Humoral autoimmune responses to the squamous cell carcinoma antigen protein family in psoriasis

Substantial evidence indicates that psoriasis is a T-lymphocyte-mediated autoimmune disease. However, longstanding data also indicate IgG and complement deposition in upper epidermis of psoriasis plaques. This led us to propose that autoantigen-autoantibody interactions in the skin may also be of pathogenic importance. Here, we have confirmed the presence of IgG in upper lesional epidermis and used high-resolution two-dimensional immunoblotting of extracts from this tissue, and laser desorption mass spectrometry of tryptic peptides, to define a series of epidermal proteins that bind IgG from psoriatic serum. The most prominent of these autoantigens are homologues of the serpin, squamous cell carcinoma antigen (SCCA), the other autoantigens identified including arginase 1, enolase 1, and keratin 10. Blood levels of IgG autoantibodies that bind to SCCA proteins were significantly higher in psoriasis than healthy controls (P=0.005), but were not detectable in sera from patients with active atopic dermatitis. To our knowledge, SCCA proteins have not previously been described as autoantigenic in animals or humans and form complexes with IgG that are associated with complement deposition. These findings expose potentially pathogenic humoral immunologic events and thus possible therapeutic targets in psoriasis.     

Epiplakin accelerates the lateral organization of keratin filaments during wound healing

BackgroundParakeratosis, the persistent presence of nuclei in the stratum corneum (SC) is associated with serious disruption of skin barrier function. Squamous cell carcinoma antigen 1 (SCCA1) is strongly up-regulated in inflamed and parakeratotic skin.ObjectiveTo find a biochemical marker for the SC barrier disruption, especially the disruption associated with parakeratosis.MethodsAn ELISA assay system was established to quantify SCCA1 in the extract of tape-stripped cornified cells. Transepidermal water loss (TEWL) and other skin parameters were measured and compared with the amount of SCCA1. Localization of SCCA1 was investigated immunohistochemically in various skin diseases with parakeratosis. Nuclei and SCCA1 on the skin surface were detected by staining of corniocytes collected on an adhesive-coated slide glass.ResultsSCCA1 showed strong up-regulation in lesional skin with psoriasis (466-fold), hayfever skin caused by Japanese ceder pollen (232-fold) and sun-exposed skin of healthy individuals (90-fold) compared to their normal sun-protected skin. The increased levels of SCCA1 were well correlated with increased values of TEWL and the number of parakeratotic cells in the SC. Furthermore, subjects with high levels of SCCA1 in the epidermis were more susceptible to barrier disruption by external stimuli, and this was accompanied with a further increase of SCCA1. We confirmed that localization of SCCA1 was limited to parakeratotic areas by using the skin surface staining technique. Immunohistochemical study also demonstrated that SCCA1 was always present at high levels in parakeratotic epidermis.ConclusionAll of our findings indicate that SCCA1 plays an important role in the induction of epidermal barrier disruption. SCCA1 may be a critical determinant of barrier function in the epidermis.     

Specificity of Human Keratinocyte X HeLa Cell Hybrid Murine Monoclonal Antibodies

The immunofluorescent staining patterns of three differentiation-specific monoclonals (HLK3, HLK7, HLK20) that display different immunofluorescent (IF) reactivity in normal and psoriatic epidermis, were examined in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), as well as other human normal epithelial and nonepithelial tissues. Similar staining patterns within epidermis were seen with HLK3 (intercellular) and HLK7 (perinuclear) in psoriasis, SCC, and BCC. HLK20 selectively stained BCC and SCC within epidermis and dermis, and was negative in psoriasis. The monoclonals did not react with nonepithelial tissues, but repetitively displayed positive, granular reactivity with simple epithelia and transitional epithelium. Stratified squamous epithelia showed IF staining similar to normal epidermis for all three monoclonals. These new monoclonal antibodies offer new investigative tools to study abnormalities in keratinocyte differentiation in benign and malignant hyperplastic skin diseases.     

Histologic distribution of staining by a monoclonal antibody (psi-3) in psoriasis and occurrence of psi-3 antigen in other cutaneous diseases

psi-3 is a monoclonal antibody that recognizes a 135,000 molecular weight structural component of maturing keratinocytes in psoriasis (the psi-3 antigen) but fails to bind to any constituent of keratinocytes in normal epidermis. This paper describes the occurrence of the psi-3 antigen in a variety of dermatopathologic conditions using immunoperoxidase (biotin-avidin-peroxidase) and immunofluorescence methods which show excellent concordance. In 35 of 36 specimens of psoriasis vulgaris, psi-3 antibody consistently immunolabels the cytoplasm of keratinocytes above the basal layer. At the edges of psoriatic plaques, psi-3 antibody staining extends for a variable distance into lesion-free epidermis. A similar pattern has been found in a certain number of other conditions described in the paper, including squamous cell carcinoma and condyloma acuminatum, but not Darier's disease, basal cell carcinoma, nor lamellar ichthyosis. In all but one condition, the outermost or basal layer of cells is never stained. The only disease in which the lowermost cell layer is stained is a lichen planus-like lesion. The occurrence of psi-3 antigen cannot be correlated with any histologic feature of psoriasis such as acanthosis, loss of the granular layer, or hyperproliferation. The antigen appears to be a unique keratinocyte constituent which is expressed in certain pathologic conditions and which is not detected by any other histologic or immunophenotyping method. It is a potentially valuable addition to the panel of antibodies available for characterizing epithelial cells.     

基因工程抗角蛋白抗体在正常皮肤和几种表皮增生性皮肤病皮损中的反应定位

目的　探索一株基因工程人源性抗角蛋白Fab抗体在正常皮肤及几种表皮增生性皮肤病皮损中的反应定位。方法　利用从噬菌体抗体库中筛选到的特异表达抗角蛋白Fab片段的质粒转化大肠杆菌 ,IPTG诱导表达出Fab抗体 ,纯化鉴定后用此抗体对正常皮肤及银屑病、鳞癌、基底细胞癌和脂溢性角化病皮损进行免疫组化染色。结果　正常皮肤表皮呈阴性染色 ,毛囊呈阳性染色 ,银屑病、鳞癌、基底细胞癌和脂溢性角化病皮损均呈现明显的阳性着色 ,其中银屑病皮损基底细胞层为强阳性。所有细胞染色部位均位于胞质 ,胞核未见着色 ,真皮为阴性。结论　该株人源性抗角蛋白Fab抗体主要与表皮组织结合 ,对银屑病等表皮增生性皮肤病的损害具有较高特异性。     

Abnormal aquaporin-3 protein expression in hyperproliferative skin disorders

Non-melanoma skin cancers (NMSCs) and psoriasis represent common hyperproliferative skin disorders, with approximately one million new NMSC diagnoses each year in the United States alone and a psoriasis prevalence of about 2% worldwide. We recently demonstrated that the glycerol channel, aquaporin-3 (AQP3) and the enzyme phospholipase D2 (PLD2) interact functionally in epidermal keratinocytes of the skin to inhibit their proliferation. However, others have suggested that AQP3 is pro-proliferative in keratinocytes and is upregulated in the NMSC, squamous cell carcinoma (SCC). To evaluate the AQP3/PLD2 signaling module in skin diseases, we determined their levels in SCC, basal cell carcinoma (BCC) and psoriasis as compared to normal epidermis. Skin biopsies with the appropriate diagnoses (10 normal, 5 SCC, 13 BCC and 10 plaque psoriasis samples) were obtained from the pathology archives and examined by immunohistochemistry using antibodies recognizing AQP3 and PLD2. In normal epidermis AQP3, an integral membrane protein, was localized mainly to the plasma membrane and PLD2 to the cell periphery, particularly in suprabasal layers. In BCC, AQP3 and PLD2 levels were reduced as compared to the normal-appearing overlying epidermis. In SCC, AQP3 staining was “patchy,” with areas of reduced AQP3 immunoreactivity exhibiting positivity for Ki67, a marker of proliferation. PLD2 staining was unchanged in SCC. In psoriasis, AQP3 staining was usually observed in the cytoplasm rather than in the membrane. Also, in the majority of psoriatic samples, PLD2 showed weak immunoreactivity or aberrant localization. These results suggest that abnormalities in the AQP3/PLD2 signaling module correlate with hyperproliferation in psoriasis and the NMSCs.     

Abnormal sequence of expression of differentiation markers in psoriatic epidermis: inversion of two steps in the differentiation program?

This immunohistologic study was undertaken to compare epidermal differentiation in normal and psoriatic skin. Although basal cells retain a normal phenotype in this disease, suprabasal layers exhibit abnormal sets of differentiation markers. The 67-kD keratin and Bd5 antigen, which are found in normal epidermis immediately above the basal layer, appear several layers higher in involved psoriatic epidermis. On the contrary, KF2 antigen, which is found in the upper spinous layers of normal epidermis, appears more precociously in psoriatic epidermis. Paradoxically, in this disease characterized by the absence of a granular layer, some markers specific for this layer in normal skin, such as involucrin and transglutaminase, appear in lower skin cell layers, while other granular markers, such as filaggrin, are either absent or found in the parakeratotic scales. These results point out the existence in psoriasis of a suprabasal cell population characterized by a set of markers that are never coexpressed in normal epidermis. The existence of this abnormal population of cells can be explained as the result of the inversion of two steps in the differentiation program. Thus, instead of an inability to express a given differentiation marker, psoriasis seems to be characterized by an abnormal sequence of expression of these markers.     

Two binding sites for Ki67 related to quiescent and cycling cells in human epidermis

The monoclonal antibody Ki67 (Ki67) binds to a nuclear antigen expressed by cycling cells of several human tissues and to the cytoplasm of the basal layer cells of squamous epithelia. We have used an immunohistochemical method to visualize the binding sites of Ki67 in normal and hyperproliferative epidermis. Cytoplasmic staining was present in the basal layer cells of normal epidermis, but was decreased in psoriatic and post-tapestripping epidermis. In sections of normal epidermis only a small minority of nuclei were positive, but sections of psoriatic epidermis and epidermis 40 and 48 h after tapestripping showed large numbers of positive nuclei in the basal and suprabasal layers. Since recent reports strongly suggest that the cell production rate is regulated by changes in the number of cycling cells, the hypothesis that Ki67 binds also in human epidermis to the nuclei of cycling cells is supported by the present findings.     

c-JUN N-terminal kinase-1 (JNK1) but not JNK2 or JNK3 is involved in UV signal transduction in human epidermis

BACKGROUND: c-Jun N-terminal kinase (JNK) plays a critical role in UV-induced apoptotic cell death. Although three isoforms are known in mammals, physiological roles of each isoform are still obscure. Furthermore, our recent findings show that serpin squamous cell carcinoma antigen (SCCA1) binds to JNK. OBJECTIVE: To determine which isoform is responsible for the UV signal transduction in human epidermis and whether SCCA1 is capable to regulate kinase activity of a specific isoform. METHODS: Immunohistochemical localization of each JNK isoform was investigated after UV irradiation in vivo and in vitro. Effect of recombinant SCCA1 on JNK kinase activity was also analyzed. RESULTS: Immunostaining for JNK1, 2 and 3 demonstrated marked elevation of JNK1 in spinous to granular cells of UV-irradiated skin, whereas they were expressed weakly in upper epidermis of the sun-protected, buttock skin. In cultured keratinocytes, only JNK1 is translocated into nucleus after UV irradiation. JNK2, which localized in the cytoplasm, or JNK3, which was confined in nucleus, remained in the same compartment after UV irradiation. We confirmed that only JNK1 mRNA was up-regulated after UV irradiation in cultured keratinocytes. In addition, recombinant SCCA1 suppressed kinase activity of JNK1 but did not affect JNK2 or JNK3 kinase activity. CONCLUSION: JNK1 is associated with UV signal transduction in human epidermis and SCCA1 is a suppressor of this process.     

Superoxide dismutase in psoriasis, squamous cell carcinoma and basal cell epithelioma: an immunohistochemical study

Monoclonal antibodies against human Cu,Zn-superoxide dismutase (SOD) and Mn-SOD were used to stain frozen sections of normal and abnormal human skin. In normal human epidermis, the Cu,Zn-SOD antibody almost exclusively stained the basal cells. Mn-SOD antibody weakly stained the whole of the epidermis but more predominantly the basal cell layer. In psoriasis, Cu,Zn-SOD antibody mainly stained the basal cells of the lowest parts of the elongated rete ridges. Basal cells corresponding to the tip of the dermal papillae were weakly stained. Mn-SOD staining was considerably decreased in the psoriatic epidermis. In squamous cell carcinoma, staining with both Cu,Zn-SOD and Mn-SOD antibodies was decreased, and single cells positive for Cu,Zn-SOD were scattered throughout the tumour nests. In basal cell epithelioma, Cu,Zn-SOD staining was intense and diffusely distributed throughout the tumour nests, while Mn-SOD staining was absent.     

Immunopathological study of proliferation-associated antigens in proliferative skin diseases

59 specimens consisting of 10 psoriasis vulgaris, 1 squamous cell carcinoma, 1 Paget disease, 3 keratoacanthomas, 1 pemphigus vulgaris, 18 cutaneous T cell lymphomas, 2 ATLs, and other skin diseases were studied by immunoperoxidase technique. We used four antibodies to demonstrate a cell proliferation-associated antigen (PC, DNA polymerase-alpha and transferrin receptor) and epidermal growth factor receptor. Our observations suggested that the expression of PC and DNA polymerase-alpha may correlate well with cell proliferation, which were demonstrated in the epidermis of psoriasis vulgaris. Primary and metastatic squamous cell carcinoma and some of psoriasis vulgaris had a positive staining for EGF-R, while normal epidermis and almost all other skin diseases were negative.     

Immunohistochemical localization of ornithine decarboxylase in human skin

The localization of ornithine decarboxylase (ODC) in human skin and cultured keratinocytes was studied with an immuno-histochemical method. ODC was found in the epidermis, hair follicles, sweat glands and errector muscles. Irritation induced by stripping or UV-B irradiation did not change the staining pattern in the epidermis. In psoriasis, the staining was most marked at the tip of the epidermal rete ridges. In basal cell carcinoma, there was a homogeneous labelling of the tumor cells and, in squamous cell carcinoma, the labelling was strong but less homogeneous. Melanoma and dermal naevus also positively stained for ODC. Cultured human keratinocytes also showed ODC positive immunofluorescence. This technique detects the ODC antigen present rather than levels of ODC activity.     

The measurement of the cell cycle time in squamous epithelium using the metaphase arrest technique with vincristine

In squamous epithelia with a single layer of germinative cells, the age distribution of cells in the cell cycle is shown to depend on the direction of the mitotic axis (i.e. a line joining the nuclei of daughter cells) relative to the plane of the basal layer. When axes are in the plane of the basal layer the age distribution is exponential; when cells divide at right angles to the plane of the basal layer, the age distribution is rectangular. When there is a ratio of vertical to horizontal axes, the age distribution is intermediate but can be calculated from knowledge of the proportion of axes in the plane of the layer. Squamous epithelia can be classified according to this arrangement of axes. When there are multiple layers of germinative cells, as in psoriasis, the age distribution is shown to be exponential to a good approximation, whatever the direction of the mitotic axes in the several layers. The importance of these observations is demonstrated by analysing metaphase arrest experiments with vincristine in the single layer of germinative cells in the mouse oesophagus, and in the several layers found in psoriatic epidermis. Choice of the wrong age distribution leads to an error of 6 h in the oesophagus and 23 h in psoriatic epidermis, when the mean cell cycle time is calculated. It is concluded that, in squamous epithelium, it is most important to know the age distribution before calculating the cell cycle time by methods involving measurement of the rate of entry of cells into mitosis or DNA synthesis.     

Localization of anionic sites in normal and psoriatic epidermis: the effect of enzyme digestion on these anionic sites

Cell surface anionic charge is known to be related to various cellular functions. Therefore, we ultrastructurally localized anionic sites in normal and psoriatic human epidermis, using poly-l -lysine-gold complex (cationic gold), to assess their possible participation in the differentiation of keratinocytes and the pathogenesis of psoriasis. In normal and psoriatic epidermis, the cell membrane of keratinocytes showed positive staining at pH 2.0. At pH 7.4 the cytoplasm and nucleus were diffusely stained, in addition to the cell membrane. In normal epidermis, the intensity of labelling on the cell membrane at pH 2.0 was strong in the basal layer and lower stratum spinosum, and decreased in parallel with differentiation of keratinocytes. In psoriatic epidermis, the intensity of labelling on the cell membrane at pH 2.0 was stronger than in normal epidermis. In normal epidermis, heparitinase digested 63% and chondroitinase ABC digested 80% of cationic labelling. This suggests that heparan sulphate and chondroitin sulphate (and/or dermatan sulphate) constitute anionic sites in normal epidermis. In psoriatic epidermis, chondroitinase ABC-sensitive anionic sites were greatly increased, whereas heparitinase-sensitive anionic sites were the same, when compared with normal epidermis. This suggests that chondroitin sulphate and/or dermatan sulphate constitute anionic sites which are increased in psoriatic epidermis.     

Hsp72 antigen expression in the proliferative compartment of involved psoriatic epidermis

Oxidative damage to growth regulatory proteins has been implicated in the aetiology of psoriasis. However, the transient synthesis of heat shock proteins has been shown to protect cells against the adverse effects of oxidative and other forms of physiological stress. This study has used an hsp72 monoclonal antibody to measure inducible 72 kDa heat shock protein expression in heat stressed normal human skin and established plaque psoriasis. Hsp72 was detected in the basal and suprabasal layer cells of heat-stressed normal skin, and in 12 involved psoriasis lesions. Hsp72 expression was not detected in unstressed normal skin or in 12 cases of uninvolved psoriasis. Immunoprecipitation and Western blotting of cell lysates from heat stressed normal skin and involved psoriasis lesions confirmed the presence of a 72 kDa polypeptide with hsp72 immunoreactivity. The MIB-1 monoclonal antibody was used to determine the proliferative fraction of normal and involved psoriastic epidermis. The Ki67 antigen was localised to the nuclei of basal and suprabasal layer cells of normal and involved psoriatic epidermis. Involved psoriatic epidermis contained a higher number of proliferating keratinocytes when compared with normal skin. The study has also demonstrated a strong correlation between hsp72 expression and keratinocyte proliferation in involved psoriatic epidermis (r=0.864, p<0.001). We believe that the 72 kDa inducible heat shock protein performs a protective function in the proliferative compartment of normal and involved psoriatic skin.     

Differentiation-associated localization of small prolne-rich rotein in normal and diseased human skin

Summary The expression of SPRR (small proline-rich protein) was investigated in normal human skin and in diseased skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma. Naevus pigmentosus, ichthyosis vulgaris and several inflammatory skin diseases, by immunohistochemical staining. A polyclonal antibody was raised against a synthetic peptide for a C-terminal common region for SPRR l and SPRR 3. In immunoblot analysis, a positive band of 18kDa was detected, which showed the presence of SPRR l in human epidermal keratinocytes. In normal epidermis, positive staining for SPRK was observed in keratinocytes in the granular layer and the uppermost or two spinous cell layers, with no staining of the other spinous or basal layers. The staining was obvious at the cell periphery, weak at the cytoplasm, and absent in the nucleus. Staining was observed in several outer layers of the follicular infundibulum to the isthmus. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, eccrine duct, melanocytes. Langerhans cells or fibroblasts. The arrectores pilorum, striated muscles, muscle layers of vessels, and myoepithelia of eccrine gland, were weakly stained. In psoriatic skin, stained keratinocytes were distributed in the spinous cell layers except for the basal layer, in ichthyosis vulgaris. SPRR was barely expressed in the uppermost living cell layers of the epidermis in epidermolytic hyperkeratosis. degenerated squamous cells widely expressed SPRR. In Darier's disease, dyskeratolic cells were clearly stained. In squamous cell carcinoma, staining was observed in keratotic cells around horny pearls. In basal cell epithelioma, naevus pigmentosus, and malignant melanoma, the tumour cells or naevus cells were not stained. The distribution of SPRR was similar to that of involucrin in normal and several diseased skin, except for ichthyosis vulgaris. We conclude that SPRR is expressed in close association with epidermal differentiation in normal skin and skin diseases. The alteration of the expression of the proteins correlated to terminal differentiation, and differs from disease to disease.     

Altered expression of occludin and tight junction formation in psoriasis

Abstract In simple epithelia, tight junctions are well developed and have barrier and fence functions. On the other hand, tight junctions are less developed in stratified epithelia. In the rodent epidermis, only maculae occludentes (i.e. focal strands or spot tight junctions) are observed in the most superficial zone of the granular cell layer. Occludin is an integral membrane protein, and is localized at tight junctions in simple epithelia. In normal epidermis, occludin is expressed at the maculae occludentes in the granular cell layer, indicating that it is associated with keratinocyte differentiation. Thus, we examined occludin expression in psoriasis, in which differentiation of keratinocytes is impaired. In psoriasis, occludin was expressed more broadly in the upper epidermis than in normal epidermis. In addition, immunoelectron microscopy showed occludin to be concentrated on the maculae occludentes in the spinous layer of psoriatic skin. These findings indicate that occludin and the formation of tight junctions are related to the proliferation and differentiation of keratinocytes, and to the pathogenesis of psoriasis. Received: 21 August 2000 / Revised: 10 December 2000 / Accepted: 3 February 2001     

Caspase-3在银屑病皮损中的表达

目的探讨银屑病皮损中凋亡调控基因天冬酰胺特异酶切的半胱氨酸蛋白酶(cysteinyl aspartate-specificprotease,Caspases)家族中Caspase-3的表达.方法采用免疫组化法检测Caspase-3在寻常性银屑病皮损中的表达.结果在正常皮肤组织中,Caspase-3的表达仅局限于表皮基底细胞层的细胞浆,在银屑病皮损中,Caspase-3表达弥散分布于增生表皮的基底细胞层、棘细胞层及颗粒细胞层;与正常表皮相比较,基底细胞层Caspase-3的表达明显减少.结论在银屑病皮损中,凋亡调控基因Caspase-3表达的定位性改变可能与银屑病发病机制有关.     

The expression of syndecan-1 in psoriatic epidermis

Psoriasis is a chronic inflammatory skin disease characterized by exaggerated keratinocyte proliferation. Current opinion indicates that psoriasis is driven by T cell-mediated immune responses targeting keratinocytes. However, psoriasis cannot be explained solely on the basis of T-cell activation, and it is likely that an intrinsic alteration in epidermal keratinocytes plays a very important role in disease expression. Syndecans comprise a major family of cell surface heparan sulfate proteoglycans. Several studies indicate their role in adhesion, cell-extracellular matrix interactions, migration, keratinocyte proliferation and differentiation, inflammation, and wound healing. To determine the expression of syndecan-1 in psoriasis, skin samples from 29 patients with fully developed psoriasis and skin samples from 14 healthy volunteer persons with no personal or family history of psoriasis were immunohistochemically examined using monoclonal antibody against syndecan-1. The expression of syndecan-1 was analyzed in whole mount section of psoriatic and non-psoriatic skin biopsies under high magnification (400x). In addition, the intensity and topography of reaction in the cell, as well as localization of positive cells in the epidermis were evaluated. Strong syndecan-1 reactivity in epidermal cells in all non-psoriatic and psoriatic samples was observed. Statistical analysis showed no significant differences between two analyzed groups (P > 0.05). In normal skin syndecan-1 was expressed in full thickness of the epidermis. The strongest reaction was observed in membranes and intercellular junctions of spinous and granular layer while basal cells showed weaker expression that was confined to cytoplasm. In psoriatic skin syndecan-1 was expressed in the membrane and intercellular junction of cells located in thickened and elongated rete ridges of the epidermis. The strongest reaction was in basal and suprabasal layers and expression diminished through spinous layer. Cells in spinous layer lose syndecan-1 expression, which is opposite pattern to normal skin. Our results suggest that aberrant skin expression of syndecan-1 may be involved in the development of psoriasis.