The effect of methotrexate on the expression of cell adhesion molecules and activation molecule CD69 in psoriasis

BACKGROUND: The mechanism of the action of methotrexate (MTX) in the treatment of psoriasis has not been completely elucidated. OBJECTIVE: To assess the effect of MTX on the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, activation molecule CD69 and T-cell phenotype in skin specimens from patients with psoriasis. METHODS: We performed an immunohistochemical analysis of the expression of T-cell phenotype and cell adhesion/activation molecules in skin biopsies from patients with psoriasis treated with a fixed dose of MTX (12.5 mg/week). To determine data on the epidermal/dermal T-cell infiltration we carried out a manual quantification. RESULTS: Skin samples prior to therapy showed a moderate to severe inflammatory infiltrate, mainly due to T lymphocytes with a helper/inducer (CD4) phenotype. Most of these cells also expressed ICAM-1 and VCAM-1. Blood vessels showed expression of E-selectin and VCAM-1, and keratinocytes were positive for ICAM-1 staining. The cell infiltrate was reduced after therapy, as well as the expression of cell adhesion molecules. However, we also noted the persistence of the T lymphocyte phenotype CD8(+), expressing the CD69 activation molecule, after the MTX treatment. CONCLUSIONS: MTX downregulates the expression of some adhesion molecules, a phenomenon that may contribute to its anti-inflammatory therapeutic effect in psoriasis. The infiltrating T cells post-treatment have an activated cytotoxic phenotype, which may suggest a pathogenic role in the continuation and/or recurrence of psoriasis.     

Comparison of changes in endothelial adhesion molecule expression following UVB irradiation of skin and a human dermal microvascular cell line (HMEC-1)

We have assessed the pattern of dermal endothelial adhesion molecule expression following broadband UVB irradiation in vivo and in vitro. Skin biopsies were taken from 4 human volunteers at baseline and at 4, 8 and 24 h post-irradiation with 2.5 minimal erythema doses of UVB. Sections were stained immunohistochemically for E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD31 and neutrophil elastase. The effect of direct UVB irradiation on E-selectin, ICAM-1 and VCAM-1 was examined in a human dermal microvascular endothelial cell line, HMEC-1. Cultured HMEC-1 were irradiated with 2.5–40 mJ/cm² of UVB, and assessed for adhesion molecule expression by immunofluorescence microscopy and fluorescence-activated cell sorter analysis. In vivo, E-selectin was minimally expressed on EC at baseline and was induced by 4 h following irradiation, P<0.01. ICAM-1 was moderately expressed at baseline and appeared mildly induced at 24 h, although this did not reach statistical significance. VCAM-1 was weakly expressed in unirradiated skin while CD31 was moderately expressed, but neither was induced by UVB irradiation. A significant neutrophilic infiltrate appeared by 8 h and was maximal at 24 h, P<0.05. Neutrophil infiltration correlated with E-selectin expression, r=0.96. In HMEC-1, ICAM-1 was upregulated at 24 h post-irradiation, with an increase in mean channel fluorescence from 100% at baseline to 145 (SD12)%> at 24 h, P<0.05. No change was seen in expression of E-selectin, VCAM-1 or CD31. These studies support the involvement of endothelial adhesion molecules E-selectin and ICAM-1 in UVB-induced inflammation. Whereas ICAM-1 is upregulated by direct irradiation of endothelial cells, E-selectin stimulation appears to be an indirect effect.     

Proliferative activity of CD8(+) T cells as an important clue to analyze T cell-mediated inflammatory dermatoses

Abstract To elucidate the pathogenesis of T cell-mediated inflammatory skin diseases, we examined the exact sites where CD8(+) T cells proliferate, correlating them with the localization of antigen-presenting dendritic cells. We performed CD8/Ki-67 double immunohistochemical staining and single staining for CD1a, CD68, and factor XIIIa on sections of paraffin-embedded tissue samples of inflammatory dermatoses in which T lymphocytes are thought to play a crucial role. The dermatoses were lichen planus (12 samples), acute graft-versus host disease (GVHD) (12 samples), chronic GVHD (10 samples), spongiotic dermatitis (8 samples) and psoriasis (7 samples). Labelling for Ki-67 among CD8(+) T cells was predominantly observed in the subepidermal lymphoid infiltrate, and was scanty in the epidermis. This suggested that proliferation of CD8(+) T cells occurred preferentially in the dermis. The labelling index for Ki-67 among dermal and epidermal CD8(+) cells was quite different among the different diseases studied (P < 0.05). They were rich in the subepidermal portion of the dermis of spongiotic dermatitis, acute GVHD and chronic GVHD, but rare in the dermis of psoriasis and lichen planus. A moderate infiltrate was also observed in lesional epidermis of spongiotic dermatitis, acute GVHD and chronic GVHD, whereas they was almost none in the epidermis of psoriasis and lichen planus. CD1a(+) dermal dendritic cells were densely distributed within the lymphoid infiltrate in the affected dermis of spongiotic dermatitis, psoriasis and lichen planus, whereas they were minimal in GVHD. These dermal dendritic cells are candidates as stimulators on T cells in the dermis. In conclusion, the proliferative status of T cells could be an important clue in the elucidation of the pathophysiology of T cell-mediated inflammatory dermatoses. Received: 13 December 2000 / Revised: 24 April 2001 / Accepted: 11 July 2001     

Changes in T-cell phenotype and adhesion molecules expression in psoriatic lesions after low-dose cyclosporin therapy

Cyclosporin is a very effective treatment for severe psoriasis, but its exact mechanism of action in this disease is not completely understood. It has been hypothesized that the drug could act through (lie inhibition of the expression of certain cell adhesion molecules on the keratinocytes prior to the reduction in the number of epidermal inflammatory cells. Several studies have focused on ICAM-1 changes on keratinocytes and endothelial cells after cyclosporin treatment in psoriatic patients but their results have been somewhat contradictory. We examined changes in T-cell markers and adhesion molecules among keratinocytes, enclothelial and inflammatory cells after low-dose cyclosporin treatment for severe psoriasis. We performed a histological and immunohistochemical study on psoriatic skin among 10 patients (7 males and 3 females; mean age 37 years) treated with low-dose (2.5 mg/kg/day) cyclosporin, prior to therapy, after 1 month, and after 3 months of treatment. The mean PASI (Psoriasis Area and Severity Index) before treatment was 23±4, 13±7 after the first month of therapy, and 8±2 at the end of the third month of therapy. Pretherapy samples showed a moderate to severe inflammatory infiltrate mainly clue to T-lymphocytes expressing a T-cell memory (UCHL-1) and helper/inducer (CD4) phenotype. Most of these cells also expressed HLA-DR and LFA-1 and ICAM-1 antigens. Alter the treatment, an overall reduction in the degree of epidermal hyperplasia was seen (p=0.01). The severity of the infiltrate was clearly reduced (p=0.05), but no significant changes in the phenotype profile were observed. Although slightly reduced, endothelial ICAM-1 expression persisted after cyclosporin therapy. Keratinocyte ICAM-1 expression was uniformly and significantly reduced after 1 month and 3 months of therapy (p=0.01). These results support the hypothesis that cyclosporin interferes with the expression of keratinocyte adhesion molecules in patients with psoriasis.     

Adhesion molecules and IL-1 costimulate T lymphocytes in the autologous MECLR in psoriasis

Membrane molecules such as CD36 (OKM5), intercellular adhesion molecule-1 (ICAM-1, CD54), gamma interferon-induced protein 10 (γ-IP10) and IL-1 are induced and/or upregulated in psoriatic epidermis. These molecules have important accessory, trafficking or signalling functions in the immune system and also play a role in the pathophysiology of psoriasis. The relevance of adhesion molecules, CD36 and epidermal IL-1 in psoriasis was studied in vitro in the autologous mixed epidermal cell-T lymphocyte reaction (MECLR). Their level of expression was quantitated in epidermal cell suspensions (ECS) from patients with psoriasis and their function was assessed by blocking with specific mAbs and antisera or by depleting CD36⁺ cells from the ECS prior to the MECLR. ECS from psoriatic lesions contained increased numbers of CD36⁺ (23±12%), ICAM-1⁺ (31±14%) and IL-1⁺ (57±21%) cells. The autologous MECLR was inhibited in saaples from all patients by mAb to CD2 (LFA-2), CD11a (LFA-1α), CD18 (LFA-1β), ICAM-1, CD58 (LFA-3) and an antiserum to IL-1β. Thus, adhesion molecules facilitate inflammation in psoriasis not only via adhesion and recruitment of T lymphocyte in psoriatic lesions, but also via activation of T cells. Furthermore CD36 molecules on psoriatic epidermal cells do not costimulate autologous T lymphocytes in psoriasis. The observed costimulatory function of IL-1β in the MECLR emphasizes its relevance in psoriasis.     

Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-α and their degranulation is associated with expression of ICAM-1 in the epidermis

Abstract The release of cytokines from cutaneous cells may be of major importance in the initiation and development of many inflammatory skin disorders. For example, tumor necrosis factor-alpha (TNF-α), which in healthy skin is found preformed only in mast cells, is able to induce the expression of several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1). Increased expression of ICAM-1 occurs in keratinocytes in lesional skin of psoriasis and atopic dermatitis (AD) and it is considered to be an important initiator of leucocyte/keratinocyte interactions in skin inflammation. We counted the mast cells showing TNF-α immunoreactivity using a double-staining method in nonlesional and lesional skin sections from 12 patients with AD and 12 patients with psoriasis. The percentage of TNF-α⁺ mast cells in lesional and nonlesional AD skin was 36 ± 22% and 21 ± 15% (P < 0.018, paired t-test), respectively, and in psoriatic skin was 16 ± 25% and 15 ± 15%, respectively (P < 0.89, paired t-test). We also cultured whole skin biopsies taken from the healthy-looking skin of psoriatic and AD patients in the presence of mast cell degranulator compound 48/80, which resulted in focal expression of ICAM-1 in the epidermis. In cultured keratinocytes, both histamine and an extract of a human mast-cell line (HMC-1) induced ICAM-1 immunostaining only in occasional cells, but the combination of histamine and the HMC-1 extract resulted in intense ICAM-1 staining in numerous cells. This enhancement of ICAM-1 staining was abolished by preincubation of the HMC-1 extract with anti-TNF-α antibody. These results suggest that the degranulation of mast cells induces the expression of ICAM-1 in keratinocytes probably via TNF-α and histamine. Received: 8 August 1997     

Effect of nerve growth factor on endothelial cell biology: proliferation and adherence molecule expression on human dermal microvascular endothelial cells

Abstract In addition to its effect on the central nervous system, nerve growth factor (NGF) appears to play a key role in the initiation and maintenance of inflammation in many organs. NGF degranulates mast cells, recruits inflammatory cellular infiltrates and activates T cells. Extravascular migration of leukocytes is initially controlled by the interaction of cell surface adhesion molecules of leukocytes and endothelial cells. A marked upregulation of NGF in keratinocytes is also observed in conditions characterized by angiogenesis such as psoriasis and wound healing. In this study we investigated the role of NGF in inflammation by studying its effects on endothelial cell proliferation and intracellular adhesion molecule expression by endothelial cells. The effect of NGF on human dermal microvascular endothelial cell (HDMEC) proliferation was measured using the hexosaminidase assay. ICAM-1 expression on HDMEC was measured by ELISA. The function of ICAM-1 was assessed by adherence of peripheral blood mononuclear cells (PBMC) to HDMEC using ⁵¹Cr-labeled PBMC. There was a significant increase in proliferation of HDMEC stimulated with NGF as compared to unstimulated HDMEC (P < 0.001). NGF-neutralizing antibody decreased the mitogenic effect of NGF significantly (P < 0.05). NGF also increased ICAM expression on HDMEC as compared to unstimulated HDMEC (P < 0.05). NGF-neutralizing antibody decreased ICAM expression on NGF-stimulated HDMEC (P < 0.05). The percentage of PBMC adherence was higher in NGF-stimulated HDMEC (P < 0.001). Anti-ICAM antibody decreased PBMC adherence. In the study reported here, the role of NGF in two important aspects of inflammation, i.e. angiogenesis and inflammatory cell recruitment at the site of inflammation, was investigated . Received: 7 December 2000 / Revised: 10 December 2000 / Accepted: 24 February 2001     

CELL INFILTRATEN PROGRESSIVE PIGMENTED PURPURA (SCHAMBERG'S DISEASE): IMMUNOPHENOTYPE, ADHESION RECEPTORS, AND INTERCELLULAR RELATIONSHIPS

Background. Progressive pigmented purpura (Schamberg's disease), a form of purpura pigmentosa chronica, is a lymphocytic capillaritis of unknown etiology and obscure pathogenesis. Our purpose was to assess the expression of cell membrane antigens (CD3, CD4, CD1a, CD36), of adhesion receptors (leukocyte function adhesion 1, LFA-1, endothelial leukocyte adhesion molecule 1, ELAM-1) intercellular adhesion molecule 1, ICAM-1), and the intercellular relationships in the early phase of the disease. Methods. Quantitative immunohistochemistry and electron-microscopy were performed on specimens of five subjects, aged 45 to 63 years. These studies were repeated in two patients after treatment with topical corticosteroid (betamethasone valerate cream 0.1%) and psoralen-ultraviolet A (puva). Results. The infiltrate consisted mainly of CD4+ lymphocytes and CD1a+ dendritic cells. Electron-microscopic investigation showed typical lymphocytes and two distinct types of dendritic cells. In the very early phase of the disease the adhesion receptors LFA-i and ICAM-1 were expressed intensely by all infiltrating cells; the adhesion receptors icam-1 and ELAM-i were expressed by endothelial cells. Close contact occured between lymphocytes and dendritic cells. After PUVA (120 J per cm²) and topical steroid therapy the infiltrate disappeared completely. Conclusions. These data suggest that a cell-mediated immune mechanism may be important in progressive pigmented purpura and that the early endothelial expression     

Immunohistochemical Studies of Infiltrating Cells in Early and Chronic Lesions of Psoriasis

The expression of surface antigens on infiltrating cells, epidermal keratinocytes, and dendritic cells in biopsy specimens from 31 patients with psoriasis was examined immunohistochemically. The specimens were divided into early-phase and chronic-phase groups and then examined in a double blind manner. Among the infiltrating cells in the epidermis, CD4-positive cells were dominant in the early phase; CD8-positive cells were dominant in the chronic phase, resulting in a markedly decreased CD4/CD8 ratio in the latter. On the other hand, among the infiltrating cells in the dermal papillae, CD4-positive cells were dominant in both the early and chronic phases; both CD4-positive and CD8-positive cells were more dominant in the chronic phase than in the early one. However, the CD4/CD8 ratios were decreased in both the dermal papillae and the epidermis in the chronic phase. CD1-positive dendritic cells (probably Langerhans cells) were more numerous in the chronic phase than in the early phase. There were no significant differences between the early and chronic phases with regard to the expression of HLA-DR and HLA-DQ antigens on the infiltrating cells. However, the HLA-DR antigens and ICAM-1 (intercellular adhesion molecule-1) were more strongly expressed on epidermal keratinocytes in the chronic phase than in the early phase. LFA-1α (lymphocyte function-associated antigen-1α)-positive cells were also significantly more numerous in the chronic phase than in the early one, consistent with the expression of HLA-DR antigens and ICAM-1 on keratinocytes mentioned above. On the other hand, VLA-4 (integrin α₄β₁) positive cells were expressed more abundantly in the epidermis in the early phase than in the chronic phase. These results suggest, first, that the chronic phase of psoriasis is as immunologically active as or more active than the early phase. Second, CD4-positive T cells are more important than CD8-positive T cells in the early phase of psoriasis; CD8-positive rather than CD4-positive T cells are more important in the chronic phase. Third, the LFA-1/ICAM-1 pathway may play an important role with regard to cell adhesion of the infiltrating cells in the psoriatic lesions in disease exacerbation or prolongation, whereas the VLA-4/VCAM-1 (vascular cell adhesion molecule-1) pathway may be more important in disease onset.     

Role of SPRED1 in keratinocyte proliferation in psoriasis

Psoriasis is a recurrent inflammatory skin disease, affecting approximately 2% of the population. Previous studies have demonstrated that psoriatic dermal mesenchymal stem cells (DMSC) stimulated keratinocyte (KC) proliferation and that psoriasis exhibited missense SPRED1 mutations. To further investigate the molecular mechanism by which psoriatic DMSC stimulate KC proliferation, and the role of missense SPRED1 mutations in psoriasis, we assessed expression levels of miRNA, and both mRNA and protein of SPRED1 in normal human epidermal keratinocyte cells (NHEK) cocultured with either psoriatic or control DMSC. Expression levels of miRNA and mRNA were determined by RNA sequencing. Expression levels of spred1 protein were assessed using western blot analysis. Moreover, the variation in SPRED1 was also examined by whole-genome sequencing in 665 psoriatic patients, and verified by Sanger sequencing. Our results showed that coculture of NHEK with psoriatic DMSC induced 32 differentially expressed miRNA, in which expression levels of miR-1 increased approximately 16-fold over control DMSC-treated NHEK (P < 0.05). Likewise, expression levels of miR-21-3p increased over twofold (P < 0.05). Moreover, coculture of NHEK with psoriatic DMSC induced marked increase in expression levels of mRNA for MAPK3, CDC25B and CDC25C, while decreasing expression levels of SPRED1 mRNA and protein in comparison with control DMSC treatment (P < 0.05 for all between cocultured with control and psoriatic DMSC). Furthermore, psoriasis displayed non-synonymous mutation of SPRED1 enriched in exon 7: c.A881T:p.Y294F (chr15:38351210). These results suggest that dysregulation and mutations of SPRED1 may participate in the pathogenesis of psoriasis, including epidermal hyperproliferation.     

Increased expression of TRAIL and its death receptors DR4 and DR5 in plaque psoriasis

TNF-related apoptosis-inducing ligand (TRAIL) is recognized as an important regulator of immune responses during infections and various autoimmune-mediated pathologies. Its role in inflammatory dermatoses is largely unknown. We aimed to investigate the expression of TRAIL and its receptors DR4 and DR5 in psoriasis vulgaris. Immunohistochemistry for TRAIL, DR4 and DR5 was performed on samples of lesional (n = 10) and non-lesional (n = 10) skin of patients with plaque psoriasis and skin of healthy volunteers (n = 10). Expression of TRAIL and its receptors was further examined by means of double immunofluorescence staining and co-localization with CD4, CD8, CD11c, CD68, CD16 and CD56 markers. Immunohistochemical staining for TRAIL was significantly enhanced in psoriatic lesional as well as non-lesional epidermis compared to the epidermis of healthy skin. Lesional epidermis also showed increased immunoreactivity for DR5. In addition, expression of TRAIL and both of its receptors was significantly increased in the dermis of lesional skin. As evidenced by double immunofluorescence, TRAIL was readily expressed by most of the examined cells of the inflammatory infiltrate in psoriatic lesions. In contrast, the expression of DR4 was found mostly among CD4+ and CD8+ cells but was only nuclear, while DR5 showed cytoplasmic staining in rare CD16+, CD56+ and CD68+ cells. According to abundant in situ presence of TRAIL and its receptors in lesional psoriatic skin, it seems that this cytokine participates in the complex interplay between keratinocytes and cells of the dermal infiltrate and thus contributes to the inflammatory cycle in psoriasis vulgaris.     

The ICAM-3/LFA-1 interaction is critical for epidermal Langerhans cell alloantigen presentation to CD4+ T cells

Intercellular adhesion molecule (ICAM)-3 is a recently described member of the immunoglobulin superfamily and, as such, is closely related to ICAM-1 and ICAM-2. All three ICAMS are cognate for the counter-receptor lymphocyte function associated antigen-1 (LFA-L CD11a/CD18). Unlike ICAM-1 and ICAM-2. ICAM-3 is constitutively expressed at high levels on resting leucocytes. We investigated the expression and function of ICAM-3 in normal skin (n= 5), as well as its expression in psoriasis (n= 4). atopic eczema (n= 4), allergic (rhus) contact dermatitis (n=3). and cutaneous T-cell lymphoma (CTCL. n=2). Five-micrometre cryostat sections of skin were stained using monoclonal antibodies to ICAM-3 and A well characterized immunoperoxidase technique. In normal skin. ICAM-3 was expressed by all cutaneous leucocytes hut most striking was the strong expression of ICAM-3 by Langerhans cells within both epidermis and dermis. This observation was confirmed by double-labelling with CD1a and negative staining with an IgG1 isotype control. In psoriasis, atopic eczema, allergic contact dermatitis, and CTCL. ICAM-3 was co-expressed on all CD1a⁺ cells, although, in psoriasis, the intensity of ICAM-3 expression was reduced. Functional blocking experiments were performed to determine whether the observed ICAM-3 expression on Langerhans cells was functionally important in antigen presentation. CD4⁺ T cells were prepared from peripheral blood and 10⁵ CD4⁺ T cells combined with 10⁵ epidermal cells harvested from keratome biopsies of normal skin of an individual allogeneic to the T-cell donor. Addition of 50 μg anti-ICAM-3 to the co-culture resulted in a consistent (50%) reduction in degree of alloantigen presentation by Langerhans cells to T cells. Inhibition was 77% of that produced by the addition of anti-LFA-1. These data indicate that ICAM-3 is constitutively expressed by Langerhans cells and is a major ligand for LFA-1 on CD4⁺ T cells during their response to Langerhans cells. Because fresh Langerhans ceils constitutively express little ICAM-1. whereas ICAM-3 is constitutively expressed at high levels, it would appear that 1CAM-3 is the dominant functional ICAM on in situ Langerhans cells in the normal epidermis.     

银屑病和扁平苔藓皮损ki-67及PCNA蛋白的表达

目的研究银屑病和扁平苔藓患者皮损表皮增殖状态，并比较ki-67和PCNA在增殖性皮肤疾病中表达的一致性。方法应用免疫组化法分别检测银屑病和扁平苔藓患者皮损处ki-67及PCNA的表达。结果正常对照组ki-67及PCNA弱阳性表达，仅在基底层有表达；扁平苔藓组ki-67及PCNA在基底层和棘层中均有阳性表达；银屑病组表皮基底层、棘层、颗粒层中角质形成细胞强阳性表达ki-67及PCNA。与正常对照组相比，扁平苔藓组、银屑病组ki-67及PCNA表达均增高（P〈0．05）；与扁平苔藓组相比，银屑病组ki-67表达增高（P〈0．05），PCNA无统计学意义。ki-67和PCNA在皮损中表达一致性差。结论ki-67和PCNA可能通过诱导角质形成细胞增殖参与了扁平苔藓和银屑病的发病，银屑病和扁平苔藓表现出不同的表皮增殖动力学状态。     

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.     

Investigation of epidermotropism in canine mycosis fungoides: Expression of intercellular adhesion molecule-1 (ICAM-1) and beta-2 integrins

In human mycosis fungoides (MF), interactions between LFA-1 (CD11a/CD18) and ICAM-1 (CD54) are involved in lymphocyte adhesion to keratinocytes. The purpose of this study was to evaluate the expression of ICAM-1, beta-2 integrins and class II major histocompatibility complex molecules (MHC II) on keratinocytes and infiltrating lymphocytes in canine MF. Sections of frozen skin biopsy specimens from normal dogs (n=3) and dogs with MF (n=17) were evaluated by immunohistochemistry for expression of ICAM-1, beta-2 integrins, and class II MHC molecules. Our results demonstrated that in canine MF, ICAM-1 was expressed variably on epidermal and follicular keratinocytes. The extent of keratinocyte ICAM-1 expression did not correlate with the degree of lymphocyte epithelial infiltration, nor with lymphocyte LFA-1 expression. This was especially evident in cases of Pagetoid reticulosis-like disease in which prominent lymphocyte epidermotropism was not accompanied by keratinocyte ICAM-1 expression. Keratinocyte class II MHC molecule expression did not correlate with keratinocyte ICAM-1 expression. In conclusion, in canine MF, the lack of statistically significant correlations between epithelial lymphocyte infiltration and keratinocyte ICAM-1 expression, and between keratinocyte ICAM-1 and lymphocyte LFA-1 staining, suggests that the LFA-1/ICAM-1 pathway is not the major adhesion mechanism between lymphocytes and keratinocytes. It is suspected that different ligands of the LFA-1 integrin (e.g. ICAM-2) or other adhesion molecules (e.g. CD2/LFA-3, VLA-1) might be involved in the epitheliotropism phenomenon in canine MF. These hypotheses cannot be evaluated in the dog at this time owing to the lack of specific monoclonal antibodies.     

Transgenic expression of human amphiregulin in mouse skin: inflammatory epidermal hyperplasia and enlarged sebaceous glands

To explore the role of amphiregulin in inflammatory epidermal hyperplasia, we overexpressed human AREG (hAREG) in FVB/N mice using a bovine K5 promoter. A construct containing AREG coding sequences flanked by 5′ and 3′ untranslated region sequences (AREG‐UTR) led to a >10‐fold increase in hAREG expression compared to an otherwise‐identical construct containing only the coding region (AREG‐CDR). AREG‐UTR mice developed tousled, greasy fur as well as elongated nails and thickened, erythematous tail skin. No such phenotype was evident in AREG‐CDR mice. Histologically, AREG‐UTR mice presented with marked epidermal hyperplasia of tail skin (2.1‐fold increase in epidermal thickness with a 9.5‐fold increase in Ki‐67⁺ cells) accompanied by significantly increased CD4+ T‐cell infiltration. Dorsal skin of AREG‐UTR mice manifested lesser but still significant increases in epidermal thickness and keratinocyte hyperplasia. AREG‐UTR mice also developed marked and significant sebaceous gland enlargement, with corresponding increases in Ki‐67⁺ cells. To determine the response of AREG‐UTR animals to a pro‐inflammatory skin challenge, topical imiquimod (IMQ) or vehicle cream was applied to dorsal and tail skin. IMQ increased dorsal skin thickness similarly in both AREG‐UTR and wild type mice (1.7‐ and 2.2‐fold vs vehicle, P < 0.001 each), but had no such effect on tail skin. These results confirm that keratinocyte expression of hAREG elicits inflammatory epidermal hyperplasia, and are consistent with prior reports of tail epidermal hyperplasia and increased sebaceous gland size in mice expressing human epigen.     

Tumour necrosis factor alpha is pro-inflammatory in normal human skin and modulates cutaneous adhesion molecule expression

Tumour necrosis factor a (TNF-α) is a potent immunoregulatory cytokine produced by many cutaneous cells, including kcratinocytes, mast cells and Langerhans cells. To explore its potential role in inflammatory skin disease, we have studied immunohistochemically the effects of intradermal recombinant human TNF-α (rHuTNF-α) on cutaneous inflammatory cells, adhesion molecules and Langerhans cells in normal human skin. Volunteers received rHuTNF-α 100U (group A), 5000 U (group B), or 100 U daily for 5 days (group C), and biopsies were taken at 6 h (groups A and B), or 6 h after the final injection (group C). An inflammatory cell infiltrate developed in all cases: following single injections of either 100 or 5000 U rHuTNF-α this was predominantly neutrophilic, whereas following multiple injections of 100 U few neutrophils were seen, although many lymphocytes (CD3⁺, CD4⁴) were present. In all groups there was an increase in cells of monocyte/macrophage lineage (CD36⁾⁺. TNF-α induced a dose- and time-dependent decrease in CDla⁺ epidermal Langerhans cell numbers and an increase in dermal CDla⁴ cells, suggesting migration of Langerhans cells away from the epidermis. TNF-α induced endothelial E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in all groups, and adhesion molecule expression by interstitial dermal dendritic cells (ICAM-1 and VCAM-1) and keratinocytes (ICAM-1) was observed. These findings indicate that TNF-α is a potent modulator of cutaneous immune function in vivo, and this central role in the cutaneous immune response suggests that TNF-α may be an attractive target for therapeutic inhibition.     

银屑病患者血清和皮损中血管内皮细胞粘附分子的对比研究

目的 探讨银屑病患者血清和皮损中4种血管内皮粘附分子表达与银屑病疾病活动性之间的关系。方法 采用ELISA法检测36例银屑病患者治疗前后和36例健康人的血清中可溶性粘附分子（sICAM-1、sICAM-3、sVCAM-1、sELAM）的浓度。同时用ABC免疫组化染色技术检测了36例银屑病患者皮损和临床治愈处皮肤粘附分子（ICAM－1、ICAM－3、VCAM－1、ELAM）的表达情况。结果 与正常人相比，银屑病患者皮损部位4种粘附分子的原位表达呈明显上调（P＜0.005），同时患者血清中4种可溶性粘附分子浓度也明显升高（P＜0.001）。经治疗后银屑病患者皮损部位4种粘附分子的原位表达明显下调（P＜0.05），同时血清中4种可溶性粘附分子浓度比前也下降（P＜0.05）；血清中4种可溶性粘附分子的浓度与银屑病疾病活动严重指数（PASI）均呈正相关，但治疗前后sVCAM-1的水平上升和下降的幅度最大，且与PASI的相关性最好。结论 血管内皮细胞粘附分子参与银屑病的发病机制；患者血清中可溶性粘附分子浓度的升高可能与皮损部位血管内皮细胞上相应的粘附分子高表达有关；血清VCAM－1的水平可以作为反映银屑病疾病活动的一个新的敏感指标。     

Foxp3+ regulatory T cells and related cytokines differentially expressed in plaque vs. guttate psoriasis vulgaris

Background Differences in the number of Foxp3+ regulatory T cells (Tregs) in lesional skin and peripheral blood and their functioning in plaque vs. guttate psoriasis have not been reported. Objectives To investigate whether there is a differential expression of Foxp3+ Tregs and a differential regulation of inflammatory cytokines in plaque vs. guttate psoriasis vulgaris. Methods The number and the percentage of Foxp3+ cells in different phases of skin lesions of patients with plaque and guttate psoriasis vulgaris were assessed by immunohistochemical staining. The expression of Foxp3 and interleukin (IL)‐17 protein in CD4 populations was measured by flow cytometry. Inflammatory cytokine production by transforming growth factor‐β1‐induced Foxp3+ Tregs was assessed in an in vitro study. The cytokines in supernatant and serum were determined by enzyme‐linked immunosorbent assay. Results The percentage of Foxp3+ CD3+ cells in the papillary layer was higher than in the reticular layer of dermis and in epidermis (P < 0·05). The numbers of Foxp3+ Tregs in skin lesions and peripheral blood were higher in plaque than in guttate psoriasis, whereas the percentage of IL‐17+ CD4+ cells was higher in guttate than in plaque psoriasis (P < 0·05). The numbers of Foxp3+ cells were positively correlated with the Psoriasis Severity Index score of skin lesions (P < 0·0001), and the percentages of Foxp3+ CD4+ cells in peripheral blood were positively correlated with the Psoriasis Area and Severity Index score of patients (P < 0·05). The inhibitory functions of Tregs to IL‐17 and IL‐6 in guttate psoriasis and to tumour necrosis factor‐α in plaque psoriasis were deficient. Conclusions Differential expression and regulatory functioning for inflammatory cytokine production by Foxp3+ Tregs may imply a different immunopathogenesis for plaque and guttate psoriasis.