Thermal injury induces expression of CD14 in human skin

Burns 28 (2002) 223–230

Thermal injury induces expression of CD14 in human skin夽 Lars Steinstraesser a,c,∗ , William Alarcon a , Ming-Hui Fan a , Richard D. Klein a , Alireza Aminlari a , Cynthia Zuccaro a , Grace L. Su b , Stewart C. Wang a a

c

Department of Trauma Burn Surgery, University of Michigan, Ann Arbor, MI, USA b Department of Medicine, University of Michigan, Ann Arbor, MI, USA Department of Plastic and Reconstructive Surgery, Ruhr University Bergmannsheil, Germany Accepted 7 January 2002

Abstract Background: Skin is equipped with an array of immune mediators aimed at fighting invading microbes. CD14 has been shown to play a key role in modulating the activation of cells by LPS. Since LPS levels within burn wounds are often found to be elevated, we sought to examine the expression of CD14 within human skin following thermal injury. Methods: Patients who sustained partial thickness burns, were recruited into the study (n = 57). Total RNA was isolated from both burn and normal (control) skin. Northern blot analysis and TaqMan RT-PCR were used to determine skin CD14 mRNA levels. Immunohistochemistry was used to localize CD14 expression in burned and normal skin. Results: Quantitative PCR showed significantly increased CD14 expression levels in the immediate post-burn period (P < 0.05 burn versus non-burn). Immunohistochemistry revealed more pronounced CD14 staining 24 h after the injury, reaching normal levels approximately 5–7 days post-burn. Conclusion: CD14 expression peaks within the first week post-burn before declining, reaching normal levels after 14 days. This loss of supranormal CD14 expression locally within the wound may contribute to a weakened host defense response 5–6 days after injury, when patients become especially vulnerable to infection. © 2002 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Immunohistochemistry; CD14; Lipopolysaccharide

1. Introduction Survival rates after thermal injury have improved dramatically during the last 25 years, but infection still represents a serious risk to the burn patients due to septic complications and the emergence of antimicrobial drug-resistant microbes. Skin is the first line of defense against invading microbes. It is equipped with an array of immune mediators, capable of recruiting inflammatory cells to enable neutralization and clearance of bacteria and fungi from the site. In burns, however, most tissue damage results from direct heat coagulation of the proteins within the skin, im夽

This research is supported in part by General Motors in accordance with an agreement between GM and the Department of Transportation, as well as by National Institute of Health, Grants HL-03803, DK-02210, GM-54911. Lars Steinstraesser is a General Motors Burn Research Scholar. ∗ Corresponding author. Ruhr University Bochum, Department of Plastic and Reconstructive Surgery, Burn Center, Buerkle-de-la-Camp-Platz 1, 44789 Bochum, Germany. Tel.: +49-234-302-6841; fax: +49-234-302-6379. E-mail address: [email protected] (L. Steinstraesser).

pairing the local innate and adaptive immune system significantly and putting the host at risk of bacterial infection [1]. Picogram amounts of microbial cell surface components such as lipopolysacharide (LPS) found in Gram-negative bacteria can induce mammalian leukocytes to secrete cytokines leading to fever, coagulation defects, lung dysfunction, kidney failure and circulatory collapse [2–10]. LPS consists of an o-specific chain, a core oligosaccharide and a lipid component, termed lipid A [11]. To prevent deleterious effects of these bacterial products on the host, it is essential to understand the mechanism of cellular activation. CD14 is the major LPS receptor and has been shown to play an integral role in LPS activation of immune cells. The binding of LPS to the CD14 receptor leads to production of pro-inflammatory cytokines such as TNF-␣, IL-1, IL-6, IL-8, IL-12, free oxygen radicals, NO, tissue factor and others, as well as anti-inflammatory cytokines such as IL-10 and TGF-␤. CD14 exists in both membrane-bound (mCD14) and soluble (sCD14) forms [12]. The membrane-bound CD14 is a 50–55 kDa receptor linked to the cell surface by a glycosyl-phosphatidyl inositol (GPI) anchor and lacks a

0305-4179/02/$22.00 © 2002 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 0 2 ) 0 0 0 3 4 - 7

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transmembrane domain [13]. This membrane-bound receptor is found on monocytes, macrophages, granulocytes and endothelial cells and mediates the activation of these cells by LPS [10,14,15]. mCD14 is considered a pattern recognition receptor for microbial antigens and in conjunction with the transmembrane toll-like receptor proteins, an important mediator of innate immune responses to infection [16–18]. sCD14 is found in serum and mediates LPS activation of non-CD14-bearing cells [14,19]. At high concentrations, recombinant sCD14 has also been shown to neutralize LPS, possibly by competing with mCD14 [20]. More recently, both the soluble and membrane-bound forms of CD14 have been found to participate in the activation of cells by infectious agents other than Gram-negative bacteria [21], including yeast and Gram-positive bacteria [22–25]. This suggests that CD14 may recognize other microbial components besides LPS on Gram-negative bacteria. Although previous investigations of sCD14 expression have concentrated on myeloid cells, we and others have shown that extramyeloid cells are also capable of expressing CD14 [26,27]. Little is known about induction of CD14 expression in vivo. In mice, it has been shown that LPS challenge induces a transient increase in CD14 mRNA levels in myeloid cells, as well as in the epithelium of the liver and kidney. In vitro LPS, TNF-␣ and IFN-␥ upregulate CD14 expression in human myeloid cells whereas IL-4 reduces it. CD14 levels can also be altered by proteolytic cleavage or phospholipase d-induced shedding of mCD14 resulting in a rise in sCD14 in serum. The role of CD14 within the site of burn injury, however, remains unclear. The goal of this study is to assess and quantify CD14 mRNA expression in burned and normal skin in order to shed light on the effect of burn injury upon local host defense mechanisms and to improve our understanding of this devastating type of injury.

2. Subjects and methods

Table 1 Study population Patients (n) Sex (n) Age Extend of injury (% BSA burned) Time interval burn-harvest (days) Number of skin samples

57 34/23 (male/female) 33.2 years (range 6–70) 23.5% (range 5–54) 6.4 days (range 1–30) 233

2.2. Bacterial counts Post-burn wound infection was objectively assessed by culturing wound homogenates from skin tissue. Skin tissue from burn wounds was weighed and homogenized in 3 ml of 0.9% normal saline using a 6 mm generator (Polytron Kinematica, Brinkmann Instruments, Westbury, NY). Following homogenization, three serial 100-fold dilutions were performed using 0.9% normal saline. A 100 ␮l aliquot from each dilution was plated on 5% sheep blood BSA plates (Fischer Scientific, Pittsburgh, PA) and incubated at 37 ◦ C for 18 h. Gram-staining was performed of representative colonies to identify the type of bacteria. The number of colonies on each plate was counted and the CFU per gram of tissue calculated for each tissue sample. 2.3. RNA Isolation and reverse transcription Wound biopsies (50–100 mg) were harvested from partial thickness burns and donor areas (normal controls), placed in labeled microcentrifuge tubes and immediately snap frozen in liquid nitrogen for preservation until RNA isolation was performed. Using TRIZOL reagent and protocol (Gibco BRL, Gaithersburg, MD), total RNA was isolated from both burn and normal (control) skin. RNA from each sample was reverse transcribed, using M-MLV reverse transcriptase RNAse inhibitor and DTT (Gibco BRL, Gaithersburg, MD); dNTP and oligo dT (Boehringer Mannhein, Indianapolis, IN) and actinomycin D (Calbiochem, La Jolla, CA) at 37 ◦ C for 1 h.

2.1. Patients

2.4. Northern blot analysis

The study population consisted of 57 patients who were admitted to the Trauma Burn Intensive Care Unit, at the University of Michigan during the 1997–1999 period. These patients had sustained partial thickness burns and subsequently required operative debridements and grafting. The clinical characteristics of the patient population studied are shown in Table 1. After informed consent was obtained, sequential full thickness tissue sections were obtained from partial thickness burned and non-injured areas at various time points. Overall, 233 tissue samples were obtained from 57 patients. The study was approved by the Human Subjects Ethical Committee of the University of Michigan in Ann Arbor (IRB).

Aliquots containing 20 ␮g total RNA were electrophorezed on a 1% agarose gel containing 3% formaldehyde. RNA was then blot transferred to GeneScreen membrane (Du Pont, NEN) and UV auto-crosslinked (UV Stratalinker 1800, Stratagene). Membranes were hybridized overnight at 42 ◦ C with DNA randomly primed with 32 P. Membranes were then washed and autoradiography carried out at −70 ◦ C with intensifying screens. After probing for CD14, membranes were stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as a control for variations in RNA loading between lanes. Relative levels of mRNA were expressed as a ratio of CD14/GAPDH.

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2.5. TaqMan fluorescent RT-PCR The limited amount of messenger RNA (mRNA) isolated from many of the tissue samples precluded conventional Northern blot analysis on all of the samples. A more sensitive assay, quantitative fluorescent TaqMan real time polymerase chain reaction (RT-PCR), was therefore used to determine skin CD14 mRNA levels. PCR amplification of synthesized cDNA from each sample (40 ng) was carried out in a final volume of 50 ␮l using paired amplification primers (0.75 ␮M) and a fluorescence-labeled oligonucleotide probe (50 nM) specific for CD14. The complete DNA sequence of the CD14 gene was obtained from the GenBank database of the National Center for Biotechnology Information of the NIH. A fluorescent probe, 30 nucleotides in length was designed to be complementary to a mid-section of the full sequence (Perkin-Elmer Applied Biosystems, Foster City, CA). Attached to the probe were the reporter (FAM) and quencher (TAMRA) molecules: (FAM)-TAG CGC TGC GCA ACG CAG GGA TGG AGA CGA-(TAMRA). The PCR primers (5 primer-AAG ATA ACC GGA ACC ATG CCT, 3 primer-GAG ATC GAG CAC TCT GAG CTT) were used under the following conditions (94 ◦ C, 0:15; 59 ◦ C, 0:45; 72 ◦ C, 0:45) × 32 cycles on a model 9600 thermal cycler (Perkin-Elmer). These primers defined the length of the final product amplicon, resulting in a sequence of 534 nucleotides (Fig. 1). The amplified PCR product (amplicon) was subcloned into a pCR2.1 plasmid vector (Invitrogen, Carlsbad, CA). Large quantities of the amplicon-containing plasmid vector were generated in competent Escherichia coli top 10 F cells (Invitrogen, Carlsbad, CA). The plasmid DNA was isolated from E. coli cells using Qiagen maxi kit and protocol (Qiagen, Santa Clarita, CA). The amplicon was removed from the plasmid using EcoRI (Boehringer Mannhein, Indianapolis, IN) and sepa-

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rated from the remaining plasmid DNA with 1.5% agarose gel electrophoresis. The amplicon DNA was isolated from the gel using the QIAquick gel extraction kit and protocol (Qiagen, Santa Clarita, CA). A sample of amplicon was digested with PstI (Boehringer Mannhein, Indianapolis, IN), a restriction endonuclease with only one cleavage site in the sequence. The generation of the designed standard template was confirmed by digestion with PstI and running the digest on a 1% agarose gel. This showed two fragments of 111 and 423 nucleotides. This amplicon served as a template to generate the standard curve for each subsequent quantitative TaqMan RT-PCR. Following amplification, the reaction mixture was analyzed using an LS-50B luminescence spectrophotometer (wavelength: excitation, 488 nm; reporter emission, 518 nm; quencher emission, 582 nm) to determine changes in fluorescence. Simultaneous quantification of amplification reactions performed on serial dilutions for known template copy numbers was used as a standard curve for back calculations of mRNA copy numbers contained in the unknowns. In all cases, R2 for the generated standard curves were >0.98 over the unknown concentration range. 2.6. Localization of CD14 Immunohistochemistry on 4 ␮m thick paraffin sections was performed using the ABC-based method [28] with the primary mouse antibody NCL-CD14-223 (Vector Laboratories, Burlingame, CA). The NCL-CD14-223 antibody recognizes the external domain of the CD14 molecule expressed on the myelomonocytic lineage, which includes monocytes, macrophages and Langerhans cells. NCL-Macro is a highly specific marker for macrophages and is useful in discriminating monocytes and macrophages from other cells. Localization of the antibody binding was visualized with NovaRed (Vector Laboratories, Burlingame, CA) which produces a

Fig. 1. CD14 amplicon for TaqMan RT-PCR. The CD14 PCR-primer (3 and 5 ) are depicted in bold. The TaqMan fluorescent probe is underlined while the cleavage site for PstI is marked. This restriction enzyme cuts the 534 bp amplicon into 423 and 111 bp fragments.

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brick red reaction product. Sections were counter-stained with hematoxylin (Vector Laboratories, Burlingame, CA), dehydrated with ethanol, cleared with Xylene and mounted with Mounting medium (Richard-Allan Scientific, Kalamazoo, MI). 2.7. Statistical analysis All data are reported as the mean ± S.E. Data were analyzed using analysis of variance and the two-tailed Student’s t-test on Statview statistical package for Apple Power Macintosh computers (Abacus Programs, Cupertino, CA). Significance was assigned at P < 0.05. 3. Results 3.1. CD14 mRNA expression The yield of RNA from the tissue samples ranged from 10–40 ␮g. On samples, where there was sufficient RNA, Northern blot analysis was performed. Fig. 2 shows a Northern blot of total RNA isolated from human burned and normal skin and probed for CD14 and GAPDH (housekeeping) mRNAs. Skin CD14 mRNA levels markedly increased following burn injury in both patients while no bands were seen for normal controls. The signal intensity for CD14 peaked on day 1 (B1), then gradually declined over the next 5–7 days (B5, B7). Probing for GAPDH showed equally distributed mRNA expression for normal and burned skin. Because we did not have adequate quantities of RNA to run a Northern on all samples, TaqMan fluorescent RT-PCR was performed on all of the samples. The average CD14 mRNA

Fig. 3. Normal and burned skin levels of CD14 mRNA. Quantitative TaqMan RT-PCR analysis of CD14 mRNA copy numbers (mean ± S.E.) from 40 ng of total RNA from burned and normal skin tissue (∗ P < 0.05 burn vs. normal).

levels (Fig. 3) in burned skin were significantly (P = 0.026) increased (54, 767 ± 9631 copy numbers) compared to normal skin (19, 923 ± 2442). CD14 mRNA expression in burns peaked within the first 7 days post-burn (P = 0.032), before declining to control levels after 14 days. Tissue samples harvested 7 days post-burn showed no difference in CD14 mRNA levels compared to normal controls (Fig. 4). 3.2. Bacterial counts Quantitative bacterial counts of burned skin tissue (Fig. 5) showed an increase, although not statistically significant (P = 0.25), in CD14 expression in skin samples with posi-

Fig. 2. Northern blot analysis of CD14 and GAPDH (housekeeping gene used as positive control) mRNA. Burned tissue (B) was harvested at different time points from two patients. A sample of normal skin (N) was obtained, as well as burned skin on days 1, 3 and 5 post-injury from Patient 1. For Patient 2, a single sample of normal skin (N) was obtained, as well as burned skin on days 1 and 7 post-injury. CD14 mRNA levels are significantly elevated 1 day after injury but decrease subsequently over the next 5–7 days.

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Fig. 4. Decline of CD14 levels in the post-burn period. Quantitative TaqMan RT-PCR analysis of CD14 mRNA copy numbers (mean ± S.E.) from 40 ng of total RNA from burned and normal skin tissue. The post-burn period was subdivided into less than 7 days, 7–14 days, and more than 14 days. Maximum CD14 expression was seen within 7 days post-burn compared to normal skin. After this initial increase, CD14 levels gradually declined, reaching baseline after day 14 (∗ P < 0.05 burn vs. normal).

tive quantitative bacterial counts as opposed to those with no growth. No growth was defined as less than 106 bacteria per gram of tissue. There was no correlation between the degree of CD14 mRNA expression in burned skin samples and the clinical outcome of the patients (i.e. survivor/non-survivor) (data not shown). 3.3. Localization of CD14 Immunohistochemistry with an anti-CD14 antibody was used to examine CD14 expression in burned and normal skin (Fig. 6). CD14 staining was detected predominantly in burned skin sections. Although, significant edema was

Fig. 5. Rise of CD14 mRNA expression in infected wounds. Quantitative wound cultures were performed on burned and normal skin tissue samples and compared with CD14 mRNA copy numbers (mean ± S.E.) obtained from these samples by TaqMan RT-PCR analysis.

present in both the dermis and epidermis in some of the burned skin sections, CD14 was clearly evident in the dermal and subdermal plexus with no staining seen in the epidermal plexus. CD14 staining was more pronounced 24 h after the injury and subsequently decreased, reaching normal levels approximately 5–7 days post-burn.

4. Discussion In the setting of infection, Gram-negative and -positive bacterial cell-wall components and endogenous phospholipids trigger inflammatory cells such as monocytes, macrophages, granulocytes and lymphocytes to produce and release pro-inflammatory cytokines. This pro-inflammatory cascade is mediated through CD14, a GPI-linked LPS receptor expressed on monocytic cells [29]. CD14 lacks a transmembrane component and therefore intracellular signaling is thought to take place via nearby toll-like receptors (i.e. Tlr4, Tlr2), with subsequent activation of NF-KappaB and mitogen-activated protein (MAP) kinases [30]. It is well-known that patients who have sustained serious burn injuries demonstrate a marked decrease in their immune status and ability to resist infection. In vitro studies of macrophages and neutrophils from burn patients show alterations in chemotaxis, phagocytosis, oxidative burst and intracellular killing capacity, which most likely contribute to an increased susceptibility to infection and sepsis in these patients [31–33]. We sought to examine the cause of this depressed immune function in the setting of thermal injury, postulating that perhaps this was related to some early event such as alteration of CD14 expression [34]. Previous studies have shown that serum CD14 levels increase in disease

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Fig. 6. Localization of CD14 expression in normal and burned human skin. Van Gieson staining of a cross-section through human skin (panel A) and burned skin (panel B). Note the tissue necrosis with swollen coagulated collagen fibers and disrupted necrotic skin appendages seen in panel B (50× magnification). Immunohistochemical staining with the anti-CD14 antibody (brick red reaction product) in normal (panel C) and burned human skin in panel D (100× magnification). Increased number of stained cells are seen in the burn group (panel B). Most of the stained cells are found perivascular in the dermal layer (arrows).

states such as sepsis and also following traumatic injury [35–37]. In our study, we chose to look at local messenger RNA expression for CD14 in burned and normal skin. We felt that this would better reflect CD14 production, within the burn wound and thus better characterize local events at the level of the tissue than serum CD14 levels, whose rise and fall reflect the generalized systemic immune response. In this study, we found that CD14 mRNA levels are significantly elevated locally in skin after thermal injury. The biological relevance of CD14 upregulation after burn injury remains unresolved. It is interesting, however, to note that CD14 performs a range of functions potentially beneficial to the host in the setting of a burn injury. For example, CD14 belongs to a group of receptors called pattern recognition receptors (PRRs). These PRRs include humoral proteins circulating in the plasma, receptors involved in signaling for phagocytosis, and various receptors present on the cells of the innate immune system [36]. CD14 itself aids in phagocytosis of bacteria by binding not only soluble lipopolysacharide (LPS) but also Gram-negative

bacteria, which are then internalized by an LBP-dependent (complement-independent) pathway [38]. In addition, it has been postulated that soluble CD14 may act as a shuttle molecule similar to lipopolysacharide binding protein (LBP), transferring LPS to HDL and thereby neutralizing its toxic effects. Very recent studies suggest that CD14 possesses a charged surface and exerts a protective function by scavenging LPS and apoptotic cells, “presenting” them to other components of the innate host defense system [39,40] for clearance. Finally, it has been recently demonstrated that baseline levels of antimicrobial peptides are greatly decreased in human burn wounds. Among their many functions, these peptides act to block the interaction of LPS with LBP [41–43]. Taking this into account, one may speculate that local CD14 expression may be upregulated in an attempt to compensate for defects in the host immune defenses within the burn wound. The possible protective role of high CD14 levels immediately after injury, is in keeping with the finding that high CD14 levels are thought to improve survival after severe tissue injury [44].

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We also demonstrated that CD14 expression peaks within the first week post-burn before declining, reached normal levels after 14 days. This loss of supranormal CD14 expression locally within the wound, may contribute to a weakened host defense response 5–6 days after injury, which correlates with when these patients become especially vulnerable to infection. It remains to be proven at this point, whether the late drop in local CD14 expression contributes to suppressed pro-inflammatory responses and pre-disposes the burn wound to further bacterial growth, impaired wound healing, and scarring which are common problems in burn injury.

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