 3

Cytokine preparations and protocols for injection, biopsy, and tissue sample fixation for the 6 horses used in experiment 3 were the same as described for experiment 2, except that the cytokine concentration was increased to 500 ng/sample for ReqIL-4 and to 1 μg/sample for ReqIL-8 and the injectate volume was increased to 250 μL/site. In addition to the 2 rows of 3 injections on the left aspect of the neck, 1 row of 3 injections was created on the right aspect of the neck for a total of 9 sites/horse. The 6 sites on the left side were sampled with a biopsy punch as described 3 hours after injection; 3 biopsy samples (1/treatment) were fixed in formalin solution for histologic analysis as previously described, and the other 3 were maintained in sterile PBSSe before being processed by flow cytometry. The sites on the right side were sampled in the same manner 6 hours after injection and were processed immediately and analyzed by flow cytometry.

Postprocedural monitoring

After each experiment was completed, a physical examination of each horse was performed to ensure that recovery from sedation was adequate and that they showed no signs of adverse effects. A physical examination was also performed every 24 hours for 3 days following collection of the samples, which included monitoring of the biopsy sites. Dexamethasonek was administered if needed for treatment of local swelling or signs of local discomfort associated with biopsy sites.

Histologic evaluation and quantification of cellular responses

Excised tissue samples destined for histologic analysis were fixed overnight in neutral-buffered 10% %%%formalin solution and embedded in paraffin. In experiment 1, the samples were cut in half perpendicular to the skin through the dermis, epidermis, and subcutaneous tissue including the biopolymer before formalin processing, and a 4-μm section near the cut edge at the center of each sample was processed with HPS stain.

For experiments 2 and 3, in an attempt to reduce data variations observed in experiment 1, 5 arbitrarily selected slides/biopsy sample were evaluated. Sections (4 μm thick) were cut at 200-μm intervals throughout each tissue sample and HPS stained. For all experiments, all slides for a given section were evaluated through a 20X objective microscope lens by a board-certified veterinary pathologist (PH) who was unaware of the treatment assignment and assessed neutrophil and mononuclear cell infiltration on a qualitative scale of 0 to 5 (0 = absent, 1 = rare, 2 = occasional, 3 = moderate, 4 = strong, and 5 = very dense). Mononuclear cell infiltration was also evaluated and scored in experiment 1 out of interest owing to the long incubation times; however, scores for these cell types were not analyzed statistically. The median neutrophil infiltration score of the 5 examined slides was used for statistical analysis of each sample in experiments 2 and 3.

Flow cytometry

The epidermis and dermis were removed from each tissue sample, and the remaining tissue was weighed and washed in sterile PBSS. Isolation of sequestrated cells from subcutaneous tissue and biopolymer was performed by enzymatic digestion (in culture mediumd with 1.2 mg of collagenase Dl and 100 μL of dispase IIm [dilution of 10 mg/mL]) with rotational movement for 2 hours in a humidified incubator at 37°C with 5% CO2 concentration. To generate a single-cell suspension, the mixtures were passed through a 70-μm cell strainer.n Cells were washed twice in cold sterile PBSS containing 0.5% bovine serum albuminm and 0.5mM EDTAj and fixed for 20 minutes in 2% paraformaldehyde solution. After 3 washes in PBSS with 0.5% bovine serum albumin, cells were stained for myeloperoxidase for 1 hour with rabbit anti-human myeloperoxidase antibodyo (dilution, 1:200) in PBSS that included a nonionic surfactantp added to a final concentration of 0.1%. All incubation steps were performed at 4°C. Cells were then washed 3 times in PBSS and incubated for 30 minutes in the dark with green fluorescent-labeled goat anti-rabbit secondary antibodyq (dilution, 1:1,000). Cells were washed twice and suspended in 400 μL of PBSS before flow cytometric acquisition of 10,000 events, and analysis was performed with a flow cytometerr and corresponding software.s Rabbit IgGs was used as a control to ensure primary antibody specificity. The mean percentage of fluorescence-positive cells was assessed for each sample. In addition to determining the proportion of cells in each sample identified as neutrophils with flow cytometry, the total cell concentration in each sample was evaluated by counting with a hemocytometer. By combining the 2 values, a final neutrophil count was identified for each tissue sample. Neutrophil counts were then normalized to the weight of the sample in milligrams prior to digestion for cell isolation.

Statistical analysis

Associations of time (the interval from injection to biopsy) and treatment (biopolymer plus ReqIL-4, ReqIL-8 [positive control], or negative control) with histologic scores for neutrophil infiltration within each experiment were evaluated with the Cochran-Mantel-Haenszel test. Slides in which the biopolymer was not visible were excluded from this analysis because the presence of recognizable biopolymer was used as a marker for successful (appropriate) sample collection by biopsy. Given the small sample size, formal evaluation of normality was not practical. We used a visual assessment of the data and looked for departures from a symmetric distribution.

Flow cytometry counts of neutrophils for samples from experiment 3 (normalized to sample weight) were log-transformed and analyzed by repeated-measures 2-way ANOVA. Fisher least significant difference tests were used to compare counts between treatments and between time points. Statistical analyses were performed with appropriate software.u,v For all comparisons, values of P < 0.05 were considered significant.

Results

Technical variables and response to injection and biopsy procedures

Once injected, sites were generally recognized by their papule-like shape and were marked with a sterile surgical marker. As the inflammatory response developed, rapid swelling of some injection sites made them difficult to distinguish from surrounding tissue. This complicated harvesting of tissue samples that included the biopolymer on some occasions, and thus, not all biopsies were considered successful on histologic examination. Fifty-four biopsies were performed in experiment 1, 36 were performed in experiment 2, and 54 were performed in experiment 3, for a total of 144 biopsies during the 3 study periods. Of 108 samples collected for histologic examination, 14 of 54 samples in experiment 1, 7 of 36 samples in experiment 2, and 2 of 18 samples in experiment 3 were considered inconclusive owing to absence of biopolymer on the examined slides. In total, 85 tissue samples were analyzed histologically, and 36 were analyzed by flow cytometry. Examples of tissues with neutrophil infiltration scores of 0 to 5 as assessed by histologic examination are provided (Figure 1).

Figure 1—
Figure 1—

Representative photomicrographs depicting qualitative histologic scoring of neutrophil infiltration (arrowheads) at local injection sites in equids after SC administration of ReqIL-4, ReqIL-8, or negative control solution, each mixed with the same biopolymer carrier. A 6-mm-diameter biopsy punch was used to collect cutaneous and subcutaneous tissues, including the biopolymer plug, at 1 injection site/treatment type/animal at selected time points, and samples were fixed in neutral-buffered 10% formalin solution prior to paraffin embedding and sectioning. Slides were excluded from analysis if biopolymer was not visible in the section. A—Absence of neutrophils (score of 0). B—Rare neutrophils (score of 1). C—Occasional neutrophils (score of 2). D—Moderate infiltration (score of 3). E—Strong infiltration (score of 4). F—Very dense infiltration (score of 5). The biopolymer (asterisk) is seen as a homogenous bright-pink substance surrounded by saffron-yellow to orange host tissue and collagen fibers. HPS stain; bar = 50 μm.

Citation: American Journal of Veterinary Research 81, 4; 10.2460/ajvr.81.4.344

The injection and biopsy procedures were well tolerated by equids, and no major complications developed. Five of the 6 equids in experiment 3 were administered dexamethasone (0.04 mg/kg, PO, once) %%%on the day after tissue samples were collected because excessive swelling and signs of discomfort during palpation around the biopsy sites were observed. The signs resolved within 48 hours after treatment. Sutures were removed 2 weeks after the biopsies were performed.

Experiment 1

There were no significant differences in neutrophil infiltration scores among the biopolymer-carried treatments (100 ng of ReqIL-4, 100 ng of ReqIL-8 [positive control], or 10 μL of DPBSS [negative control]) 6 hours (P = 0.66), 48 hours (P = 0.88), and 7 days (P = 0.73) after injection (1 sample/treatment/equid at each time point) and no significant effect of time on these scores within treatments (P = 0.79, P = 0.62, and P = 0.08 for ReqIL-4, ReqIL-8, and DPBSS, respectively; Figure 2). In addition, neutrophil infiltration subjectively appeared similar for all treatments on visual assessment, slightly decreasing as time increased after injection. Mean scores for mononuclear cell infiltration were subjectively similar among treatments within each time point but appeared to increase with increasing time after injection.

Figure 2—
Figure 2—

Scatterplots of histologic scores for neutrophil (A) and mononuclear cell (B) infiltration into injection site tissue samples collected at various time points after SC injection of biopolymer (15 mg/mL) mixed with 100 ng of ReqIL-4 (squares), 100 ng of ReqIL-8 (positive control; triangles), or 10 μL of DPBSS (negative control; circles) in the lateral aspect of the neck in 6 equids (experiment 1). Infiltration scores for cells of each type were assigned from 0 (absent) to 5 (very dense) as depicted for neutrophils in Figure 1. The total injection volume for each treatment was 100 μL/site. Each point represents the cellular infiltration score for 1 sample/equid. Slides where no biopolymer was visible were excluded from the analysis. Horizontal lines depict the mean for all samples for a given treatment at the specified time point.

Citation: American Journal of Veterinary Research 81, 4; 10.2460/ajvr.81.4.344

Some samples were so densely infiltrated by inflammatory cells that the tissues and biopolymer were undistinguishable. Finally, a moderate inflammatory cell infiltration was observed in samples obtained at the sites of the negative control injections.

Experiment 2

On the basis of results from experiment 1, experiment 2 protocols were adjusted so that the biopolymer concentration was decreased, the amounts of ReqIL-4 and ReqIL-8 were increased, culture medium instead of DPBSS was added to the biopolymer as a negative control, and the biopsies were performed 3 and 12 hours after injection (1 sample/treatment/ equid at each time point) as described. Subjectively, neutrophil infiltration appeared somewhat greater in experiment 2 than in experiment 1. However, neutrophil infiltration scores did not differ significantly among biopolymer-carried treatments (250 ng of ReqIL-4, 250 ng of IL-8, or 10 μL of culture medium) 3 hours (P = 0.33) or 12 hours (P = 0.92) after injection (Figure 3). Neutrophil infiltration score variability was subjectively less pronounced among the 3 treatments in this experiment, compared with results for experiment 1.

Figure 3—
Figure 3—

Scatterplots of histologic scores for neutrophil infiltration into injection site tissue samples 3 and 12 hours after SC injection of biopolymer (8 mg/mL) mixed with 250 ng of ReqIL-4 (squares), 250 ng of ReqIL-8 (triangles), or 10 μL of the same culture medium used for dilution of cytokines (negative control; circles) in the lateral aspect of the neck in 6 equids (experiment 2). See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 81, 4; 10.2460/ajvr.81.4.344

Experiment 3

On the basis of results for experiments 1 and 2, the protocols for experiment 3 were modified so that the amounts of cytokines were increased, the overall injection volumes were increased, and all biopsies for histologic analysis (1 sample/treatment/equid) were performed 3 hours after injection as described. One animal was excluded from the neutrophil infiltration score analysis owing to poor-quality slides for 2 of 3 injection site tissue samples. Statistical analysis for samples from the remaining 5 equids revealed a significant (P = 0.049) difference in neutrophil infiltration scores among the 3 biopolymer-carried treatments (500 ng of ReqIL-4, 1 μg of ReqIL-8, or 10 μL of %%%culture medium); however, paired comparison of the 2 treatments with the highest (ReqIL-8) and lowest (culture medium) mean scores revealed a nonsignificant (P = 0.059) difference (Figure 4). Neutrophilia in response to ReqIL-4 and ReqIL-8 appeared more consistent than that observed for the negative control treatment. Subjectively, the overall scores for each treatment in experiment 3 were lower than the corresponding scores at the same time point in experiment 2.

Flow cytometric analysis of tissue samples 3 hours and 6 hours after injection (1 sample/treatment/time point for each equid) revealed considerable variability in neutrophil counts among individual animals after control and ReqIL-4 treatments, and all data were log-transformed for analysis (Figure 5). The ANOVA revealed a significant (P = 0.025) difference among treatments, and the Fisher least significant difference test revealed a significant (P = 0.032) difference in neutrophil counts between ReqIL-8 and the negative control treatment 3 hours, but not 6 hours (P = 0.054), after injection. Neutrophil counts after ReqIL-4 treatment did not differ from those for the negative control treatment 3 hours (P = 0.92) or 6 hours (P = 0.73) after injection.

Figure 4—
Figure 4—

Scatterplots of histologic scores for neutrophil infiltration into injection site tissue samples 3 hours after SC injection of biopolymer (8 mg/mL) mixed with 500 ng of ReqIL-4, 1 μg of ReqIL-8, or 10 μL of the same culture medium used for dilution of cytokines (negative control) in the lateral aspect of the neck in 6 equids (experiment 3). Results for 1 animal were excluded from analysis because of poor-quality slides. The total injection volume for each treatment was 250 μL/site. The Cochran-Mantel-Haenszel test indicated a significant (P = 0.049) difference in scores among groups; however, the greatest difference between treatments did not meet significance criteria in pairwise comparisons (P = 0.059). NC = Negative control. See Figure 2 for remainder of key.

Citation: American Journal of Veterinary Research 81, 4; 10.2460/ajvr.81.4.344

Figure 5—
Figure 5—

Scatterplots of log-transformed neutrophil counts in injection site tissue samples as determined by flow cytometry 3 hours (A) and 6 hours (B) after SC injection of biopolymer mixed with ReqIL-4, ReqIL-8, or culture medium in experiment 3. Results of ANOVA revealed a significant (P = 0.025) difference in mean neutrophil counts among treatments; pairwise comparison with the Fisher least significant difference test indicated mean neutrophil counts (normalized to sample weight prior to enzymatic digestion for preparation of cell suspensions) were significantly greater for the positive control (ReqIL-8) treatment than for the negative control treatment 3 hours (P = 0.032), but not 6 hours (P = 0.054), after injection. See Figures 2 and 4 for key.

Citation: American Journal of Veterinary Research 81, 4; 10.2460/ajvr.81.4.344

Discussion

In the study reported here, a biopolymer delivery system was used to investigate the ability of ReqIL-4 to recruit neutrophils into subcutaneous tissues of equids. The results suggested that, at the amounts tested, ReqIL-4 did not trigger neutrophil chemotaxis in the subcutaneous tissues of healthy equids; however, our findings also indicated that use of the described biopolymer-cytokine preparation was not a useful means of evaluating this response. Unlike findings in other species,23,27 the biopolymer itself (diluted in DPBSS or culture medium) induced an inflammatory response in most equids in the present study, which could have limited our ability to measure neutrophil migration in response to ReqIL-4 if the effects were subtle.

To characterize the inflammation process on a cellular level, we developed a protocol intended to create a local, cytokine-rich microenvironment that could easily be monitored, would allow reliable tissue retrieval for examination, and would not alter the inflammation process. In preliminary experiments, we evaluated the effects of ID administration of ReqIL-4 and ReqIL-8 without use of a biopolymer carrier (data not shown). However, 10 ng of these cytokines resulted in signs of discomfort, profuse sweating, and shivering in a horse approximately 30 minutes after injection. Studies30–32 have similarly reported adverse effects following IV or SC administration of IL-4 but at doses greater than those we investigated. In human patients, predominant signs included flu-like symptoms, gastrointestinal upset, lethargy, transient hypotension, headache, and increased circulating concentrations of liver enzymes, but more severe signs of toxicosis such as vascular leakage syndrome, epistaxis, and hallucinations have also been reported in people.30–32 We aimed to limit the systemic distribution of these cytokines to attenuate such adverse effects. The biopolymer we chose, supplemented with specific cytokines and mediators, including IL-823 and other cytokines,33–35 lipopolysaccharide,27 basic fibroblast growth factor, and hepatocyte growth factor,36 provides an appropriate microenvironment for the recruitment of various cell infiltrates, including neutrophils, when implanted in mice.27 The same biopolymer was used for in vivo experiments in dogs,21 rabbits,22 and leeches (Hirudo medicinalis)23 and was reported to be well tolerated. The matrix offered attractive properties; it maintains a fluid state between 2°C and 6°C, allowing the addition of cytokines, and starts to gel rapidly at temperatures > 10°C, forming a solid pellet after SC administration that can be harvested. However, at the undiluted concentration of 20 mg/mL, the biopolymer is highly viscous and does not become free-flowing unless diluted. As suggested by the manufacturer,37 we added culture medium to facilitate mixing of cytokines into the biopolymer. We anticipated that the biopolymer would be a convenient vehicle for administration of ReqIL-4 and ReqIL-8, allowing a safer use of these cytokines in equids.

Because IL-4 itself fails to induce direct neutrophil migration,15,17 we expected that the release of mediators by resident cells in response to IL-4 would lead to neutrophil recruitment. These mediators could have autocrine and paracrine effects on neutrophils in the inflammatory milieu,38 perpetuating the ongoing recruitment and activation of neutrophils in affected tissues, as found in the airways of heaves-affected horses.6,7,9,10 In addition to our previous findings linking neutrophils and IL-4,5,12 IL-4 mRNA expression by cells present in bronchoalveolar lavage fluid of heaves-affected horses develops concurrently with neutrophil recruitment.7,8 Taken together, this led us to believe that IL-4 and neutrophils play key roles in the pathogenesis of equine asthma. We believed that ReqIL-8, a potent chemoattractant for neutrophils,28,29 would trigger a rapid and massive neutrophilic infiltrate into tissues surrounding the injection sites at earlier time points and that a neutrophilic response to ReqIL-4 would develop later and have lesser intensity. Overall, cellular infiltrate was consistent with what is normally described39: neutrophils migrated toward the biopolymer soon after injection, consistent with the acute phase, followed by mononuclear cells taking on the role of predominant cells by 7 days after injection even though neutrophils remained present. However, the neutrophilic infiltration we observed was milder than expected, even in response to ReqIL-8. We studied these inflammatory responses by histologic evaluation 6 hours, 48 hours, and 7 days after injection of ReqIL-4 (100 μg/site), ReqIL-8 (100 μg/site), and the negative control treatment (DPBSS) in experiment 1, as chemokines are known to diffuse rapidly from the biopolymer into culture medium into which the biopolymer pellet is incubated, where diffusion carries on steadily for ≥ 7 days.23 A possible explanation for the observed lack of neutrophilic inflammation in this experiment was that infiltration might have been commencing at the 6-hour time point and could already have declined at 48 hours; this consideration led us to perform biopsies for histologic examination 3 and 12 hours after injection in experiment 2.

In experiment 2, the quantity of cytokines injected was increased to 250 ng/site to enhance neutrophil chemotaxis. Even though earlier time points were evaluated, compared with the methods for %%%experiment 1, the neutrophil responses to ReqIL-4 and ReqIL-8 were not significantly different from that for the negative control. The increased amount of cytokines at each site subjectively resulted in a general increase in neutrophil infiltration, compared with that in experiment 1. However, histologic examination of 5 slides/sample instead of 1, while reducing score variability, prevented us from making conclusions related to dose effects. Lastly, replacing DPBSS with culture medium for dilution of the biopolymer in negative control treatments and diluting the biopolymer to a lower concentration did not attenuate neutrophil infiltration in the negative control samples.

The histologic neutrophil scoring method had its limitations, as it relied on subjective assessment of tissue sections. Moreover, a proportion of slides were excluded from histologic analysis owing to the absence of biopolymer, which further reduced the sample size. Initially, we expected formation of a compact and well-defined biopolymer plug between layers of subcutaneous tissue, as reported to occur in mice and annelids.23,27 In most samples, however, the biopolymer was intertwined with collagen fibers and dispersed throughout the tissue. Neutrophils were observed lodged in the biopolymer as well as in the surrounding subcutaneous tissue. We opted to consider the entirety of each slide to determine neutrophil score, as peripheral neutrophils were likely migrating toward the biopolymer when the biopsy was performed. The variable distribution of the biopolymer and neutrophils on each slide was one of the reasons we chose to incorporate flow cytometry in experiment 3. Flow cytometry offered the advantage of direct cell counts, thus eliminating subjective bias and allowing assessment of neutrophils in the entire tissue sample. Also, in an attempt to limit the number of samples excluded from the analysis because biopolymer was not detected on the slides, we chose to inject larger volumes of biopolymer (250 μL instead of 100 μL) in experiment 3. Finally, since an immunogenic response to the biopolymer was thought to be responsible for infiltration of tissues with inflammatory cells in negative control samples, we chose to shorten the time between injection and assessment. With these changes, sampling was improved, as only 2 of 18 samples were inconclusive on histologic examination. Nevertheless, histologic neutrophil infiltration scores for the ReqIL-4 and ReqIL-8 treatments (at concentrations increased to 500 ng and 1 μg/ sample, respectively) did not differ significantly from those for the negative control treatment in the final analysis. Flow cytometric evaluation identified the only significant difference in neutrophil infiltration between groups, with greater neutrophil counts for the ReqIL-8 treatment than for the negative control treatment at the 3-hour time point. The findings for experiment 3 suggested that flow cytometry is more useful than histologic analysis for detection of neutrophil migration into local tissues with the treatment methods used in this study. In retrospect, it would have been interesting to compare results of histologic and flow cytometric analyses in experiments 1 and 2, but the tissue samples were no longer available.

Interestingly, histologic scores for samples after all 3 treatment types in experiment 3 were generally lower than those found in experiment 2 at the same time point (3 hours after injection). Greater cytokine concentrations in experiment 3 would be expected to trigger a more potent neutrophil response than in experiment 2, but studies40,41 have shown that the cellular response to chemokines often follows a bell curve. Just as low doses fail to generate a response, high doses or steep gradients of IL-8, for example, not only decrease the chemotactic neutrophil response, but can induce a persistent, directionally biased movement away from that chemokine, known as chemorepulsion.42,43 It is also possible that the larger volume of injected biopolymer reduced the contact area between cytokines and subcutaneous tissues, diminishing the expected response. In planning future investigations, it should also be considered that local swelling and signs of discomfort in 5 of 6 equids in experiment 3 may have resulted from the increase in cytokine concentrations in this experiment, compared with the concentrations used in experiments 1 and 2.

The reasons for the failure of ReqIL-4 to induce a neutrophilic response significantly different from that resulting from the negative control treatments were unclear. The controversial yet pleiotropic biological effects of IL-4 complicated our analysis. Indeed, study44–49 findings have characterized IL-4 as either an activator or inhibitor of neutrophil function. This cytokine generally has anti-inflammatory properties in studies44–46 conducted on neutrophils activated with microbial compounds or proinflammatory cytokines in vitro. However, recent findings by our group suggest that stimulation of equine neutrophils with a low concentration of ReqIL-4 (40 U/mL) for 5 hours induces a mixed inflammatory profile characterized by increased expression of IL-8 and tumor necrosis factor-α and decreased IL-1β.15 In people and in mice, both neutropenic32,47 and neutrophilic30,48 responses, often in an indirect manner,16 have been reported following IL-4 administration, whereas another study49 did not find altered neutrophil trafficking in airway tissues or bronchiolar lavage fluid of mice after IL-4 administration. These divergent results may have been partly attributable to the state of cell activation prior to IL-4 administration; considering that human patients enrolled in clinical trials30–32 were confirmed as having malignancy or were HIV seropositive, it is possible that their clinical conditions, prescribed medications, or both altered neutrophil and IL-4 interactions. Combined, these findings suggest that several cytokines and chemotactic factors are involved in the recruitment of neutrophils and that resident cells, a state of preactivation, or the milieu in which interactions take place may influence neutrophil responses. Indeed, neutrophils isolated from heaves-affected %%%horses show increased responsiveness and alterations in gene expression relative to neutrophils of healthy horses.15 Similar findings have also been reported in neutrophils isolated from human patients with atopic dermatitis, compared with nonatopic patients.50,51 Because our study was conducted with healthy horses and ponies, it is possible that the response to ReqIL-4 would be different in equids with disease.

The apparent reaction to the biopolymer in the present study is another aspect to consider when evaluating our results, as infiltrations of neutrophils and other leukocytes were observed in tissues following negative control injections. The extent to which this may have influenced interactions between ReqIL-4 and neutrophils was unknown. Considering that the positive control treatment with ReqIL-8 also resulted overall in a modest neutrophilic response, which was unexpected for the potent neutrophil chemoattractant,13,14 interference by the biopolymer must be considered in addition to the possibility that the amounts of ReqIL-4 used in these preparations were insufficient to trigger local neutrophil infiltration. First, the biocompatibility or immunogenicity of the biopolymer in equids has not been formally studied. A proteomic analysis of this product52 revealed a complex mixture consisting of structural proteins, growth factors and their binding proteins, and other types of proteins. Matrices extracted from living cells, such as the one selected for the present study, are commonly used for growing cells that are sensitive to culture conditions, probably because of the presence of essential growth factors.53 However, component variations can be observed among batches of this matrix, and signaling pathways participating in cell growth or cell recruitment may be altered, potentially affecting experimental outcomes.52 The murine origin of the biopolymer is a second aspect to consider. In vivo studies are frequently conducted with mice, and reports of immunologic rejections of the biopolymer have not been found in the literature for this species. Toxic effects in the eyes of rabbits have been reported,54 and development of excessive fibrous tissue proliferation and macrophage infiltration when the product is placed in diffusion chambers of a bio-artificial endocrine pancreas suggests a possible lack of biocompatibility in dogs,55 in contrast to results of previous studies in these same species.21,22 In the latter investigations, the biopolymer was used for reasons other than the assessment of the inflammation process, and therefore, conflicting results could be attributed to the context of the assays or having no information available regarding an inflammatory cell infiltration that may have been present. Given that small sample sizes were used in the aforementioned studies as well as in the present study, the data should be interpreted cautiously, particularly in regard to conclusions about biocompatibility in species other than mice. In our short-term study, local inflammatory reactions to the diluted biopolymer following SC injection were common, suggesting that the product used is not an appropriate vehicle, at least for assessing inflammatory responses to agents such as cytokines in horses and ponies, and that additional studies are needed to investigate immune system interactions with the biopolymer in equids.

Acknowledgments

This manuscript represents a portion of a thesis submitted by Dr. Godbout to the Université de Montréal's Faculté de Médecine Vétérinaire and Département des Sciences Cliniques as partial fulfillment of the requirements for a Master of Science degree.

Funding was provided by a discovery grant from Natural Sciences and Engineering Research Council of Canada (JPL No. RG-PIN-2014-06-198) and a scholarship from le Fonds du Centenaire.

The authors declare that there were no conflicts of interest.

The authors thank Guy Beauchamp for statistical analysis.

ABBREVIATIONS

DPBSS

Dulbecco PBSS

HPS

Hematoxylin-phloxine-saffron

IL

Interleukin

ReqIL

Recombinant equine interleukin

Th

T helper

Footnotes

a.

ReqIL-4, Pierce, Rockford, Ill.

b.

ReqIL-8, Pierce, Rockford, Ill.

c.

Matrigel matrix—growth factor reduced, high concentration (20 mg/mL), Corning, Bedford, Mass.

d.

RPMI 1640, Invitrogen, Burlington, ON, Canada.

e.

Invitrogen, Burlington, ON, Canada.

f.

Fisher, Nepean, ON, Canada.

g.

Bayer Animal Health, Mississauga, ON, Canada.

h.

Vetoquinol, Lavaltrie, QC, Canada.

i.

Integra Miltex biopsy punch, Fisher, Nepean, ON, Canada. j. Supramid 0, Serag-Weissner, Naila, Germany.

k.

Dominion, Winnipeg, MB, Canada.

l.

Roche Diagnostics, Laval, QC, Canada.

m.

Sigma-Aldrich, Oakville, ON, Canada.

n.

CellTreat 70μm cell strainer, CellTreat, Pepperell, Mass.

o.

Dako, Burlington, ON, Canada.

p.

Triton X-100, Sigma-Aldrich, Oakville, ON, Canada.

q.

Alexa Fluor 488, Invitrogen, Burlington, ON, Canada.

r.

CellQuest Pro software, BD Biosciences, Mississauga, ON, Canada.

s.

FACSCalibur instrument, BD Biosciences, Mississauga, ON, Canada.

t.

I-1000, Vector Laboratories, Burlingame, Calif.

u.

Prism 5, Graphpad software, La Jolla, Calif.

v.

SAS/STAT, version 9.4, SAS Institute Inc, Cary, NC.

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Contributor Notes

Dr. Vargas’ present address is Faculté de Médecine, Université de Montréal, Montréal, QC H3T 1J4, Canada.

Dr. Godbout's present address is Service Vétérinaire Équin Dr. Richard Beaudoin, St-Jean-Sur-Richelieu, QC J2X 4B6, Canada. Dr. Bullone's present address is the Dipartimento di Scienze Veterinarie, Università Degli Studi di Torino, 10095 Grugliasco TO, Italy.

Address correspondence to Dr. Lavoie (jean-pierre.lavoie@umontreal.ca).