Partial characterization of phospholipase C activity in normal, psoriatic uninvolved, and lesional epidermis

Arachidonic acid (AA), the precursor of prostaglandins and leukotrienes, can be directly liberated from membrane phospholipids by phospholipase A2 or indirectly by phospholipase C. One or both of these enzymes may be responsible for the increased content of AA found in psoriatic lesional epidermis. Keratome biopsies were obtained from normal and psoriatic individuals. After homogenization and sonication, a 10,000 g supernatant was used as the enzyme source. The activities of both phospholipase A2 and C were assayed in each sample using phosphatidylcholine and phosphatidylinositol, respectively, as substrates. Phospholipase A2 activity was found to be significantly higher than normal in both uninvolved and lesional psoriatic epidermis. In contrast, phospholipase C activity was significantly higher than normal in only the psoriatic plaque on the basis of wet weight (p less than 0.001), protein (p = 0.01), and DNA (p = 0.004) content. Phospholipase C activity in pmol diacylglycerol formed/min/microgram DNA was: normal 4.96 +/- 0.80, n = 13; uninvolved 7.29 +/- 1.06, n = 18; plaque 14.44 +/- 2.50, n = 18. Analysis (pH profile, calcium requirement, substrate specificity, and saturation kinetics) of pooled epidermal extracts showed no inherent differences in phospholipase C from normal and psoriatic epidermis, suggesting either a higher concentration or the presence of an activated form of the enzyme in psoriatic plaque. Since phospholipase C activity, in contrast to phospholipase A2 activity, is elevated only in lesional epidermis, it is possible that this enzyme contributes to AA accumulation observed in this tissue.     

The uninvolved psoriatic epidermis

Normal control and apparently unaffected skin from psoriatics have been compared quantitatively for their acid phosphatase activity. The unaffected skin from psoriatics shows a more rapid reaction, which may be related to a lysosomal defect. No marked differences were detected in the dehydrogenase reactions in these two tissues. However, in frankly involved psoriatic skin it has previously been shown that there is a marked increase in glucose-6-phosphate dehydrogenase activity in the transitional zone. In the present report an increase in glycero-phosphate dehydrogenase activity has been demonstrated in the lower portion of psoriatic epidermis.     

The activity of caspase-1 is increased in lesional psoriatic epidermis

Caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the proinflammatory cytokine IL-18, a cytokine known to play an important role in the pathogenesis of psoriasis. The purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress-induced activation of caspase-1 and IL-18. Interestingly, increased caspase-1 activity in lesional compared with non-lesional psoriatic skin was seen. In vitro experiments in cultured human keratinocytes demonstrated anisomycin-induced, p38 mitogen-activated protein kinase (p38 MAPK)-dependent increased secretion of procaspase-1 and active caspase-1. Furthermore, anisomycin increased the mRNA expression of IL-18 through a p38 MAPK-dependent but caspase-1-independent mechanism, reaching a maximum level after 12 hours of stimulation. Finally, anisomycin caused a rapid (4 hours) increase in the secretion of proIL-18 and active IL-18. Secretion of active IL-18 was mediated through a p38 MAPK/caspase-1-dependent mechanism, whereas secretion of proIL-18 was mediated by a p38 MAPK-dependent but caspase-1-independent mechanism. These data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that IL-18 secretion is regulated by a p38 MAPK/caspase-1-dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis.     

寻常性银屑病皮损表皮中Smad7表达的检测及其临床意义

目的:探讨Smad7基因及其蛋白在寻常性银屑病皮损区表皮中的表达及其意义。方法:采用逆转录(RT)-PCR和SP免疫组化法分别检测寻常性银屑病皮损和正常对照皮肤中Smad7的表达。结果:寻常性银屑病皮损中Smad7表达水平上调。与正常对照皮肤相比,寻常性银屑病皮损区表皮角质形成细胞Smad7的免疫组化染色显著增强(P<0.01)。结论:寻常性银屑病皮损区表皮Smad7的过度表达可能是通过阻断转化生长因子(TGF)-β信号转导,从而有助于银屑病皮损区表皮过度增生。     

The phospholipid pattern in the involved and the uninvolved psoriatic epidermis

Summary The phospholipid pattern of the involved and the uninvolved epidermis from 48 psoriatic patients and of the normal epidermis from 23 healthy controls was determined by thin-layer chromatography. A higher amount of total phospholipids was found only in the involved psoriatic epidermis, whereas significant alterations in the phospholipid pattern were observed in the psoriatic lesion and in the lesion-free epidermis. Namely, the decrease of phosphatidylserine and the increase of phosphatidylinositol were identically present in both the involved and the uninvolved psoriatic epidermis. It seems likely that these alterations in the phospholipid pattern in psoriasis may be related to subclinical alterations of the epidermis in this disease.Supported in part by the Deutsche Forschungsgemeinschaft, Grant 674/1     

comparison between DNA synthesis and mitosis in uninvolved and involved psoriatic epidermis and normal epidermis

Biopsies have been taken from the involved and uninvolved skin of eleven patients with psoriasis and the normal skin of six patients without psoriasis, 1 h after in vivo injection of 10 μCi of ³H-thymidine. Mitotic counts and counts of labelled cells (synesizing DNA) after autoradiography have been carried out. The ratio of mitoses to labelled cells was 1: 25 in clinical psoriasis, 1: 190 in uninvolved psoriatic epidermis and 1: 450 in normal epidermis. The number of labelled cells in clinical psoriasis was approximately 12 times greater than normal epidermis. However, the number of labelled cells in uninvolved psoriatic epidermis was approximately twice that of normal. These results imply an alteration in the components of the cell cycle in both involved and uninvolved psoriatic epidermis.     

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.     

In situ localization of IFN-gamma-positive cells in psoriatic lesional epidermis.

Interferon-gamma (IFN-gamma) is believed to be an important mediator in the cytokine cascade of psoriasis. Lesional T cells in the epidermis may play a role in psoriasis. We examined whether IFN-gamma-producing T cells were present in the epidermis of psoriasis in situ by immunohistochemical techniques. Mixtures of CD4+ T cells and CD8+ T cells were found to be present in the papillary dermis and the epidermis of the psoriatic lesions. CD8+ T cells seemed to be dominant in the epidermis. Considerable amounts of IFN-gamma-positive cells were detected in infiltrates of the papillary dermis. IFN-gamma-positive cells were found to be present in the epidermis. The pattern of IFN-gamma staining appeared to be a combination of intracellular staining in mononuclear lymphoid cells and extracellular deposition in the surrounding areas. The staining was considered to be highly specific because it could be completely blocked by preabsorption with recombinant IFN-gamma. Our data suggest that psoriatic epidermal T cells produce and secrete IFN-gamma within the lesion and that these T cells are involved in the pathogenesis of psoriasis.     

The effect of PUVA therapy on glycoprotein biosynthesis in uninvolved psoriatic epidermis

Protein and N-linked glycoprotein biosynthesis was studied in the uninvolved epidermis of patients with psoriasis by the incorporation of radiolabelled leucine and mannose prior to and during PUVA treatment. Analysis of the polyacrylamide gel electrophoresis (PAGE) patterns of the 3[H]-labelled proteins and glycoproteins showed that the major changes in untreated uninvolved psoriatic epidermis compared to normal epidermis were: (a) a shift towards the synthesis of low-molecular-weight glycoproteins; (b) the absence of a 48-kDa peak labelled with mannose; (c) the appearance of 3[H]-mannose-labelled peaks at 40-36 kDa. PUVA treatment gradually changed the PAGE profile back more towards that expected for normal epidermis, with the reintroduction of a 52-48-kDa glycoprotein and reduction of the peaks in the 40-34-kDa region. This effect was dependent on uninterrupted treatment. The PUVA-treated PAGE profiles were compared to those expected in skin tumours (i.e. increased 3[H]-mannose-labelled peaks at 95 and 40-34 kDa with an absence of 62-kDa peaks). It appeared that these criteria were not seen generally as a result of PUVA treatment. However, the results indicate that tumour development may be possible if a patient responds to PUVA treatment by showing an increased peak at 95 and 40-34 kDa in association with a loss of an 3[H]-mannose-labelled peak at 62 kDa.     

Surface glycoproteins of cultured human keratinocytes from normal and uninvolved psoriatic epidermis

Surface glycoproteins of cultured human keratinocytes from normal skin and uninvolved psoriatic epidermis, isolated by the suction blister method, were studied by two different methods. Cells were cultured on collagen-coated culture dishes and showed a fibrillar keratin-specific staining by immunofluorescence. Surface labelling experiments using the neuraminidase/galactose oxidase/sodium borohydride method (which labels the penultimate galactose moieties of glycoproteins) revealed one major glycoprotein with M_r 53 kD (kilodaltons) both in normal keratinocytes and in keratinocytes from uninvolved psoriatic skin. The periodate/sodium borohydride method (which labels the terminal sialic acids in glycoproteins) by contrast revealed three major glycoproteins, with M_r 53 kD to 63 kD, in normal keratinocytes but only a single major glycoprotein, with M_r 53 kD, in keratinocytes from uninvolved psoriatic skin. Treatment of cultured keratinocytes with etretinate appeared to restore the normal pattern of surface glycoproteins in uninvolved psoriatic keratinocytes.     

Alefacept therapy reduces the effector T-cell population in lesional psoriatic epidermis

Alefacept, a LFA-3/IgG1 fusion protein, interferes with the activation and proliferation of T cells by binding to the CD2 receptor on their surfaces. The clinical efficacy of this drug has been demonstrated in chronic plaque psoriasis. We performed a single-center, open-label study to investigate the immunohistochemical effects in psoriatic lesional skin. A group of 11 patients with plaque psoriasis all received 12 weekly doses of 7.5 mg alefacept intravenously. Skin biopsies were obtained at baseline and on days 8, 43 and 92, and were evaluated by digital image analysis after immunohistochemical staining. After completion of treatment, 8 out of the 11 patients experienced a reduction in PASI of 50% or more compared to baseline. Immunohistochemical analysis displayed a gradual decrease in the number of cutaneous T cells during therapy, with a significant reduction in epidermal CD8⁺ cells and dermal CD4⁺ cells on day 92. Patients with a reduction in PASI of 50% or more after therapy had a clearance of effector/memory T cells from the epidermis, in contrast to patients with a reduction in PASI of less than 50%. These findings support the hypothesis that effector/memory T cells play a prominent role in the pathogenesis of psoriasis, and that alefacept is capable of reducing these cells in lesional psoriatic skin.     

Initial hyperproliferation and incomplete terminal differentiation of cultured human keratinocytes from lesional and uninvolved psoriatic skin

Human epidermal keratinocytes (KCs) were isolated from lesional and from uninvolved skin of 8 patients with chronic plaque-like psoriasis and from the normal skin of 8 healthy volunteers. Primary KC cultures, grown on 3T3 cell feeder layers, were examined over a period of 4 weeks and their plating efficiency, colony growth area, DNA synthesis and ultrastructural cell differentiation were evaluated. Psoriatic KCs formed colonies one day earlier than non-psoriatic controls and proliferated faster during the first 2 weeks, as assessed by the mean colony growth area and 3H-thymidine incorporation. After 4 weeks, however, no significant differences were observed between the in vitro proliferation parameters of normal and psoriatic KCs. At the ultrastructural level, cultures of lesional psoriatic KCs consisted of more cell layers with adherent transitional cells and incomplete formation of cornified envelopes, even after 4 weeks, while KCs from uninvolved psoriatic skin were characterized by a transient delay of in vitro maturation. These results indicate that the characteristic hyperproliferation of psoriatic KCs may only be maintained over a short period of primary culture, whereas defective terminal differentiation of lesional psoriatic KCs was maintained throughout the culture period.     

Decreased membrane-bound ATP-hydrolytic activity in psoriatic epidermis

Previous studies from our laboratory suggested that architectural alterations of the cell membranes may have a major significance in the pathogenesis of psoriasis. Consequently, the membrane-bound Mg⁺⁺ activated ATP-hydrolytic activity was investigated in normal and psoriatic epidermis under the electron microscope. The present study revealed that, in contrast to normal epidermis, only minimal ATP-hydrolytic activity is present on the cell membranes of psoriatic keratinocytes. This finding may reflect either a diminished attachment of substrate on the altered cell surface or an enzyme defect of the cell membrane. In both cases the reduced interaction between ATP and membrane-bound ATP-hydrolysing enzymes represents a functional membrane disorder of the psoriatic keratinocyte, presumably resulting in an insufficient utilization of ATP for active transport mechanisms on the cell surface.     

Increased phosphatidylinositol kinase activity in psoriatic epidermis

Phosphatidylinositol (PI) kinase is activated by growth factors, such as epidermal growth factor (EGF), and is thought to be involved in cellular proliferation. Psoriasis is a hyperproliferative epidermal disease in which EGF receptor expression is altered and phospholipase C activity is increased. Considering the potential importance of growth factor stimulated phosphoinositide metabolism in the genesis of abnormal growth, we measured PI kinase activity in epidermal keratome biopsies from normal skin and the lesional and nonlesional skin of psoriatic patients. The PI kinase activity in 10 psoriatic involved plaques was increased 6.7-fold (Vmax = 67.1 +/- 23.9 pmol formed/min/mg protein +/- SE) when compared with 11 normal epidermal biopsies (Vmax = 10.0 +/- 1.3 pmol/min/mg protein, p less than 0.025). Similar results were noted when enzyme activity was standardized using DNA content. The apparent Km of PI kinase for ATP in involved psoriatic biopsies (0.45 +/- 0.14 mM) was also significantly (p less than 0.025) increased compared with normals (0.11 +/- 0.02 mM). The PI kinase activity in 11 biopsies of nonlesional psoriatic epidermis was not statistically different from normal epidermis. Both psoriatic and normal PI kinases required Mg++ and were inhibited by Ca++. The polyamine, spermine, a known activator of PI kinase in other tissues, stimulated normal but not psoriatic epidermal PI kinase. Both normal and psoriatic PI kinase activities had an apparent mol wt of 85,000. Increased synthesis of phosphoinositides by PI kinase in psoriatic tissue may provide more substrate for phospholipase C; a key enzyme in growth factor-mediated signal transduction.     

Alterations in the desquamation-related proteolytic cleavage of corneodesmosin and other corneodesmosomal proteins in psoriatic lesional epidermis

Background  Desquamation occurs after proteolysis of corneodesmosomal proteins, including corneodesmosin (CDSN), by proteases of the kallikrein family, particularly KLK7. Impaired desquamation is one of the features of psoriasis, and psoriasis-associated single nucleotide polymorphisms of the CDSN gene may potentially modify the proteolysis of the encoded protein.
Objectives  To test whether the proteolysis of CDSN and other corneodesmosomal components is altered in psoriatic epidermis.
Methods  Total protein extracts obtained by tape-stripping of nonlesional and lesional skin from 11 patients were compared by immunoblotting experiments.
Results  An almost intact form of CDSN that has never been observed previously in the normal upper stratum corneum was detected in the lesional skin extracts, showing an altered proteolytic processing of the protein. This form was also observed in the nonlesional skin extracts, but in lower amounts. For most patients, increased amounts of desmoglein 1, plakoglobin and of high molecular weight fragments of desmocollin 1 were detected in the lesional skin. For most of them, similar amounts of KLK7 were immunodetected in both nonlesional and lesional skin extracts. No particular differences were observed related to the psoriasis type, the HLA-Cw6 status of the patients or any particular CDSN polymorphisms.
Conclusions  We detected a near full-length form of CDSN that has not been previously observed in normal stratum corneum. The results suggest a reduced degradation of all corneodesmosomal proteins in psoriatic lesions which probably reflects the persistence of corneodesmosomes.     

Direct immunofluorescence in lesional and uninvolved skin in dle

Direct immunofluroscence was evaluated in 16 patients with DLE including 1 case of lupus profundus and 4 with disseminated DLE. A BMZ band with multiple immunoreactants was demonstrated in lesional skin in 14 patients (87.5%), while 2 had only single immunoreactants. C3 and lgM were the commonest reactants followed by lgG, lgA and fibrinogen. A perifollicular prominence was seen in several patients. Four patients showed a band in uninvolved skin. This may indicate a potential to develop SLE. ANA was also positive in 5 patients but ds DNS was negative.     

Elevated phosphorylase kinase activity in psoriatic epidermis: correlation with increased phosphorylation and psoriatic activity

Summary To determine whether abnormal activity of a calmodulin-containing enzyme which catalyses phosphorylation reactions may play a pathogenetic role in psoriasis, the presence and activity of phosphorylase kinase (PK) in human epidermis were determined in patients with untreated/active psoriasis (n =10), treated/resolving psoriasis (n= 10), and non-psoriatic controls (n= 10). Biopsies were taken from involved and uninvolved skin for PK, organic phosphorus, and inorganic phosphate estimation, and light and electron microscopy. The enzyme was present in involved and uninvolved skin of every patient in the study. PK activity (units/mg protein) was significantly higher in active psoriasis than in resolving psoriasis and controls. PK activity correlated directly with organic phosphorus levels, and inversely with the extent of cellular glycogenolysis measured by the depletion of glycogen granules within the keratinocytes. The study demonstrates that PK is present in both psoriatic and normal epidermis, with significantly higher levels in active psoriasis. Furthermore, higher levels of PK activity, glycogenolysis and phosphorylation are associated with increased clinical psoriatic activity. We conclude that PK, a calmodulin-containing enzyme, is involved in regulating calcium-dependent phosphorylation events in human epidermis, and disturbance of its activity may play a key role in the clinical manifestations of psoriasis.     

Membrane-bound phospholipase C activity in normal and psoriatic epidermis

We report the quantification of a membrane-bound phospholipase C in human epidermis which is active against the physiologically relevant substrate, phosphatidylinositol 4,5-bisphosphate. The level of this enzyme is significantly increased in the psoriatic lesion, both on a weight and protein basis. Etiological implications of this observation are discussed.