|Year : 2018 | Volume
| Issue : 1 | Page : 61-66
Immunohistochemical expression of human β-defensin-2 in verruca vulgaris
Amany M Abdel-Latif1, Abeer Abd El-Hakam Hodeib1, Safinaz H El-Shorbagy2, Shimaa A.W Shaheen1
1 Department of Dermatology and Venereology, Tanta University Hospital, Tanta, Egypt
2 Department of Pathology, Tanta University Hospital, Tanta, Egypt
|Date of Submission||25-May-2017|
|Date of Acceptance||21-Jan-2018|
|Date of Web Publication||26-Jul-2018|
Shimaa A.W Shaheen
Department of Dermatology and Venereology, Faculty of Medicine, Tanta University, Kafr El-Zyat Hospital, Tanta, 31511
Background Verruca vulgaris is the commonest viral cutaneous infection that is caused by human papilloma virus. There are more than 150 genotypically different types of human papilloma virus. Types 1, 2, 4, 27, and 57 were found with verruca vulgaris. Human β-defensins (hBDs), a family of small antimicrobial peptides, are important effector molecules of innate immunity, possessing antimicrobial, anti-inflammatory, and immunomodulatory activities.
Aim The aim of this work was to investigate the immunohistochemical expression of hBD-2 in verruca vulgaris lesion in a trial to elucidate its possible role in the pathogenesis of the disease.
Patients and methods This study included 15 lesional skin specimens of 15 patients with typical clinically and histopathologically diagnosed verruca vulgaris (group I). Seven specimens of nonlesional normal skin of contralateral sides of seven of these patients (group II) in addition to other seven normal skin specimens from seven healthy subjects of matched age and sex (group III) served as controls. All specimens were studied histopathologically using hematoxylin and eosin stain and immunohistochemically for detection of hBD-2 expression.
Results All lesional skin specimens (group I) showed positive expression of hBD-2. Twelve (80%) specimens showed positive expression in all epidermal layers [immunoreactivity (IR) 3]. Two (13.3%) specimens showed positive expression in the stratum corneum only (IR 1), and one (6.7%) specimen showed positive expression in the stratum corneum and stratum granulosum (IR 2). Groups II and III showed no expression of hBD-2 (IR 0). There was highly significant difference between patients and controls (P<0.001).
Conclusion There was positive immunohistochemical expression of hBD-2 in lesional skin specimens taken from patients with verruca vulgaris, whereas there was no expression in normal skin specimens taken from the patients and the volunteers. This may indicate a possible role of hBD-2 in the pathogenesis of verruca vulgaris and also in the immunity against it.
Keywords: human beta defensin -2 expression, human papilloma virus, verruca vulgaris
|How to cite this article:|
Abdel-Latif AM, Abd El-Hakam Hodeib A, El-Shorbagy SH, Shaheen SA. Immunohistochemical expression of human β-defensin-2 in verruca vulgaris. Tanta Med J 2018;46:61-6
|How to cite this URL:|
Abdel-Latif AM, Abd El-Hakam Hodeib A, El-Shorbagy SH, Shaheen SA. Immunohistochemical expression of human β-defensin-2 in verruca vulgaris. Tanta Med J [serial online] 2018 [cited 2019 Feb 22];46:61-6. Available from: http://www.tdj.eg.net/text.asp?2018/46/1/61/237624
| Introduction|| |
Human skin represents the first barrier against invading microorganisms. Although it is constantly exposed to these microbial threats, invasive infections and pathological disorders are rather rare and are usually locally limited. One reason for this natural resistance might be apart from the physical barrier, the release of various antimicrobial agents like antimicrobial peptides (AMPs) .
AMPs are small cationic peptides that protect their hosts against a vast array of microorganisms. These peptides are produced by several species including bacteria, insects, plants, and vertebrates . They exert their effects mostly by interacting with the membrane of the microorganism and lead to lysis and subsequently killing of the pathogen. In human skin, the most important AMPs are the group of human β-defensins (hBDs) including hBD 1–4, cathelicidin/LL-37, S100 protein, psoriasin, ribonuclease 7 (RNase 7), lysozyme, antileucoprotease, and dermcidin . Some of these peptides are expressed constitutively like psoriasin and RNase 7. Others like hBD-2 and hBD-3 are also inducible by proinflammatory cytokines and bacterial molecules. The inducibility varies among different defensins .
AMPs, which are synthesized in the skin at sites of potential microbial entry, provide a soluble barrier that acts as an impediment to infection. In case of infection, AMPs expression in the skin is up-regulated owing to increased synthesis by keratinocytes and deposition from degranulation of recruited neutrophils. Constitutive and inducible expression of human cathelicidin, as well as hBD 1–3, has been observed in epidermal keratinocytes .
hBD-2 is an endogenous inducible AMP that is found in the skin. Expression of hBD-2 is up-regulated in the lesions of some dermatoses such as psoriasis, acne vulgaris, and dermatophytosis. However, it has been seen in ductal epithelia of sweat glands and in hair follicles under normal conditions . The antimicrobial range of hBD-2 encompasses the Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia More Details coli), the yeasts (Candida albicans and Malassezia furfur) , and viruses [herpes simplex virus, human papilloma virus (HPV), and others] . Defensins might have the capacity to control viral replication by modulating the inflammatory response and serve as a bridge between the innate and the adaptive immune systems .
The aim of this work is to investigate the immunohistochemical expression of hBD-2 in verruca vulgaris, to throw the light on its possible role in the pathogenesis of the disease.
| Patients and methods|| |
This study was carried on 15 patients with verruca vulgaris, from which 15 lesional skin specimens (group I) were taken. In addition, seven nonlesional normal skin from contralateral sides of bodies of seven patients (group II) were taken. They were recruited from the Outpatient Clinic of Dermatology and Venereology Department, Tanta University Hospitals. Seven normal skin specimens were taken from seven healthy volunteers of matched age and sex who were obtained from Plastic Surgery Department, Tanta University Hospitals (group III). Groups II and III served as controls.
Group I included 10 female and five male patients. Their ages ranged from 19 to 42 years, with mean±SD of 29.80±8.01 years. Group II included five female and two male patients. Their ages ranged between 20 and 40 years, with mean±SD of 33.71±6.80 years. Group III included five female and two male individuals. Their ages ranged from 20 to 40 years, with mean±SD of 26.57±7.11 years.
The following were the inclusion criteria:
- Patients who have clinical symptoms and signs of verruca vulgaris.
- Persons who agreed to join the study and signed informed consents.
The following were the exclusion criteria:
- Patients having secondary infected verruca vulgaris.
- Patients who have other systemic or skin diseases.
- Patients who received current topical or systemic treatment for verruca vulgaris or other conditions during the previous 4 weeks.
- Patients who are pregnant or lactating.
- Patients who are cigarette smokers.
All participants included in this study were subjected to the followings:
- Complete history taking, which includes personal history, present history (onset, course, and duration of the disease), family history of warts, and past history of similar condition.
- General and dermatological examination.
- Digital photography of the lesions.
- Routine laboratory investigations including complete blood count, random blood glucose level, and liver and renal function tests.
- Skin biopsies were taken after informed written consent. A punch biopsy specimen of 4 mm was taken under local anesthesia from lesional skin of each patient. Seven nonlesional skin biopsy specimens were obtained from contralateral sides of seven patients. Other seven normal skin specimens were obtained from seven subjects during plastic surgical operations. Then the biopsies were immediately fixed in 10% formalin and embedded in paraffin, to be processed for hematoxylin and eosin staining, histopathological examination, and immunohistochemical staining  for hBD-2.
- Evaluation of immunostaining  by light microscopic examination of the slides. The result is considered positive when brownish stain is observed in the cytoplasm and/or nuclei of the keratinocytes. Scoring of skin samples was performed using a semiquantitative scoring system to evaluate the immunoreactivity (IR) of the epidermal layers against hBD-2:
- IR 0=no expression.
- IR 1=expression in the stratum corneum.
- IR 2=expression in the stratum corneum and granulosum.
- IR 3=expression in all epidermal layers.
| Results|| |
Clinical results were reported in [Table 1] and [Table 2].
|Table 1 Comparison between the studied groups regarding demographic data|
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Group I showed positive expression of hBD-2 in all of the studied specimens. Twelve (80%) specimens showed positive expression in all epidermal layers (IR 3) ([Figure 1]). Two specimens (13.3%) showed positive expression in the stratum corneum only (IR 1) ([Figure 2]) and one (6.7%) specimen only showed positive expression in the stratum corneum and granulosum (IR 2). Groups II and III showed no expression of hBD-2 (IR 0).
|Figure 1 Section of verruca vulgaris showing expression of human β-defensin-2 in all epidermal layers (immunoreactivity 3) (streptavidin biotin, ×200).|
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|Figure 2 Section of verruca vulgaris showing expression of human β-defensin-2 in stratum corneum only (immunoreactivity 1) (streptavidin biotin, ×100).|
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There was highly significant change in the expression of hBD-2 in lesional skin (group I) when compared with nonlesional skin of patients (group II) and also when compared with normal skin of volunteers (group III) (P<0.001) ([Table 3]).
|Table 3 Comparison between the studied groups regarding immune reactivity against human β-defensin-2|
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| Discussion|| |
hBD-2 are inducible in keratinocytes by bacterial infection, cytokines [interleukin (IL)-1α, IL-1β and tumor necrosis factor-α], the state of differentiation , and calcium and phorbol 12-myristate 13-acetate (PMA). This hBD-2 can be inhibited or suppressed by retinoic acid pretreatment, indicating that retinoic acid is an important regulator of the innate immune system in epidermis .
The aim of this study was to investigate the immunohistochemical expression of hBD-2 in skin lesions of verruca vulgaris in a trial to elucidate its possible role in the pathogenesis of the disease.
Regarding immunohistochemical results, the current study reported positive expression of hBD-2 in lesional skin of patients with verruca vulgaris (group I) and no expression in nonlesional skin of patients (group II) and normal skin of volunteers (group III). There was highly significant increase in the expression of hBD-2 in lesional skin (group I) when compared with nonlesional skin of patients (group II) and also when compared with normal skin of volunteers (group III) (P<0.001). This expression is observed from the stratum corneum to the stratum basale.
In accordance with our study, Meyer-Hoffert et al.  studied the IR for hBD-2 and hBD-3 in 20 biopsy specimens of verrucae vulgaris and 15 tissue samples of condylomata acuminata. In their study, specimens have been shown to be positive for hBD-2 displaying expression from the stratum corneum to the stratum spinosum. Normal skin tissue exhibited no staining of both hBD-2 and hBD-3, with the exception of two cases.
In the current study, groups II and III showed negative expression of hBD-2 whereas all cases of group I showed positive expression. In contrast to that, Wittersheim et al. , who studied expression of AMPs in healthy skin of different parts of the body, reported that hBD-2 was barely expressed in healthy skin. This hBD-2 IR was detected more frequently in older individuals. The possible cause of higher hBD-2 IR ascended with increasing age could be age-dependent skin changes including a reduced barrier function as reported by Cerimele et al. , and an accumulated ultraviolet exposure, a hypothesis supported by hBD-2 induction upon acute and chronic skin barrier disruption and ultraviolet radiation as reported by Gläser et al.  and Harder et al. .
In the current study, different biopsies sites in control groups taken from legs and forearms showed negative expression whereas biopsies sites in patient group taken from hands, feet, and forearms showed positive expression of hBD-2 without increase in certain site. In accordance to that, Wittersheim et al.  reported that hBD-2 was barely expressed in healthy skin with no increased in certain sites in contrast to psoriasin expression which was found in localizations with a high density of sebaceous glands and hair follicles, and RNase 7 expression was found in forehead, cheek, lumbar region, and buttock.
Chong et al.  have reported an increase of hBD-2 mRNA and IR in lesions of recurrent respiratory papillomatosis caused by HPV 6 and 11 . Kreuter et al.  have also reported that hBD-2 expression was shown to be significantly up-regulated in HPV-associated anal skin lesions (anal intraepithelial neoplasia) of both HIV-positive and negative patients. Erhart et al.  have reported that hBD-2 is significantly elevated at both the mRNA and protein levels in vulvovaginal HPV-induced lesions. Our findings are in concordance with these reports implicating a functional role of hBD-2 in papilloma viral infection at different body localizations.
As papilloma viruses complete their replication cycle in terminally differentiated cells and release progeny virions through desquamation of the epithelial surface, there is relatively little exposure of viral antigens to the mechanisms of immune surveillance. Therefore, infection with HPV tends to be more persistent than with other microbes. However, the vast majority of HPV infections in immune competent hosts are eventually resolved, as evidenced by the high rate of remission of primary genital HPV infection. This suggests that most infected hosts are capable of mounting an effective immune response against HPV infections .
Regarding hBD-2 induction in HPV, Harder et al.  reported that hBD-2 has been up-regulated during keratinocytes differentiation. Sherman et al.  reported that pathogenetic studies of HPV and keratinocytes interaction that have been performed mainly in HPV type 16 showed that the E6 gene of HPV-16 has been shown to inhibit calcium-induced differentiation in cultured keratinocytes. Pim et al.  have reported that defensin upregulation might not be caused by enhanced differentiation of keratinocytes. It remains speculative whether upregulation of hBD-2 in HPV skin diseases occurred indirectly by locally produced proinflammatory cytokines or directly by viral molecules. Yang et al.  reported that the expression might be induced directly as the induction occurred at the upper epidermal layers. Moreover, HPV-induced lesions did not elicit significant levels of tissue inflammation. It is also imaginable that the virus has evolved to induce expression of AMPs to prevent bacterial infections in the lesions. Inhibition of bacterial infection could help retard the focusing of cell-mediated immunity on the lesions, which is thought to be responsible for inducing lesional regression .
In epidermal keratinocytes, hBD-2 is secreted in lamellar bodies  which are lipid-containing vesicles secreted into the intercellular space. These vesicles make the skin impermeable to water and generate the antimicrobial barrier in the epidermis . β-Defensins have different effects on keratinocytes according to their level. Basal levels of β-defensins are present in epithelial cells in the absence of an inducing stimulus that may play a role in maintaining a noninflammatory environment before an immune response has been elicited perhaps by neutralizing the effects of continual low-level exposure to commensal and pathogenic microbial antigens. Defensins are expressed at a high level at the site of pathogen entry resulting in a proinflammatory response involving the chemoattraction of macrophages and other immune cells. As the danger is neutralized and defensins and other proinflammatory molecules decrease, defensins may then have a role in resolving inflammation .
At 10 μg/ml, hBD-2 mediates keratinocyte migration, and at 5–40 μg/ml, it stimulates keratinocytes proliferation. This migratory and proliferative effects to keratinocytes occur under the control of epidermal growth factor receptor and signal transducer and activator of transcription-1 and 3 activation. At 30 μg/ml, it stimulates keratinocytes to produce proinflammatory cytokines and chemokines such as IL-18, IL-6, IL-10, interferon γ-induced protein 10, monocyte chemoattractant protein-1, macrophage inflammatory protein-3α, and regulated upon activation, normal T-cell expressed and secreted, the production of which is through the G protein and phospholipase C signaling pathway .
hBD-2 has been shown to be microbicidal to a variety pathogens including bacterial, fungal, and enveloped and nonenveloped viruses such as HIV type 1, adenovirus, and HPV. The direct antiviral mechanisms of defensins include direct targeting of viral envelopes, glycoproteins, and capsids in addition to inhibition of viral fusion and postentry neutralization. Binding and modulation of host cell surface receptors and disruption of intracellular signaling by defensins can also inhibit viral replication .
hBD-2 links innate and adaptive immunity by attracting memory T cells and recruiting macrophages and immature dendritic cells (DCs) . This recruitment has been shown to occur via direct binding and activation of the chemokine receptors CCR6 and CCR2 . However, it has been reported that although hBD-2 can induce the chemotaxis of macrophages, it only weakly recruits DCs. This effect is independent of CCR6 . Overall, the ability of hBD-2 to control immune cell chemotaxis and therefore to regulate immune response to viral infection is well established, even if there remains debate about the specific cell subsets that are recruited . hBD-2 can bind DNA and promote its uptake by plasmacytoid DCs leading to interferon production .
Dendritic cells or LCs are believed to be essential for the initiation of adaptive immune response against HPV infection. DCs have been shown to interact with HPV virions and virus-like particles and thus play a role in inducing protective immunity against primary HPV infection ,,. Hence, the demonstrated role of DCs in HPV immunity together with the influence of hBDs on their recruitment makes it important to investigate the role of hBDs in papilloma virus infections. Expression of hBD-2 is elevated in HPV-infected tissue, and this might facilitate host defense against papilloma virus infection .
This expression of hBD-2 suggests that defensins might contribute to innate and adaptive immune responses targeted against papilloma virus infection. This observation is relevant to vaccine development and could provide a rationale for the development of defensin-based therapy for HPV using exogenous defensin preparations or by enhancing the production of defensins in target epithelial surfaces .
| Conclusion|| |
From these results, it can be concluded that there was positive immunohistochemical expression of hBD-2 in all lesional skin specimens taken from patients with verruca vulgaris (group I), whereas there was no expression in normal skin specimens taken from the patients (group II) and the volunteers (group III). This indicates a possible involvement of hBD-2 in the pathogenesis of verruca vulgaris. hBD-2 might also contribute to innate and adaptive immune responses targeted against papilloma virus infection in verruca vulgaris.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]