Distribution of p230, an α-spectrin-related polypeptide in normal and psoriatic epidermis and in cultured human keratinocytes

We studied the localization of p230, an immunoanalogue of erythroid alpha-spectrin, in normal and psoriatic human epidermis and in cultured human keratinocytes. In immunofluorescence microscopy of frozen sections of normal skin a bright cytoplasmic staining was seen in the cortical area of the keratinocytes. Similar staining was also seen in lesional and uninvolved areas of psoriatic epidermis. The pericytoplasmic localization of p230 could also be seen in cultured human keratinocytes: a lamina-like reticular staining was seen mostly confined to the ventral cytoplasmic aspect and to junctional areas of the cells. Immunoblotting of electrophoretically separated polypeptides of epidermal cells revealed a distinct polypeptide of Mr 230 kD. The results indicate that alpha-spectrin-like polypeptides form a major cytoskeletal framework in human epidermal cells in both normal and psoriatic skin.     

Effect of polyamine antimetabolites on cultured human keratinocytes from normal and uninvolved psoriatic skin

We describe the effect of two polyamine antimetabolites on polyamine and macromolecule synthesis of cultured human keratinocytes obtained by suction blisters from normal skin and the uninvolved skin of psoriatic patients. The concentrations of spermidine and spermine steadily increased during the culture of normal keratinocytes in vitro, whereas the putrescine concentration showed only a transient rise at the beginning of the active growth phase. Treatment with difluoromethylornithine decreased the concentrations of putrescine and spermidine in both normal and uninvolved psoriatic keratinocytes, but had no effect on either DNA or protein synthesis. Methylglyoxal bis(guanylhydrazone) marginally decreased the levels of spermidine and spermine and significantly inhibited the DNA and protein synthetic activities. Pretreatment of uninvolved psoriatic keratinocytes with difluoromethylornithine enhanced the accumulation of methylglyoxal bis(guanylhydrozone), resulting in a profound inhibition of cellular macromolecule synthesis. This synergistic was not seen in normal keratinocytes. Thus, although no statistically significant difference was observed between the cells derived from normal and uninvolved psoriatic epidermis, the psoriatic keratinocytes appeared to be more sensitive to the action of polyamine antimetabolites. The inhibition of DNA and protein synthesis by methylglyoxal bis (guanylhydrazone) was prevented by concomitant treatment with spermidine.     

Secretion of urokinase and tissue-plasminogen activator by epidermal cells in the presence of psoriatic fibroblast-conditioned medium.

The present study examined secretion of urokinase and tissue-plasminogen activator by epidermal cells in the presence of psoriatic or uninvolved skin fibroblast-conditioned medium. Using zymographic analyses, a 54kD lysis band and a small 110kD band derived from urokinase could be detected in the harvest fluid from keratinocytes treated with both psoriatic and uninvolved fibroblast-conditioned medium, as well as very weak lysis bands of 63kD and 120kD derived from tissue-plasminogen activator in the harvest fluid treated with psoriatic fibroblast-conditioned medium, but not with uninvolved fibroblast-conditioned medium.     

银屑病患者角质形成细胞VEGF表达的研究

目的　研究银屑病发病与血管内皮生长因子 (VEGF)的关系 ,探讨银屑病可能的发病机制。方法　①用免疫组化法检测银屑病患者皮损和非皮损处皮肤、正常健康人皮肤及体外培养的银屑病患者和正常人角质形成细胞(KC)VEGF的表达 ;②用双抗体夹心ELISA法检测银屑病患者及正常人KC培养上清液中VEGF含量。结果　①银屑病皮损处VEGF表达明显高于非皮损处和正常人皮肤 (P均 <0 .0 0 1) ,非皮损处与正常人皮肤VEGF表达也有显著性差异 (P <0 .0 5 ) ;②体外培养的银屑病皮损处和非皮损处KCVEGF表达明显高于正常人 (P均 <0 .0 0 1) ;银屑病皮损处KC与非皮损处KC相比VEGF表达也有显著性差异 (P <0 .0 5 ) )。结论　VEGF可能参与银屑病的发病。     

The response of distant uninvolved psoriatic skin to standardised injury is not different from that in normal skin.

The epidermis of uninvolved psoriatic skin is characterised by a slight hyperproliferation and an increase in inflammatory parameters, whereas no differentiation abnormalities are seen. Data with respect to the response of distant uninvolved psoriatic skin to standardised injury are not uniform. In this study, a recently developed multiparameter flow cytometric assay was used to compare the response to tape stripping of uninvolved psoriatic and normal skin. With this method, a parameter for proliferation, differentiation and inflammation was measured simultaneously. Concerning these parameters, no statistically significant differences were found between uninvolved psoriatic skin and normal skin. The mechanism that underlies hyperproliferation in distant uninvolved psoriatic skin does not indicate an intrinsic abnormality in keratinocytes. Inflammatory signals might play a role in this process.     

RANTES expression in psoriatic skin, and regulation of RANTES and IL-8 production in cultured epidermal keratinocytes by active vitamin D3 (tacalcitol)

The chemokine RANTES is a chemoattractant for eosinophils, T lymphocytes of memory phenotype and monocytes, suggesting that it plays an important part in chronic inflammatory and allergic diseases. In various types of cells, RANTES production is markedly induced by tumour necrosis factor (TNF)-α and interferon (IFN)-γ in combination. Psoriasis vulgaris is a chronic cutaneous inflammatory disease. Cytokines and chemokines produced by T cells and epidermal keratinocytes, such as interleukin (IL) 8, are involved in the pathogenesis of psoriasis. T-cell clones obtained from psoriatic skin have been shown to produce the Th1 cytokine IFN-γ. In addition, abnormal expression of proinflammatory cytokines including TNF-α has been observed in psoriatic lesions. These reports led us to hypothesis that psoriatic skin could provide epidermal keratinocytes with TNF-α and IFN-γ, so that keratinocytes could produce RANTES. In this study, we addressed the question as to whether RANTES was involved in psoriasis vulgaris. Immunohistochemistry of skin biopsies showed RANTES was present in the intercellular spaces between epidermal keratinocytes, in the fully developed lesions from the middle to the edge of psoriatic plaques, but not in the perilesional uninvolved and healthy control skin. Further, we confirmed the production of RANTES, together with IL-8, by cultured normal human epidermal keratinocytes, using an enzyme-linked immunosorbent assay. Stimulation with TNF-α and IFN-γ in combination synergistically increased the RANTES production in this system. These results clearly demonstrate the expression of RANTES in psoriatic lesions and suggest the involvement of this chemokine in the outcome of cutaneous inflammatory diseases. Tacalcitol (1α,24(R)-dihydroxyvitamin D₃), an active vitamin D₃ analogue, inhibited RANTES and IL-8 production in cultured normal epidermal keratinocytes. This result indicates that active vitamin D₃ is effective in the regulation of chemokine production by epidermal keratinocytes, which may partly account for its action as an antipsoriatic drug.     

CELL PROLIFERATION AND CYTOKINE INDUCTION BY TNF-�� OF PSORIATIC KERATINOCYTES ARE NOT DIFFERENT FROM NORMAL KERATINOCYTES IN VITRO

Background:

Recent studies indicate that various cytokines including tumor necrosis factor-α (TNF-α) play an essential role in the induction and maintenance of psoriatic lesion.

Aims:

To compare the cell proliferation of keratinocytes by various cytokines and TNF-α-induced cytokine secretion among normal keratinocytes, uninvolved, and involved keratinocytes.

Methods:

The keratinocytes from normal skin, uninvolved, and involved psoriasis were cultured in the presence of IL-6, IL-8, epidermal growth factor (EGF), hepatocyte growth factor (HGF), transforming growth factor-α (TGF-α) epiregulin, amphiregulin, and TNF-α and then MTT assay for keratinocytes proliferation was performed. Furthermore, TNF-α-induced secretion of IL-6, IL-8, EGF, HGF, TGF-α, epiregulin, and amphiregulin were compared among these keratinocytes.

Results:

IL-6, IL-8, EFG, TGF-α, epiregulin, and amphiregulin, but not TNF-α increased keratinocyte proliferation of normal, psoriatic uninvolved, and involved skin. The increased cell proliferation by these cytokines and growth factors were not different among the keratinocytes derived from normal skin, uninvolved, and involved psoriasis. The significant induction of TNF-α increased IL-6, IL-8, EGF, HGF, TGF-α, epiregulin, and amphiregulin, but the increase in these cytokines and growth factors were not different among normal skin, uninvolved, and involved psoriasis.

Conclusion:

Cell proliferation by various cytokines and growth factors and TNF-α-induced cytokine secretion are not different between normal and psoriatic keratinocytes.     

Increased DNA synthesis of uninvolved psoriatic epidermis is maintained in vitro

Clinically uninvolved psoriatic epidermis shows increased DNA synthesis in vivo. We have studied the DNA synthesis of cultured keratinocytes from uninvolved psoriatic skin. Trypsinized epidermal cells were plated on plastic dishes pre-coated with bovine collagen type I. In initial studies, normal human serum was found to be superior to fetal bovine in supporting the growth of human epidermal keratinocytes. Furthermore, keratinocyte cultures established in the presence of normal human serum produced large keratin proteins (68,000 daltons) indicating that the terminal steps in cell differentiation can occur in vitro. In subsequent experiments keratinocyte cultures were grown in medium supplemented with 10% normal human serum. Confluent cultures of keratinocytes from uninvolved psoriatic epidermis had an increased DNA synthesis determined both as the incorporation of [3H]thymidine and as the autoradiographic labelling index. The DNA synthesis of both normal and psoriatic keratinocyte cultures increased in response to incubation in medium with 10% psoriatic serum. The ability of keratinocytes from uninvolved psoriatic epidermis to maintain an increased DNA synthesis suggests the presence of an inherent defect within the population of epidermal keratinocytes in psoriasis. Such a culture system can be used as an in vitro model for the study of psoriasis.     

Growth-inhibitory effects of 1,25-dihydroxyvitamin D3 on normal and psoriatic keratinocytes

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the growth and DNA synthesis of cultured human keratinocytes obtained from involved and uninvolved psoriatic epidermis and normal epidermis were studied. Treatment with 10(-8) M and 10(-7) M of 1,25(OH)2D3 inhibited cell growth as follows: 58.5 +/- 19.3% and 21.3 +/- 13.6% in normal keratinocytes (n = 6); 43.8 +/- 22.8% and 17.8 +/- 12.3% in psoriatic uninvolved keratinocytes (n = 4); 51.7 +/- 18.2% and 13.2 +/- 6.4% in psoriatic involved keratinocytes (n = 6). Inhibition was virtually complete at 10(-6) M. DNA synthesis was also inhibited by 10(-8) M, 10(-7) M and 10(-6) M of 1,25(OH)2D3 as follows: 70.0 +/- 8.3%, 59.0 +/- 6.8% and 16.7 +/- 4.0%, respectively, in normal keratinocytes (n = 3); 78.5 +/- 13.5%, 51.5 +/- 25.5% and 24.5 +/- 21.5%, respectively, in psoriatic uninvolved keratinocytes (n = 2); and 69.3 +/- 14.5%, 41.3 +/- 19.1% and 14.8 +/- 11.2%, respectively, in psoriatic involved keratinocytes (n = 4). These results indicate that 1,25(OH)2D3 functions as a growth inhibitor for cultured human keratinocytes derived from both normal and psoriatic skin.     

Studies on the plasma membrane of normal and psoriatic keratinocytes. 6. Cell surface and shed glycoproteins

Quantification and characterization of [3H]-fucose-labeled cell surface glycoproteins are reported. Two approaches have been compared; first the analysis of glycoprotein shed spontaneously into the medium during incubation of keratinocytes in vitro, and second the study of material released by exposure of the cell to proteolytic enzymes. It is shown that psoriatic keratinocytes "shed" glycoprotein more rapidly than normal, although the material is of similar molecular weight (mainly "biantennary" transferrin type glycopeptides). By contrast, the percentage of glycoprotein released by proteolysis of psoriatic keratinocytes is normal, but the molecular weight distribution of the labeled glycopeptides is markedly altered. The abnormal turnover and composition of fucose-labeled glycoproteins from the cell surface may be related to the loss of growth control in psoriatic epidermis.     

Basal keratinocytes from uninvolved psoriatic skin exhibit accelerated spreading and focal adhesion kinase responsiveness to fibronectin

We previously proposed that the keratinocyte hyperproliferative state in psoriatic skin results from a combination of T cell cytokine interaction with basal keratinocytes that exist in a primed state. We now provide evidence that basal keratinocytes from psoriatic uninvolved skin are in a preactivated state with regard to their interaction with fibronectin. Freshly isolated basal keratinocytes (K(1)/K(10)(-)) from non-lesional psoriatic skin demonstrated a significantly higher percentage of spreading cells 1 h after plating on fibronectin-coated plates than keratinocytes isolated from normal skin (p =0.0002). No differences were observed on collagen-laminin-coated plates, however. The keratinocyte spreading on fibronectin-coated plates involved alpha 5 beta 1 and alpha V beta 1 integrins. To address the potential signaling cascades that may respond to integrin changes in psoriatic keratinocytes, focal adhesion kinase changes were assessed. The percentage of keratinocytes from psoriatic uninvolved skin that exhibit positive focal adhesion kinase staining was significantly greater than the percentage from healthy volunteers after 1 h incubation on fibronectin (p =0.006). Additionally, focal adhesion kinase isolated from uninvolved psoriatic keratinocytes had a greater degree of tyrosine phosphorylation. Thus, the proliferative effect of fibronectin in combination with T cell lymphokines on psoriatic uninvolved basal keratinocyte progenitors may be due to abnormal in vivo integrin-driven focal adhesion kinase activity and downstream signaling.     

Immunolocalization of adhesion molecules in psoriatic arthritis, psoriatic and normal skin

Adhesion molecule expression in synovial membrane obtained from patients with psoriatic arthritis (PA) has previously been compared with rheumatoid arthritis (RA). Although expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was similar in both psoriatic and rheumatoid synovium, in contrast, little or no endothelial leucocyte adhesion molecule-1 (ELAM-1) was observed in psoriatic synovium. In the present study, the expression of ICAM-1. ELAM-1 and VCAM-1 was examined in the involved and uninvolved skin from patients with PA (n= 15), patients with psoriasis (Ps) but no arthritis (n= 5) and in normal skin (n= 4). ICAM-1 was intensely expressed on endothelium and keratinocytes of involved skin from patients with Ps with or without arthritis. There was constitutive expression of ICAM-1 on endothelium only in uninvolved and normal skin. In contrast, ELAM-1 expression was restricted to endothelial cells; it was widespread and intense in involved skin, but was minimal in uninvolved and normal skin. VCAM-1 was expressed on endothelium, and also on some dendritic cells in involved psoriatic skin. There was minimal VCAM-1 staining on endothelial cells in uninvolved and normal skin. In conclusion, in involved psoriatic skin from patients with and without arthritis ICAM-1, ELAM-1 and VCAM-1 expression is up-regulated on vascular endothelium, and ICAM-1 is expressed on keratinocytes. However, ELAM-1 and VCAM-1 expression seen in dermal vessels is not found in psoriatic synovial vessels. These differences suggest a mechanism for controlling cellular traffic in Ps and in PA.     

Detection of nitric oxide and nitric oxide synthases in psoriasis

Biopsies from psoriasis lesions and clinically uninvolved skin of eight patients and five normal subjects were studied by immunocytochemistry with computerized image analysis for the presence of endothelial, neuronal and inducible isoforms of nitric oxide synthase. Endothelial nitric oxide synthase was expressed in the endothelium and weakly in some keratinoctyes. Its expression was not significantly different in psoriasis. Inducible nitric oxide synthase, however, was absent from normal skin but was significantly upregulated in psoriatic lesional skin, focally in keratinocytes but to the greatest extent in the papillary dermis and to a lesser extent in clinically uninvolved psoriatic skin. Inducible nitric oxide synthase staining was greatest in the more severe lesions and correlated with the inflammatory infiltrate (CD3-positive cells) and with keratinocyte proliferation (Ki-67-positive cells). In normal skin, neuronal nitric oxide synthase was expressed only in keratinocytes in the granular layer and eccrine sweat glands. However, in psoriasis and clinically uninvolved skin the neuronal form was present through all levels of the epidermis. Direct measurement of nitric oxide production from the skin surface revealed a tenfold increase in the lesions of 16 psoriatic patients compared with their nonlesional skin, and this nitric oxide production was inhibited by topical betamethasone. Received: 4 March 1996     

Distribution of glycoproteins containing fucose in normal and psoriatic keratinocytes

Electron microscope autoradiography has been used to compare the fucose-containing glycoproteins of normal and psoriatic epidermis. Normal keratinocytes and uninvolved psoriatic cells have most of their fucose on the plasma membrane, whereas involved psoriatic cells retain more within the cytoplasm. The fucose distribution in other epidermal cell types is also described.     

Increased lysophosphatidylcholine content in lesional psoriatic skin

Various cell stimuli occur via activation of phospholipase A₂, which hydrolyses polyunsaturated fatty acids from the sn-2 position of membrane phospholipids, resulting in the formation of polyunsaturated fatty acids and lysophospholipids. The level of lysophospholipids is determined by the balance between phospholipase A₂ activity and the rate of catabolism of the lysophospholipids. One of the lysophospholipid classes, lysophosphatidylcholine, has been shown to stimulate certain leucocyte activities which are of importance for the induction and maintenance of inflammation. In addition, it has been demonstrated that phospholipase A₂ activity is increased in psoriatic skin. In the present study, we analysed the levels of lysophosphatidylcholine, by thin layer chromatography, in lesional psoriatic skin, uninvolved psoriatic skin and normal skin. The lysophosphatidylcholine content, expressed as μmol lysophosphatidylcholine/μmol phosphatidylcholine, was 1.55, 0.21 and 0.12% in lesional psoriatic skin, uninvolved psoriatic skin and normal skin, respectively. The level of lysophosphatidylcholine was significantly elevated in lesional compared with uninvolved psoriatic skin (P= 0.004) and normal skin (P= 0.002). The increased lysophosphatidylcholine levels in psoriatic skin indicate that the phospholipase A₂ activation is not accompanied by a corresponding increase in the activity of enzymes catabolizing lysoPC. If present in biologically active concentrations, lysophosphatidylcholine may contribute to the induction and maintenance of the inflammatory and immunological processes occurring in lesional psoriatic skin.     

Psoriatic keratinocytes are resistant to tumor necrosis factor alpha's induction of mRNA for the NMDA‐R2C subunit

Psoriatic individuals demonstrate accelerated healing and the Koebner phenomenon, suggesting that psoriatic proliferation of keratinocytes is not inhibited appropriately after skin injury. Serial analysis of gene expression in TNFα‐exposed keratinocytes shows the greatest alteration in expression of NMDA‐R2C. Expression of the NMDA receptor is altered in diseased skin containing TNFα, and TNFα plays a prominent role in psoriasis. An abnormality in induction of NMDA‐R2C by TNFα in psoriatic keratinocytes may explain their lack of growth inhibition. We compared the capacity of TNFα to induce expression of NMDA‐R2C in normal and psoriatic (involved and uninvolved) keratinocytes in vitro. After 72 h of incubation with TNFα, normal keratinocytes demonstrated a significant induction of NMDA‐R2C mRNA compared with control cultures, whereas psoriatic keratinocytes showed no induction. In an in vitro model of wounding (scratches on monolayers), TNFα inhibited migration/proliferation of keratinocytes only at the edge of NMDA‐R2C expressing wounded monolayers of normal keratinocytes.     

Immunohistochemical localization of lipocortins in normal and psoriatic human skin

The distribution of lipocortin I, a steroid-induced inhibitory protein of phospholipase A₂, was examined in normal and psoriatic human skin. Using immunoblotting analysis with specific antibody against human lipocortin I purified from human placenta, lipocortin I was detected as a 37 kDa protein in cultured epidermal cells, whole skin and epidermis. In the dermis and stratum corneum, lipocortin I was only weakly detectable by Western blotting. In contrast to normal skin, much less lipocortin I was detected by Western blotting analysis in psoriatic skin. Using immunoperoxidase immunohistochemical analysis, lipocortin I was demonstrated in the cytoplasm of keratinocytes in the upper and middle layers of the epidermis and in some infiltrating cells in the dermis in normal skin. In involved psoriatic skin, by contrast, lipocortin I was almost undetectable in the epidermis, although it was demonstrated in some infiltrating cells in the dermis. No immunostaining of lipocortin I was observed in the stratum corneum of normal or psoriatic skin. These results, together with the finding that phospholipase A₂ activity is higher in psoriatic epidermis than in normal epidermis, suggest that lipocortin I plays an important role in the regulation of differentiation and proliferation of epidermal keratinocytes.     

Localization of elevated transforming growth factor-alpha in psoriatic epidermis

Biopsies of involved and uninvolved skin from five patients with plaque psoriasis and normal skin from four healthy volunteers were investigated for steady-state quantities of TGF-alpha RNA and protein by in situ hybridization and immunohistochemistry. Increased levels of TGF-alpha RNA were found only in the high-level keratinocytes of involved psoriatic skin (p less than 0.001). Elevated levels of TGF-alpha protein were seen in both the high-level and basal layers of involved psoriatic skin compared to uninvolved psoriatic and normal control skin. Elevated TGF-alpha gene expression is thus implicated in the hyperproliferation of keratinocytes and possibly the altered maturation pattern seen in psoriasis.