In 2011, approximately 450,000 burns required medical treatment in the United States.1 Along with age and concomitant inhalation injury, the extent and depth of a burn wound are critical factors that determine survival after thermal injury.2,3 Thermal injuries cause severe tissue damage not only because of the injury itself but also through other mechanisms, such as ischemia and dehydration. Uninhibited apoptotic tissue loss in the zone of stasis can extend the size and the depth of the original defect and worsen outcomes.4,5
When tissue damage is present, various factors, such as adenosine triphosphate (ATP), are released into the surrounding tissues. As a damage-associated molecular pattern molecule, extracellular ATP can initiate a cascade leading to a robust and tissue- damaging inflammatory response even in the absence of a pathogen.6 A recent review documented examples in which extracellular ATP inhibition by application of ATP hydrolyzing enzymes attenuated the inflammatory response in stroke models, airway inflammation, dermatitis, among other disease states.7 Furthermore, adenosine diphosphate and adenosine monophosphate (AMP) are broken down to adenosine, which can decrease TNF-α expression.8 Previously, TNF-α, an inflammatory marker associated with various inflammatory conditions, had been found to be increased in animal burn models and in human patients suffering burns.9–12
Although inflammation is an integral component of wound healing, the rate and quality of wound healing can be limited by excessive and ongoing leukocyte infiltration.13 The localized expression of cytokines and chemokines by inflammatory cells, like neutrophils, proceeds to activation of matrix metalloproteinase and eventual wound matrix destruction.14 The disruption of tissue leads to the release of cellular component that can activate the innate immune response via toll-like receptors (TLRs). In particular, activation of TLR-3, which recognizes ds-RNA typically associated with virus-infected cells and necrotic cells, leads to an interferon response.15,16
On the basis of this background, early inhibition of extracellular ATP in burns is an attractive therapeutic target. We hypothesize that by hydrolyzing ATP at the burn site, we can decrease neutrophil infiltration and tissue necrosis to improve cell proliferation and tissue healing. The purpose of this study is to determine the effect of removing ATP-dependent signaling on neutrophil infiltration and tissue necrosis in a partial-thickness mouse burn model.
All experiments used 8- to 10-week-old male C57Bl/6 mice (20–25 g; Harlan Laboratories, Oxford, MI). All animals were housed in standard cages with food and water available ad libitum in a specific pathogen-free facility. Animals were allowed to acclimatize for 1 week before experimentation. Experiments were performed in accordance with National Institutes of Health guidelines, and prior approval was obtained from the University of Michigan Animal Care and Use Committee.
To study the effects of apyrase on burn wounds, the mice were anesthetized with pentobarbital (Lundbeck Inc., Deerfield, IL) delivered at a dose of 65 mg/kg intraperitoneal, and then placed in a custom-made insulated mold with a rectangular opening to expose approximately 30% of the TBSA. The mice were immersed in 60°C water for 18 seconds to produce a partial-thickness dermal burn. Control animals underwent the same preparation but were immersed only in room temperature water (20°C). The mice were immediately dried. Buprenorphine (0.01 mg/kg, Buprenex; Reckitt Benckiser Pharmaceuticals Inc., Richmond, VA) was administered by subcutaneous injection every 12 hours for the first 72 hours after burn injury. The mice were killed at either 24 hours (inflammatory phase) or 5 days (proliferative phase) after burn injury to harvest skin for histologic analysis and RNA and protein extraction.
Local Application of Apyrase Enzyme
Where indicated, burn animals subjected to burn injury received local treatment with apyrase (New England Biolabs, Ipswich, MA) diluted in phosphate-buffered saline (PBS) to a concentration of 400 mU/ml.17 Enough apyrase or PBS alone to cover the burn region (200 μl) was applied topically to the burn wound immediately after injury. The site was then covered with TegadermTM (3M, St. Paul, MN).
The ATP-free swab (BBL CultureSwab; Becton Dickinson, Franklin Lakes, NJ) was used to scrape the full area of burn site, and was then put into a 15 ml Corning centrifuge tube containing 2 ml of 1× PBS buffer (with 0.1 mM EDTA). The tube was vortexed vigorously for 1 min to release ATP from swab. ATP level was measured by using Bactiter Glo Microbial Cell Viability Assay (Promega, WI) according to the manufacturer’s instructions. A microtiter plate reader (Synergy HT, BioTek, VT) was used for measuring the luminescence, and deoxy-ATP (Promega, WI) was used for the standard curve.
Skin tissue harvested from all mice 24 hours and 5 days after the burn injury were formalin fixed and paraffin embedded before sectioning. After staining, images of bright field low-powered fields (×10) were obtained from all slides in regions adjacent to the site of injury at equal distances within the wound using a Nikon E-800 upright microscope (Nikon, Melville, NY) connected to a Olympus DP-71 camera (Olympus, Center Valley, PA).
The extent of tissue necrosis was qualitatively assessed from hematoxylin and eosin slides from the 24-hour and 5-day time points by noting the deepest layer expressing signs of cell death, including decreased eosinophilia, loss of cell architecture, vacuolization, cell disruption, and karyolysis as previously described.18 In both the burn and burn + apyrase groups, regions of the burn were examined.
Neutrophil infiltrate and activation in skin sections from both time points were evaluated by staining for neutrophil elastase, a neutrophil-specific marker (naphthol AS-D chloroacetate esterase kit; Sigma-Aldrich, St. Louis, MO). Naphthol AS-D chloroacetate is a substrate for neutrophil-specific elastase. Three investigators blinded to study group counted the number of neutrophils in each randomly selected field.
The proliferative capacity of skin tissue was appraised from 5-day sections by immunohistochemical staining of Ki-67 (1/500; Abcam, Cambridge, MA) as had been established previously.19 Images of Ki-67 stained sections were analyzed by semiautomating the approach.20 Two independent and blinded investigators used Photoshop CS6 (Adobe, San Jose, CA) to count positive-staining pixels in 10 high-powered fields (×40) taken from random sites. Any pixels not within a nucleus were excluded.
Real-Time Reverse-Transcription Polymerase Chain Reaction
Total RNA was extracted from skin harvested 5 days after injury using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions and was spectrophotometrically quantified ([λ] = 260 nm). A total of 1.0 µg of the RNA extract was reverse transcribed to cDNA synthesis using random primers and MultiScribe™ reverse transcriptase (Applied Biosystems, Carlsbad, CA) following the manufacturer’s instructions. Quantitative real-time polymerase chain reaction was performed using the Smart Cycler (Cepheid, Sunnyvale, CA) and Power Sybr GreenTM PCR master mix (ABI, Foster, CA). The primers for mouse genes of interest are found in Table 1. Gene amplification was measured in terms of the cycle threshold (CT) value, which was automatically determined by the cycler software. The obtained CT value was normalized with the CT value of the housekeeping gene, GAPDH, according to the established ΔΔCT method.
Skin samples (100 mg) were homogenized in radioimmunoprecipitation assay lysis buffer (Sigma) with protease inhibitor (Thermo Scientific) to extract protein. The homogenized samples underwent shaking for 30 minutes at 4°C. The supernatant was collected after centrifugation at 14,000g for 15 minutes.
Western blot analysis of protein expression and kinase phosphorylation was performed according to established protocols. Briefly, equal amounts of protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were probed with primary antibody overnight at 4°C. Horseradish peroxidase–linked antirabbit secondary antibodies (1:2000; Cell Signaling Technologiy, Danvers, MA) were used in conjugation with electrochemiluminescence to visualize the protein bands on autoradiography films. Anti–nuclear factor (NF)-κβ (1:1000), anti–phospho-NF-κβ (1:500), anti–interferon regulatory factor (INF)-3 (1:1000), anti–phospho-IRF-3 (1:1000) and α-tubulin (1:1000) were from Cell Signaling Technology. Sample loading was assessed with α-tubulin.
Analysis was performed using SPSS (Version 20; IBM, Armonk, NY). Unless indicated otherwise, data are expressed as means ± SD. Statistical analysis was performed by Kruskal–Wallis one-way analysis of variance followed by pairwise comparison using Mann–Whitney U tests. For comparison of extracellular ATP levels, Student’s t-test was used. Differences with P < .05 were considered to be statistically significant.
Apyrase Decreases Extracellular ATP in the Wound Site
The first step was to determine whether indeed apyrase would mitigate extracellular ATP levels at the burn wound site. To investigate ATP levels, we performed an ATP assay to quantify the amount of extracellular ATP (nmol/cm2). Interestingly we saw a spike of ATP levels in the first few hours after burn injury, which was absent in our control group. When analyzing the effect of apyrase we noticed a dramatic decrease in extracellular ATP compared with the burn injury at these early time points demonstrating its ATP hydrolyzing effect (Figure 1). Having demonstrated the powerful effect of apyrase on ATP levels at the burn site, we next set out to investigate how this decreased ATP would affect inflammation and tissue necrosis.
ATP Hydrolysis Reduces Extent of Necrosis in Partial-Thickness Burn
Next, we investigated the effects of burn injury and inhibition of ATP-dependent signaling on tissue necrosis at 24 hours and 5 days postinjury. Necrosis was less extensive in the burn + apyrase group when compared to the burn group at 24 hours and 5 days. At 24 hours after thermal injury, acute, locally extensive fat necrosis and steatitis (fat inflammation) were visible in the hematoxylin and eosin skin sections from mice in the burn group whereas only moderate focal fat necrosis and mild steatitis was present in comparable sections from mice in the burn + apyrase group (Figure 2). This pattern persisted even at 5 days after the thermal injury.
ATP Hydrolysis Decreases Neutrophil Quantity and Activation
To measure neutrophil infiltration and activation, skin tissue sections (24 hours and 5 days) were stained for neutrophil elastase (Figure 3A). The data showed that at 24 hours after burn injury, the burn group had greater neutrophil infiltration than the burn + apyrase group (P < .001; Figure 3B). These differences in infiltration and activation were not apparent in sections from 5 days after injury (data not shown). Thus, during the acute inflammatory phase, apyrase appears to decrease neutrophil infiltration and activation.
ATP Hydrolysis Increases Cell Proliferation at the Burn Site
Although apyrase acts only on extracellular ATP, it was important to assess its effects on intracellular processes since ATP is the energy currency of cells. Therefore, the effects of extracellular ATP on cell proliferation were measured by analyzing Ki-67 staining of skin tissue sections at 5 days after injury (Figure 4A). The cells that stained positively were found at the base of the epidermis and surrounding hair follicles. Inflammatory cells in the dermis also caused non-specific signaling due to their endogenous peroxidase activity and thus were not included in the analysis. The data showed a tendency for higher signal in the burn + apyrase group’s epidermal region compared to the other two groups (Figure 4B; n.s.). We demonstrate that apyrase does not cause negative effects in the tissues ability for regeneration.
ATP Hydrolysis Decreases TNF-α and IFN-β Gene Expression
The effects of apyrase on the expression of inflammatory markers after injury were examined. On the basis of quantitative real-time polymerase chain reaction, we found that both TNF-α (P < .05) and IFN-β (P <.001) expression 24-hours postinjury in the burn + apyrase group were decreased compared with the burn group (P < .05; Figure 5A, B). Interleukin-23 also tended to increase in the burn group; however, the differences were not significant (Figure 5C). These changes in gene expression demonstrate that ATP hydrolysis has a local effect on inflammatory cytokine activation. Interestingly, macrophage inflammatory protein (MIP)-2, a neutrophil chemoattractant, was increased in the burn + apyrase group although the effect was not significant (Figure 5D).
To explore the downstream effects of the changes in cytokine signaling, we looked at activation of the NF-κβ pathway through Western blotting. In general, a decrease in phosphorylation of NF-κβ in tissue lysates from the burn + apyrase group when compared with the burn group was observed (Figure 6).
In an effort to elucidate the mechanism leading to decreases in cytokine expression, we looked at phosphorylation of IRF-3 as a downstream effect of changes in Ca2+flux. Protein kinase C (PKC) activity is calcium dependent and required for IRF-3 activation. We found that there was no difference in IRF-3 activation among the groups (Figure 6).
Our study established the preliminary results that in a mouse partial-thickness scald burn injury, apyrase decreased neutrophil infiltration and activation. Additionally, compared with burn samples the extent of tissue necrosis was attenuated by the application of apyrase (Figure 2). These results support previous findings that extracellular ATP plays an important role in determining neutrophil activity, from migration to secretion of enzymes. Previous studies have shown that ATP is required to direct neutrophils migration21,22 as well as to modulate the expression and release of proteolytic enzymes.23,24 Although neutrophil migration to a site of injury is beneficial to remove necrotic debris, the released neutrophilic enzymes are not specific for necrotic cells, and therefore can also act to damage healthy surrounding tissues. Thus, modulation of ATP concentrations at the site of a burn wound could improve wound healing.
As stated previously, levels of TNF-α are increased after burns and are associated with patient outcomes.25,26 TNF-α can stimulate neutrophil adhesion and activation.27–29 Thus, the decrease in neutrophil infiltration and relative lower intensity in the burn + apyrase group is expected, considering the lower TNF-α levels present in the skin.
Extracellular ATP acts on P2X and P2Y purinergic receptors on the cell membranes. Activation of P2X receptors, causes a calcium influx whereas P2Y downstream signaling is mediated by G-protein–coupled receptors.7,21 The mechanism behind the decrease in TNF-α expression might be because of attenuated activation of the purinergic receptors, specifically those in the P2Y subset, and the increased levels of adenosine at the site of apyrase application. P2Y11 and P2Y13 receptors are coupled to the Gi/o signaling pathway inhibiting adenylyl cyclase and decreasing cyclic-AMP (cAMP) levels.30 By hydrolyzing ATP and adenosine diphosphate, apyrase may decrease the activation of this pathway and thus lead to more production of cAMP. It has previously been established that cAMP can reduce TNF-α production by macrophages.31 Therefore, it is possible that the decrease in TNF-α is because of tempered inhibition of cAMP formation. Furthermore, an increase in calcium flux has also been associated with increased TNF-α expression.32 Apyrase can decrease the calcium flux through two possible mechanisms: greater activation of the adenosine A3 receptor33 and reduced activation of P2X receptors.
IFN-β, which is typically associated with viral infections, is also expressed in the presence of cellular damage.34 TLRs are central to pathways leading to IFN-β expression. In particular, TLR-3 recognizes ds-RNA typically associated with virus-infected cells and necrotic cells.15,16 By applying apyrase at the site of injury, the extent of cellular necrosis is attenuated resulting in less endogenous TLR-3 ligands. Thus, expression of IFN-β is expected to fall.
Calcium flux can moderate the activation of IFN-β and NF-κB via TLRs.35,36 Thus, the P2X class of purinergic receptors, which are ion channels, could possibly be involved in the reduction of IFN-β expression and NF-κB phosphorylation after apyrase treatment through this mechanism. The reduction of extracellular ATP by the application of apyrase causes a decline in the calcium current through the P2X receptors. PKC activity is calcium dependent and required for IRF-3 activation necessary for IFN-β production. Consequently, as PKC activity falls, less IFN-β is transcribed.37 Future studies in a larger population of animals will be performed to further verify these findings.
Although it may seem counterintuitive to rid the injury of an energy molecule like ATP, we found that there was no difference in the proliferative capacity of the tissue as demonstrated by Ki-67 signaling. This suggests two things: 1) the topical administration and dose of apyrase is not affecting the intracellular stores of ATP and 2) by diminishing the amount of cell death signaling, the neighboring cells to the apyrase-treated injury may be primed to channel their energy to reparative processes.
This study sets a foundation to further explore the benefits of ATP modulation of inflammation in burn wounds. Several questions not answered by this project need to be addressed before apyrase can be recommended for clinical use. Primarily as this is a pilot study, these results should be verified with a larger sample size for each group. Future studies should also include more indices of wound healing such as the rate of healing, the magnitude of the overall wound, and the quality of the healed wound. Furthermore, it is important to keep in mind that this study only looked at short-term effects of apyrase treatment. Thus, it is imperative that long-term studies be done to determine the ultimate outcome on wound healing and scarring. Although the present study is based on a simple murine model, one should note that the composition of mouse skin is different than that of larger animals, including humans. Additionally, more work should be done to clarify the mechanism of apyrase’s effect on the inflammatory response by looking at adenosine signaling.
In conclusion, local topical application of apyrase mitigates the inflammatory response caused by a burn injury in a mouse model. This finding suggests a possible mechanism to improve burn wound care. Modulation of the inflammatory response could reduce tissue damage and allow for more rapid healing, especially within the zone of stasis. In the future, ATP hydrolyzing enzymes could be used as topical inflammatory regulators to quell the injury caused by inflammation.
We thank Dr. J. Erby Wilkinson and the University of Michigan Pathology Cores for Animal Research staff for their assistance with histology and its evaluation.
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