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(Journal of Leukocyte Biology. 2003;73:448-455.)
© 2003 by Society for Leukocyte Biology

Accelerated wound closure in neutrophil-depleted mice

Julia V. Dovi^,*, Li-Ke He and Luisa A. DiPietro^*,

Departments of
^* Microbiology and Immunology and
Surgery, Burn and Shock Trauma Institute, Loyola University Medical Center, Maywood, Illinois

Correspondence: Luisa A. DiPietro, Loyola University Medical Center, 2160 S. 1st Ave., BSTI, Building 110, Maywood, IL 60153. E-mail: ldipiet{at}lumc.edu

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The infiltration of neutrophils into injured tissue is known to protect wounds from invading pathogens. However, more recent studies suggest that neutrophils might inhibit the wound repair process. To investigate the role of neutrophils in wounds, mice were neutrophil-depleted by injection with rabbit anti-mouse neutrophil serum. Remarkably, epidermal healing, measured by wound closure, proceeded significantly faster in neutropenic than control mice (77.7+14.2% vs. 41.2+0.9%, P<0.02 at day 2). Dermal healing was not affected by neutrophil depletion, as neither collagen deposition nor wound-breaking strength was significantly different between neutropenic and control mice. As the delayed repair of diabetic individuals exhibits robust inflammation, the effect of neutrophil depletion on diabetic wound healing was investigated. Similar to the observations in wild-type mice, wound closure was accelerated by nearly 50% in neutropenic, diabetic mice. The results suggest that although neutrophils may provide protection against infection, they may retard wound closure.

Key Words: healing • keratinocytes • inflammation • diabetic

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Upon wounding, a sequence of events is set in motion culminating in tissue repair. Three overlapping phases can be distinguished: an inflammatory phase, characterized by neutrophil and macrophage infiltration; a proliferative phase, dominated by collagen deposition and angiogenesis; and a maturation phase, involving resolution of inflammation and scar maturation [¹ ]. As part of the inflammatory phase, a massive infiltration of neutrophils, which is greatest at day 1 post-injury, into the subepidermal region of the wound bed can be observed. Starting within minutes after injury, neutrophils are thought to protect the host from infection by combating an invading microorganism and clearing cellular debris. During this process, activated neutrophils secrete a battery of bioactive substances, such as proteases and reactive oxygen intermediates, which can lead to serious tissue damage. This is most clearly demonstrated in nonhealing wounds in which resolution of inflammation is often delayed [² ,³ ]. Despite numerous correlations between excess inflammation and wounds that heal poorly or not at all, it is not well understood how neutrophils influence normal healing. In the absence of infection or underlying medical conditions of the wounded individual, neutrophils are considered neutral to healing.

Our overall goal is to elucidate the role of neutrophils and their products in wound healing. In this study, we investigated healing of murine full-thickness dermal excisional and incisional wounds following the depletion of neutrophils.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals
BALB/c mice were obtained from Harlan Sprague Dawley (Indianapolis, IN). Diabetic mice (C57BLKS/J-m +/+Lepr^db) were purchased from The Jackson Laboratory (Bar Harbor, ME). Females, 6–10 weeks of age, were used throughout this study. Mice were kept in cages of four with water and food ad libidum. Animal protocols used in this study were reviewed and approved by the Loyola University Institution Animal Care and Use Committee (Maywood, IL).

Antisera and monoclonal antibody (mAb)
Rabbit anti-mouse neutrophil serum was purchased from Intercell Technologies (Hopewell, NJ). The rabbit preimmune serum was kindly provided by Dr. Katherine L. Knight. The hybridoma RB6-8C5, which was originally developed by Dr. Robert Coffman (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA), secretes a monoclonal rat anti-mouse Gr-1 antibody [⁴ ,⁵ ]. This clone was a kind gift from Dr. C. Brown (University of Michigan, Ann Arbor). The anti-Gr-1 antibody was purified by precipitation of the hybridoma culture supernatant with ammonium sulfate as described elsewhere [6]. Precipitated protein was subsequently dialyzed against phosphate-buffered saline (PBS) and was then subjected to affinity protein A chromatography (Protein G Sepharose 4 Fast Flow, Amersham Pharmacia Biotech, Piscataway, NJ). Concentrated antibody was eluted from the column, and the concentration was determined by spectrophotometry at 280 nm.

Fluorescence-activated cell sorter (FACS) analysis
A total of 3 ml murine blood was collected by cardiac puncture into heparinized tubes. Red blood cells were lysed by addition of Qiagen EL erythrocytes lysis buffer, according to the manufacturer’s instructions (Qiagen, Valencia, CA). White blood cells (5x10⁵) were resuspended in 0.5 ml PBS + 1% bovine serum albumin (BSA) to block nonspecific binding. Cells were then incubated with 1 µg/ml monoclonal anti-Gr-1 antibody or rabbit anti-mouse neutrophil serum diluted 1:100 in 0.5 ml for 30 min on ice. Cells were washed in two changes of PBS/BSA and subsequently resuspended in 0.5 ml of the appropriate secondary antibody solution. For the RB6-8C5-stained cells, a fluorescein isothiocyanate (FITC)-conjugated goat anti-rat immunoglobulin G (IgG; Sigma Chemical Co., St. Louis, MO) was used (1:32, final concentration 31.3 µg/ml). For the rabbit anti-mouse neutrophil serum-labeled cells, a FITC-conjugated goat anti-rabbit antibody (Sigma Chemical Co.) was used (1:80, final concentration 12.5 µg/ml). After a 30-min incubation on ice, cells were washed in two changes of PBS/BSA and two changes of PBS. Analysis was performed on a FACScaliber (Becton Dickinson, San Jose, CA).

In vivo neutrophil depletion
To induce neutropenia, female BALB/c mice were injected intraperitoneally (i.p.) with 150 µl rabbit anti-mouse neutrophil serum or rabbit preimmune serum. Successful depletion of peripheral neutrophils was confirmed by differential counts of blood smears prepared from the injected animals. Blood was collected by removing 2 mm of the tail tip from anesthetized mice. To prepare blood smears, 2 µl murine blood was pipetted onto a 45° tilted glass slide and was then evenly distributed by dragging a second glass slide over the first one. Blood smears were air-dried for 30 min and stained with Giemsa Wright stain (Sigma Chemical Co.) for 1 min. All slides were then covered with an equal volume of distilled water and incubated for an additional 10 min. Slides were then rinsed with distilled water and air-dried. A total of 200 cells per slide was counted, and the percent neutrophils was determined.

Excisional injury model and tissue collection
Sixteen hours post-injection of rabbit anti-mouse neutrophil or control serum, neutropenic and control mice were anesthetized with Halothane (Halocarbon Laboratories, River Edge, NJ) and shaved on their dorsum. A dermal biopsy punch (Acu Punch, Acuderm, Ft. Lauderdale, FL) was used to produce six full-thickness dermal excisional wounds. Each wound measured 3 mm in diameter and extended through the panniculus carnosus on the dorsal skin of each mouse. At days 1, 2, 3, and 5 post-wounding, mice were killed, and the wounds were excised. None of the wounds displayed any signs of infection. Wound tissue was embedded in Tissue Freezing Medium (Triangle Biomedical Science, Durham, NC) and was frozen at -70°C for biochemical analysis. Some day-1 wounds were embedded in paraffin for preparation of 1 µm sections.

Analysis of wound myeloperoxidase (MPO)
The measurement of MPO levels in tissue has been shown to reflect neutrophil content. MPO content in wound tissue was determined using previously described methods [⁷ ,⁸ ]. Briefly, wounds were homogenized in 2 ml 20 mM phosphate buffer, pH 7.4. Homogenates were centrifuged at 12,000 g for 45 min, and the supernatant was decanted. The pellets were resuspended in 1 ml 50 mM phosphate buffer containing 10 mM EDTA and 0.5% hexadecyltrimethylammonium bromide (HTAB). After a freeze-thaw cycle, the samples were briefly sonicated and incubated at 60°C for 2 h. The samples were centrifuged at 500 g for 10 min, and the supernatant was transferred to 1.5 ml tubes and directly subjected to analysis or stored at -20°C. For analysis, a standard curve ranging from 0 to 3.0 units/ml MPO was generated. Aliquots of samples (50 µL) or standards were placed in 12 x 75 mm glass tubes with 500 µl assay buffer (0.1 M phosphate buffer, pH 5.4, 1% HTAB, 0.43 mg/ml 3,3',5,5'-tetramethylbenzidine). The reactions were started by the addition of 50 µl 15 mM H₂O₂, incubated at 37°C for 15 min, and stopped with 1.0 ml cold 0.2 M sodium acetate, pH 3.0. The absorbance of each sample and standard was read at 655 nm within 10 min of completion. All samples and standards were tested in duplicates.

Analysis of re-epithelialization
To analyze the degree of re-epithelialization, 10 µm sections were prepared from frozen, embedded wounds. Sections were then stained with hematoxylin and eosin (H&E; Sigma Chemical Co.) and visualized under 100x power using a light microscope. The wound width covered by epithelium as well as the total wound width were measured and used to calculate % re-epithelialization with the formula: % re-epithelialization = re-epithelialized wound width x 100/total wound width. The analysis of wound closure was conducted in a blinded manner.

Incisional injury model and wound disruption strength
Mice were injected with the rabbit anti-mouse neutrophil or control serum, as described above. Sixteen hours post-injection, the mice were anesthetized by i.p. injection of Ketamine (Abbott Laboratories, North Chicago, IL) and Xylaject (Phoenix Pharmaceutical, St. Joseph, MO). The fur of the ventral and dorsal skin was removed by shaving, and the skin was washed briefly with 70% isopropanol. A 2-cm incisional wound extending through the dermis and the panniculus carnosus was prepared in the paraspinal region and closed using sterile surgical clips. After 5 days, the mice were lightly anesthetized with Halothane to remove the surgical clips. At days 7 and 14 post-injury, wounds were harvested. From each wound, two strips perpendicular to the incision were excised and subjected to tensiometry. In this procedure, sequentially increased tension was applied to the excised wound strips. Wound disruption strength is defined as the load value of wound disruption. The tensiometer used was designed and built by the Department of Surgery and Instrument Models Facility at the University of Vermont (Burlington).

Collagen content
The collagen content of wounds was assessed by determining the amount of hydroxyproline present [⁹ ]. Frozen wound tissue was hydrolized in 2.0 ml 6 N HCl for 3 h at 130°C or overnight at 110°C. The reaction was stopped with 2.5 N NaOH and diluted 40-fold with dH₂O. A 0.05 mol/L N-chloro-p-toluene-sufonamide solution (1 ml) was added to 2 ml of the diluted hydrolysate and incubated for 20 min at room temperature. Perchloric acid (1 ml 3.15 mol/L) was added to the samples and incubated for an additional 5 min at room temperature. p-Dimethylaminobenzaldehyde (1 ml 20%) was then added followed by a 20-min incubation at 60°C. The samples were cooled with cold tap water, and the absorbance was measured at 557 nm. The hydroxyproline content was determined by comparison to a standard curve. All reagents in this assay were purchased from Sigma Chemical Co.

Macrophage staining
For analysis of macrophage content, sections from the central portion of each wound were stained with MOMA-2, a rat mAb specific for murine macrophages (Serotec, Raleigh, NC). Histologic sections were fixed in acetone for 30 min and pretreated with 0.3% H₂O₂ in methanol to block endogenous peroxidase. After three 5-min washes in PBS, sections were incubated with normal mouse serum (1:10; Harlan Sprague Dawley) for 30 min. Sections were incubated in primary antibody MOMA-2 (1:500) for 30 min, washed three times in PBS, and incubated in secondary antibody (biotinylated mouse anti-rat Ig, 13.0 µg/ml; The Jackson Laboratory) for another 30 min. Subsequently, sections were incubated with avidin-biotin-horseradish peroxidase complex (Vector Laboratories, Burlingame, CA) for 30 min and were again washed in PBS. Color development was performed with 3,3'-diaminobenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for 10 min, and sections were counterstained with Gills hematoxylin (Sigma Chemical Co.). To quantitate macrophages, MOMA-2-positive cells were counted in five random high-power fields (HPF) within the wound bed. The analysis was conducted in a blinded manner.

Statistical analysis
Data were analyzed using GraphPad Prism, version 2.01 (GraphPad Software, San Diego, CA). The means and SEM were calculated for each data set. A one-way or two-way ANOVA followed by an unpaired t-test was used for comparison of groups. Values of P< 0.05 were considered statistically significant.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Rabbit anti-mouse neutrophil-serum characterization
The specificity of the rabbit anti-mouse neutrophil serum was confirmed by comparing the staining of murine white blood cells with the rabbit anti-mouse neutrophil serum to that of the monoclonal anti-Gr-1 antibody (RB6-8C5). Gr-1 is highly expressed on neutrophils and at low levels on monocytes [¹⁰ ]. Binding of the two primary reagents was detected with subsequent staining with FITC-conjugated secondary antibodies and then analyzed by flow cytometry. Populations of neutrophils, monocytes, and lymphocytes were identified by characteristic appearance in the forward- and side-scatter during flow cytometry. Consistent with reports in the literature [¹⁰ ], the Gr-1 antigen was expressed at high levels on granulocytes and only at low levels on monocytes, and lymphocytes were negative for Gr-1 staining (Fig. 1A ). The FACS profile of the rabbit anti-mouse neutrophil serum-stained cells was virtually identical (Fig. 1A and 1B) . These data suggest that the rabbit anti-mouse neutrophil serum binds to epitopes highly expressed on murine neutrophils.

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Figure 1. Flow cytometric analysis of the rabbit anti-mouse neutrophil serum specificity. White blood cells (5x105) were stained with (A) rabbit anti-mouse neutrophil (solid line) or a rabbit preimmune serum (dotted line); (B) a monoclonal rat anti-mouse Gr-1 (Ly6G) antibody (solid line) or an isotype-matched, irrelevant mAb (dotted line).

Time course of neutrophil depletion after rabbit anti-mouse neutrophil-serum injection
To determine the time course of neutrophil depletion after a single injection of 150 µl rabbit anti-mouse neutrophil serum, blood was collected from the tail of rabbit anti-mouse neutrophil serum-treated and control BALB/c mice over a 5-day period. Injection (i.p.) of rabbit anti-mouse neutrophil serum but not rabbit preimmune serum resulted in 85% reduction of peripheral neutrophil counts (Fig. 2 ). The number of neutrophils returned to control levels at approximately day 5 post-injection (Fig. 2) .

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Figure 2. Time course of peripheral neutrophil depletion. Mice (n=6) were injected i.p. with 150 µl rabbit anti-mouse serum. Blood was collected from the tail every day for 5 days, and the number of neutrophils was determined by differential counts of blood smears. The bars indicate the means of neutrophil counts from six animals. The lines above the bars indicate the SEM.

Wound histology
To determine if the absence of neutrophils affects the overall microscopic wound architecture, the histologic appearance of sections from day 1 wounds of neutropenic and control mice was compared. Sections of day 1 wounds were selected for analysis, as neutrophil infiltration peaks at this time point in our wound-healing model (Fig. 3A ). Specimens of neutropenic and control mice displayed a layer of orthokeratinized, stratified, squamous epithelium. In the center of the excisional wound, a fibrous clot had formed, which appeared consistently thinner in wounds of neutropenic mice. As expected, wounds from neutropenic mice contained a much less exuberant infiltrate of neutrophils, typically observed below the epidermis, when compared with wounds from control mice (Fig. 3B) . The overwhelming majority of cells condensed in the blood clot above the epidermis appeared to be lymphocytes, which most likely became passively trapped in this location during hemostasis. It is interesting that the wound tissue obtained from neutropenic mice appeared much less disorganized compared with that of control mice. From this initial examination of overall histologic appearance, it appeared that the absence of neutrophils altered wound healing in a positive manner.

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Figure 3. Wound histology. (A) Kinetics of inflammatory cell infiltration. At 0.5, 1, 2, 3, 5, and 7 days after injury, wounds were harvested and were snap-frozen for the analysis of MPO as an indicator of neutrophil content, or wound sections were subjected to immunohistochemistry for the analysis of macrophage infiltration. The data are represented as percent of maximal cellular infiltration. The bars indicate the standard error of the mean. N. S., Not significant. (B) Histology of wound sections from control and neutrophil-depleted mice. Wounds of control (left panel) and neutropenic (right panel) mice were collected at day 1 post-wounding and embedded in paraffin. Sections (4 µm) were prepared and stained with H&E. Sections are shown at x50 original magnification. In each panel, the section is oriented with the epidermal side to the top of the figure. In the left panel, a prominent neutrophilic infiltrate is seen in the wound from the control mouse. The inset depicts an area below the regenerating epidermis x1000 original magnification, and an arrow (left panel insert) points to infiltrating neutrophils. In contrast, wounds of neutropenic mice (right panel) show very little inflammation. The inset, at 1000x original magnification, again depicts the subepidermal region. The arrowhead (right panel) points to a keratinocyte within the regenerating epidermis that is migrating toward the middle of the wound. As shown, relatively few inflammatory cells are seen in the subepidermal region of the wounds from neutropenic mice.

Re-epithelialization
As early as day 1, all of the wound sections obtained from neutropenic mice displayed onset of re-epithelialization, as evident by keratinocytes that had migrated over the wound bed (Fig. 3B) . In some cases, the epithelium had progressed over one-third of the width of the wound bed in wounds of neutropenic mice. In contrast, no evidence of the initiation of re-epithelialization was present in any day-1 post-injury wound sections of control mice. This histologic finding suggested that re-epithelialization might be improved in wounds of neutrophil-depleted mice. To explore this possibility, we quantified and compared wound re-epithelialization for each of two wounds from each of six mice per group (neutropenic and control) and at each time point (days 1, 2, 3, and 5 post-injury). Comparing re-epithelialization of wounds from neutropenic and control mice, we found that wounds of neutropenic mice were significantly further re-epithelialized on days 2 and 3 post-wounding compared with those of control mice (day 2: 77.7±14.2% vs. 41.2±0.9%, P<0.02, n=6; day 3: 91.7±8.3% vs. 56.0±10.6%, P<0.04, n=6; Fig. 4 ). These data suggest that neutrophils retard wound closure during normal healing.

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Figure 4. Effect of neutrophil depletion on wound re-epithelialization. BALB/c mice were injected with rabbit anti-mouse neutrophil (solid bars) or control serum (open bars) and were wounded with a 3-mm dermal biopsy punch. After 1, 2, 3, and 5 days, the wounds were harvested and sectioned. Wound sections were stained with H&E, and re-epithelialization was quantified as described in Materials and Methods. The bars indicate the means of percent re-epithelialization. The lines above the bars indicate the SEM. *, Not significant; **, P < 0.02; ***, P< 0.05.

Collagen content
Cutaneous wound healing includes not only the healing of the epidermal layer, in which keratinocytes regenerate the barrier function of the skin, but also healing of the dermal layer, mediated by fibroblasts and additional cells infiltrating the wound bed. Dermal healing leads to restoration of tensile strength of the tissue as a result of the deposition of structural proteins such as collagen. Clearly, both of these healing events can occur concomitantly, but whether epidermal and dermal healing is intimately linked is not known. Thus, to more fully understand the role of neutrophils in wound healing, we examined the effects of neutropenia on healing of the dermal layer.

To determine if neutrophil depletion affects the collagen content present in the wound, we compared the amount of collagen in wounds of neutropenic and control mice. The hydroxyproline content, indicative of collagen presence, was not significantly different between neutrophil-depleted and control mice (Fig. 5 ). These data suggest that neutrophils do not affect the collagen content in the wound bed.

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Figure 5. Assessment of collagen content in wounds of neutrophil-depleted and control mice. The collagen content in day 3 and day 5 wounds of control (open bars) and rabbit anti-mouse neutrophil serum-injected mice (solid bars) was determined by measuring the hydroxyproline content. The bars indicate the means of collagen content in micrograms per wound. The lines above the bars indicate the SEM.

Wound disruption strength
Wound closure is one of the most critical events during the healing process, as it restores the skin’s barrier function. However, the ultimate success of healing is also dictated by the ability of the previously injured skin to withstand tensile forces. If sufficient tissue strength cannot be restored, the wound may dehisce upon challenge with tension.

To determine whether neutrophils also influence the regeneration of tissue strength, we studied the effects of neutropenia on the development of wound disruption strength in an incisional wound model. In this model of a clean incision, approximated by sterile surgical clips, the least amount of epithelial damage occurs. This wound closely resembles a surgical incision and allows for quantification of dermal healing. Measurements of wound disruption strength provide highly quantifiable estimates of the efficacy of subepidermal repair.

Quantifying wound disruption strength, we found no significant difference between the neutropenic and control groups (day 7: 100.3±5.9 g compared with 90.9±10.6 g, P>0.4, n=12; day 14: 281.1±14.6 g compared with 270.6±15.7 g, P>0.6, n=12; Fig. 6 ). These results suggest that neutrophils do not affect the regeneration of tissue strength during normal wound healing.

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Figure 6. Effect of neutrophil depletion on wound disruption strength (wds) in grams (gr). Incisions through the panniculus carnosus (2 cm) were prepared on the back of control (open bars) and neutrophil-depleted (solid bars) mice. Wounds were excised at days 7 and 14, and two strips orthogonal to the incision were excised. Both strips were subjected to tensiometric analysis, and the average of both values for each mouse was determined. The bars indicate the means of wound disruption strength in grams. The lines above the bars indicate the SEM. P > 0.05 (days 7 and 14).

Macrophage infiltration into the wound space
Macrophages are thought to play an integral role for a successful outcome of wound healing through the generation of growth factors that promote cell proliferation and protein synthesis, as well as components of the extracellular matrix (ECM) [^{11 12 13 14} ]. To determine if an increase in macrophage infiltration might account for the acceleration of re-epithelialization observed in neutropenic mice, we quantified the number of macrophages in wound sections of neutropenic and control mice. Day 3 was chosen as time of analysis, as macrophage infiltration is known to peak at day 3 in this model [¹⁵ ], and re-epithelialization was significantly different between neutropenic and control mice at day 3. The number of macrophages infiltrating into the wound bed was not significantly different between the neutropenic and control group of mice (45.5±10.7 vs. 25.3±4.2) when analyzed by unpaired two-tailed t-test (P=0.13; Fig. 7 ). This result suggests that accelerated re-epithelialization observed in neutropenic mice does not involve an alteration in macrophage infiltration.

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Figure 7. Effect of neutrophil depletion on macrophage infiltration into the wound bed. Day 3 wounds of neutrophil-depleted (solid bar) and control (open bar) mice were sectioned (10 µm) and stained with a monoclonal anti-MOMA antibody to detect macrophages. The number of MOMA-positive cells was counted in five HPF per slide. The bars indicate the mean macrophage number in five HPF. The lines above the bars represent the SEM. P > 0.05.

Re-epithelialization of wounds from diabetic mice
Numerous wounds that heal poorly or not at all display a prolonged infiltration of neutrophils. Wounds of diabetic individuals as well as diabetic mice display such a poor healing phenotype. It has been suggested that the prolonged inflammatory phase may retard wound closure in diabetic mice [³ ,¹⁶ ]. If this hypothesis were true, then depletion of neutrophil should alleviate the healing impairment. To test this prediction, we examined wound re-epithelialization in diabetic mice that had been injected with rabbit anti-mouse neutrophil or control serum. We found that wound re-epithelialization of neutropenic, diabetic mice was significantly accelerated. In fact, by day 5, re-epithelialization was completed in neutropenic, diabetic mice, and the epithelium of control mice covered only 50% of the wound width (P<0.0001, n=6; Fig. 8 ). The alleviation of the healing impairment after neutrophil depletion of diabetic mice suggests that the prolonged persistence of neutrophils participates in the healing retardation seen in diabetic mice.

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Figure 8. Effects of neutrophil depletion on wound re-epithelialization of diabetic mice. Diabetic C57BLKS/J-m +/+Leprdb mice were injected with a rabbit anti-mouse neutrophil (solid bar) or control (open bar) serum and wounded with a 3-mm dermal biopsy punch. After 5 days, the wounds were harvested and sectioned (10 µm). Wound sections were stained with H&E, and re-epithelialization was quantified as described in Materials and Methods. The bars indicate the means of percent re-epithelialization. The lines above the bars represent the SEM. P< 0.0001.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Wound healing is a complex biological process that integrates the function of a variety of cell types. The manner by which a distinct cell population modulates the function of another cell population has been of great interest. Specifically, understanding how cells of the immune system influence nonimmune cell function is critical, as numerous reports link excess infiltration of inflammatory cells to chronic healing. Although normal healing is characterized by a transient inflammatory response [¹ ], chronic wounds often contain a nonresolving inflammatory response [³ ,¹⁶ ,¹⁷ ]. In many cases, this may be directly or indirectly the result of an imbalance of bioactive substances. For instance, Wetzler et al. [¹⁶ ] demonstrated a sustained induction of chemokines during murine diabetic healing, which results in a prolonged persistence of neutrophils at the wound site.

Despite growing interest in how inflammatory cells contribute to the pathology of nonhealing wounds and immune-mediated tissue damage, little is known about how neutrophils affect normal healing. In the present study, we used a neutropenic mouse model of normal wound healing to determine if neutrophils affect the repair process. We found that 1) wounds of neutrophil-depleted mice exhibited significantly accelerated re-epithelialization, 2) neutrophil depletion had no effect on wound disruption strength or collagen content, 3) neutrophil depletion did not influence the number of macrophages at the wound site, and 4) the healing impairment of re-epithelialization displayed in diabetic mice was alleviated by neutrophil depletion. These results suggest that in an uninfected wound, neutrophils retard wound closure by inhibiting re-epithelialization but not the overall dermal repair. Additionally, in case of poorly healing wounds with excess neutrophils at the wound site, neutrophils may contribute significantly to the impaired healing phenotype.

Apart from their role to control infection, neutrophils have previously been regarded as a cell type with minor influence on healing of uninfected wounds. This concept is largely derived from a study conducted by Simpson and Ross in 1972 [¹⁸ ], which investigated the effects of neutrophil depletion on wound healing in a guinea pig model. In this study, no discernable differences in fibrin content or distribution were observed in wounds of neutropenic and control guinea pigs. Further, no differences in percent wound volume occupied by macrophages or in macrophage phagocytosis were seen. Using a similar approach of neutrophil depletion to study the effects of neutropenia on wound healing in a murine model, we obtained results consistent with but also extending the study conducted by Simpson and Ross [¹⁸ ]. In the murine study described herein, we also did not find significant differences in collagen deposition, wound disruption strength, or macrophage infiltration in wounds of neutropenic mice. Apart from these parameters of healing that take place in the dermal layer of the skin, our study complements the study conducted by Simpson and Ross [¹⁸ ] by investigating epidermal healing in the absence of neutrophils. The fact that re-epithelialization, but not any of the processes in the dermis, was accelerated by neutrophil depletion strongly suggests a direct effect of neutrophil presence on keratinocyte function. The nature of a possible neutrophil-keratinocyte interaction remains to be characterized. Our results also suggest that epidermal and dermal healing are not necessarily linked, as epithelialization, but not the regeneration of tensile strength, was accelerated in the absence of neutrophils.

In the current study, we chose a murine model of cutaneous healing in which components of the repair process, in particular, infiltration of inflammatory cells, have been extensively studied. Although our data suggest an overall negative influence of neutrophils in normal wound healing, previous study of neutrophil function supports both a positive [¹⁹ ] and a negative [²⁰ ] role for neutrophils in normal tissue repair. Neutrophils produce a variety of growth factors that could promote repair of injured tissue, among them interleukin-8 [²¹ ] and vascular endothelial growth factor [²² ]. On the contrary, neutrophils produce many bioactive substances that can oppose the repair process by inducing further tissue damage. For instance, within the wound, neutrophils secrete a battery of proteases that can convert wound cytokines to an active or inactive form and can induce substantial tissue damage. For example, the proteases neutrophil elastase and PR-3 are capable of cleaving elastin and a variety of ECM proteins, including fibronectin, laminin, vitronectin, and collagen IV. Apart from serving as a supporting scaffold, the ECM influences cellular function such as migration and proliferation. Briggaman et al. [²³ ] demonstrated that incubation of skin with neutrophil elastase leads to separation of the dermal and epidermal layers. It is interesting that the addition of neutrophils to a monolayer of keratinocytes leads to a protease-dependent keratinocyte detachment [²⁴ ].

An important caveat to our experimental approach is the use of a polyclonal rabbit anti-mouse neutrophil serum as a depleting agent. This antiserum appears to exhibit low reactivity to monocytes, which could lead to a decrease in monocyte numbers. However, we did not observe a difference in the numbers of macrophages at the wound site (Fig. 7) . In fact, although not statistically significant, there appears to be a trend to more macrophages infiltrating the wound bed of neutrophil-depleted mice when compared with control mice. As macrophages play a critical role in wound healing, additional macrophages would be expected to accelerate wound healing [¹¹ ]. One interesting possibility is that an enhancement in macrophage function, favorable to healing, in tandem with the depletion of neutrophils, accounts for the accelerated re-epithelialization observed in neutrophil-depleted mice.

Taken together, these reports in the literature and our own findings lead to speculate that neutrophils at the wound site directly inhibit keratinocyte migration and possibly proliferation. In wounds, neutrophils localize to the subepidermal layer and are, hence, in close contact with keratinocytes. Thus, neutrophil-derived proteases could degrade ECM, fibrin clot components, and other proteins at the site of keratinocyte migration and proliferation. This proteolysis could lead to keratinocyte detachment. As keratinocytes absolutely require contact to the ECM components for migration and proliferation [²⁵ ], a now, at least partly, unattached keratinocyte would not be able to mediate re-epithelialization until the cell regained attachment. The above-described observation that keratinocytes detach in the presence of neutrophils in vitro supports this possibility.

It is possible that the extent of degradation of ECM components by neutrophil-derived proteases at the site of injury may not actually lead to keratinocyte detachment but may still impede keratinocyte migration. Rather, a limited amount of proteolysis of provisional matrix components in the vicinity of keratinocytes could stall re-epithelialization, as keratinocytes lose their directionality (Fig. 9 ). Pilcher et al. [²⁶ ] proposed a model in which interaction and then cleavage of collagen I is used by keratinocytes to determine their direction during re-epithelialization. The proteolysis of type I collagen, mediated by keratinocyte-derived collagenase-1, may serve as a "molecular compass" for migrating keratinocytes to move forward. Based on Pilcher’s model of keratinocyte migration [²⁶ ], a degradation of provisional matrix components by neutrophil-derived proteases could stall re-epithelialization even if keratinocytes are not detached.

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Figure 9. Model of keratinocyte migration in the presence or absence of neutrophils. (A) Shortly after wounding, a massive infiltration of neutrophils into the wound bed occurs. These inflammatory cells secrete a battery of proteases such as MMP8 (purple spheres), which cleaves type I collagen. As a result, high-affinity interactions between the keratinocytes and the cleaved collagen will not form, and thus, keratinocyte migration could become less efficient. (B) In the absence of neutrophils, however, keratinocytes can use high-affinity interactions with native type I collagen to control the direction of their migration during re-epithelialization. Initially, keratinocytes extend and form new contacts with native type I collagen (magnification). The production and activity of collagenase-1 by keratinocytes (green spheres) allow these cells then to break away from those interactions that tether them and are incompatible with migration. This process of interacting with and then cleaving type I collagen enables the migrating keratinocytes to determine and maintain their directionality during the process of re-epithelialization.

If neutrophils inhibit the vital process of restoration of the skin’s barrier function, then why does the inherently negative, massive neutrophil infiltration at the wound site occur? One teleologic explanation for prominent neutrophil infiltration into wounds, even into those inflicted under sterile surgical conditions, is their protective function from microbial infection. The very characteristic of the innate-immune system is to react to an insult in an antigen-independent manner. The first line of defense may be critical in recovering from a wound inflicted by the tooth of a wild animal but may, conversely, be unnecessary and even troublesome in recovering from a wound inflicted by a sterile surgical instrument.

In summary, we have shown that neutropenia, induced by injection of a rabbit anti-mouse neutrophil serum, accelerates the rate of wound epithelial closure without altering the overall quality of the dermal healing process. This is also true for wounds of diabetic mice, in which excess inflammation appears to participate in healing impairment. Thus, although the inflammatory phase is a vital response to tissue injury, a subset of inflammatory cells may in fact retard wound closure. Further detailed studies on the mechanism of neutrophil-mediated delay in healing will be necessary. Ultimately, re-evaluating the necessity of complete neutrophil function in a setting of sterile surgical wound healing may be indicated.

 
We thank Dr. K. L. Knight for providing the rabbit preimmune serum, Dr. C. Brown for providing the RB6-8C5 clone, Mrs. M. K. Olsen for help with paraffin sections, Mrs. P. Simms for help with the FACS analysis, Dr. M. W. Lingen for help with histologic analysis, and Mr. A. J. Dovi for help with graphic illustrations.

Received August 21, 2002; accepted December 5, 2002.

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