Expression of 1,25-dihydroxyvitamin D3 receptors in normal and psoriatic skin.

Increasing evidence suggests an immunoregulatory function of the potent steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) which has been successfully applied for treatment of psoriasis. The skin is both a site of production and a target of 1,25(OH)2D3. In vitro, 1,25(OH)2D3 inhibits proliferation and stimulates differentiation of keratinocytes. We investigated the in situ expression of vitamin D-receptors (VDR) in normal and psoriatic skin by immunochemical methods. The VDR were visualized using the monoclonal antibody (MoAb) 9A7g to the VDR and the labeled avidinbiotin technique. Immunoreactivity was consistently confined to nuclei in all skin biopsies. In normal skin specimens (n = 10) VDR antigens were expressed in keratinocytes of all epidermal layers (except those of the stratum corneum) and in cells of the epidermal appendages. Double labeling experiments with MoAb to cluster-defined antigens indicated that melanocytes and approximately 75% of Langerhans cells exhibit 1,25(OH)2D3 receptors in normal skin biopsies (n = 5). Depending on their localization in skin compartments 42-62% of CD11b+ positive macrophages and 45-75% of CD3+ T lymphocytes expressed VDR. Non-lesional psoriatic skin specimens (n = 8) revealed nearly identical staining patterns. Lesional psoriatic skin specimens (n = 8) exhibited a significant increase of VDR expression both in basal and suprabasal epidermal layers as measured by computer-assisted morphometry and showed a remarkable change of the immune cell pattern: the densitity and proportion of VDR positive T lymphocytes and macrophages were higher in the epidermal and the perivascular papillary loop compartment. These in vivo findings strongly support the hypothesis that 1,25(OH)2D3 modulates immune response and cell proliferation/differentiation in human skin.     

Expression of serotonergic receptors in psoriatic skin

Psoriasis appears to be influenced by stress, which causes release of adrenal hormones. Serotonin, or hormonal actions on serotonin and serotonin receptors, may have a role in psoriasis. Distribution of serotonin receptors was studied in involved and noninvolved skin in patients with psoriasis and compared to normal skin, by using immunohistochemistry and antibodies to 5-HT1A, 5-HT2A and 5-HT3 receptors (R). There was a decreased (P<0.001) number of 5-HT1AR positive cells, the majority being tryptase positive, in involved and noninvolved psoriatic papillary dermis, compared to normal skin. 5-HTlAR expression was also found in the upper part of the epidermis, on vessel walls and on melanocytes. 5-HT2AR expressing papillary mononuclear cells, CD3 positive, were increased (P<0.001 and P<0.01, respectively) in involved and noninvolved psoriatic skin, compared to normal skin, an increase (P<0.01) also being found in the involved compared to noninvolved skin. Expression of 5-HT3R could be found in the basal epidermal layer of noninvolved but not in the involved skin of psoriasis, where it was only found in the acrosyringium. The present findings are compatible with the 5-HT1A and 5-HT2A receptors having antagonistic functions, and raise the possibility of using receptor specific drugs in the treatment of psoriasis.     

Expression and localization of peroxisome proliferator-activated receptors and nuclear factor kappaB in normal and lesional psoriatic skin

Abnormal epidermal proliferation and differentiation characterize the inflammatory skin disease psoriasis. Here we demonstrate that expression of PPARdelta mRNA and protein is markedly upregulated in psoriatic lesions and that lipoxygenase products accumulating in psoriatic lesions are potent activators of PPARdelta. The expression levels of NF-kappaB p50 and p65 were not significantly altered in lesional compared with nonlesional psoriatic skin. In the basal layer of normal epidermis both p50 and p65 were sequestered in the cytoplasm, whereas p50, but not p65, localized to nuclei in the suprabasal layers, and this distribution was maintained in lesional psoriatic skin. In normal human keratinocytes PPAR agonists neither impaired IL-1beta-induced translocation of p65 nor IL-1beta-induced NF-kappaB DNA binding. We show that PPARdelta physically interacts with the N-terminal Rel homology domain of p65. Irrespective of the presence of agonists none of the PPAR subtypes decreased p65-mediated transactivation in keratinocytes. In contrast p65, but not p50, was a potent repressor of PPAR-mediated transactivation. The p65-dependent repression of PPARdelta- but not PPARalpha- or PPARgamma-mediated transactivation was partially relieved by forced expression of the coactivators p300 or CBP. We suggest that deficient NF-kappaB activation in chronic psoriatic plaques permitting unabated PPARdelta-mediated transactivation contributes to the pathologic phenotype of psoriasis.     

Expression of transferrin receptor in normal human skin, psoriatic skin and various skin tumors

The distribution of transferrin receptor in normal human skin and its expression in psoriatic skin and various skin tumors have been investigated. Immuno-peroxidase staining was performed on biopsy specimens using monoclonal OKT 9 antibody, which reacts with transferrin receptor. Normal human skin showed positive staining with OKT 9 in eccrine glands and outer root sheaths of the hair. Sebaceous glands were also strongly positive. The basal layer stained very weakly. Psoriatic skin expressed OKT 9 strongly in the epidermis, especially in the area of the rete ridge. In squamous cell carcinoma, Bowen's disease, and malignant melanoma, a widespread and strong labelling reaction was found. Basal cell epithelioma and genital Paget's disease were partially and moderately positive in their staining pattern. No such positive staining pattern could be found in either nevus cell nevi or seborrhoic keratosis. These findings indicate that, in normal skin, transferrin receptor exists in eccrine glands, sebaceous glands, and outer root sheaths of the hair in greater amounts. High amounts of expression of this receptor in psoriatic epidermis and malignant tumors suggests that immunohistochemical demonstration of transferrin receptor parallels the proliferating activity of the tissue or tumor of the skin and may provide an useful aid for detecting such conditions.     

Chromosome studies of normal and psoriatic skin fibroblasts.

A method for obtaining large numbers of fibroblasts from punch biopsies of skin tissue and a procedure for the visualization of chromosomes from these cells are presented in detail. The chromosomes are well separated, and fine details of their structure are seen. A study was made of 275 cells from apparently normal and from involved areas of skin from patients with psoriasis. No changes either in the number or in the fine structure of the chromosomes were observed.     

Interleukin-8 receptors in normal and psoriatic polymorphonuclear leukocytes.

Polymorphonuclear leukocyte (PMNL) infiltration is an important characteristic in psoriatic lesions. The proinflammatory 8-kD peptide interleukin-8 (IL-8) is present in psoriatic scales and possesses a high chemotactic activity on human neutrophils, which may relate to its role in psoriasis. Its chemotactic activity is mediated via specific receptors on PMNL. The goal of our work was to ascertain whether PMNL infiltration in psoriasis can be accounted for by functional abnormalities of the circulating PMNL due to alterations in the IL-8 receptor density or affinity (or both). Results of radioligand binding studies performed in 10 psoriatic patients, 10 patients with atopic eczema and 11 normal controls showed no difference in receptor affinity (Kd) between the groups. However, a slight but significant elevation in IL-8 receptor density was seen on PMNL from psoriatic individuals (31,230 +/- 3,237 binding sites per cell) compared to those from normal volunteers (24,152 +/- 2,643) and atopic eczema patients (24,092 +/- 2,743). Increased number of IL-8 receptors may, besides elevated cutaneous IL-8 concentrations, contribute to the intraepidermal accumulation of PMNL in psoriasis.     

Mast cells in psoriatic skin are strongly positive for interferon-gamma

The increased number and early activation of cutaneous mast cells is a typical feature of psoriatic inflammation. Interferon-gamma (IFN-gamma) is believed to be one of the important mediators in the cytokine cascade of psoriasis. Human mast cells have been previously reported to release various cytokines upon stimulation including interleukin (IL) -4, IL-5, IL-6, IL-8, IL-13 and tumour necrosis factor-alpha. Here we report that human mast cells synthesize also IFN-gamma at mRNA and protein level and that the number of IFN-gamma producing mast cells is significantly increased in the psoriatic skin. IFN-gamma immunoreactivity in mast cells was demonstrated by staining non-lesional and lesional skin sections from 21 patients with psoriasis. Ten patients with atopic dermatitis (AD) and five healthy persons served as control groups. The percentage (mean +/- SD) of IFN-gamma + mast cells in lesional compared with non-lesional psoriatic skin was 67 +/- 18% vs. 44 +/- 17% (P < 0.0001, paired t-test), respectively, but only 9 +/- 6% vs. 10 +/- 7% in corresponding skin samples of AD. In the skin of healthy controls, only 12 +/- 12% of the mast cells were IFN-gamma +. Using immunoelectron microscopy, we confirmed the ultrastructural localization of IFN-gamma within the granules of mast cells in psoriatic skin. In addition, stimulation of a human mast cell line HMC-1 with phorbol myristate acetate (PMA) (100 nmol/L) for periods of 2-24 h induced expression of IFN-gamma mRNA, which peaked at 24 h. When HMC-1 cells were stimulated with PMA (100 nmol/L) for periods of 0-3 days, the cells released IFN-gamma protein, peaking on day 1. These results provide further evidence for the important role of mast cells in the pathogenesis of psoriasis.     

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.     

Cytophotometric studies of epidermal proliferation in psoriatic and normal skin.

Cytophotometric measurements of single-cell DNA content were used to study human epidermal cell proliferation in vivo. It was found that Feulgen-DNA profiles can be used to assess proliferative activity in involved, uninvolved, and nonpsoriatic skin. Profiles of involved psoriatic skin were bimodal as is characteristic of actively proliferating populations. This was due to the presence of cells with twice (2C--presynthetic) or four times (4C--post-synthetic) the amount of DNA of the gametes, separated by the intermediate values of cells undergoing scheduled DNA synthesis. Profiles of uninvolved psoriatic as well as nonpsoriatic epidermis were unimodal with the majority of cells containing a 2C amount of DNA incating relatively low levels of proliferative activity. The observed variations in proliferative activity of these samples are discussed in terms of two alternative models. Since radioisotopes are not required, this technique presents a useful approach to studying human epidermal proliferation in vivo.     

Comparison of bacterial microbiota in skin biopsies from normal and psoriatic skin

Microorganisms have been implicated in the pathogenesis of psoriasis. Previous studies of psoriasis and normal skin have used swabs from the surface rather than skin biopsies. In this study, biopsies were taken from 10 patients with psoriasis and 12 control subjects from unmatched sites. Samples were analysed with massive parallel pyrosequencing on the 454 platform targeting the 16S rRNA gene and the variable regions V3–V4. The samples grouped into 19 phyla, 265 taxon and 652 operational units (OTUs) at 97% identity. A cut-off abundance level was set at 1%. The three most common phyla in both normal and psoriasis skin were Firmicutes (39% psoriasis, 43% normal skin), Proteobacteria (38% psoriasis, 27% normal skin) and Actinobacteria (5% psoriasis, 16% normal skin, p = 0.034). In trunk skin, Proteobacteria were present at significantly higher levels in psoriasis compared to controls (52 vs. 32%, p = 0.0113). The commonest genera were Streptococci in both psoriasis (32%) and normal skin (26%). Staphylococci were less common in psoriasis (5%) than in controls (16%), as were Propionibacteria (psoriasis 0.0001669%, controls 0.0254%). Both Staphylococci and Propionibacteria were significantly lower in psoriasis versus control limb skin (p = 0.051, 0.046, respectively). This study has shown some differences in microbiota between psoriasis and normal skin. Whether these are of primary aetiological significance, or secondary to the altered skin of psoriasis remains to be determined.     

Expression of the T-cell activation antigens CD27 and CD28 in normal and psoriatic skin

Activated T lymphocytes are thought to be involved in the pathogenesis of psoriasis. From studies with peripheral blood T lymphocytes it is known that T cells show a decrease in membrane expression of CD27 molecules during continuous antigenic stimulation. The T-cell activation molecule CD28 is thought to be involved in the transduction of an antigen-non-specific costimulatory signal. Therefore, in order to elucidate further the pathogenesis of psoriasis we studied the expression of CD27 and CD28, together with CD4, CD8 and CD45RA in this benign inflammatory dermatological disease. We used immunohistochemical techniques to determine absolute numbers of T lymphocytes and expression of these T-cell activation and T-subset-specific molecules in normal (n= 7), uninvolved perilesional (n= 7) and lesional psoriatic (n= 7) skin. We found that not only lesional but also clinically uninvolved perilesional skin showed an increased number of T cells. Further, immunohistochemical studies showed that CD27 is expressed by a minority of normal skin T cells, while in lesional psoriatic skin, expression was even lower, and almost absent in perilesional skin sections. In contrast to normal skin, both perilesional and lesional psoriatic skin contained no CD28 positive T cells. In lesional psoriatic skin, however, T cells showed predominantly the CD4 phenotype, while in perilesional skin CDS positive T cells were dominant. Two conclusions were reached: first, the absolute number of T cells, their CD27, CD28 and CD45RA expression, and the influx of CD8 positive T cells, indicate that perilesional psoriatic skin is different from normal and lesional psoriatic skin; and secondly, the data on CD27 and CD28 suggest that not only lesional but also perilesional psoriatic skin is subject to continuous antigenic stimulation, thus leading to decreased CD27 and CD28 expression on skin T cells.     

Expression, subcellular localization and cytokinic modulation of Toll-like receptors (TLRs) in normal human keratinocytes: TLR2 up-regulation in psoriatic skin

The aim of the research described here was to investigate the expression of Toll-like receptors (TLRs) in normal human keratinocytes, to study its modulation by proinflammatory cytokines, and to characterize the function of the latter within the epidermis. Our results demonstrate that normal human keratinocytes may present an intra-cytoplasmic expression of TLR2, TLR3, and TLR4. Exposure of keratinocytes to IFN-gamma and TNF-alpha increased intra-cytoplasmic expression and led to partial translocation at the cell surface. Keratinocyte activation by TLR2, TLR3, and TLR4 ligands led to the nuclear translocation of NF-kappab and the release of proinflammatory cytokines TNF-alpha and IL-8. In immunochemistry analysis, psoriatic skin showed a strong over-expression of TLR2 in the epidermis compared with normal skin. Our results thus demonstrate large TLR expression in keratinocytes and the functionality of TLRs 2, 3, and 4. TLR2 over-expression in psoriatic skin provides new insights into TLR implication in the pathogenesis of psoriasis, through inappropriate stimulation by infectious or endogen ligands.     

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.