Psoriasis is a chronic inflammatory disorder in which both genetic and immunologic factors play a role 1. The role of T-helper (Th1) cells in the initiation and maintenance of psoriatic lesions has been documented. Activated T cells in the region of the dermal–epidermal junction are believed to drive the hyperplastic proliferative response through Th1 cytokines including tumour necrosis factor α (TNF-α), interferon γ and interleukins (ILs) 2–4.
Leptin, a 16 kDa adipocyte-derived hormone, is involved in the regulation of a wide range of biological responses including energy homeostasis, haematopoiesis, neuroendocrine function and immune responses 5,6. In addition, leptin mediates proliferative and antiapoptotic activities in different cell types including T cells, macrophages and eosinophils 7,8. Leptin exerts its biologic actions through the activation of its cognate receptors, which belong to the type I cytokine receptor superfamily. The leptin receptor (leptin R) is primarily expressed in the hypothalamus, but it is also expressed by peripheral blood mononuclear cells, vascular endothelial cells, smooth muscle cells, osteoblasts and fibroblasts 9.
Leptin links the proinflammatory Th1 immune response to the nutritional status and the energy balance as both CD4 and CD8 T lymphocytes express leptin R 10. Moreover, several reports have shown that leptin stimulation leads to enhanced production of TNF-α and IL-6 by monocytes and IL-2 and interferon γ by T lymphocytes 11,12.
The role of leptin in psoriasis is still debatable, with some studies reporting hyperleptinaemia in psoriasis 13 and the presence of the leptin gene 2548 G/A polymorphism in psoriasis with metabolic syndrome 14 and others not reporting such an association 15.
The aim of the current study was to evaluate the level of leptin and leptin R mRNA in lesional psoriatic skin and to determine the effect of PUVA, which is a standard therapeutic tool for the treatment of psoriasis, on the values of leptin and leptin R mRNA in psoriatic patients in an attempt to shed some light on the role of leptin in the pathogenesis of psoriasis.
Patients and methods
Twenty-one patients with chronic plaque psoriasis and 15 age-matched, sex-matched body weight-matched healthy volunteers, who served as controls, were recruited from the Dermatology Outpatient Clinic, Kasr El-Aini Hospital, Cairo University, in the period from 2008 to 2010; an informed consent was obtained from each of the participants.
Patients with psoriasis that covered more than 30% of the body surface area, justifying treatment with PUVA therapy, were included. Children younger than 12 years of age, and pregnant and lactating women were excluded. Patients with pustular, erythrodermic or arthropathic psoriasis and chronic liver disease were excluded. Patients who had received PUVA or systemic treatment for psoriasis in the past 6 weeks and those who had used any topical medication other than emollients 2 weeks before presentation were also excluded.
All patients recruited were subjected to the following: at the first visit, complete personal and medical history was assessed, and routine laboratory investigations, general and skin examination were carried out; an ophthalmologic examination was also performed. The baseline pretreatment psoriasis area and severity index (pre-PASI) was calculated for all patients before the initiation of therapy and at the end of 10 weeks post-treatment (post-PASI). A 4 mm punch skin biopsy was obtained from a psoriatic lesion before the initiation of therapy and at the end of 10 weeks. Each biopsy was divided into two parts. The first part was placed in lysis buffer for protein extraction for the measurement of leptin by enzyme-linked immunosorbent assay (ELISA) and the second part was placed in lysis buffer for RNA extraction and the measurement of leptin R mRNA by reverse transcriptase PCR, and then stored at −70°C.
The patients received PUVA therapy three times/week for 10 weeks (30 sessions) in a PUVA 1000 (Waldmann GmbH, Villingen-Schwenningen, Germany) cabin equipped with an integrated UV radiometer equipped with F85/100 W fluorescent lamps that emits UV light in the wavelength range of 315–400 nm, with a peak emission at 355 nm. The patients received 0.7 mg/kg 8-methoxypsoralen 2 h before phototherapy. If thick scales were present, topical keratolytics were prescribed. The Ultraviolet A (UVA) dose was started at 2 J/cm2 for skin types IV and V and 1 J/cm2 for skin type III. The dose of UVA was increased by 0.5 J/cm2 every other session until mild erythema was achieved, and then the dose was fixed 16. Liver function tests and ophthalmologic examination were repeated every 4 weeks for the patients.
Measurement of leptin level
Leptin was examined using a quantitative ELISA. The kit is manufactured and distributed by (Quantikine R&D System Inc., Minneapolis, Minnesota, USA). Tissue leptin was measured by homogenizing 30 mg of tissue specimen in 1 ml lysis buffer for protein extraction. This buffer contained 0.0625 mol/l Tris buffer (pH 6.8), 2% SDS, 3% 2-mercaptoethanol, 10% glycerol, 10 μg/ml aprotinin and 1 mmol/l phenyl methyl sulphonyl fluoride (Sigma, St Louis, Missouri, USA). After cell lysis, the homogenate was centrifuged at 8000 rpm for 20 min at 4°C. The supernatant was kept frozen at −70°C until analysis 17.
Reverse transcriptase polymerase chain reaction detection of leptin receptor gene expression
Total RNA was extracted from skin tissue homogenate according to the procedure of Chomczynski and Sacchi 18. The content and purity of RNA were measured using an ultraviolet spectrophotometer (A260/A280 ratio of 1.8–2.0). First-strand cDNA was synthesized from total RNA, which was reverse transcribed with the Moloney murine leukaemia virus RT (Promega, Madisson, Wisconsin, USA). The reaction tubes were incubated at 37°C for 60 min, further denatured at 95°C for 10 min and quick-chilled on ice. cDNA obtained from different RNA samples was amplified in 2 mmol/l MgCl2, PCR buffer (Promega), 0.2 mmol/l deoxynucleoside triphosphates and 50 pmol of specific primer pairs to 2 U of Taq DNA polymerase (Promega). For the semiquantitative PCR, the newly synthesized DNA sample was mixed with the housekeeping β-actin primer pairs plus the leptin R primer pairs in the same tube. All PCRs were carried out using a hot-start program (60°C for 10 min). The reaction tubes were heat denatured at 95°C for 60 s, annealed at 55°C for 60 s and the primer was extended at 72°C for 90 s. The reaction parameters were programmed in a Programmable Thermal Controller (Thermolyne-Temp-Tronic, Dubuque, Iowa, USA) and repeated for 30 cycles. The final step was set at 72°C for 10 min and then 4°C indefinitely. PCR products were electrophoresed on 2% agarose gels, stained with ethidium bromide, using the DNA marker (GeneRuler 100 bp DNA Ladder, 100–1000 bp, Thermo Fisher Inc., Webster, Texas, USA), and visualized using an ultraviolet transilluminator. Semiquantification was performed using the gel documentation system (BioDO Analyser, Biometra, Gottingen, Germany). According to the amplification procedure, the relative expression of each studied gene (R) was calculated according to the following formula: densitometrical units of each studied gene/densitometrical units of β-actin.
The oligonucleotide primers sequence (according to GenBank Accession number: G15320 T67103).
- Forward: 5′-GAAGATGTTCCGAACCCCAAGAAT-3′
- Reverse: 5′-CTAGAGAAGCACTTGGTGACTGAA-3′
- Forward: 5′-TCA CCC TGA AGT ACC CCA TGG AG-3′
- Reverse: 5′-TTG GCC TTG GGG TTC AGG GGG-3′
Data were described statistically in terms of mean±SD, frequencies and percentages when appropriate. Comparisons between groups were carried out using the χ2-test and Fisher’s exact test for qualitative variables. Independent-samples t-test was performed for normally distributed quantitative variables. The Mann–Whitney U-test was used for quantitative variables, not normally distributed. Correlations were performed to test for the linear relations between variables. The significance level was set at P less than 0.05. Statistical analyses were performed using a software package statistical package for the social sciences, version 15.0 for Windows (SPSS Inc., Chicago, Illinois, USA).
This study included 21 (11 women and 10 men) patients with psoriasis vulgaris whose age ranged between 14 and 68 years, with a mean of 41.1±17.07 years. Fifteen (eight women and seven men) healthy volunteers whose ages ranged between 23 and 60 years, with a mean of 40.8±10.93 years, served as controls. The mean body weight of the patients was 73.95±13.47 kg and that of the control individuals was 70.73±13.046 kg. There was no significant difference between the patient group and the control group in terms of age, sex and body weight (P=0.949, 0.955 and 0.488, respectively).
Psoriasis area and severity index score
The mean pre-PASI was 14.85±6.03, whereas the post-PASI was 5.32±2.82. The post-PASI score was significantly lower than the pre-PASI (P<0.001). The mean percentage of reduction in the PASI score was 56.2%.
The baseline level of pretreatment leptin in patients ranged from 5.17 to 16.50 ng/mg, with a mean of 9.80±3.39 ng/mg. At the end of 10 weeks, the level of post-treatment leptin in patients ranged from 4.93 to 10.3 ng/mg, with a mean of 7.15±1.88 ng/mg (Table 1).
In the control group, leptin levels ranged from 0.67 to 6.38 ng/mg, with a mean of 2.39±1.55 (Fig. 1). The control values were significantly lower than the pretreatment and post-treatment leptin levels in the patient group (P<0.0001) in both (Table 1).
Leptin levels decreased significantly at the end of 10 weeks in comparison with its baseline levels, where the P value was less than 0.0001 (Table 1). The mean percentage of reduction in leptin levels for patients was 23.3%.
Before treatment, there was a positive correlation between the PASI score and leptin (r=0.564, P=0.008) in the patient group; this correlation was still present between pretreatment PASI and post-treatment leptin (r=0.647, P=0.002; Fig. 2).
Pretreatment levels of leptin showed a positive correlation with their post-treatment levels (r=0.733, P=0.0001). The reduction in leptin levels did not correlate with the reduction in the PASI score (r=0.332, P=0.142).
Leptin receptor mRNA
The baseline level of pretreatment leptin R. mRNA in patients ranged from 0.62 to 3.1, with a mean of 1.48±0.81. At end of 10 weeks, the level of post-treatment leptin R mRNA in patients ranged from 0.42 to 1.92, with a mean of 0.92±0.42 (Table 1).
In the control group, leptin R mRNA ranged from 0.1 to 1.31, with a mean of 0.46±0.35 (Fig. 1). These values were significantly lower than the pretreatment and post-treatment leptin R mRNA levels in the patient group (P<0.0001, P=0.001), respectively (Table 1).
Leptin R mRNA levels decreased significantly at the end of 10 weeks in comparison with its baseline levels, where the P value was 0.001 (Table 1). The mean percentage of decrease in leptin R mRNA for patients was 28%.
In the patient group, before treatment, there was a positive correlation between the PASI score and leptin R mRNA (r=0.634, P=0.002). This correlation was still present between pretreatment PASI and post-treatment leptin R mRNA (r=0.511, P=0.018; Fig. 2).
Pretreatment leptin R mRNA levels showed a positive correlation with their post-treatment levels (r=0.566, P=0.008). The reduction in leptin R mRNA did not correlate with the reduction in the PASI score or the reduction in leptin levels (r=0.419, P=0.059; r=0.263, P=0.249), respectively.
There was a positive correlation between pretreatment leptin and pretreatment leptin R mRNA levels (r=0.552, P=0.009).
In this work, significantly elevated lesional leptin and leptin R mRNA values were found in psoriasis skin compared with the normal skin of control individuals. These values showed a significant reduction after PUVA therapy. The observation of high tissue leptin and leptin R mRNA levels points to the possible role of leptin in the pathogenesis of psoriasis. In agreement with our findings, hyperleptinaemia was reported to be associated with psoriasis in several studies 13,19,20. Contrasting results have been found in other studies by Özdemir et al.21 and Aktan et al.22, who failed to find elevated serum leptin levels in their psoriatic patients. The difference between their results and ours may be methodical as the previously mentioned studies had assessed serum leptin values, whereas this study had assessed tissue leptin levels. Leptin plays an important role in inflammatory processes involving T cells and has been reported to modulate Th1 cell activity 23,24. Leptin was shown to activate monocytes and macrophages and to potentiate proinflammatory cytokine production as TNF-α, IL-6, and IL-9 and direct T-cell differentiation to the Th1 phenotype 25–27. Furthermore, leptin has been shown to stimulate keratinocyte proliferation, the expression of adhesion molecules and angiogenesis 28–30. More recently, leptin was shown to exert an antiapoptotic effect on human dendritic cells, licensing them towards Th1 priming and promoting DC survival 31. Because of these diverse immunological effects, leptin was suggested to play a role in the pathogenesis of psoriasis, especially when associated with obesity, metabolic syndrome and cardiovascular disease 32.
In our work, pretreatment and post-treatment leptin and leptin R mRNA levels showed a positive correlation with the pretreatment PASI scores. This means that leptin and leptin R levels could be considered as markers or indicators of the severity of psoriasis. Our results were in agreement with those of Cerman et al.32, who found elevated serum and tissue leptin and leptin R in severe psoriasis when compared with mild–moderate psoriasis and normal controls, and suggested that leptin might serve as a marker of severity in psoriasis as well as a cofactor augmenting the inflammatory response. They also suggested that leptin may be a pathogenetic factor contributing towards the chronicity of psoriasis 32. Moreover, several studies investigating leptin in immune-mediated inflammatory diseases have shown results similar to ours. Elevated serum leptin levels were found in patients with active rheumatoid arthritis 33 and in 35 patients with Behcet syndrome 34, showing a positive correlation with disease activity in both conditions.
The current work found a positive correlation between pretreatment and post-treatment leptin and leptin R mRNA values. This means that patients with higher pretreatment leptin and leptin R values had higher respective post-treatment values.
Moreover, a positive correlation was found between pretreatment leptin levels and leptin R mRNA values, indicating that high leptin levels were associated with higher levels of its corresponding receptor.
Leptin and leptin R mRNA were significantly reduced after PUVA therapy. Whether this reduction is a direct effect of PUVA therapy or a sequel to the improvement in psoriasis remains unclear. We suggest that this decrease in leptin levels might be related to the efficacy of PUVA in the treatment of psoriasis as published work has hypothesized that proinflammatory mediators in psoriasis stimulate leptin expression, which leads subsequently to metabolic dysregulation. IL-1, IL-6 and lipopolysaccharides were shown to regulate leptin mRNA expression and circulating leptin levels 35. Furthermore, leptin has been reported to be produced by inflammatory regulatory cells, suggesting that leptin expression could trigger or participate in inflammatory processes through direct paracrine or autocrine action 36. It is well known that PUVA’s mechanism of action in psoriasis stems from its toxic effects on activated lymphocytes 37; thereby, it could be speculated that PUVA may result in the reduction of leptin through its effect on T lymphocytes. Moreover, PUVA has been shown to reduce macrophages, dendritic cells and epidermal Langerhans’ cells in psoriatic lesions 38. Accordingly, PUVA would presumably lead to the downregulation of the proinflammatory signals that upregulate leptin expression.
In contrast to our findings, a recent study reported a reduction in serum resistin, but not serum leptin in psoriatic patients after phototherapy 39. The difference between their results and our work might be attributed to methodical variations, as the authors evaluated serum leptin and resistin levels before and after phototherapy by ELISA.
In the current work, there was no significant correlation between the reduction in leptin and the reduction in the PASI score in our patients following PUVA therapy. Thus, we could surmise that although leptin might play a role in the therapeutic efficacy of PUVA, it cannot be considered a marker of treatment response or therapeutic efficacy.
A limitation in our study was our inability to study leptin and its receptor levels in perilesional and normal skin, as well as in the serum of psoriatic patients. This would have provided a more global view of the pathogenic effects of leptin and might have provided more insights into the suspected causal versus association relation between leptin and psoriasis.
This study found elevated tissue leptin and leptin R mRNA in lesional skin in psoriasis that was associated with more severe psoriasis, suggesting that leptin might be considered as a marker of severity in psoriasis vulgaris. It also showed a significant reduction in their levels after PUVA, suggesting that decreased leptin might be related to the clinical efficacy of phototherapy in psoriasis. It is still unclear whether an association or a causal relationship exists between leptin and psoriasis. The effects of leptin on immune cells and keratinocytes within psoriatic lesions need to be further studied. Moreover, drugs targeting the proinflammatory effects of leptin might provide new adjuvant therapeutic approaches in psoriasis.
Conflicts of interest
There are no conflicts of interest.
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