Expression And Function Of Neutrophil Elastase And Trappin-2 Protein In Psoriasis

Background and objectivesPsoriasis is a common, recurrent, chronic inflammatory skin disease, which greatly influences the physical and mental health of patients. Therefore, psoriasis has long been a focus of research in dermatology. T lymphocyte-mediated autoimmune reaction is considered as a putative mechanism of this disease. However, this mechanism cannot thoroughly explain clinical findings of psoriasis.Psoriasis vulgaris is the commonest type of psoriasis, which is pathologically characterized by Munro microabscess in skin lesions, i.e., aggregation of polymorphonuclear neutrophils(PMNs) in the stratum corneum. Munro microabscess exists only in parakeratosis areas in which the mitosis rate of keratinocytes increases significantly. Clinically, there are both"chronic"and"acute"areas in the same skin lesion of psoriasis vulgaris. In the"chronic"region, infiltration of large numbers of T lymphocytes in the dermis and migration of these cells toward the epidermis are observed microscopically. In the"acute"area, Munro microabscess is observed microscopically. The"chronic"area recovers quickly, while the"acute"area is refractory to treatment and recovers slowly. Such a clinical and histological"heterogeneity"suggests PMNs play a role in pathogenesis of psoriasis vulgaris. In psoriasis pustulosa, there are spongiform pustules (Kogoj abscess) with infiltration of many PMNs. The keratinocyte proliferation level is higher in psoriasis pustulosa than in psoriasis vulgaris. The role of PMNs in the pathogenesis of psoriasis has not been investigated thoroughly. Although, it was suggested that non-infective inflammatory reaction dominated by PMNs is mediated by T lymphocytes, the role of PMNs in the pathogenesis of psoriasis also should be further investigated.PMNs are one of the major effector cell types involved in non-specific inflammatory reactions. Over recent years, PMNs have been found to produce various immunoregulating proteases and synthesize and secrete highly active cytokines. Of these immunoregulating proteases, neutrophil elastase (NE) is a focus of interest. As a member of the serine protease/chymotrypsin superfamily, NE plays a role in tissue injury, secretion promotion, and augmentation of inflammation. It has been shown that NE plays a key role in such diseases as acute lung injury. An imbalance between NE and endogenous protease inhibitors is widely recognized as a mechanism of these diseases. NE stimulates keratinocyte proliferation through the EGFR signal pathway, which partially explains the accelerated proliferation of skin lesions with PMNs infiltration in psoriasis vulgaris. NE also acts as a proinflammatory factor, inducing the expression of multiple cytokines, forming complex cytokine networks, and resulting in cascade reactions and augmentation of inflammatory reactions. Trappin-2 protein is an endogenous NE inhibitor mainly secreted by keratinocytes and epithelial cells, which can effectively suppress NE activity through degrading NE. Trappin-2 is not expressed in the normal skin, but in inflammatory skin diseases and certain skin tumors. Trappin-2 has been shown to promote the differentiation of keratinocytes and tumor cells, and can be used as a differentiation marker for keratinocytes and certain tumor cells. Trappin-2 suppresses cell proliferation through suppressing the NE activity, and also directly suppresses the proliferation of certain tumor cells. Trappin-2 plays an anti-inflammatory role in such diseases as lung injury, and reduces the release of cytokines such as IL-6 and IL-8. Hence, trappin-2 can be utilized to suppress excessive inflammatory reactions. The balance between NE and trappin-2 is crucial for cell proliferation and inflammatory reaction.No systematic research on the role of NE and trappin-2 in psoriasis and the relationship between NE and trappin-2 and the pathogenesis and severity of psoriasis has been reported up to now. Hence, we investigated the expression of NE and trappin-2 in psoriasis, and the role of NE and trappin-2 in the psoriasis proliferation and inflammation, so as to elucidate the pathogenesis of psoriasis and find new therapeutic targets and methods of psoriasis.Methods and results1.The expression levels of NE and trappin-2 in serum and skin lesions of patients with psoriasis vulgaris and psoriasis pustulosa were quantitatively analyzed by enzyme linked immunosorbent assay (ELISA). The results demonstrated that both the NE and trappin-2 levels in the serum and skin lesions were significantly higher in the psoriasis group than in the control group (P<0.01), and the serum levels of NE and trappin-2 were positively associated with the activity of psoriasis vulgaris (Pearson correlation coefficient=0.632, 0.541, P<0.01). The NE level was significantly higher in the psoriasis pustulosa group than the psoriasis vulgaris group. But the trappin-2 level did not increase correspondingly in the psoriasis pustulosa group, suggesting an imbalance between NE and trappin-2 expression levels in the psoriasis pustulosa group.2.In order to test if NE and trappin-2 participate in the regulation of psoriasis proliferation, we constructed the trappin-2 eukaryotic expression vector pIRES2-EGFP- trappin-2, and transfected human HeLa cells with the vector using liposome to obtain trappin-2 overexpressed supernatants (97ng/ml). Then NE and trappin-2 overexpressed supernatants were used to treat HaCaT cells and the transwell psoriasis organ culture model. MTT and 3H-TdR incorporation assay demonstrated that NE significantly promoted HaCaT cell proliferation (P<0.01) in a dose-dependent manner within a certain range of NE concentration. Sivelestat, a potent NE inhibitor, effectively blocked the proliferation-promoting effect of NE. Immunohistochemical staining demonstrated that 10 IU/L NE elevated the Ki67 and p53 expression levels in the transwell psoriasis organ culture model (P<0.01, P<0.05), suggesting the significant proliferation-promoting effect of NE in psoriasis.In addition, MTT and 3H-TdR incorporation assay indicated significant, dose-dependent differences between HaCaT cells treated with trappin-2 overexpressed supernatants and the control group (P<0.01). 97ng/ml trappin-2 effectively lowered the Ki67 and p53 expression levels in psoriasis lesions (P<0.01), suggesting psoriasis proliferation suppression by trappin-2.3.The IL-6 and IL-8 expression levels in the culture supernatants were determined by ELISA, ICAM-1 expression on HaCaT cells and PBMCs of psoriasis patients was detected by Western blotting, and ICAM-1 expression in the transwell psoriasis organ culture model was detected immunohistochemically. The results demonstrated that NE alone did not enhance IL-6 and IL-8 secretion or ICAM-1 expression by HaCaT cells, but significantly increased the expression levels of IL-6, IL-8 and ICAM-1 under the stimulation of TNF-α(P<0.01). NE also elevated the expression levels of these inflammatory factors both on PBMCs and in skin lesions of psoriasis patients (P<0.01). Hence, NE may act as an inflammation-enhancing factor in psoriasis and contribute the inflammatory progress of psoriasis.In addition, trappin-2 overexpressed supernatants and TNF-αtogether effectively reduced IL-6 and IL-8 secretion and ICAM-1 expression by HaCaT cells (P<0.05). Trappin-2 overexpressed supernatants alone lowered the expression levels of IL-6, IL-8 and ICAM-1 on PBMCs and in skin lesions of psoriasis patients (P<0.01 or P<0.05). Thus, trappin-2 may act as an inflammatory suppressing factor in psoriasis and slow down the inflammatory process of psoriasis.Conclusions1.The NE and trappin-2 expression levels of serum and skin lesion of psoriasis patients are significantly higher than those in the control group, and partially correlated positively with the activity of psoriasis. Moreover, there is an imbalance between NE and trappin-2 expression levels in psoriasis pustulosa.2.Treatment of HaCaT cells and the transwell psoriasis organ culture model with NE and trappin-2 overexpressed supernatants indicates significant proliferation-promoting effect of NE and certain proliferation suppressing effect of trappin-2.3.NE is not a typical inflammatory factor. But as an inflammation-enhancing factor, it increases the expression levels of IL-6, IL-8 and ICAM-1 in psoriasis. Trappin-2 attenuates inflammatory reactions through lowering the expression levels of IL-6, IL-8 and ICAM-1 in psoriasis, thus helping recovery of the disease.This study provides an experimental basis for evaluating the role of NE/trappin-2 in the pathogenesis of psoriasis and for finding new therapeutic targets.