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. Author manuscript; available in PMC: 2014 Sep 2.
Published in final edited form as: J Burn Care Res. 2014 Jan-Feb;35(1):54–61. doi: 10.1097/BCR.0b013e31829b36d6

ATP hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice

Jill Bayliss 1,*, Sara DeLaRosa 2,*, Jianfeng Wu 3, Jonathan R Peterson 2, Oluwatobi N Eboda 2, Grace L Su 1,3, Mark Hemmila 2, Paul H Krebsbach 5, Paul S Cederna 2, Stewart C Wang 2, Chuanwu Xi 4, Benjamin Levi 2
PMCID: PMC4152376  NIHMSID: NIHMS618942  PMID: 23877144

Abstract

Objective

Extracellular ATP, present in thermally-injured tissue, modulates the inflammatory response and causes significant tissue damage. We hypothesize that neutrophil infiltration and ensuing tissue necrosis would be mitigated by removing ATP-dependent signaling at the burn site.

Methods

Mice were subjected to 30% total-body- surface-area partial-thickness scald burn by dorsal skin immersion in a water bath at 60°C or 20°C (non-burn controls). In the treatment arm, an ATP hydrolyzing enzyme, apyrase, was applied directly to the site immediately after injury. Skin was harvested after 24 hours and 5 days for hematoxylin and eosin stain (H&E), elastase, and Ki-67 staining. TNF-α and IFN-β expression were measured through qRT-PCR.

Results

At 24 hours, the amount of neutrophil infiltration was different between the burn and burn + apyrase groups (p<0.001). Necrosis was less extensive in the apyrase group when compared to the burn group at 24 hours and 5 days. TNF-α and IFN-β expression at 24 hours in the apyrase group was lower than in the burn group (p <0.05). However, Ki-67 signaling was not significantly different among the groups.

Conclusions

Our results support the role of extracellular ATP in neutrophil activity. We demonstrate that ATP hydrolysis at the burn site allays the neutrophil response to thermal injury and reduces tissue necrosis. This decrease in inflammation and tissue necrosis is at least partially due to TNF-α and IFN-β signaling. Apyrase could be used as topical inflammatory regulators to quell the injury caused by inflammation.

Keywords: ATP hydrolysis, burn wound, neutrophils, wound healing, extracellular ATP, inflammatory response, apyrase

INTRODUCTION

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 due to 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 (DAMP), 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, ADP and AMP are broken down to adenosine, which can decrease TNF-α expression.8 Previously, TNF-α, an inflammatory marker associated with various inflammatory conditions, has been found to be elevated in animal burn models and in human patients suffering burns.9–12

While 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 (MMPs) 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

Based on 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.

METHODS

Animals

All experiments used 8- to 10-wk-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 Institute of Health guidelines and prior approval was obtained from the University of Michigan Animal Care and Use Committee.

Burn procedure

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 i.p., and then placed in a custom-made insulated mold with a rectangular opening to expose approximately 30% of the total body surface area (TBSA). The mouse was immersed in 60°C water for 18 s 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 h for the first 72 h after burn injury. The mice were euthanized 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 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 Tegaderm™ (3M, St. Paul, MN).

ATP Assay

The ATP-free swab (BBL CultureSwab, BD, NJ) was used to scrape all the area of burn site, and was then put into a 15 ml Corning centrifuge tube containing 2 ml of 1 x 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 dATP (Promega, WI) was used for the standard curve.

Histology

Skin tissue harvested from all mice 24 hours and 5 days after the burn injury were formalin fixed and paraffin embedded prior to sectioning. Following staining, images of bright field low-powered fields (10x) 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 H&E 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 (40x) 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 following injury using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions and was spectrophotometrically quantified ([lambda] = 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 manufacturers instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the Smart Cycler (Cepheid, Sunnyvale, CA) and Power Sybr Green™ 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.

Table 1.

Sequences used in real-time polymerase chain reaction.

Gene Primer Sequence
GAPDH Forward: 3′-TCA ACA GCA ACT CCC ACT CTT CCA-5′
Reverse: 3′-ACC CTG TTG CTG TAG CCG TAT TCA-5′
TNF-α Forward 3′-GG ACA GTG ACC TGG ACT GT-5′
Reverse 3′-CTC CCT TTG CAG AAC TCA GC-5′
IFN-β Forward 3′-AGC TCC AAG AAA GGA CGA ACA T-5′
Reverse 3′-GCC CTG TAG GTG AGG TTG ATC T-5′
IL-23 Forward 3′-TGT GCC CCG TAT CCA GTG T-5′
Reverse 3′-CGG ATC CTT TGC AAG CAG AA-5′
MIP-2 Forward 3′-CTC CAG ACT CCA GCC ACA CTT C-5′
Reverse 3′-ACT TTT TGA CCG CCC TTG AGA-5′
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Western Blotting

Skin samples (100 mg) were homogenized in RIPA lysis buffer (Sigma) with protease inhibitor (Thermo Scientific) to extract protein. The homogenized samples were incubated at 4° C while shaking for 30 minutes. The supernatant was collected after centrifugation at 14000-x g 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 SDS-PAGE and transferred to PVDF membranes. The membranes were probed with primary antibody overnight at 4°C. Horseradish peroxidase–linked anti-rabbit secondary antibodies (1:2,000; Cell Signaling Technologiy, Danvers, MA) were used in conjugation with electrochemiluminescence to visualize the protein bands on autoradiography films. Anti-NF-κβ (1:1000), anti-phospho-NF-κβ (1:500), anti-IRF-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.

Statistical Analysis

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 t test was used. Differences with p < 0.05 were considered to be statistically significant.

RESULTS

Apyrase decreases extracellular ATP in the wound site

The first step was to determine if 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 (nmole/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 to the burn injury at these early time points demonstrating its ATP hydrolyzing effect (Fig. 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.

Figure 1.

Figure 1

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Extracellular ATP levels on wound surface along with time after burn. At time point 0, n = 3; other time points, n > 6. Student t test was used for statistic analysis, p< 0.01 (***) and p<0.05 (*).

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 post-injury. Necrosis was less extensive in the burn + apyrase group when compared to the burn group at 24 hours and 5 days. At 24 hours following thermal injury, acute, locally extensive fat necrosis and steatitis (fat inflammation) were visible in the H&E 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 (Fig. 2). This pattern persisted even at 5 days after the thermal injury.

Figure 2.

Figure 2

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Extent of necrosis is reduced by application of apyrase to thermally injured skin. A. Representative images of H & E stains of control, burn and burn + apyrase groups at 24 hours (n=6 for each group) and 5 days (n=6 for each group) taken at 20X magnification. (Arrow points to steatitis. Stars indicate fat necrosis. Scale bars = 100 μm.)

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 (Fig. 3A). The data showed that at 24 hours following burn injury, the burn group had greater neutrophil infiltration than the burn + apyrase group (p<0.001, Fig. 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.

Figure 3.

Figure 3

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Apyrase reduces neutrophil numbers in thermally injured skin. A) Elastase stain (40X) done on skin sections collected 24 hours after injury from control, burn or burn + apyrase groups (n=4). Circles note location of positively-staining neutrophils while inserts are enlargements (total 160x) of neutrophils in the sections. B) Quantification of neutrophil infiltration was done by identifying positively staining cells in randomly selected 10x fields (*** p < .001).

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 following injury (Fig. 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 (Fig. 4b, n.s.). We demonstrate that apyrase does not cause negative effects in the tissues ability for regeneration.

Figure 4.

Figure 4

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Apyrase has no effect on cell proliferation after a burn injury. (A) Representative 10X images of sections stained for Ki-67 5 days after burn injury. Arrows point to positively staining nuclei. Cells in the dermis were not included to minimize the risk of confounding the results with inflammatory cell peroxidases. Double arrows point toward the burn wound. (Scale bars = 100 μm.) (B) Quantification of Ki-67 stain. (C) Negative and (D) positive control for Ki-67 stain.

ATP hydrolysis decreases TNF-α and IFN-β gene expression

The effects of apyrase on the expression of inflammatory markers after injury were examined. Based on qRT-PCR, we found that both TNF-α and IFN-β expression 24-hours post-injury in the burn + apyrase group were decreased compared to the burn group (p< 0.05, Fig. 5A and 5B). IL-23 also tended to increase in the burn group, however the differences were not significant (Fig. 5C). These changes in gene expression demonstrate that ATP hydrolysis has a local effect on inflammatory cytokine activation. Interestingly, MIP-2, a neutrophil chemoattractant, was increased in the burn + apyrase group although the effect was not significant (Fig. 5D).

Figure 5.

Figure 5

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Effect of apyrase on cytokine gene expression. A and B) Apyrase decreases TNF-α and IFN-β gene expression after a burn injury (* p < 0.05). D) No difference exists in MIP-2 and IL-23 expression as a result of apyrase application.

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 to the burn group was observed (Fig. 6).

Figure 6.

Figure 6

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Effect of apyrase on NF-κβ and IRF-3 signaling pathways. Protein extracted from skin samples was fractionated via SDS-PAGE and probed for activation of downstream signaling pathways of TNF-α and interferon β. Representative bands after overnight incubation with corresponding primary antibodies. Bands are from same membrane and lanes.

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 (Fig. 6).

DISCUSSION

Our study established preliminary results that in a mouse partial thickness scald burn injury, apyrase decreased neutrophil infiltration and activation. Additionally, compared to burn samples the extent of tissue necrosis was attenuated by the application of apyrase (Fig. 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 While 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 elevated following 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 due to 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 cAMP levels.30 By hydrolyzing ATP and ADP, 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 due to 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 Toll-like receptors (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 following 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.

While 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. While 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 due to 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.

Acknowledgments

Source of Funding

The research is partially supported by the NIH grant (R01GM098350-02) to C.X. and S. W.

We would like to thank Dr. J. Erby Wilkinson and the University of Michigan Pathology Cores for Animal Research staff for their assistance with histology and its evaluation.

Sara De La Rosa is a Howard Hughes Medical Institute Medical Research Fellow.

Footnotes

Conflicts of Interest: None of the authors have a financial interest in any of the products, devices, or drugs mentioned in this manuscript.

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