A Study On The Role Of Cysteinyl Leukotrienes And Their Receptors In The Pathogenesis Of Eczema

Background and objectivesEczema is a common allergic inflammatory dermatosis, with the clinical characteristics of severe itching, polymorphism rash, exudation and tending to chronic relapse. Due to the unclear aetiology and pathogenesis, the therapeutic options of eczema are relatively limited.Now most of the present studies of eczema are limited on the balance of T-helper cell subgroups and on seeking for potential allergens. Although some progress has been achieved, eczema is still in a distressing condition and seriously affects patients'life quality. Since 2000,inflammatory mediators especially leukotrienes(LTs) in eczema lesion are increasingly and deeply concerned. Cysteinyl leukotrienes(CysLTs), the final inflammatory mediators in a wide range of diseases, which biological activities reach far beyond bronchoconstriction, telangiectasia, glandular secretion, are also important modulators of cytokine having extensive interaction between variety of other inflammatory mediators. Moreover, CysLTs may participate in the proliferation and differentiation control of some cells including several tumor cells. Therefore, CysLTs play a pivotal role in lots of inflammatory diseases. However, in different tissues and different diseases, the biological activity of CysLTs is quite different, showing that there must exist some positive links with the distribution and function of cysteinyl leukotriene receptors(CysLTRs). To explore the role of CysLTs in the pathogenesis of eczema, we should pay special attention to revealing the expression and function of their receptors in the skin. The CysLTs,including LTC4, LTD4 and LTE4, trigger contractile and inflammatory processes through the specific interaction with cell surface receptors belonging to family of the G protein-coupled receptor (GPCR). Pharmacological studies have identified two classes of CysLTRs (CysLTR1 and CysLTR2) based on their sensitivity to CysLTR1 selective antagonists. The genes of human CysLTR1 and CysLTR2 have been cloned in 1999 and 2000. The two receptors share only 38% in identity in amino acid. CysLTR1 is highly expressed in spleen, peripheral blood leukocytes, interstitial lung macrophages and airway smooth muscle, while CysLTR2 is mostly expressed in heart, adrenals, placenta, spleen, peripheral blood leukocytes and less strongly in brain. So far, the distribution and function of CysLTRs in skin are rarely reported in documents.For quite a long time the biological behavior changes of peripheral blood mononuclear cells (PBMC) in eczema and the resulting loss of balance of Th subsets are more concerned. But keratinocyte cell(KC) is a major component of epiderm cells. For a long time, the conventional view maintains that KC only sets a barrier between human body and the external environment. Recent studies find that KC not only plays an important role in regulating the process of skin inflammation, but also works as non-specific antigen-presenting cell. This biological behavior changes of KC will have a direct impact on the incidence and prognosis of a large number of dermatosis, Therefore, KC has become an important target for treatment.If we realize the changes in the levels of CysLTs in patients with eczema, and make clear the expression and function of their receptors in skin tissue or even in KC, we would make some contribution to get a better understanding to the pathogenesis of eczema. Methods and results1. Enzyme-linked immunosorbent assay and reverse phase high performance liquid chromatography are adopted to detect the levels of LTs and cytokines in peripheral blood, and LTs in urine and skin lesions of 10 patients with subacute eczema and 10 healthy volunteers. Meanwhile eczema patients accept clinical evaluation and receive mizolastine 10mg once a day for 5 days individually. The above markers were assayed again after treatment.The levels of serum LTB4, LTC4, IL-2, IFN-γand urinary LTE4 and skin tissue LTB4, LTC4, LTE4 levels in patients are significantly higher than those in healthy volunteers (P<0.05). Mizolastine significantly reduced serum LTB4 and IFN-γlevels as well as skin lesion LTB4, LTC4, LTE4 concentrations. These results suggest that LTs involve in the pathogenesis of eczema and mizolastine clearly reduces LTs levels in skin lesion. The Pearson's linear correlation test indicates that the levels of serum IFN-γreduced congruously with that of serum LTB4 and there is strong correlation between them(r=0.844,P <0.01);Serum LTB4 reduced congruously with that of serum LTB4 and there is strong correlation between them(r=0.674,P <0.01). LTs may amplify the skin imflammation through interaction of IFN-γ.2. Reverse transcriptase polymerase chain reaction and immunohistochemical techniques are adopted to detect the expression of CysLTRs in skin lesions of patients with subacute eczema and psoriasis vulgaris.CysLTR1 and CysLTR2 simultaneously exist in human skin and locate in epidermis, sebaceous follicles, hair follicles, sweat glands,blood vessels and smooth muscle. The expression of CysLTR1 and CysLTR2 in eczema and psoriasis is significantly higher than that in normal skin (P<0.05). But the intensity of expression shows no significant difference between the two subtype cysteinyl leukotriene receptors(P >0.05).3. A non-contact coculture system of KC and PBMC is set up by separating of polycarbonate membrane to watch the synthesis of leukotrienes in KC under the existence of PBMC.In descending order, levels of LTB4 in culture supernatant of KC are KC with PBMC of eczema patients, KC with PBMC of healthy volunteers and KC separate. Meanwhile, We also observed that IL-2 and IFN-γcan also stimulate the synthesis of LTB4 in KC separate (P<0.01). The synthesis of leukotrienes in KC can be promoted by PBMC through water-soluble cytokines such as IL-2 and IFN-γ.4. Immunofluorescence and immunoblot are used to confirm the expression of CysLTRs in KC. In order to understand the function of CysLTRs in KC, Fura-2/AM is used to assay the intracellular calcium flow and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolliumbromide (MTT) is used to detect the ability of cell proliferation.It is confirmed for the first time that CysLTR1 and CysLTR2 express in human keratinocytes,mainly in plasma membrane,cytoplasm and some nuclear. After CysLTRs activation by LTD4,nuclear translocation of CysLTRs and increasing of Ca2+ in cell and cell proliferation are observed.However, the antagonists of CysLTRs—MK571,BAYu9773 can weaken such effection. The phenomena suggest that CysLTs may promote the proliferation of keratinocyte through the nuclear translocation of their receptors.Conclusion1. LTs involve in the pathogenesis of eczema as potent inflammatory mediators.2. KC can not only synthesize LTs but also express CysLTRs, so that it is not only the major component of epiderm but also a target cell of CysLTs. Moreover, the synthesis of LTs and expression of CysLTRs in KC are both enhanced in inflammatory state.3. In inflammatory state, nuclear translocation of CysLTR1 may promote the proliferation of KC.These experiments help us to reveal what roles CysLTs and CysLTRs play in eczema, and provide experimental data for seeking a new therapeutic target of eczema.