After injury, neutrophil migration can be initiated by a arge number of skeletal muscle cell–derived products (15,27). In vitro studies have shown that after mechanical (scrape) injury, human myotubes release factors promoting neutrophil chemotaxis (30). However, neutrophil chemotaxis occurred without an elevation in any of the known migratory factors measured in that study (interleukin 8 [IL-8], tumor necrosis factor α [TNF-α], and transforming growth factor β [TGF-β]), suggesting that other factors are involved, or that several factors may function synergistically to achieve optimal migratory capacity.
Studies in various models, as recently reviewed (19), have shown that—similar to neutrophil accumulation (23,28)—the accumulation of type M1 macrophages exacerbates injury, whereas a predominance of anti-inflammatory macrophages (in particular type M2c) in tissue is associated with repair (2,8). It was initially thought that neutrophils were responsible for macrophage chemotaxis because their accumulation in injured tissue precedes that of macrophages. However, this was subsequently reported to be inaccurate because macrophages can accumulate in muscles depleted of neutrophils, as demonstrated in a model of muscle injury induced by lengthening contraction (25).
Most research on skeletal muscle injury has focused on the roles of neutrophils and macrophages once they have already entered the injured area (20,25). In contrast, chronic inflammatory diseases with or without flare-ups (e.g., juvenile arthritis) are monitored through changes in the monocyte/macrophage phenotypes in blood samples (21). In the context of muscle injury, the activation and recruitment of blood monocytes to the inflammatory sites provides an important source of tissue macrophages (32). It is therefore of interest to investigate potential anti-inflammatory effects of interventions in the circulatory compartment.
Many plant-derived compounds have anti-inflammatory properties and can modulate the cytokine system (5). For example, resveratrol, one of the most researched grape-derived antioxidants, has several anti-inflammatory properties, which include (a) inhibition of pro-inflammatory cytokine, IL-8 and IL-6, production (11) and (b) partial inhibition of activated immune cells. Guabiju extracts were also investigated for their ability to inhibit neutrophil chemotaxis toward lipopolysaccharides, an effect which the authors attributed to their antioxidant properties (1). A different plant-derived antioxidant, quercetin, has been reported to possibly have “natural anti-inflammatory effects” (10). In two previous studies by our group, administering grape-seed–derived proanthocyanidolic oligomers (PCO), either daily for 2 wk before or immediately after experimental skeletal muscle contusion injury, neutrophil infiltration into the injured area was limited (18,22). In the experimental group, PCO also seemed to result in earlier macrophage appearance in tissue.
The aim of the current study was to determine whether in vivo treatment with PCO might have effects on neutrophils and monocytes/macrophages, specifically in the blood compartment, which may alter their migratory capacity. We hypothesized that neutrophil migration capacity would be reduced despite the presence of chemotactic factors and that PCO may favor a switch in macrophage phenotype already in circulation.
MATERIALS AND METHODS
Fifty-five adult, male Wistar rats, weighing approximately 280 g, were used in this study. All rats were housed in groups of five in standard rat cages and fed rat chow and tap water ad libitum. All animals were exposed to a 12-h light–12-h dark cycle (lights on at 6:30 a.m.), ambient temperature controlled at 21°C, and rooms ventilated at 10 changes per hour. All experimental protocols were approved by the Animal Research Ethics Committee of Sub-Committee B of Stellenbosch University and adhered to the American College of Sports Medicine animal care standards. It is important to note that the current study reports on findings from a complete new set of experimental animals subjected to the protocol as described in the next section, and not from analysis of existing samples as an extension of a previously published study.
Preceding the onset of the supplementation protocol and contusion injury, 50 of the 55 experimental rats were randomly divided into four groups. These groups consisted of a noninjured control placebo group (C-PLA; n = 5), a noninjured control PCO group (C-PCO; n = 5), with the other 40 rats exposed to either chronic placebo (I-PLA; n = 20 or chronic PCO supplement (I-PCO; n = 20), as well as experimental contusion injury. Postinjury, five rats from each group were sacrificed per time point—12 h, 1 d, 3 d, and 5 d. The remaining five animals were not supplemented or injured and were used for harvesting control neutrophils to be used in the in vitro neutrophil migration study.
Experimental rats, with the exception of the animals used for neutrophil isolation, were orally gavaged with either 20 mg·kg−1·d−1 of PCO (dissolved in 0.9% saline) or 0.9% saline (PLA) for the duration of the experimental procedure. Control (C) rats were gavaged for 2 wk without injury, and contusion injury groups received PCO or PLA for 2 wk before injury as well as up to 5 d after injury (depending on sacrifice time points). The chosen dose was derived from the daily dose for humans as recommended by the supplier (140 mg·d−1), with dose translation adjustment as previously described (26), to ensure that the dose was physiologically appropriate.
The PCO supplement (Oxiprovin™, Brenn-O-Kem, Wolseley, South Africa) is a hydrophilic extract from the seeds of Vitis vinifera L., all harvested from locally cultivated vines (Western Cape/Winelands, South Africa). The extract (dry powder) typically contains 45% PCO and less than 5% monomers (with the remainder constituted by the long chain sugars and glycosides attached to the oligomers).
Induction of experimental muscle contusion injury.
Hind limb contusion was produced by a noninvasive drop-mass injury jig, as previously described (22). Briefly, this technique entails dropping a weight of 200 g from a height of 50 cm onto the medial surface of the right gastrocnemius muscle of isoflurane anesthetized rats. This injury model simulates a mild contusion or bruise injury such as might commonly be sustained in contact sports, with no added infection or surgical trauma. The results obtained using this model have much broader relevance than just to contact sport specifically though because the literature is in agreement that most muscle injuries—actually most injuries in any tissue—have an inflammatory component, irrespective of the specific cause.
At the selected time points postinjury, rats were killed by pentobarbitone sodium overdose and whole blood collected from the right ventricle via cardiac puncture. Heparinized whole blood samples (Vacutainer, BD Vacutainer systems, Plymouth, UK) were analyzed for total and differential white blood cell count using automated analysis (CellDyne 3700CS Haematology Analyzer, Abbott Diagnostics, Fullerton, CA) and for macrophage subpopulations using flow cytometry (for more details, see next section). These analyses were done within 4 h after collection. Plasma was also analyzed for myeloperoxidase (MPO) concentration using ELISA (Hycult Biotech cat no. HK105; Whitehead Scientific, South Africa). Freshly collected plasma was used to prepare 20% serum conditioned media for use in the in vitro neutrophil migration assay. These plasma samples—from each group and each time point—were also analyzed for TNF-α, IL-6, and IL-10 concentrations by ELISA.
Immunophenotypic characterization of macrophages by flow cytometry.
The determination of different subsets of leukocytes was accomplished by flow cytometry (BD FACSAria™ Cell Sorter; BD Biosciences, San Jose, CA) with three-color analysis. Whole blood was incubated for 30 min at 4°C with (a) fluorescein isothiocyanate (FITC)-conjugated anti-His48 to identify neutrophils (29), (b) Texas Red–conjugated anti-F4/80 to identify total macrophages (29), (c) FITC-conjugated anti-CD68 to identify M1 macrophages, and (d) allophycocyanin-conjugated anti-CD163 to identify M2c macrophages (2). M2c macrophages can also be identified by their production of TGF-β and IL-10. However, because of logistic reasons, this could not be assessed, and cytokine analysis was limited to circulating levels. Red blood cells were lysed for 5 min at room temperature (BD Lysing Solution; BD Biosciences), resuspended in 2 mL phosphate-buffered solution and immediately analyzed.
The neutrophil population was set as standard reference population for counting (“gate”), and M1 and M2c macrophage phenotype counts were calculated per 5000 neutrophils. Macrophage counts were then expressed as the number of cells per microliters, taking the total number of neutrophils and blood volume into consideration.
Neutrophil migration assay.
A standardized population of neutrophils were isolated from pooled blood collected from five additional animals (i.e., subjected to no intervention) within 4 h of collection, making use of a two-step process involving Ficoll-Histopaque-1077 (Sigma) and BD Cell lysis solution (BD Biosciences), which consistently yielded >95% neutrophils.
The neutrophil migration assay was performed using a modified Boyden chamber (4) and Transwell inserts. Briefly, isolated neutrophils were added to 3-μm pore size polycarbonate inserts (Falcon; BD Biosciences) (2 × 105 cells per well) and stimulated for 15 min at 37°C by addition of 100 ng·mL−1 recombinant granulocyte colony stimulating factor and 20% conditioned plasma from either control or PCO-supplemented rats collected at different time points after injury. RPMI 1640 media containing the chemotactic factor, N-formylmethionyl-leucylphenylalanine (fMLP) (1 × 10−7 M), was added to the bottom wells of a 24-well plate (BD Biosciences) and also allowed to incubate for 15 min. The inserts were then placed into their respective wells, and neutrophils were allowed to migrate for 2 h at 37°C. Although neutrophil chemotaxis assays use shorter incubation protocols to track single cell locomotion, end point assays such as ours commonly incubate for 2 h or longer (17,34). Because the purpose of the current study was to investigate effects of PCO on leukocytes themselves using appropriate controls, it was decided to not include variations of the model previously reported, such as coating the insert filter with an endothelial monolayer or collagen (7,16,35).
As recommended in an earlier review of migration assays (33), migrated neutrophils were calculated as adherent cells on the bottom of the insert filter plus cells adherent to the bottom well. Nonmigrated cells were represented by cells adhered to the top of the insert filter. For quantification, cells were fixed and immediately stained with anti-His48 (the neutrophil marker) and Hoechst (nuclear marker) and visualized with a fluorescent microscope (Olympus Cell-R System; Soft Imaging Systems, Tokyo, Japan).
Immunofluorescent image collection with the Olympus Cell-R System.
Neutrophils adherent to the top and bottom of the insert filter, as well as to the bottom of the well, were visualized with an IX-81 inverted fluorescence microscope equipped with an F-view-II cooled CCD camera (Soft Imaging Systems). Using a Xenon-Arc burner (Olympus Biosystems GMBH) as light source, images were excited with the 360- or 472-nm excitation filter for 4,6-diamidino-2-phenylindole and FITC, respectively. The emission filter was a ultraviolet/blue/green triple-band pass emission filter cube (chroma). The cells on the bottom surface of the culture well (migrated and adherent) were evenly distributed in all samples, and thus only one 20× image was taken. Using this image, an area of 79.2 μm2 of the total area of 606.5 mm2 (well) was analyzed for number of neutrophils. Because of the conical shape of the insert, there was a greater density of cells in the peripheral regions and a lower even distribution of cells toward the center of the insert. Images were thus taken in the central region (an area of 79.2 μm2 of the total area of 132.67 mm2, insert), as well as a similar area in the top and outer half of the insert. The microscope focus was repositioned to visualize cells on the bottom of the insert filter, images taken, and cells viewed and counted in the same relative regions on the bottom surface of the insert filter. Hoechst staining for nuclei was used to assist counting cells stained for His48.
Data were analyzed using a factorial ANOVA with Fisher post hoc tests to assess differences between subgroups. All data are presented as means ± SEM, with P < 0.05 regarded as statistically significant. All statistical analyses were done using the computer software Statistica version 9 (StatSoft Software, Tulsa, OK).
Leukocyte Response in Circulation
A main effect of time (P < 0.05) was observed in both PLA and PCO (Fig. 1A), with both groups showing an early increase in blood neutrophil counts at 12 h after injury. Although counts had returned to baseline levels by day 1 after injury in PLA, the increased count remained elevated at this time point in PCO (P < 0.05), only returning to baseline levels by day 3. For plasma MPO, there was an ANOVA main effect of treatment (P = 0.005); although PLA MPO decreased significantly on day 1 after injury, the same time point showed a significant transient increase in PCO MPO, with levels significantly higher than all PLA values, as well as PCO on day 3 after injury (Fig. 1B).
PLA showed a significant increase in blood M1 macrophage counts that occurred relatively late, peaking on day 5 after injury (Fig. 2A), whereas M1 macrophage counts in PCO peaked significantly earlier (day 3) and had already returned to baseline by day 5 postinjury. In both groups the increases were approximately five to sixfold. Overall, the M2c macrophage count range was lower than that of M1 macrophages. Furthermore, PLA displayed a significant early decrease in M2c macrophage number at 12 h and 1 d after injury, after which counts returned to baseline (Fig. 2B). At none of the time points assessed did PLA M2c macrophage counts increase significantly above baseline. In contrast, in PCO, M2c macrophage numbers were maintained over early time points, with a significant increase evident on day 5 after injury. This increase was 2.5-fold higher than the preinjury level for the PCO group and significantly higher than the levels determined at all other time points assessed. On day 5, PCO M2c macrophage counts were also significantly higher when compared with PLA.
In control rats supplemented with PCO for 2 wk—and not subjected to injury—macrophages expressing both M1 and M2c were significantly lower than in PLA (Fig. 2C). In response to injury, double-labeled macrophages in PLA were decreased for the duration of the study protocol, only returning toward baseline levels by day 5 postinjury. In contrast, double-labeled macrophages in PCO increased from baseline and significantly so on both days 1 and 5 postinjury.
Plasma Cytokine Response
PCO supplementation significantly affected all cytokines assessed (Fig. 3). TNF-α levels were significantly decreased when compared with PLA, in a manner independent of injury (ANOVA main effect of treatment, P < 0.0001). Neither group exhibited a significant response over time. A similar result was observed for IL-6, but with levels only detectable in PLA (ANOVA main effect of treatment, P < 0.0001). In PLA, a time effect was also observed (ANOVA, P < 0.0001), with peaks in IL-6 on days 1 and 5 postinjury. Although IL-10 decreased significantly from baseline in response to injury in the PLA group (all time points assessed), IL-10 levels in PCO showed a transient but significant peak at 12 h postinjury. As a result, PCO IL-10 was significantly higher (more than twofold higher) compared with PLA IL-10 at this time point.
Altered In vitro Neutrophil Migration
Under control conditions, that is, with no injury to affect the plasma used to condition neutrophils, no difference in migration is evident between PLA and PCO (Fig. 4). After injury, striking effects of PCO on in vitro neutrophil migration capacity were evident, with all significant effects occurring on day 1 postinjury. Results obtained by immunocytochemistry and fluorescence microscopy clearly illustrate a major increase in neutrophil migration through a 3-μm pore filter in response to injury in PLA. This was largely blocked by PCO. Indeed, on average, ≈70% fewer migrated neutrophils per area were found adherent to the bottom of the insert filter (Fig. 4B), and ≈23% fewer neutrophils adhered to the bottom of the culture well in PCO when compared with PLA at day 1 post-injury (Fig. 4C).
When muscle is injured as a result of an active stretch or contusion injury, myocytes and other cell types within muscle tissue release chemotactic factors, which result in specific immune cell mobilization and attraction to the injured site (6,13,24,27). The current study focused specifically on effects of PCO on neutrophils and monocyte/macrophages in the blood compartment, as well as immune modulating effects of some of these (as yet unidentified) chemotactic factors or immune modulators present in the plasma of rats treated with PCO. A particular strength is that the results reported here support earlier findings by our group achieved using the same in vivo rodent model protocol (in a different set of rats), providing testimony for the robustness and repeatability of our model.
We have previously demonstrated that PCO presupplementation resulted in significantly fewer neutrophils in the injured area and border zone in a study using the same injury protocol (22). This suggested either a lesser degree of mobilization or a lesser capacity for extravasation. Here we provide several lines of evidence suggesting that mobilization (activation) was not altered but that circulating leukocyte properties were changed after PCO treatment.
First, neutrophil mobilization into the circulation 12 h after injury was similarly elevated with PCO or PLA supplementation, but neutrophil numbers in circulation remained elevated on day 1 postinjury only in the PCO group—this time point corresponding with a transient increase in plasma MPO concentration in PCO only. Previously, resveratrol supplementation was reported to inhibit neutrophil adhesion to stimulated human umbilical vein endothelial cells via inhibition of the TNF-α–induced expression of adhesion molecules ICAM-1 and VCAM-1 on endothelial cells (3,12). These studies did not investigate effects of the plant-derived antioxidant on neutrophils themselves.
The current study used an in vitro migration model where the insert filter was not coated with an endothelial cell monolayer, to specifically exclude any role of or effect on endothelial cells. One day postinjury was the critical period at which differences between groups was evident in circulatory neutrophils. This was supported by in vitro results obtained in our modified model for neutrophil migration. There are several possible explanations for this finding: in vivo treatment of PCO resulted in a reduced sensitivity to chemotactic signals relative to PLA as a result of decreased activation of neutrophils. This is unlikely though because changes in MPO levels paralleled the changes seen in neutrophil counts on day 1 after injury. Alternatively, similar to results reported for endothelial cells, PCO treatment resulted in altered adhesion molecule expression on activated neutrophils, inhibiting their capacity for migration. It is important to note that all neutrophils were isolated from uninjured, nonsupplemented animals. Therefore, any effects seen occurred as a result of incubation in the conditioned plasma collected from injured rats at different time points. Although it is a limitation of the current study that adhesion molecules expressed on neutrophils were not analyzed, factors involved in chemotaxis were assessed.
Pro-inflammatory cytokines (such as TNF-α, IL-1β, and IL-6) are released as a result of injury and can activate chemotaxis of granulocytes and monocytes (31). TNF-α specifically is responsible for the release of chemotactic factors such as cytokine-induced neutrophil chemoattractant (CINC-1, equivalent to rat IL-8), an important mediator promoting neutrophil migration and also further exacerbating inflammation (9). Previous studies have shown that proanthocyanidin supplementation inhibited TNF-a and CINC-1 (but not IL-6 and IL-10) levels in pleural exudates in a model of carrageenan-induced pleurisy in rats, suggesting that proanthocyanidin might be either directly or indirectly responsible for reducing neutrophil migration (14). The current study shows that both TNF-α and IL-6 concentrations in circulation were substantially decreased in PCO relative to PLA at all time points, which cannot explain the effect on neutrophils on day 1 postinjury. However, the early peak in IL-10 concentration in PCO at 12 h postinjury may suggest that PCO treatment alters the cytokine profile in circulation to a largely anti-inflammatory one, which may at least in part explain the decreased migratory capacity for neutrophils. IL-10 is known to be an early response anti-inflammatory cytokine. Because IL-10 no longer differed between groups or from other time points on day 1 postinjury, the effector is likely to be downstream of IL-10.
PCO treatment also had significant effects on this cell type and its subpopulation distribution. Previous work by our group has shown through immunohistochemical analysis of injured rat muscle that PCO results in faster resolution of total macrophage number within tissue—peak macrophage infiltration occurred on days 3 and 5 postinjury but had returned to baseline numbers by day 7. The current study indicated a much earlier, transient M1 macrophage response in circulation after PCO treatment versus PLA (day 3 vs day 5), followed by a relatively earlier peak in circulating M2c macrophage numbers in PCO on day 5 postinjury, whereas PLA failed to have an M2c macrophage response within the period assessed. These data suggest that chronic PCO supplementation may mobilize M1 monocytes into circulation within 3 d. An important finding is that a relatively higher number of M1 monocytes appeared to differentiate into M2c macrophages while still in circulation. From the literature, it is doubtful whether macrophages with an M2 phenotype are able to infiltrate the injured area from circulation. Current opinion seems to be that only nondividing, pro-inflammatory monocytes infiltrate the injured area from circulation (2). From absolute counts of PCO macrophage subpopulations in circulation, it is clear that a significant portion of M1 cells mobilized into circulation by day 3 were not present as M1 in circulation by day 5—the time point at which PLA M1 cells peaked in circulation. M1 macrophages in tissue switch to an anti-inflammatory phenotype in the injured area after having fulfilled their phagocytic role (2). Our data suggest that in PCO, a large portion of M1 macrophages may convert to M2 while in circulation, which only occurs in an environment favoring resolution of inflammation.
In conclusion, data suggest that in the context of skeletal muscle injury, PCO exerts major effects on immune cells in circulation, in particular affecting both neutrophils and monocytic cells. Specific effects included lower two-dimensional, transmembrane migration of neutrophils and early mobilization of M1 macrophages.
This study was funded by the South African Medical Research Council and the Stellenbosch University Subcommittee B.
The authors declare no conflict of interest. They confirm that they do not have a professional relationship with the manufacturing company and that the results of the current study do not constitute endorsement of the product by the American College of Sports Medicine.
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