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Does the antimicrobial peptide, granulysin, play a role in decreasing the incidence of secondary bacterial infection in psoriasis?

Mahgoub, Doaa M.a; Nagui, Noha A.a; Rashed, Leilab

Journal of the Egyptian Women's Dermatologic Society: January 2011 - Volume 8 - Issue 1 - p 50–54
doi: 10.1097/01.EWX.0000392823.59573.fc
Original Articles
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Background Although the skin is constantly challenged by microbes, it is rarely infected. Cutaneous production of antimicrobial peptides is a primary system for protection. Patients with psoriasis, a chronic, hyperproliferative, and noninfectious skin disease, suffer fewer cutaneous infections than would be expected. Granulysin is a recently identified antimicrobial protein expressed in cytotoxic T cells, natural killer cells, and natural killer T cells. It has been shown that granulysin contributes toward the defense mechanisms against mycobacterial infection.

Objective To estimate the tissue level of granulysin in psoriatic patients versus controls aiming to prove its possible role as antimicrobial peptide in preventing secondary bacterial infection in psoriasis.

Patients and methods This study included 15 patients with psoriasis and 15 healthy volunteers as controls. Five millimeter punch skin biopsies were taken from all patients and controls and assessed for granulysin by enzyme-linked immunosorbent assay technique.

Results Granulysin level in psoriatic patients was significantly higher compared with that of normal controls (P=0.001). Correlation between granulysin level and Psoriasis Area and Severity Index score showed a statistically significant positive correlation (P=0.001).

Conclusion High tissue level of granulysin in psoriatic patients may participate in the pathogenesis of psoriasis and provide an explanation for the relative immunity of psoriatic plaques against both gram-positive and gram-negative bacterial infections.

aDepartments of Dermatology

bBiochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt

Correspondence to Doaa Mohamed Mahgoub, Assistant Professsor of Dermatology, Faculty of Medicine, Cairo University. 18 Salah El Din Street, Zamalek, Cairo, Egypt Tel: +0105670009; fax: +33052633; e-mail: amelnashar@hotmail.com

Received June 10, 2010

Accepted August 15, 2010

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Introduction

Psoriasis is a chronic inflammatory and proliferative papulosquamous skin disease [1] of unknown etiology [2]. It has been considered as an autoimmune T cell-mediated disorder in which adaptive immune responses predominate over those of nonantigen-specific innate immunity. The immune response in psoriasis is Th1-mediated and associated with local neutrophilic infiltration [3].

Human skin is constantly challenged by microbes but is rarely infected. Cutaneous production of antimicrobial peptides (AMPs) is a primary system for protection, whereas the expression of some AMPs increases in response to microbial invasion [4].

Psoriatic patients, surprisingly, suffer from fewer cutaneous infections than would be expected. This observation led us to the hypothesis that a local ‘chemical shield’ in the form of antimicrobial proteins provides psoriatic skin with resistance to infection [1].

Host defense peptides (HDPs), also known as AMPs, play an important role in the innate immune system as antimicrobial and immunomodulating agents and present an important link between the innate and adaptive immune response [5,6]. Owing to their multiple functions, they are considered as promising agents for new therapeutic approaches in infectious diseases and wound healing [7,8].

HDPs show great variance of effects and interactions. One major function of HDPs is to inactivate microbes, including bacteria, fungi, parasites, and viruses through multiple direct effects on their membranes [9,10].

AMPs are predominantly small cationic polypeptides that are classified together on account of their capacity to inhibit the growth of microbes. As effectors of innate immunity, AMPs directly kill a broad spectrum of bacteria, fungi, and viruses. In addition, these peptides modify the local inflammatory response and activate mechanisms of cellular and adaptive immunity. Cathelicidins and defensins comprise the major families of AMPs in the skin [6].

These AMPs not only act as endogenous antibiotics but are also involved in immune and/or inflammatory responses by attracting several types of cells to infection or inflammation sites and by stimulating cytokine and chemokine production, thus accelerating angiogenesis and promoting wound healing [2].

Granulysin is a cytolytic and proinflammatory molecule first identified by screening genes expressed ‘late’ (3–5 days) after the activation of human peripheral blood mononuclear cells. Granulysin is present in cytolytic granules of cytotoxic T lymphocytes and natural killer (NK) cells. Granulysin is made in a 15-kDa form that is cleaved into a 9-kDa form at both the amino and the carboxy terminals. The 15-kDa form is constitutively secreted, and its function remains poorly understood. The 9-kDa form is released by receptor-mediated granule (exocytosis). Nine kilo Dalton granulysin is broadly cytolytic against tumors and microbes, fungi/yeast, and parasites. It kills the causative agents of both tuberculosis and malaria. Granulysin is also a chemoattractant for T lymphocytes, monocytes, and other inflammatory cells, and activates the expression of a number of cytokines, including regulated upon activation, normal T cell expressed and secreted, monocyte chemoattractant protein 1, monocyte chemoattractant protein 3, macrophage inflammatory protein 1α, interleukin (IL)-10, IL-1, IL-6, and interferon α. Small synthetic forms of granulysin are being developed as novel antibiotics [11].

Granulysin and NK-lysin are members of the saposin-like protein family. The members of this family share several structural properties, including six similarly located cysteines forming three disulfide bridges with the same cysteine pairings [12].

Granulysin is a recently identified antimicrobial protein expressed in cytotoxic T cells, NK cells, and natural killer T cells. It has been shown that granulysin contributes toward the defense mechanisms against mycobacterial infection [13]. Granulysin is a cationic protein that can kill bacterial pathogens through disruption of microbial membrane integrity [14].

Granulysin expression is observed in some bacterial conditions. A large number of granulysin positive T cells were observed in psoriasis; the skin condition associated with high resistance to secondary infection. This is in contrast to the very few number of granulysin-positive T-cells observed in atopic dermatitis (AD); the skin condition frequently associated with gram-positive bacterial infection, which suggests that lesional granulysin level may determine the relative risk of secondary infection [15].

Tewary et al. [16] reported that both 9-kDa and 15-kDa forms of recombinant granulysin induced in-vitro chemotaxis and activation of both human and mouse dendritic cells, recruited inflammatory leukocytes including antigen presenting cells in mice, and promoted antigen-specific immune responses on coadministration with an antigen. Granulysin-induced antigen presenting cells recruitment and activation required the presence of TLR4. The observed activity of recombinant granulysin was not because of endotoxin contamination.

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Patients and methods

Patients

This study included 15 patients with psoriasis: seven males and eight females; recruited from the Outpatient Clinic of the Dermatology Department of the Kasr Al-Aini Hospital between the period October 2008 and April 2009. Their age ranged from 14 to 63 years. The Psoriasis Area and Severity Index (PASI) score was calculated for each patient to determine disease severity.

Fifteen healthy volunteers (nine males and six females) were chosen as controls. Their ages ranged from 20 to 60 years. Neither of them had a history of skin or systemic disease nor a family history of psoriasis.

A written informed consent was taken from all patients and controls before starting the study.

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Method

All patients were subjected to full history taking, clinical examination, and grading of disease severity using the PASI score.

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Measurement of tissue granulysin

Sample collection and preparation

A 5-mm punch skin biopsy was taken from all patients and controls and assessed for granulysin by enzyme-linked immunosorbent assay (ELISA) technique. Each skin biopsy was divided into two parts: one was stained with haematoxylin and eosin for confirmation of the diagnosis and the other was processed for granulysin estimation. Assessment of granulysin level was made using approximately 50 mg of the biopsy tissue, which was homogenized in 1 ml lysis buffer containing 0.0625 mol/l Tris buffer (pH 6.8), 2% sodium dodecyl sulfate, 3% β-mercaptoethanol, 10% glycerol, 100 mmol/l sodium fluoride, 10 μgm/ml aprotinin, and 1 mmol/l phenylmethylsulfonylflouride (Sigma, Saint Louis, Missouri, USA) for protein extraction.

Skin biopsies were homogenized using a tissue homogenizer. After cell lysis the homogenate was centrifuged at 10 000×g for 20 min at 4°C and the supernatant was stored at −80°C until performance of the assay.

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Measurement of tissue granulysin level by enzyme-linked immunosorbent assay

The supernatant was examined for granulysin using quantitative sandwich ELISA (R'D Systems Europe, Ltd., UK). The principal of this sandwich ELISA-based assay is the capture of granulysin molecules from samples to the wells of a microtiter plate coated with a monoclonal antigranulysin antibody. This is followed by washing of unbound materials from samples and binding of a second biotinylated monoclonal antigranulysin antibody to the captured molecules. Washing of unbound materials is repeated followed by binding of stretavidin–horseradish peroxidase conjugate to the immobilized biotinylated antibody and washing of excess free enzyme conjugates. Quantification of immobilized antibody–enzyme conjugates by monitoring horseradish peroxidase activities in the presence of the substrate 3,3′,5,5′-tetramethylbenzidine. The enzyme activity is measured spectrophotometrically by the increased absorbance at 450–590 nm to the amount of captured granulysin in the unknown sample; the latter can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of granulysin [12].

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Statistical analysis

The data were coded and entered on an IBM compatible computer (IBM Co., New York, New York, USA) using the statistical package for social sciences (SPSS) version 12.0. The descriptive statistics were computed to summarize the mean and standard deviation for quantitative variables and percentage for qualitative variables. The Fisher's exact test was used to assess differences between qualitative variables whereas Mann–Whitney test was used for quantitative variables. Difference between studied groups was considered statistically significant at a P value of less than 0.05. Correlations were done to test the relation between variables.

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Results

This study included 15 patients with psoriasis and 15 healthy age-matched and sex-matched controls. The patients were seven males and eight females with an age range of 14–63 years (mean: 35.13±16.10 years). None of the studied patients had been receiving systemic steroids for at least 2 months before the study. Grading of disease severity was done according to the PASI score using parameters of extent, course, and intensity.

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Granulysin level in skin biopsies

The mean granulysin level in the skin biopsies of psoriatic patients was 2390.78±1086.21 pg/dl and in healthy control individuals was 1202.82±292.89 pg/dl. These values denote that granulysin level in psoriatic patients was significantly higher compared with that of controls (P=0.001) (Table 1, Fig. 1).

Table 1

Table 1

Figure 1

Figure 1

Correlation between granulysin level and the PASI score showed a statistically significant positive correlation (P=0.001) (Table 2, Fig. 2). No statistically significant difference was found in mean granulysin level in skin biopsies of male and female psoriatic patients (P=0.652). In addition, no statistically significant correlation was found between the granulysin levels in the skin biopsies of the patients and duration of disease (P=0.330) (Table 2). Finally, no statistically significant correlation was found between the granulysin levels in the skin biopsies of the patients and their age (P=0.285) (Table 2).

Table 2

Table 2

Figure 2

Figure 2

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Discussion

Over years of observation clinicians have noticed that some inflammatory skin diseases are prone to bacterial skin infections such as AD. In contrast, despite open wounds and cracks, cutaneous infection is uncommon in psoriasis. It is possible that certain in-vivo factors including AMPs are responsible for this relative susceptibility versus resistance to infection [5].

Lesions of psoriasis are heavily colonized with Staphylococcus aureus [17], but secondary bacterial infection is extremely rare in psoriatic lesions. Several hypothesis, including antimicrobial activity of the lesional cytokine [1], defensin expression by keratinocytes [9,18], and increased number of natural killer T cells in lesions [19] have been proposed to explain the unique phenomenon of resistance to bacterial infection in psoriatic plaques.

A series of postoperative deaths as a result of sepsis were caused by an anesthetist with psoriasis who was a carrier of S. aureus [8].

AMPs are small molecular weight proteins with broad-spectrum antimicrobial activity against bacteria, viruses, and fungi. These peptides are usually positively-charged and have both hydrophobic and hydrophilic sides that enable the molecule to be soluble in aqueous environments and also enter lipid-rich membranes. Once in a target microbial membrane, the peptide kills target cells through diverse mechanisms. Cathelicidins and defensins are major groups of epidermal AMPs. Decreased levels of these peptides have been noted in patients with AD whereas increased levels have been noted in psoriatic patients. In addition to important antimicrobial properties, growing evidence indicates that AMPs have been shown to be important in diverse functions such as angiogenesis, wound healing, and chemotaxis. As the knowledge of AMP biology expands, the precise role and relevance of these peptides will be better elucidated [20].

Discovery of defensin has provided new understanding about the local epithelial defense system [21]. Defensin is produced by epithelial cells including keratinocytes in psoriatic plaques [18]. Defensin has a potent antimicrobial activity against gram-negative bacteria and Candida but not against gram-positive organisms such as S. aureus. Increased amount of granulysin in psoriatic plaques provides a supportive mechanism to the resistance of psoriatic plaques against both gram-positive and gram-negative bacterial infections [22].

Granulysin as one of the AMPs has a broad-spectrum antimicrobial activity, killing bacteria, fungi, and parasites. Granulysin also functions as a chemoattractant and proinflammatory activator [6].

Endsley et al. [14] suggested that augmentation of granulysin expression by cytotoxic lymphocytes, or therapeutic application of granulysin peptides, could constitute important strategies for protection against select agent bacterial pathogens. Investigations of the microbial surface molecules that determine susceptibility to granulysin may identify important mechanisms that contribute to pathogenesis.

In recent years, granulysin has become a focus of interest more for its antimicrobial activity in a number of chronic inflammatory skin diseases. As the role of granulysin as AMP in the pathogenesis of psoriasis is recently highlighted, this study tried to figure out this role and how far is its importance in the pathogenesis of the disease and its possible role in rarity of secondary bacterial infection in psoriasis despite the cracks and deep fissures of psoriatic lesions. ELISA technique was used in this study to measure the level of granulysin in skin biopsies of psoriatic patients and controls. This study assessed the level of granulysin in skin biopsies of 15 patients with psoriasis and 15 normal healthy controls. These findings showed a highly statistically significant increase in granulysin level in skin biopsies of psoriatic patients when compared with those of controls. These results agreed with the earlier findings of Raychaudhuri et al. [15] who measured the level of granulysin in cryosections of skin biopsies of psoriatic patients, normal control individuals, patients with AD, and nummular eczema. They found a strong expression of granulysin in the lymphmononuclear infiltrates and dermal dendritic cells in psoriatic plaques compared with those of controls or of AD. It is important to note that increased number of granulysin-positive T cells was observed in psoriasis, where secondary bacterial infection is extremely rare. Very few granulysin-positive T cells were observed in AD and nummular eczema, two conditions frequently associated with gram-positive bacterial infections. These observations suggested that lesional granulysin levels may determine the relative risk of secondary infection in certain inflammatory conditions of the skin [15].

These data suggested the possible role of granulysin in the pathogenesis of psoriasis and in decreasing liability of secondary bacterial infections among psoriatic patients.

To our knowledge, no other studies on granulysin in psoriasis were carried out, but many other AMPs were studied and showed similar results. Gambichler et al. [23] found higher expression of human β-defensin (hBD)-1, hBD-2, hBD-3, and psoriasin in pemphigus vulgaris (n=35) and AD (n=42) patients when compared with controls (n=18), whereas psoriatic lesions showed significantly higher mRNA hBD-2 levels than lesions of AD.

Ong et al. [9] showed better immune response against S. aureus in patients with psoriasis caused by a higher human cathelicidin (LL-37) expression level, whereas patients suffering from AD showed decreased expression. These findings may provide an explanation for the susceptibility of patients suffering from AD to skin infection compared with patients with psoriasis [24].

In addition, Frohm et al. [20] found that LL-37 is induced in human keratinocytes of patients with psoriasis, lupus erythematosus, and contact dermatitis.

Harder et al. [25] and Nomura et al. [26] proved that hBD-2 and hBD-3 are also upregulated in keratinocytes of psoriatic lesions. The increased expression of AMPs in psoriasis patients correlates with a low rate of secondary infection in those patients.

De Jongh et al. [27] also found an overexpression of many antimicrobial proteins in keratinocytes from psoriatic skin compared with AD skin. They concluded that psoriatic epidermis expresses high levels of host defense proteins compared with AD epidermis, and this phenomenon appears to be specific for these proteins. It remains to be investigated whether this is caused by genetic polymorphisms in pathways leading to an epidermal antimicrobial response, or by differences in the cellular infiltrate in psoriasis compared with AD.

This study not only showed that there was no statistically significant correlation between the levels of granulysin and age of psoriatic patients, but also showed that there was no statistically significant difference in granulysin levels between male and female patients of psoriasis.

In this study, correlation of granulysin level in skin biopsies of psoriatic patients with the PASI score of these patients showed a statistically significant positive correlation (P=0.001). Correlation of granulysin level in skin biopsies of psoriatic patients with duration of disease was not significant (P=0.330). However, no earlier published data on those two parameters was found.

In conclusion, this study showed a significant increase in the granulysin level in the skin biopsies of psoriatic patients compared with normal controls. Moreover, a significant positive correlation between the level of granulysin in psoriatic patients and the PASI score was found, that is, the more the increase in the PASI score, the more is the increase in granulysin.

This high level of granulysin may participate in the pathogenesis of psoriasis and provide an explanation for relative immunity of psoriatic plaques against both gram-positive and gram-negative bacterial infections.

There is no conflict of interest.

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Keywords:

antimicrobial peptides; granulysin; psoriasis

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