Upregulation of human β-defensin-3 and cathelicidin LL-37 in Kaposi’s sarcoma

Background: Kaposi’s sarcoma (KS) is a rare neoplasm of lymphatic endothelial cells. Human herpes virus 8 (HHV-8) is considered to be a necessary, but not sufficient causal agent of KS and additional cofactors remain unknown. In this study we evaluated the expression of human β defensin (HBD)-3 and LL-37 in cutaneous lesions of KS in comparison to the healthy skin of normal subjects. Methods: We performed a quantitative immunohistochemical study of HBD-3 and LL-37 on skin lesions from 18 patients having KS, and on healthy skin from 12 normal controls. Results: HBD-3 and LL-37 were significantly upregulated in epidermal and dermal specimens of all KS patients in comparison to normal skin of healthy controls. The immunostaining score of dermal HBD-3 was significantly higher in nodular lesions (9.6 ± 2.4) versus plaque lesions (4.1 ± 2.2), P = 0.001. Also the immunostaining score of dermal LL-37 was significantly higher in nodular lesions versus plaque lesions (P = 0.001). Conclusions: We have demonstrated for the first time that HBD-3 and LL-37 are significantly upregulated in lesional skin of KS in comparison to the skin of healthy controls. The obtained data suggest a possible involvement of these antimicrobial peptides in the pathogenesis of KS. However, the biological significance of HBD-3 and LL-37 in KS lesions needs further research.

KS is strongly associated with human herpes virus 8 (HHV-8), which is implicated in the pathogenesis of all forms of KS 3 . HHV-8 is present in the vast majority (> 90%) of spindle cells and in the neoangiogenic vessels 4-6 . It is considered to be a necessary, but not sufficient causal agent of KS. Besides immunosuppression and AIDS, additional cofactors remain unknown 7 .
Ensoli et al. 8 speculated that early stage KS is a reactive inflammatory angiogenic process that may be triggered or enhanced by infection with HHV-8 with many lymphocytes and monocytes infiltrating the lesion. These cells produce inflammatory cytokines including interferon-y (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-2, IL-6 and others 9 . The inflammatory cytokines induce the recruitment of circulating cells into tissues, induce the production of angiogenic factors that mediate angiogenesis and edema and activate endothelial cells to acquire the phenotype of KS spindle cells 10 .
Antimicrobial peptides (AMPs) are an evolutionarily conserved component of the innate immune system that defend against invading bacteria, viruses and fungi through membrane or metabolic disruption 11 . This diverse group of peptides is separated into several classes. Defensins and cathelicidin (also known as LL-37) are considered the two main groups of AMPs in human skin 12 . It was confirmed that human β defensin (HBD)-3 and LL-37 demonstrated the ability to inhibit viral infection, affecting both enveloped RNA and DNA viruses and non-enveloped viruses 13-21 . Moreover, beside their role in protecting the host from the invasion of pathogens, HBD-3 and LL-37 have immunomodulatory properties in inflammatory skin diseases as psoriasis 22,23 and rosacea 24 .
Based on the antiviral and immunomodulatory effect of HBD-3 and LL-37, as well as the angiogenic role of LL-37 25 , this study was carried out to assess the expression of the two main antimicrobial peptides (HBD-3 and LL-37) in skin lesions of KS in comparison to normal skin from healthy subjects. A lesional skin biopsy (intact lesion without evidence of secondary infection) was obtained from each patient. Biopsy from 12 normal subjects served as controls. They were obtained from normal skin beside benign neoplasms such as melanocytic naevus, infundibular cyst and lipoma of age, sex and localization-related subjects. Informed consent was taken from all participants. The study protocol was approved by the ethical committee of the College of Medicine of Mansoura University.

Immunohistochemistry
All specimens were fixed in formalin 10% and sections from paraffin blocks (3-4 um) were cut on glass slides for routine hematoxylin and eosin stains as well as immunohistochemical staining using an bindirect avidin-biotin-peroxidase method. Endogenous peroxidase -We have also replaced the panels I in Figure 1 so that they now have the same resolution (x400) as panel J. We have also included arrows onto panels E and I in Figure 1 to indicate positive immunohistochemical staining of HBD-3 and LL-37 in endothelial and spindle shaped cells.

See referee reports
activity was blocked with 0.6% H 2 O 2 . After blocking, sections were incubated at room temperature for 60 minutes with antibodies to HBD-3 using rabbit polyclonal antibody (Catalog number D2444; Sigma Aldrich, St. Louis, USA) at a dilution of 1:2 and with human LL-37 monoclonal antibody (Catalog number HM2070; Hycult Biotech, Frontstraat 2a, 5405 PB Uden Netherland) at a dilution of 1:500. Diaminobenzidine (DAB) reaction was used for visualization, followed by a hematoxylin counterstain. Negative controls for all studies were obtained by omission of the primary antibodies of an adjacent section to assess the degree of non-specific staining. All slides were examined by Olympus light microscope.

Immunohistochemical analysis of HBD-3 and LL-37
Immunostaining results of HBD-3 and LL-37 were evaluated in four layers of the epidermis and the proposed score was modified from Meyer-Hoffer et al. 20 as follows: 0, none; 1, stratum corneum only; 2, stratum corneum and stratum granulosum; 3, stratum corneum, stratum granulosum and stratum spinosum; 4, whole epidermis. Next an intensity score was assigned, which represented the average intensity of positive epidermal cells as follows: 0, none; 1, weak; 2, moderate; 3, intense staining. Both scores were then added to obtain a total epidermal score which ranged from 0 to 7. Skin samples of psoriasis that are known to exhibit high expression of HBD-3 and LL-37 in the epidermis were used as positive controls. Immunostaining results of dermal lesions of KS were scored as previously described 29 . Immunoreactivity of HBD-3 and LL-37 were evaluated in the two basic component of this disease (spindleshaped cells and endothelial cells of newly formed vessels). The percentage of positive cells was graded from 0 to 4 as follows: 0, zero to 10%; 1, 11 to 33%; 2, 33 to 66%; 3, 67 to 90%; and 4, 91 to 100%. Specimens were considered immunopositive when more than 10% of cells showed clear evidence of immunostaining. The intensity of immunostaining was rated as follows: 0, none; 1, weak; 2, moderate; and 3, intense. Because KS lesions frequently showed significant intra-specimen heterogeneity, a score was calculated in which the percentage positive rating was multiplied by the intensity rating. Each component of the lesion was scored independently and the results were added up. The score was calculated on 6 to 10 representative high-power fields after examination of the totality of lesion present in one section for each case. Sweat glands served as internal positive controls for LL-37. Inflammatory cells (monocytes and macrophages) served as internal positive controls for both studied AMPs. However, specimens from healthy subjects served as negative controls. Slides were scored by two independent and trained researchers (the authors).

Statistical analysis
Data were analyzed using SPSS version 16. Qualitative variables were presented as numbers and percentages. Quantitative variables were presented as mean ± SD (median) and a Mann-Whitney test was used for group comparison. Spearman's correlation coefficient was used to calculate correlation between variables in patients with KS. P ≤ 0.05 was considered statistically significant.

Results
The mean age of the patients (fourteen males and four females) was 63.9 ± 7.4 years (ranged from 55 to 82 years). The age and sex-matched control group were eight males and four females with a mean age of 63.2 ± 3.7 years (ranged from 45 to 67 years). All investigated patients had classic KS without clinical evidence of immunodeficiency and no history of immunosuppressive drug intake. They had normal total and differential leukocytic count, normal blood chemistry and negative HIV serology. Fourteen KS patients were classified as stage I (≤ 15 cutaneous lesions) and 4 patients as stage II (> 15 cutaneous lesions) (Table 1). No patients had mucosal lesions, lymphadenopathy or clinical or radiological features suggesting visceral involvement and none had gut nodules as determined by endoscopy (which was only done to stage II KS patients).
Immunohistochemical staining of HBD-3 and LL-37 were generally cytoplasmic, and membranous staining was also seen. There was significant upregulation of HBD-3 and LL-37 in both epidermal and dermal specimens of all studied patients in comparison to normal skin of healthy controls. The expressions of HBD-3 and LL-37 were seen in the epidermis as well as in the dermis (neoformed vessels, spindle cells and inflammatory cells) ( Figure 1D, 1H) with different scores of immunoreactivity in KS lesions (Figure 1). Also positive immunohistochemical staining for LL-37 was seen in the sweat glands of KS lesions ( Figure 1C). HBD-3 and LL-37 were absent in the epidermis and dermis of control normal skin ( Figure 1G, 1K). Only three sections of control normal skin showed weak staining within the stratum corneum for HBD-3 and only two sections for LL-37 within stratum granulosum.

Discussion
Recently many reports about the antiviral effect of defensins and cathelicidin have been published. The antiviral activity of HBD-3 has been reported against herpes simplex virus (HSV) 13 , vaccinia virus 14 , and HIV 15 . Similarly LL-37 is found to inhibit replication of vaccinia virus 16 , kill HSV 17 , is effective against adenovirus 18 and inhibits HIV-1 replication in peripheral blood mononuclear cells 19 . Furthermore, HBD-3 expression was shown to be upregulated in human papillomavirus induced lesions 20,21 .
In our study we investigated for the first time the expression of HBD-3 and LL-37 in KS. They were significantly upregulated in epidermal and dermal (neoangiogenic vessels, spindle cells and inflammatory cells) regions of all studied KS lesions in comparison to normal skin of healthy subjects. These findings suggest a potential role of HBD-3 and LL-37 in the pathogenesis of KS. Furthermore, we found that the expressions of these AMPs were increased with progression of KS lesions from plaque stage to nodules. The stage-related differences that we found in HBD-3 and LL-37 expression add more support to the possible role of these AMPs in the progression of KS.
The induction of HBD-3 and LL-37 in KS may be in response to infection with HHV-8 that is implicated in the pathogenesis of all forms of KS 3 . HBD-3 and LL-37 might exhibit antiviral activity against HHV-8 as shown for the related HSV 13,17 .
In addition to functioning as direct antimicrobial compounds, AMPs can function as chemokines 30 . It was found that LL-37 increases natural killer cell proliferation by activating the Toll like receptor 9. LL-37 increases proinflammatory cytokines at the dendritic cell level, promoting CD4 + TH1 cell responses 31 . LL-37 can synergize with IL-1β to increase the production of cytokines, such as IL-6, IL-8 and IL-10, and chemokines, such as the cc-chemokine ligand 2 32 . Similarly to cathelicidin, HBD-3 has chemoattractant properties on different cell types such as T lymphocytes and dendritic cells 33 . Furthermore, HBD-3 and cathelicidin induce the production of diverse chemokines and cytokines such as monocyte chemotactic protein-1, macrophage inflammatory protein-3, interferon-inducible protein-10, IL-1, IL-6, IL-8, IL-10 and TNF-α mainly in keratinocytes 34,35 . It remains unclear whether KS itself is a true malignancy, a reactive proliferation or both 28 . Whatever the nature of KS, HBD-3 and IL-37 may serve as a growth factor for KS. This is supported by the finding that the expression of these AMPs were increased with progression of KS lesions from plaque stage to nodules in our study. Recent evidence suggests cathelicidin LL-37 to be a putative growth factor for various human cancers 40-43 . Similarly, HBD-3 may play an important role in the development and progression of oral cancer. HBD-3 stimulated the expression of tumor-promoting cytokines, including IL-1α, Il-6, Il-8 and TNF-α in macrophages 44 . These cytokines are also important in the progression of KS 36 . Further study to elucidate the hypothesis that HBD-3 and LL-37 serve as progression factor for KS is therefore recommended. It is not known whether upregulation of HBD-3 and LL-37 in KS lesions is signaled indirectly by locally produced proinflammatory cytokines or directly by HHV8 molecules. HBD-3 is upregulated by proinflammatory cytokines, TNF-α, IFN-γ and IL-1β 22 . However IL-6 is a potent inducer of LL-37 45 . Interestingly, these inflammatory cytokines are abundant in KS lesions 9 . Furthermore, the possibility of induction of these AMPs by HHV-8 protein can be supported by the finding of increased expression of HBD-3 and LL-37 with progression of KS lesions from plaque into nodular stage in our study. Similarly, increased load of HHV-8 has been found with progression of KS lesions 5 . Further study is recommended to verify this point.

Conclusions
HBD-3 and LL-37 are for the first time shown to be significantly upregulated in KS skin lesions as compared with skin of healthy controls. The obtained data suggest a possible contribution of HBD-3 and LL-37 in the innate and adaptive immune response target against HHV-8. Another possibility is the potential involvement of these antimicrobial peptides in the pathogenesis of KS. However, the biological significance of HBD-3 and LL-37 in KS lesion needs further research.

Consent
Written informed consent for publication of clinical details was obtained from the patients.
Author contributions HF: participated in study design, collecting data, clinical evaluation, participated in pathological evaluation, interpretation of data, writing the manuscript, and i deciding to submit the manuscript for publication. MMA: participated in collecting data, reviewed the pathologic material, photographed the slides, analysis, revision and approval of final and revised manuscript draft, and in deciding to submit the manuscript for publication. AHG: analysis and interpretation of data. All authors read and approved the final manuscript.

Competing interests
The author(s) declared that they have no competing interests.

Grant information
The author(s) declared that no grants were involved in supporting this work.