Abd El Kawy, Faten A.a; Abd El Azeem, Amera A.a; Abd El-Aziz, Ghada A.a; Yahea, Rehaba; El-Badawy, Nafissab
Psoriasis is a chronic, inflammatory skin disorder, affecting 2–3% of the world’s population, and is characterized by altered proliferation and differentiation of keratinocytes (KCs) 1,2.
The exact pathogenesis of psoriasis is not well understood, but growing evidence suggests the role of both innate and adaptive immune responses 3. Autoimmunity and microbial agents have been suggested to be of pathogenic evidence in the disease. Psoriasis might be considered to be a genetically determined and easily triggered state of an otherwise dormant innate immunity 4.
Toll-like receptors (TLRs) are a family of highly conserved glycoprotein pattern recognition receptors 5. They play a crucial role in the innate immune response to microbial invaders. These receptors are expressed on immune cells, such as monocytes, macrophages, dendritic cells, and granulocytes. Importantly, TLRs are not only expressed by peripheral blood cells, but their expression has been demonstrated in airway epithelium and skin, which are important sites of host–pathogen interactions 6.
Activation of TLR triggers a complex signal transduction cascade that induces the production of inflammatory cytokines and costimulatory molecules, thus initiating innate and adaptive immunity 5. Considering the role of TLRs in the innate immune response and in triggering inflammation, it has been suggested that they may be involved in psoriasis, in the recognition of exogenous, microbial products, or either of self-ligands such as fibronectin or heat shock protein (HSP) 7. The expression of TLRs has been studied, and conflicting results have been obtained. TLR1 is highly expressed in the basal KCs of normal skin, whereas TLR5 is exclusively expressed in the basal layer. In contrast, in lesional psoriatic skin, strong TLR1 staining is observed in the KCs of the upper epidermis, whereas TLR5 is downregulated in the basal KCs 8,9.
The aim of this work was to evaluate the expression of TLR2 in lesional psoriatic skin and to determine the correlation between TLR2 expression and the duration and severity of disease.
Patients and methods
Twenty patients with psoriasis vulgaris who had not received any systemic therapy for psoriasis before and had stopped the topical medication at least 6 weeks before the study were included in this study. They were diagnosed on the basis of typical clinical and histopathological features. Twenty age-matched and sex-matched volunteers were included as controls. They did not have any dermatological or systemic diseases. All patients and controls were recruited from the Outpatient Dermatology Clinic of Al Zahra University Hospital (Cairo, Egypt), during the period from September 2009 to April 2010. An informed written consent was obtained from the participants or their legal guardians before their participation in this study.
Each patient was subjected to full history taking, clinical examination, and psoriasis area and severity index (PASI) scoring 10.
A 4-mm punch skin biopsy specimen was taken from the plaques of every psoriatic patient and control.
Cutaneous biopsy was fixed in 10% buffered formalin, and treated with ascending grades of ethyl alcohol and xylene. Two paraffin sections (5 μm thickness) were obtained; one was stained with hematoxylin and eosin for histopathological examination and the other was immunostained for TLR2.
Immunohistochemical staining procedures were carried out in the 20 patients and the 20 controls. Paraffin sections to be immunostained were mounted on positively charged slides (Menzel-Glazer-Polysine, Menzel GmbH and CoKG; Braunschweig, Germany). Purified mouse polyclonal antibodies to TLR2 [TLR2 (N-17); Santa Cruz Biotechnology Inc., California, California, USA; sc-8689; Autogen Bioclear UK Ltd, Calne, Wiltshire, United Kingdom] were used as the primary antibodies to detect TLR2 in the specimens. The Mouse ABC staining system (Santa Cruz Biotechnology Inc.; sc 2023; Autogen Bioclear UK Ltd) was also used. The sections were counterstained with hematoxylin for 5–10 s. The sections were examined under a light microscope (Olympus CX41; Spach Optics, Rochester, New York, USA) using different magnifications.
Interpretation of the immunostaining results
On light microscopic examination, the epidermis was divided into basal, suprabasal, and upper layers. Immunohistochemical analysis was performed semiquantitatively according to Chaiyarit et al. 11.
The epidermis was evaluated for the site of the immunoreactive cells. The intensity of staining was graded as follows: 0=negative staining, 1=mild staining, and 2=moderate staining.
The dermis was evaluated for staining of immunoreactive cells, their grades as well as the presence or absence of stained blood/vessels. Cells were graded as follows: 0=no immunoreactive cells stained and 1=positive immunoreactive cells. Blood vessels were graded as follows: 0=negative staining and 1=staining.
The statistical analysis of data was performed using the statistical package for social science version 16 on Windows XP (IBM Corporation, New York, New York, USA). The data were described as frequency and percentage for qualitative data and mean±SD for quantitative data. The analysis of data was performed using Student’s t-test to test the statistically significant difference for quantitative data and the χ 2-test for qualitative data. The mutual correspondence between the two values was assessed using Pearson’s correlation coefficient. P value was considered significant if it was 0.05 or less at a confidence interval of 95% 12.
The study included 20 patients with chronic plaque-type psoriasis. There were 11 men and nine women. Their age ranged from 4 to 47 years (mean±SD: 23.70±13.26 years). The duration of the disease varied from 1 to 21 years (mean±SD: 6.30±4.82 years). The PASI score ranged from 8 to 46 (mean±SD: 26.90±11.13). Nine of the 20 patients (45%) reported a family history of psoriasis. The control group included 10 men and 10 women. Their age ranged from 9 to 49 years (mean±SD: 29.95±11.67 years).
Immunohistochemical results of the skin
There was positive immunohistochemical staining in the basal and suprabasal layers in all 20 controls (100%), whereas for psoriatic skin, staining in the basal and suprabasal layers was positive in eight cases (40%). There was negative immunohistochemical staining in the upper epidermal layers in all 20 controls (100%), whereas for psoriatic skin, the staining was positive in 18 cases (90%). For the controls, no specimens showed positive TLR2 staining in all epidermal layers, whereas for psoriatic skin, seven cases (35%) showed TLR2 staining in all layers with variable intensities that decreased toward the basal layer (P<0.01) (Table 1, Figs 1 and 2).
There was no correlation between TLR2 staining intensity in the epidermis and either the duration of the disease (r=−0.204, P>0.05) or the PASI scoring (r=0.151, P>0.05).
For normal control skin, 19 of the 20 specimens (95%) showed moderate TLR2 staining of the epidermis, whereas only one specimen (5%) showed mild staining and the staining of KCs was cytoplasmic in all normal control specimens. In psoriatic skin, only six specimens (30%) showed a moderate TLR2 staining of the epidermis and 14 specimens (70%) showed mild staining; the staining of KCs was cytoplasmic in 18 specimens (90%) and both cytoplasmic and nuclear in two specimens. This shows that controls had a higher intensity of TLR2 compared with psoriatic patients, with a high statistically significant difference between both the groups (P<0.01) (Table 2).
Only one control (5%) showed positive dermal staining for TLR2, whereas in psoriatic skin, 11 specimens (55%) showed positive dermal staining for TLR2; thus, psoriatic skin has a higher frequency of TLR2 expression in the dermal infiltrate compared with normal skin, with a high statistically significant difference between both groups (P<0.01). There was no statistically significant difference between both the groups in terms of staining of dermal blood vessels for TLR2 (P>0.05) (Table 3, Figs 3 and 4).
Psoriasis is a multifactorial polygenic inflammatory skin disease characterized by epidermal proliferation and an altered KC differentiation pathway 13. The exact pathogenesis of psoriasis is not well understood, but growing evidence suggests the role of both innate and adaptive immune responses 3. TLRs have found to be important pattern recognition receptors that are involved in recognizing components of microbial pathogens and initiating and instructing cutaneous immune responses. TLRs may contribute to the increased levels of antimicrobial peptides and cutaneous immune response in psoriatic skin 14. TLRs have attracted particular attention because of their main fold functions in the regulation and linking of immune and inflammatory processes 15.
The present study has demonstrated moderate cytoplasmic TLR2 staining in the basal and suprabasal epidermal cell layers in most of the control cases. This is in agreement with Baker et al. 9, who reported that TLR2 was highly expressed in normal epidermis with increased cytoplasmic staining in basal KCs. In contrast, Curry et al. 16 demonstrated that in normal human skin, TLR2 was detected on epidermal Langerhans cells and dermal dendritic cells, with a weak expression by basal layer KCs. TLR2 was focally expressed on the plasma membrane of mid-epidermal KCs. In addition, Begon et al. 15 reported that TLR2 was consistently expressed in KCs of normal skin predominantly in the lower layers. Staining was strong and diffuse throughout the cytoplasm for TLR2.
The present study showed that in the epidermis of psoriatic skin, TLR2 was more highly expressed on KCs of the upper epidermis rather than the basal layer. A statistically significant difference was detected on comparing the site of expression of TLR2 between psoriatic skin and normal skin. The pattern for TLR2 was either cytoplasmic or both cytoplasmic and nuclear. This is in agreement with Baker et al. 9, who reported that TLR2 was more highly expressed on KCs of the upper epidermis than on those of the basal layer in plaque psoriasis. Begon et al. 15 reported that the intracytoplasmic localization was confirmed by immunohistochemistry that revealed a strictly cytoplasmic paranuclear distribution of TLR staining.
The current study revealed a cytoplasmic pattern of immunostaining of TLR2 in all samples of normal skin. The intracytoplasmic localization observed in KCs contributes to the hyporesponsiveness of these cells to microbial stimulation 9. Baker et al. 9 reported that unexpectedly, staining of KC nuclei by the TLR2 antibody was observed in psoriatic skin, but was absent in normal skin. Evidence for the presence of TLRs in cell nuclei has not been previously reported, except by Baker et al. 9, who failed to find any appropriate explanation for this finding. Further studies are required to explain the validity of this observation.
Wu et al. 17 studied TLR2 and TLR4 expression in KC after incubation with mouse anti-K16 antibodies to induce psoriasis and they reported an increased expression of TLR2 and TLR4 mRNA in cultured KCs by quantitative real-time PCR and flow cytometry. In contrast to our results, Matsumoto et al. 18 reported that TLR2-mediated function of KCs was reduced in psoriatic skin because of the reduced expression of TLR2 on their surface, suggesting that the impaired innate immune function is one of the factors responsible for the development or exacerbation of psoriasis.
The present study showed a highly significant difference between normal and psoriatic skin in TLR2 expression in the dermal inflammatory infiltrate, which may be related to the disease activity. Whether this finding is primary in the pathogenesis of psoriasis or only reflects secondary reactive changes induced by inflammation could not be determined. Further studies are required to clarify this issue. In the current study, TLR2 immunostaining in dermal blood vessels was positive in only two psoriatic skin biopsies and was negative in all normal skin of the controls. Baker et al. 9 detected TLR1 and to a lesser extent TLR2 expression in dermal blood vessels. In addition, another study of cultured human dermal microvessel endothelial cells demonstrated strong TLR1, 3, 4, and 5, but weak TLR2 mRNA expression and lack of staining with anti-TLR2 antibody 15.
It has been reported that TLR2 was overexpressed in KCs of psoriatic skin, and this overexpression could result from an inappropriate stimulation by TLR2 ligands expressed on KCs surface or it might be a consequence of the proinflammatory skin environment in psoriatic skin 15. In the light of previous results for antigenic stimulation in psoriasis, the role of two TLR2 ligands, streptococcus and HSPs, was discussed. Streptococcus, as a Gram-positive bacterium, is a natural TLR2 ligand 19,20. Begon et al. 15 hypothesized that a facilitated TLR2 stimulation by streptococcus could lead to an excessive inflammatory response. TLR2 stimulation by endogenous HSPs, or streptococcus HSPs, may prolong the inflammatory response and contribute to the pathogenesis of psoriasis. TLR2 overexpression could be an indirect sign of this continual stimulation. Considering the fact that the total numbers of bacteria isolated from psoriatic plaques are significantly higher than those on adjacent normal skin and that colonization of lesional skin of psoriasis by Staphylococcus aureus has been demonstrated in 20–50% of patients with psoriasis 21, upregulation of TLR2 expression in the upper layers of the epidermis in psoriatic lesions might occur in response to the presence of Gram-positive bacteria on the horny layer 9. Psoriasis is characterized by increased KCs proliferation and infiltration of inflammatory cells through the release of cytokines 22. Accordingly, the modulation of TLR2 expression observed in psoriasis might be secondary to the altered KC pathway or might result from the effect of proinflammatory cytokines such as tumor necrosis factor--α and interferon-γ present in this disease 15.
The present study has shown that TLR2 is expressed in the epidermis of the normal skin, where they probably play a protective role against infection by various microbial agents. Moreover, modulation of TLR2 expression has been demonstrated in the skin lesions of psoriasis, where TLR2 is more highly expressed on KCs of the higher epidermis than the basal layer compared with normal skin. Accordingly, TLR2 may play an essential role in the pathogenesis of psoriasis. In addition, there was no significant correlation between TLR2 expression and the severity and the duration of the disease. Further studies are required to clarify the role of this group of innate receptors in the pathogenesis of psoriasis as this may offer a new preventive and therapeutic strategy through TLR2 antagonists for psoriasis.
In conclusion, TLR2 overexpression in psoriatic skin provides new insights into the role of TLR in the pathogenesis of psoriasis, through inappropriate stimulation by infectious or endogenous ligands. Furthermore, TLR2 targeted by specific pharmacologic TLR2 antagonists may offer new therapeutic modalities and preventive approaches for psoriatic patients.
Conflicts of interest
There are no conflicts of interest.
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