The Study Of Association Between Psoriasis Vulgaris And IL-20, IL-20 Receptor And IL-15

Epidermal overexpression of interleukin - 20, - 20R and - 15 mRNA in psoriatic skinIntroductionA structural, profile - based algorithm was used to identify interleukin 20 (IL -20) , a novel IL -10 homolog. Chromosomal localization of IL -20 led to the discovery of an IL - 10 family cytokine cluster. Overexpression of IL -20 in transgenic (TG) mice causes neonatal lethality with skin abnormalities including aberrant epidermal differentiation. Recombinant IL -20 protein stimulates a signal transduction pathway through STAT3 in a keratinocyte cell line, demonstrating a direct action of this ligand. An IL - 20 receptor was identified as a heterodimer of two orphan classâ…¡cytokine receptor subunits. Both receptor subunits are expressed in skin and are dramatically upregulated in psoriatic skin. Taken together, these results demonstrate a role in epidermal function and psoriasis for IL -20, a novel cytokine identified solely by bioinformatics analysis. To study association between psoriatic with IL - 20, IL - 20 receptor and IL - 15, we determined expression of their in psoriatic lesions by situ hybridization.Materials and MethodsObjectsNormal human skin:Twenty biopsies of normal skin were obtained from trunk and extremities. No tumor or inflammation involved in the biopsies and all biopsies were exluded of subcutis.Psoriatic lesions from skin diseases:Psoriatic lesions from 35 patients with psoriasis. All were performed diag- nosis confirmation by clinical, pathologic and immunopathologic features, etc.Treatment of samples1. Fixation and embedding of tissues2. Preparation of thin sections:The sections (6μm thick) were made using a rotary microtome. Each sectionwas stretched on the surface of a drop of DEPC - treated water mounted on a 3 - aminopropyltriethoxysilane - coated glass slides. Air - dried sections were directly used for in situ hybridization.PrehybridizePrehybridize each section for over night at 37℃, by carefully adding 20 ml of hybridization buffer onto the glass slide.Hybridization1. Put 1.0μg/ml of probe on each slide. Assume all probes are approximately 0.1μg/μl. Therefore, use 1.0μl probe in 100.0μl of hybridization mix for each slide. Place 50.0μl on each half slide, spread a piece of parafilm through the probe drop to evenly cover the section, and slowly place the cover slip on top (try to avoid any bubbles).2. Place slides in a moist chamber overnight at 70 C. Moist chambers; put a piece of Whatman paper and broken 5 ml pipets (at 2ml and 5. 5ml) into the box, to form a platform for the slides. Pour enough moist chamber solution into the box to cover the bottom but dont fill it over the pipette base. Tape the box shut with yellow sticky tape.3. Place a beaker of ddH₂O in the hybridization oven with the moist chambers to keep the humidity level stable. This will prevent the moist chamber solution from evaporating over night.4. Drop an aliquot of 4 SSC ( containing 0. 1% SDS) onto the glass slides and incubates it at 50 C for 30 min to rinse hybridization buffer.5. Replace the hybridization buffer once at 50 C for 30 min with 1 SSC (containing 0.1% SDS).6. Replace once at 50 C for 30 min with 0. 5 SSC ( containing 0. 1% SDS).7. Digest the unhybridized RNA probe with RNase A at a final concentra- tion of 2 5 ng/ml.Important note: the final RNase A concentration is a critical factor to obtain an image with a low nonspecific background and clear specific signals.8. Replace SDS at room temperature for 10 min with PBS.9. Cover the sections on the glass slides with blocking solution at 37 C for 30 min.Post - Hybridization Washes1. Add the antibody to the blocking solution on the glass slides at a ratio of 1:5000 and incubate the antibody solution at 37 C for 1 h.2. Replace the blocking solution three times at RT for 10 min with bufferâ… .3. Replace the blocking solution two times at RT for 10 min with bufferâ…¡.4. Drop DAB (25 mg/ml) on the sections and incubate RT for 30 min.5. Replace the substrates with distilled water.6. The images of the hybridized sections were observed by microscopy.Statistical analysisStatistical comparisons between different groups of subjects were performed with SPSS10.0, and data are expressed in means s. They were compared with group of PV patient and group of controls using a paired Student t test. A value of P <0.05 was taken as significant.ResultsStained with brown in the cytoplasm was determined as positive (+). The intensity of staining were divided into four degrees: (-)no positive (cell number value < 5%); (+)weak staining (cell number value > 5%); (+ +)moderate strong positive (cell number value > 50);(+ + +) very strong positive (cell number value >65% )The expression of IL - 20, IL - 20R and IL - 15 mRNA was semiquantitatively assessed by Meta Morph Imaging System depended on gray values. Enumeration methods: The density of positive - staining cell in the epidermis was then calculated as follows, positive - staining cell/mm length of epidermal surface and (or) positive - staining cell /mm² of epidermis (positive - staining cells were counted on 20 consecutive fields at 400 magnification) , measured by micrometer.The expression levels of IL -20, IL -20R and IL-15 mRNA in the epidermis of normal and psoriatic groups were analyzed with Student's t - test.IL - 20 Expression is higher in psoriasis than normalIL - 20 secretion was significantly higher in psoriatic groups than normal groups (As shown in Table 1). DisscusionInterleukin (IL) - 10 is an important immunoregulatory cytokine. One of its main biological function seems to be the limitation and termination of inflammatory responses. Remarkably, a relative deficiency in IL - 10 expression is found in psoriasis, a frequent inflammatory skin disease, characterized by a type 1 cytokine pattern. Induction of IL - 10 expression was found by conventional antipsoriatic therapies, suggesting that IL -10 may be a key cytokine in psoriasis and that application of this cytokine may have therapeutic effects. In first clinical trials over 3-7 weeks in patients with established psoriasis IL -10 was well tolerated and clinical efficient. In a long term trial in patients with psoriasis in remission, IL - 10 therapy decreased the incidence of relapse and prolonged the disease free interval. Laboratory investigations suggest that IL - 10 exerts its antipsoriatic activity by effects on different cell populations including antigen pre - senting cells and T - cells. IL - 10 led to a lasting type 1/ type 2 cytokine balance shift. Direct effects of IL -10 on keratinocytes, however, are unlikely to have contributed to the clinical response, since IL - 10 unresponsiveness of keratinocytes was found in vitro. IL - 10 seems to have major importance in psoriasis. Further investigations are necessary to determine whether its application may represent a promising new therapeutic approach.Interleukin -20 is a new member of the IL - 10 family binding and signaling through the IL - 20R1/IL - 20R2 heterodimer, while IL -20 also bind to the IL - 20R2/IL -22R1 heterodimer. Using in situ hybridization we have stud- ied mRNA expression of IL - 19, 20, and 24 and their related receptor chains in skin from psoriatic patients before and during short - term treatment with either oral cyclosporine A or topical calcipotriol. In untreated lesions IL - 19 and IL -20 mRNA was expressed focally in epidermis above the dermal papillae, whereas IL - 24 was expressed in mononuclear cells in the dermal infiltrate. The expression of IL -19 and 20 mRNA was confined to the basal and suprabasal keratinocytes. No expression of IL - 19 and 20 mRNA could be detected in uninvolved psoriatic skin. Treatment with cyclosporine A and calcipotriol resulted in disappearance of the IL - 19 and 20 mRNA. Expression of mRNA for the receptor chains IL -20R1 and IL -20R2 was found throughout the psoriatic epidermal layer, whereas IL -22R1 mRNA was predominantly expressed in the superficial part of the psoriatic epidermis. These findings show that IL - 19 and IL -20 are synthesized by a distinct population of keratinocytes. It remains to be clarified whether IL - 19 and IL - 20 are implicated in the pathogenesis of psoriasis.The interleukin - 10 (IL - 10) cytokine family consists of several viral and human homologs that exhibit distinct receptor binding specificities. In the present study, the complex between interleukin -19 (IL-19) and its physiological receptor - the interleukin - 20 receptor alpha - chain (IL - 20R1) - was modeled. The most prominent feature of this complex is an extended binding interface formed by a long loop of IL -20R1 and a bulge region of IL - 19. The two regions exhibit complementary charges and have no structural counterparts in the IL - 10/IL - 10R1 complex but show some resemblance to the complex between interferon - gamma (IFN - gamma) and its receptor. Sequence comparison of the three cytokines (IL-19, IL - 20, IL - 24)that bind the IL - 20R1 reveals a considerable conservation of the length of the interacting loops. One residue suggested to play a key role in receptor binding specificity is a conserved glutamate. The binding interface of IL -20R1 is rich in aromatic residues while the interfaces of its cytokine ligands are mainly formed by more flexible aliphatic amino acids. This structural feature might play an important role for the specific recognition of a single receptor chain by three different cytokines. Comparison of the ligand/receptor interfaces in the A IL - 10/IL - 10R1, B IL - 19/IL -20R1 and C IFN - gamma/receptor complexes. The translucent Connolly surfaces of the receptor and the ligand are shown in yellow and white, respectively. The backbone of the receptor and ligand are highlighted by a red and blue/green tube, respectively. For clarity, only one domain of the intertwined dimes is shown for IL -10 and IFN - gamma. Arrows denote the location of helix B and the corresponding structural elements in IL -19 and IFN - gamma as well as the location of receptor loop L2. As evident from B and C an extended interaction surface is created in the IL - 19/IL - 20R1 and IFN - gamma/receptor complexes by the interaction of this structural element with a long loop of the respective receptor.Using in situ hybridization we have studied mRNA expression of IL - 20,-20R, and - 15 and their related receptor chains in skin from psoriatic patients. We analyzed the frequency of positive - staining cell in the epidermis and density of linearity. The significant association between patients with psoriasis and the expression of IL -20, -20R, and - 15 at position was established (13.502 9.613 vs 1.512 1.503; 11.540 8.493 vs 1.362 1.406; 9.512 6.503 vs 1.339 1.011) (P<0.01, P<0.01 and P<0.05). And we found that patients with psoriasis had a higher frequency of the positive of staining (P < 0. 01) compared to the normal group. Our data indicate that IL -20, -20R, and-15 should have a role in determining susceptibility to psoriasis. The possible role of the studied psoriatic patients in the regulation of the expression of IL-20, - 20R, and -15 is unknown yet and needs further studies.