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Ann Surg. Jan 2002; 235(1): 113–124.
PMCID: PMC1422403

Interleukin-1α and Interleukin-6 Enhance the Antibacterial Properties of Cultured Composite Keratinocyte Grafts

Abstract

Objective

To determine whether the antibacterial properties of cultured composite keratinocyte grafts can be enhanced by cytokines that stimulate the innate immune response.

Summary Background Data

Use of composite grafts of cultured keratinocytes has been limited because of their susceptibility to burn wound microorganisms as a result of their lack of a vasculature and immune cells when transplanted. Moreover, use of topical antimicrobial agents is limited with these composite grafts because of cytotoxic effects. Keratinocytes, like all epithelial cells in the body, maintain a natural defense mechanism called the innate immune system. Some components of this system can be induced by cytokines.

Methods

The innate immune response of cultured composite keratinocyte grafts treated with various cytokines was assessed indirectly by measuring the levels of mRNA encoding antimicrobial peptides (human beta defensin-1 and -2, LL-37, and antileukoprotease) and antimicrobial proteins (lysozyme, bactericidal/permeability-inducing protein, and phospholipase A2) by reverse transcription–polymerase chain reaction and directly by measuring the ability of keratinocytes to inhibit the growth of added bacteria (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus).

Results

Treatment with interluekin-1α increased mRNA levels of antimicrobial peptides in keratinocytes on plastic dishes and in composite grafts. Interleukin-6 increased mRNA levels of antimicrobial proteins in composite grafts only. When added to composite grafts, both cytokines increased antibacterial activity against E. coli, P. aeruginosa, and S. aureus. Moreover, interleukin-1α and interleukin-6 did not impair the formation of a differentiated epidermis in vitro or after transplantation of the composite grafts.

Conclusions

Treatment with interleukin-1α or interleukin-6 of cultured composite keratinocyte grafts stimulates the innate immune response of keratinocytes, enhances the antibacterial properties of these grafts, and may better prepare them to combat infections in contaminated burn wounds.

Tissue-engineered skin substitutes are a promising alternative for the permanent closure of burn wounds. 1,2 Methods are well established for the culture of large numbers of human epidermal keratinocytes as well as dermal fibroblasts, and several approaches have been developed to combine these cells with different analogs of the dermis. 3–5 Many of these cultured skin grafts have been evaluated in the clinic with some success. 6–10 Although a promising approach, none perform as well as a split-thickness skin graft. Graft failure resulting from infection is a problem and serious limitation and is related to the fact that at the time of grafting, cultured skin grafts do not contain immune cells, nor are they vascularized. Moreover, it has been shown that cultured skin grafts are more sensitive than split-thickness skin grafts to many topically applied antimicrobial agents. 11–14

The innate immune system, which is distinct from the adaptive or acquired immune response, provides a first line of host defense against a broad spectrum of microorganisms. 15–17 In contrast to acquired immunity, the innate immune system does not require recognition of specific antigens and can mount a more immediate response to pathogens. Components of the innate immune system are varied and include cells such as macrophages and neutrophils as well as antimicrobial peptides and proteins produced by various epithelia of the body. 16–19

Keratinocytes of the epidermis have been shown to produce a variety of peptides and proteins with antimicrobial activity. Proteins with antibacterial activities include phospholipase A2 and lysozyme, 20,21 and antimicrobial peptides include human beta defensin-1 (HBD-1), human beta defensin-2 (HBD-2), a member of the cathelicidin family (LL-37), and antileukoprotease. 22–26 Expression of some of these antimicrobial peptides and proteins is constitutive, whereas others are induced during inflammation or by specific cytokines. 22–26

Cultured skin grafts are produced ex vivo under aseptic conditions; however, soon after transplantation, they encounter a wide spectrum of microorganisms found in the typical burn wound site. To determine whether cultured skin grafts could be better prepared for this abrupt transition, we investigated whether cytokines could be used to stimulate the innate immune response of cultured composite keratinocyte grafts before transplantation.

METHODS

Culture of Human Keratinocytes

Human keratinocytes were isolated from neonatal foreskins following the method of Rheinwald and Green. 27 Keratinocytes were cocultured with 3T3-J2 mouse fibroblasts (originally provided by H. Green, Harvard Medical School, Boston, MA) that had been pretreated with 15 μg/mL mitomycin C (Boehringer Mannheim, Indianapolis, IN). Culture medium was changed every 3 days with a 3:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) (Life Technologies, Gaithersburg, MD) and Ham’s F12 medium (Life Technologies), supplemented with 10% fetal bovine serum (JRH Bioscience, Lenexa, KS); adenine, 1.8 × 10−4 (Sigma Chemical, St. Louis, MO); cholera toxin, 10−10 mol/L (Vibrio cholera, type Inaba 569B, Calbiochem, La Jolla, CA); hydrocortisone, 0.4 μg/mL (Calbiochem); insulin, 5μg/mL (Novo Nordisk, Princeton, NJ); triiodo-l-thronine, 2 × 10−9 mol/L (Sigma) and penicillin–streptomycin, 100 IU/mL:100 μg/mL (Boehringer Mannheim). With the first medium change, 100 ng/mL mouse epidermal growth factor (EGF) (Collaborative Biomedical Products, Bedford, MA) was added. Cells were subcultured by first removing the fibroblast feeder layer with 5 mmol/L ethylenediaminetetraacetic acid (EDTA) and treating the keratinocytes with trypsin-EDTA. 3T3 fibroblasts were routinely passaged in DMEM (high glucose) supplemented with 10% bovine calf serum (Hyclone, Logan, UT) and penicillin–streptomycin (100 IU/mL:100 μg/mL) and incubated at 37°C with 10% CO2.

Keratinocytes were passed onto 60-mm dishes (1 × 106 cells/dish) for reverse transcription–polymerase chain reaction (RT-PCR) or onto 96-well plates (1 × 104 cells/well) for antibacterial assays and cultured in serum-free keratinocyte growth medium (KGM) (Clonetics, San Diego, CA) supplemented with 0.5 μg/mL hydrocortisone, 0.4% bovine pituitary extract, 0.1 ng/mL EGF, and 50 μg/mL:50 ng/mL gentamicin–amphotericin B (Clonetics). Gentamicin–amphotericin B was excluded from the medium for antibacterial assays.

Preparation of Composite Keratinocyte Grafts

Human cadaver skin, cryopreserved and negative for cytomegalovirus, human immunodeficiency virus, and hepatitis B virus, was obtained from Shriners Burns Hospital Skin Bank. Acellular dermis was prepared by exposing the skin to three rapid freeze–thaw cycles in liquid nitrogen to devitalize the cells and incubating at 37°C for 1 week in sterile phosphate-buffered saline with 100 μg/mL gentamicin, 10 μg/mL ciprofloxacin, 2.5 μg/mL amphotericin B, and 100 IU/mL:100 μg/mL penicillin–streptomycin. After 1 week, the epidermis was separated from the dermis and the dermis was maintained in antibiotic cocktail at 4°C for an additional 4 weeks.

Keratinocytes were seeded onto the papillary side of acellular dermis using methods similar to those previously described, 28 with slight modifications of media as described by Ponec et al. 29 The acellular dermis was cut into 1-cm2 pieces, and each piece was placed on a 35-mm tissue culture dish, papillary side up. Second-passage cultured keratinocytes were seeded onto each piece of dermis (2.5 × 102 cells/piece) in a seeding medium composed of DMEM/F12 (3:1), fetal bovine serum 1%, cholera toxin 10−10 mol/L, hydrocortisone 200 ng/mL, insulin 5 μg/mL, ascorbic acid 50 μg/mL (Sigma), and penicillin–streptomycin 100 IU/mL:100 μg/mL. The next day, culture medium was changed to priming medium, which was the same as seeding medium but supplemented with bovine serum albumin 24 μmol/L (Sigma), fatty acid cocktail (oleic acid 25 μmol/L, linoleic acid 15 μmol/L, arachidonic acid 7 μmol/L, palmitic acid 25 μmol/L) (Sigma), l-carnitine 10 μmol/L (Sigma), and l-serine 1 mmol/L (Sigma). Grafts were maintained in this medium submerged for an additional 2 days. At day 4, the grafts were placed on a stainless-steel mesh in a 35-mm dish and were raised to the air–liquid interface for 7 days. The air–liquid interface medium was composed of serum-free priming medium supplemented with 1 ng/mL EGF (Collaborative Biomedical Products). The medium was changed every 2 days. When composite grafts were used for antibacterial assays, penicillin–streptomycin was not added to the medium.

Treatment of Keratinocytes in Culture and Composite Grafts

Confluent keratinocyte cultures in 60-mm tissue culture dishes for RT-PCR or in 96-well plates for antibacterial assay were treated with 100 ng/mL of the following agents: lipopolysaccharide (from Escherichia coli serotype 055:B5; Sigma), tumor necrosis factor alpha (TNF-α), interleukin-1 alpha (IL-α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), or interferon gamma (IFN-γ) (human recombinant; Sigma) for 24 hours. Experiments done with low (50 ng/mL) or high (200 ng/mL) doses of cytokines gave similar results in the RT-PCR and antibacterial assays. Composite grafts in 35-mm dishes were also exposed to the same agents on the sixth day of air–liquid interface for 24 hours.

RNA Isolation and RT-PCR

Total RNA was extracted from keratinocytes cultured in 60-mm tissue culture dishes and from the composite grafts using the Clontech nucleospin kit (Clontech, Palo Alto, CA), according to the manufacturer’s instructions. cDNA synthesis and PCR amplification were made with a GeneAmp RT-PCR kit (Perkin Elmer, Branchburg, NJ). The primers for the HBD-1, HBD-2, LL-37, antileukoprotease, lysozyme, bactericidal/permeability-inducing protein (BPI), and phospholipase A2 were based on published sequences (Table 1). 20–25,30,31 Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (Clontech) was used as control amplimer set in PCR reactions.

Table thumbnail
Table 1. PRIMERS USED FOR RT-PCR

For each mRNA, RT-PCR was performed using a Perkin Elmer DNA Thermal Cycler 480 with the thermal cycle profiles shown in Table 1. Analysis of PCR reactions was done in 1.5% agarose gel and visualized by ethidium bromide staining. The results were quantified by scanning the light intensities of each band using a Fluor-S Imager coupled to Multi-Analyst/PC Software (Bio-Rad Laboratories, Hercules, CA). Levels of mRNA were calculated relative to G3PDH mRNA levels and the value of peptide/G3PDH expression in untreated controls was set as 1.

Antibacterial Assays

E. coli (ATCC 43827), Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 29213) were used for antibacterial assays. Bacteria were maintained on Luria Berlani agar (E. coli), Tripticase Soy Agar (S. aureus), and Nutrient Agar (P. aeruginosa) (Sigma) plates, and organisms from a single colony were inoculated in 10 mL related broth (Sigma) and cultured overnight on a platform shaker at 300 rpm at 37°C. Standard curves for each type of bacteria were created by plating various dilutions on agar plates for counting colonies and measuring the optical densities of these dilutions at 650 nm. Bacterial concentrations were calculated by using the relationships C = (6.82 × 108 cfu/mL) × A650 for E. coli, C = (6.26 × 108 cfu/mL) × A650 for S. aureus, and C = (4.28 × 108 cfu/mL) × A650 for P. aeruginosa, where C is the bacterial concentration (cfu/mL) and A650 is the absorbance at 650 nm.

To test the antimicrobial properties of cultured cells, second-passage keratinocytes were cultured in 96-well tissue culture plates (2 × 104 cells/well) in serum-free KGM without antibiotics. When the cells reached confluence, they were treated with the stimulants stated above for 24 hours. Afterward, 10 μL of 10−1 or 10−5 serial dilutions (1.62 × 107 and 0.5 × 104 bacteria, respectively) of E. coli from an overnight culture were added to each well. Plates were maintained at 37°C on a plate shaker at 300 rpm and optical densities at 650 nm were determined every 2 hours using a Thermomax microplate reader (Molecular Devices, Menlo Park, CA).

To test the antimicrobial properties of composite grafts, a bacterial inoculation model was created. The surface of the grafts was exposed for 5 seconds to a 2-mm-diameter metal probe held at 140°C to destroy the epidermal barrier. Into this well-like area in the center of the graft, 3.2 × 104E. coli, 6.5 × 104P. aeruginosa, or 4.6 × 104S. aureus in 2 μL phosphate-buffered saline was inoculated. The grafts were then maintained at 37°C and 10% CO2 in an incubator for 24 hours. At the end of the incubation, the grafts were removed from the steel meshes and placed in plastic tubes containing 0.5 mL sterile phosphate-buffered saline. The grafts were homogenized with a tissue homogenizer (Fisher Scientific, Pittsburgh, PA) for 2 minutes. The homogenizer was rinsed with alcohol and phosphate-buffered saline between each homogenization. Serial dilutions from homogenates were made and 100 μL of each dilution was plated onto appropriate agar plates. After 24 hours of incubation at 37°C, the number of colony-forming units was determined.

Transplantation of Composite Keratinocyte Grafts

Five- to eight-week-old male NIH Swiss nu/nu mice, weighing 25 to 30 g (Massachusetts General Hospital), were anesthetized with an intraperitoneal injection of 2,2,2,-tribromoethanol (0.58 mg/g) (Aldrich, Milwaukee, WI). A 1-cm2 full-thickness defect was created on the right subscapular area of each mouse and composite grafts were placed onto the wounds without stitches. Grafts were closed with Telfa no-stick gauze (Kendall, Mansfield, MA), Tegaderm polyurethane occlusive dressing (3 mol/L, St. Paul, MN), and 3 mol/L Sports Band-Aid, respectively. Telfa dressing and the Band-Aid were sutured to the surrounding mouse skin to protect the grafts from displacement. A 1-inch-wide elastic adhesive tape (Johnson & Johnson, Skillman, NJ) was wrapped around the mouse to cover the dressings. Grafts were harvested at 3, 7, and 14 days, three grafts for each group, photographed, and fixed in formalin for histologic analysis.

Histology

Composite grafts in vitro at day 7 of air–liquid interface and excisional biopsies from grafts transplanted onto mice at 3, 7, and 14 days were fixed overnight in 10% buffered formalin at room temperature. After washing, tissues were dehydrated in ethanol (70%, 80%, 90%, 95%, 100%), cleared in xylene, infiltrated, and embedded in paraffin. Sections (6 μm), cut on a rotary microtome, were mounted on slides and stained with Harris hematoxylin (1.5 minutes) and aqueous eosin (1.5 minutes) and mounted in Permount. Images were obtained using a Spot camera coupled to a Nikon Eclipse E800 microscope.

Data Collection and Statistical Analysis

SigmaPlot and SigmaStat (Jandel Scientific, San Rafael, CA) were used to calculate the mean ± standard error of the mean for each data point and for statistical analyses. Data collected from the antibacterial assay on cells on plastic were tested for significance with analysis of variance, and P < .01 was considered significant. For data from the antibacterial assay on composite grafts, Tukey’s test was used for pairwise comparisons and P < .01 was considered significant.

RESULTS

Levels of mRNA Encoding Antimicrobial Peptides Can Be Upregulated by Cytokine Treatment of Cultured Keratinocytes and Composite Keratinocyte Grafts

To determine whether the levels of mRNA encoding antimicrobial peptides could be upregulated, we isolated RNA from keratinocytes cultured on plastic that had been stimulated with various cytokines and assayed by semiquantitative RT-PCR for mRNAs encoding the following antimicrobial peptides: HBD-1, HBD-2, LL-37, and antileukoprotease. Control unstimulated keratinocytes expressed mRNAs for all four antimicrobial peptides (Fig. 1). HBD-1 and LL-37 mRNA levels were upregulated by lipopolysaccharide; HBD-2 levels were unaffected by lipopolysaccharide but were upregulated by IL-1α and TNF-α. Levels of antileukoprotease mRNA were constitutive and unchanged by any of the cytokines tested.

figure 15FF1
Figure 1. Cytokines increase mRNA levels of antimicrobial peptides in cultured keratinocytes. Reverse transcriptase–polymerase chain reaction was used to determine mRNA levels of human beta defensin-1 and -2, LL-37, and antileukoprotease in cultured ...

Previously, we and others have shown that keratinocytes seeded on acellular dermis and cultured at the air–liquid interface form a stratified and differentiated epidermis in vitro. 4,32 To determine whether mRNA levels of antimicrobial peptides could also be upregulated in cultured composite keratinocyte grafts, we used the RT-PCR assay to monitor the effects of the same cytokines. Like cells on plastic, HBD-1, HBD-2, and antileukoprotease mRNAs were present in nonstimulated control grafts; however, there was little if any evidence of LL-37 mRNA (Fig. 2). IL-1α increased HBD-1 and HBD-2 mRNA levels and was the only factor to induce LL-37. TNF-α and IL-6 had minor effects on HBD-2, as did IFN-γ on HBD-1 expression. IL-4 and IL-10 appeared to downregulate HBD-2 mRNA levels, as did IL-8 on HBD-1 expression. Similar to the results with cultured cells, antileukoprotease mRNA was also expressed in control composite grafts, and the levels were unchanged by any of the agents tested.

figure 15FF2
Figure 2. Cytokines increase mRNA levels of antimicrobial peptides in cultured composite grafts. Reverse transcriptase–polymerase chain reaction was used to determine mRNA levels of human beta defensin-1 and -2, LL-37, and antileukoprotease in ...

Antibacterial Properties of Cultured Keratinocytes and Cultured Composite Keratinocyte Grafts Can Be Enhanced

To determine whether cytokine stimulation of cultured keratinocytes could increase their antimicrobial properties, we tested their ability to inhibit the growth of E. coli. Keratinocytes cultured in 96-well plates were treated with various cytokines, and then each well was inoculated with either a high dose or low dose of E. coli. As an additional control, bacteria were also inoculated into growth medium without keratinocytes. Figure 3 shows the growth curves of the high and the low doses of E. coli. For both high and low doses, growth of bacteria was greatest in growth medium without keratinocytes. The presence of untreated keratinocytes depressed the late growth phase of the high dose of bacteria as well as the initial and late growth phases of the low dose of bacteria. This inhibition of bacterial growth was enhanced if the keratinocytes were pretreated with lipopolysaccharide, TNF-α, IL-1α, or IFN-γ. Pretreatment with these agents inhibited the initial and late phases of growth for both doses of bacteria. This effect was statistically significant for all cytokines in the low dose of bacteria and for IL-1α and IFN-γ in the high dose of bacteria. IL-4, IL-6, IL-8, and IL-10 had no effect (data not shown).

figure 15FF3
Figure 3. Cytokines increase the antibacterial activity of cultured keratinocytes. Cultured keratinocytes were inoculated with a high dose (1.62 × 107 bacteria/mL) (top level) or a low dose (0.5 × 104 bacteria/mL) (bottom level) of Escherichia ...

To determine whether cytokine treatment could also augment the antibacterial properties of cultured composite keratinocyte grafts, we inoculated cytokine-stimulated grafts with 3.2 × 104E. coli. After 24 hours of incubation at 37°C, control grafts had an average of 8.7 × 108 bacteria/mL of extract (Fig. 4). When pretreated with IL-1α, this number decreased dramatically to 3.5 × 106 (P < .01). IFN-γ, IL-6, TNF-α, and lipopolysaccharide were also able to stimulate the antibacterial response and decreased bacteria number to 6.3 × 106, 6.6 × 106, 2.2 × 107, and 5.1 × 107, respectively (P < .01).

figure 15FF4
Figure 4. Cytokines increase the antibacterial activity of cultured composite keratinocyte grafts. Composite grafts were inoculated with 3.2 × 104Escherichia coli and the number of viable bacteria (colony-forming units [cfu]) was ...

Interleukin-6 Increases mRNA Levels of Other Antimicrobial Proteins in Cultured Composite Keratinocyte Grafts

Interleukin-1α and IL-6 both stimulate the antimicrobial response, but IL-6 had little effect on mRNA levels of the antimicrobial peptides tested. To determine whether IL-6 or IL-1α could be stimulating the expression of other antimicrobial genes in cultured composite grafts, we used RT-PCR to measure the mRNA levels of BPI, lysozyme, and phospholipase A2 (Fig. 5). Control, untreated grafts expressed mRNAs for all three proteins and IL-6 upregulated mRNA levels of lysozyme and phospholipase A2, but not BPI. Levels were unchanged by IL-1α treatment.

figure 15FF5
Figure 5. Interleukin-6 increases mRNA levels of antimicrobial proteins in composite keratinocyte grafts. Reverse transcriptase–polymerase chain reaction was used to determine the mRNA levels of bactericidal-/permeability-inducing protein, lysozyme, ...

Interleukin-1α and Interleukin-6 Stimulate the Antibacterial Response to S. aureus and P. aeruginosa

To determine whether the IL-1α- or IL-6-mediated stimulation of the antibacterial response could be used to protect against more common burn wound pathogens, we tested S. aureus and P. aeruginosa in our infection model. Composite grafts were pretreated with IL-1α or IL-6 for 24 hours and then inoculated with either 4.6 × 104S. aureus or 6.5 × 104P. aeruginosa (Fig. 6). IL-1α and IL-6 pretreatment significantly inhibited the growth of S. aureus and P. aeruginosa (P < .01).

figure 15FF6
Figure 6. Composite keratinocyte grafts stimulated with interleukin-1α or interleukin-6 show antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Composite grafts treated with interleukin-1α or interleukin-6 ...

Cytokine Treatment Influences Formation of the Epidermis

In addition to their effects on the antimicrobial response, many of the cytokines tested could also influence keratinocyte growth and differentiation. To determine the effect of cytokine treatment on the formation of the epidermis in vitro, control and cytokine-treated grafts were fixed in formalin at days 7 and 10 for histology (Fig. 7). Untreated grafts formed a stratified and differentiated epidermis complete with basal, spinous, granular, and cornified layers. In contrast, the epidermis of the grafts treated with lipopolysaccharide, TNF-α, and IFN-γ was significantly perturbed versus controls. In contrast to these agents, IL-1α- and IL-6-treated grafts formed a well-developed and differentiated epidermis comparable to controls.

figure 15FF7
Figure 7. Interleukin-1α and interleukin-6 have minimal effects on the formation of epidermis. Composite grafts of keratinocytes on acellular dermis were either untreated, control (a), or treated with lipopolysaccharide (b), tumor necrosis factor ...

Interleukin-1α or Interleukin-6 Pretreatment Does Not Impair Transplantation

To determine whether IL-1α or IL-6 pretreatment had any effect on the ability of these composite keratinocyte grafts to be transplanted, we tested them in athymic mice. Cultured composite grafts pretreated with IL-1α or IL-6 in vitro were transplanted to full-thickness wounds on the back of athymic mice. Grafts were harvested at 3, 7, and 14 days after transplantation. At each time point, graft take of control grafts was comparable to that of treated grafts, with a slight improvement in wound closure and epithelialization in IL-1α-treated grafts (data not shown). Histologic examination showed that 3 days after the transplantation, the acellular dermis was beginning to be repopulated with fibrovascular cells of the host, and this process increased at later time points (Fig. 8). At day 3, the inflammatory response found beneath the grafts of IL-1α- and IL-6-treated grafts was greater than in control grafts. This difference subsided by day 7. All grafts had a stratified, keratinized, and differentiated epidermis at day 3 and the later time points. As seen in vitro, the epidermis of the IL-1α-treated grafts was slightly thicker than control or IL-6-treated grafts at day 3, and this persisted to day 14.

figure 15FF8
Figure 8. Histology of composite keratinocyte grafts after transplantation. Control (a, b, c), interleukine-1α-treated (d, e, f), or interleukin-6-treated (g, h, i) composite grafts were transplanted to full-thickness excisional wounds on athymic ...

DISCUSSION

In this study, we investigated the potential use of cytokines to enhance the antimicrobial properties of cultured composite keratinocyte grafts. Composite grafts of cultured keratinocytes combined with analogs of the dermis are potential alternatives to split-thickness skin grafts, especially in extensively burned patients who have limited donor sites for grafting. However, clinical experience has shown that they are more susceptible to burn wound microorganisms and graft failure than split-thickness autografts. Cultured grafts are prepared under rigorously aseptic conditions and then transferred to the burn wound, which is potentially contaminated with a variety of microorganisms. We asked whether they could be better prepared to combat infections by prior treatment with cytokines that stimulate innate immunity. The innate immune response was assessed indirectly by measuring the levels of gene expression of several peptides/proteins with well-known antimicrobial activities and directly by measuring the ability of keratinocytes to inhibit the growth of added bacteria. Our results show that treatment with either IL-1α or IL-6 upregulates the levels of mRNA encoding antimicrobial peptides/proteins and significantly enhances the ability of cultured composite keratinocyte grafts to inhibit the growth of gram-negative and gram-positive bacteria. Moreover, IL-1α or IL-6 treatment did not impair the ability of these grafts to be transplanted.

The information provided by the complementary assays that we used to measure the innate immune response provided clear answers regarding the importance of IL-1α and IL-6 in the enhancement of the antimicrobial state. In addition, these assays revealed the complex nature of the innate immune response of epidermal keratinocytes. Using RT-PCR, it was obvious that of the cytokines tested, there were clearly some that were able to increase the levels of mRNA encoding antimicrobial peptides (lipopolysaccharide, TNF-α, IL-1α, and IFN-γ) and others that had little if any effect on their mRNA levels (IL-4, IL-8, IL-10). When further tested in two antibacterial assays (keratinocytes cultured on dishes or keratinocytes on acellular dermis), the cytokines that upregulated levels of mRNA encoding antimicrobial peptides also enhanced the antibacterial effect, whereas the cytokines that did not were unable to stimulate the antibacterial response. The only exception to this correlation was IL-6, which showed little if any upregulation of levels of mRNA encoding antimicrobial peptides but was able to significantly enhance the antibacterial response of a composite keratinocyte graft.

The four antimicrobial peptides tested showed distinct patterns of expression in response to cytokine treatment. In our experiments, mRNA levels of antileukoprotease remained constitutive regardless of cytokine treatment. This is consistent with a report that antileukoprotease expression by cultured keratinocytes was constitutive and unaffected by added TNF-α or IFN-γ. 25 However, another study showed that antileukoprotease expression was increased during inflammation and wound healing. 26 In contrast to antileukoprotease, mRNA levels of HBD-1, HBD-2, and LL-37 in our study fluctuated in response to certain cytokine treatments. HBD-2 expression by cultured keratinocytes has been reported to be upregulated by TNF-α as well as by contact with bacteria, 23 and expression of LL-37 was shown to be absent in normal skin but elevated in inflamed skin.

Levels of mRNA of the four antimicrobial peptides also showed differences in response to whether the keratinocytes were cultured as a typical monolayer or as part of a composite graft. When seeded on the surface of acellular dermis and cultured at the air–liquid interface, keratinocytes differentiate and form a stratified epidermis complete with basal, suprabasal, granular, and cornified layers. 4 Antileukoprotease mRNA levels remained constitutive regardless of whether the keratinocytes were cultured as a monolayer or as part of a composite graft. In contrast to antileukoprotease, there was a significant change in the mRNA levels of LL-37 by monolayer cells versus stratified/differentiated cells. As a monolayer, LL-37 mRNA levels were evident even in the absence of a stimulating cytokine, whereas when the cells were part of a composite keratinocyte graft, LL-37 baseline mRNA levels were significantly reduced but could be dramatically upregulated by treatment with IL-1α. Changes in expression could be explained by any number of differences between monolayer keratinocytes and stratified/differentiated keratinocytes, including alterations to gene expression as well as the presence of suprabasal and granular cells in the differentiated skin substitutes. Likewise, these differences could explain our observation that IL-6 was unable to stimulate an antibacterial response in monolayer cells but was able to stimulate the antibacterial response of composite grafts.

Although antimicrobial peptides are an important component of the innate immune response of keratinocytes and are capable of killing gram-positive and gram-negative bacteria, viruses, fungi, and protozoa, 19 they are not the only molecules that participate in the innate immune response. It is well known that other molecules participate in innate immune responses, such as inorganic molecules, fatty acids, carbohydrates, 16,19 and possibly other unidentified antimicrobial peptides and proteins. From our data, we cannot conclude which molecules are responsible for the enhanced antibacterial state of composite keratinocyte grafts after IL-1α or IL-6 treatment. The differences in levels of mRNA encoding antimicrobial peptides we observed suggest that IL-1α and IL-6 may stimulate the expression of different molecules, and it would be interesting to see whether combined cytokine stimulation could further increase the antimicrobial state.

Both IL-1α and IL-6 are expressed by keratinocytes. IL-1α is synthesized without a signal peptide; thus, most of it is retained inside the cell unless it is released by injury to the epidermis. 33 In contrast, IL-6 is synthesized with a signal peptide and is actively secreted in very small amounts, and levels of IL-6 are induced by IL-1, TNF-α, and IL-4. 34 IL-1α and IL-6 are known to be proinflammatory cytokines, and in addition to stimulating keratinocyte proliferation and migration, both stimulate keratinocytes to increase their synthesis of cytokines as well as chemokines. 34–38 These actions are thought to be a crucial link between innate and acquired immunity 39 and may also contribute to the ability of cytokine-treated grafts to combat infections by recruitment and stimulation of components of the acquired immune system.

For transplantation purposes, the effects of some cytokines we tested were unacceptable for further consideration even though they enhanced the antibacterial response. For example, TNF-α and IFN-γ both stimulated the antibacterial response of composite grafts; however, growth and differentiation of the in vitro epidermis were significantly perturbed. These cytokines also suppressed keratinocyte proliferation in culture (data not shown). Likewise, lipopolysaccharide was unacceptable because although it stimulated the antibacterial response of composite grafts, it has well-known toxic effects. IL-1α and IL-6 treatment did not impair the growth or differentiation of the in vitro epidermis and did not adversely affect the ability of treated composite grafts to be transplanted.

Cultured composite grafts of cultured keratinocytes on dermal analogs provide a ready supply of skin substitute for patients with massive skin loss. However, its use is limited because of poor graft take and susceptibility to infection. In this study, we showed that antimicrobial properties of cultured composite keratinocyte grafts could be enhanced with cytokine treatment by increasing their innate immunity. IL-1α and IL-6 are effective at enhancing the antimicrobial properties of cultured grafts and do not adversely effect epidermal formation.

Graft failure from infection is a significant limitation to the use of cultured skin grafts in the treatment of the burned patient. Use of parenteral antibiotics has a limited impact on this problem and there are significant limitations to the use of topical antibiotics, which may be toxic to cultured keratinocytes 11,12,14 and may promote the growth of antibiotic-resistant strains in the immune-compromised burn patient. In this study, we showed that IL-1α or IL-6 treatment of cultured skin grafts stimulates innate immunity, enhances the antibacterial properties of these grafts, and may improve their performance in contaminated burn wounds.

Acknowledgments

The authors thank Robert Crowther of the Shriners Morphology Core for his help with histology.

Footnotes

Supported in part by the NIH (HD-28528) and the Shriners Hospitals for Children.

Correspondence: Jeffrey R. Morgan, PhD, Shriners Hospital for Children, 51 Blossom St., Boston, MA, 02114.

E-mail: jmorgan@sbi.org

Accepted for publication April 18, 2001.

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