Studies On The Mechanism Of Inflammatory Cells And Matrix Metalloproteinases In Skin Photoaging

Photoaging is described as a premature skin aging due to repeated exposure to ultraviolet,histologically characterized by excessive deposition of degenerative elastotic material and basophilic degeneration of collagen,and accompanied by degradation of typesⅠandⅢprocollagen. This impairment is thought to result from breakdown of collagen by increased matrix metalloproteinases(MMPs) expression,which may involve multiple materials such as cells,cytokines and metabolites,but the mechanisms and pathogenesis of photoaging are not well understood.Previous evidence has indicated that the ultraviolet irradiation promotes MMPs production by activation of multiple cell surface receptors on keratinocytes and fibroblasts and altered downstream signal transduction pathways,among which MMP-1,-3 and -9 are the most significant.In recent years the effect of infiltrating cells in the photoaging process has been noticed,showing histological increase in inflammatory infiltrate in sun-exposed skin.In order to further explore whether and how the inflammatory cells involved in the process of photoaging,the number of infiltrating cells and expression of MMPs·in sun-exposed and sun-unexposed skin would be compared,and the induction of MMPs and the effect on the human fibroblasts after activation of peripheral blood mononuclear cell(PBMC) would be studied,which providing a fundamental basis for occurrence,prevention and treatment of photoaging.The paper consists of three chapters.ChapterⅠHistological manifestation of infiltrating cells in sun-exposed and sun-unexposed skinObjective To investigate the effect of infiltrating cells on the photoaging by comparing types and number of infiltrating cells in sun-exposed and sun-unexposed skin.Methods The expression of CD3,CD45RO and CD68 was detected by immunohistochemical staining in 46 paraffine samples from extensor forearm(sun-exposed) and upper-inner arm(sun-unexposed) skin of 23 healthy female volunteers,and the number of positive cells was counted. The above data were analyzed by paired-samples t test and Pearson correlation analysis.Results The mean±SD number of positive cells for CD3,CD45RO and CD68 in the sun-exposed skin sections(48.91±13.173,46.83±12.915 and 85.43±22.346 cells/mm~2,respectively) was significantly higher than that in the sun-unexposed skin sections(40.61±11.571,38.00±10.109 and 73.48±16.208/mm~2,respectively)(the two former P＜0.01,the latter P＜0.05, respectively).The number of positive cells for CD3 and CD45RO in the sun-exposed skin sections increased significantly with age(r=0.557,0.555, all P＜0.01),but the number was uncorrelated with age in the sun-unexposed skin,and the number of positive cells for CD68 was uncorrelated with age in the sun-exposed and sun-unexposed skin.Conclusions The number of positive cells for CD3,CD45RO and CD68 in the sun-exposed skin sections were significantly higher than that in the sun-unexposed skin sections,and the number of positive cells for CD3 and CD45RO in the sun-exposed skin sections was positively correlated with age,implying that T lymphocytes and macrophages may play a role in the process of photoaging.ChapterⅡExpression of MMPs in sun-exposed and sun-unexposed skin Objective To study the expression of MMP-1,-3 and -9 in sun-exposed skin and -unexposed skin as well as its significance in the mechanism of photoaging.Methods Skin samples were resected from the extensor side of forearm(sun-exposed area) and upper-inner arm(sun-unexposed area) of 23 healthy female volunteers.The expression of MMP-1,-3 and -9 was detected by immunohistochemical staining in 46 skin samples.Immunoreactivity intensity distribution index(IRIDI) based on the intensity of immunoreactivity and proportion of immunoreactive cells was calculated to assess the expression of MMP-1,-3 and -9.Wilcoxon signed ranks test, Mann-Whitney U-test and Spearman rank correlation analysis were performed. Results MMP-1,-3 and -9 were expressed in both sun-exposed and -unexposed skin.The average IRIDI value for MMP-1,-3 and -9 was 7.70(range,3 to 12),9.22(range,6 to 12),8.30(range,6 to 12) in sun-exposed skin,and 4.26(range,2 to 6),5.39(range,2 to 9),4.04(range,1 to 6) in sun-unexposed skin,respectively;significant difference existed between sun-exposed and -unexposed skin in the three parameters(all P＜0.01).A significant increase was observed in the average IRIDI value for MMP-1,-3 and -9 in sun-exposed skin vs.sun-unexposed skin in women aged above 50 years(9.17 vs 4.75,10.58 vs 6.42,8.92 vs 4.33,respectively,all P＜0.05). In women younger than 50 years,the average IRIDI value for MMP-1,-3 and -9 was 6.09(range,3 to 8),7.73(range,6 to 9),7.64(range,6 to 12) in sun-exposed skin,significantly higher than that in sun-unexposed skin[3.73 (range,2 to 6),4.27(range,2 to 8),3.73(range,1 to 6),all P＜0.05]. Increased IRIDI scores of MMP-1,-3 and -9 were noticed in sun-exposed skin in women aged above 50 years vs.those younger than 50 years,but there was no statistical difference in MMP-1 or MMP-9 between the two aged groups in sun-unexposed skin(all P＞0.05).The IRIDI scores of MMP-1,-3 and -9 were positively correlated with age(r=0.656,0.691,0.742,P＜0.01) in sun-exposed skin,but the IRIDI scores of MMP-1 and MMP-9 uncorrelated with age in sun-unexposed skin.Conclusions There is an elevated expression of MMP-1,-3 and -9 in sun-exposed skin vs.sun-unexposed skin,hinting that these three MMPs play a role in the occurrence and development of photoaging, but their biological mechanism may be different.ChapterⅢActivation of PBMC and the effect of conditioned media(CM) on the cultured human fibroblastsObjective To study the effect of different stimulator on the induction of MMPs from PBMC,and the effect of conditioned media on human fibroblasts' proliferation and the induction of MMPs.Methods PBMC were isolated from human venous blood and stimulated by PHA or antibodies against CD3 and CD28,simultaneously RPMI1640 medium with 10%fetal calf serum serving as negative stimulator,after which CM were obtained,then CM were added to human skin fibroblasts.Cells' proliferation was measured by MTT assay,the level of IL-6 and MMPs in the supernatant media was detected by ELISA,and the expression of MMP-1 and MMP-3 mRNA was measured by RT-PCR. Analysis of variance(One-Way ANOVA) was mainly used.Results There were significant differences in different stimulated cells about the proliferation of PBMC,the level of IL-6,MMP-3 in the supernatant media (all P＜0.05),and the expression of MMP-1 mRNA(P＜0.05),showing the highest level in cells stimulated by PHA and lowest level in untreated cells.But the expression of MMP-3 mRNA was negative,and there were no MMP-1 and MMP-9 proteins in the supernatant after activation.Using different concentrations of CM to stimulate cultured fibroblast,the level of MMP-3 in different fibroblasts' supernatant media increased with stimulus concentration of CM,but was lower than that in baseline CM.There were no significant differences in different treated fibroblasts about the proliferation,MMP-1 and MMP-3 mRNA expression(all P＞0.05),and no MMP-1 and MMP-9 proteins in the supernatant after stimulation.Conclusions MMP-1 mRNA could be expressed and MMP-3 secreted from PBMC after activation.The supernatant media obtained from PBMC activation have no capacity to improve the induction of MMP-1,-3 and -9 from fibroblasts,suggesting that the inflammatory cells may be involved in the process of photoaging by production of MMPs themselves.