Programmed cell death of keratinocytes in infliximab-treated plaque-type psoriasis

BACKGROUND: Tumour necrosis factor (TNF)-alpha blockade using infliximab, a chimeric anti-TNF-alpha antibody, is an effective treatment for plaque-type psoriasis, inducing remission in about 80% of patients. OBJECTIVES: To examine infliximab-induced programmed cell death (PCD) of keratinocytes in psoriatic plaques on serial skin biopsy samples. METHODS: Five patients with moderate to severe plaque-type psoriasis received infliximab infusions intravenously (5 mg kg(-1)) at weeks 0, 2 and 6. Biopsies of nonlesional and lesional skin (days 0, 5, 14 and 21) were obtained. Conventional microscopy was used to examine the morphology of the psoriatic keratinocytes. In situ detection of apoptosis was performed by electron microscopy and by immunohistochemical staining with anti-p53 and anti-caspase-3 antibodies. Results Infusion of infliximab induced a clinical response in all five patients with psoriasis, with a mean Psoriasis Area and Severity Index improvement of 24.8% already at day 5. This was accompanied by significant histopathological changes in the skin biopsy samples after infliximab treatment. Light and electron microscopic evaluation revealed apoptosis-like morphological changes in lesional keratinocytes, i.e. nuclear condensation, chromatin fragmentation and cytoplasmic vesiculation, visible already after the first infusion. These damaged keratinocytes stained positively for p53, but not for active caspase-3. CONCLUSIONS: The effects of infliximab in psoriasis extend beyond merely anti-inflammatory actions, and may include caspase-independent PCD of lesional keratinocytes. The PCD of keratinocytes may be an important mechanism that could explain at least in part the rapid and sustained therapeutic effect of infliximab in psoriasis.     

Matrix metalloproteinase-19 is expressed by keratinocytes in psoriasis

Keratinocyte hyperproliferation, inflammatory infiltrates, neoangiogenesis and alterations in cytokine levels are hallmarks of psoriatic skin. Matrix metalloproteinases (MMPs) have been associated with the remodeling of the extracellular matrix during inflammation, neovascularization, and malignant transformation. We have previously shown that particularly MMP-12 is abundantly expressed by macrophages and MMP-9 in macrophages and neutrophils of psoriatic lesions. In this work the expression of two novel metalloproteinases, MMP-19 and MMP-28, was investigated in psoriatic lesional and non-lesional skin. MMP-19 protein was detected by immunohistochemistry in 28/29 samples in keratinocytes in the same regions as Ki67 (marker of proliferating keratinocytes) and p63 (marker of keratinocyte stem cells). Immunosignaling was also seen in endothelial cells and fibroblasts. Furthermore, MMP-19 mRNA was upregulated in psoriatic keratinocytes and skin as assessed by quantitative real-time polymerase chain reaction. In lichen planus and lichenoid chronic dermatitis, MMP-19 staining was found in keratinocytes in areas where the basement membrane was abnormal. MMP-28 was not detected in psoriatic or non-lesional skin. Our results suggest that keratinocytes as well as the previously reported cell types (smooth muscle, endothelial and macrophages) can express MMP-19 in psoriasis and lichen planus. Upregulation of MMP-19 in keratinocytes may be influenced by changes in the architecture of the basement membrane zone.     

Nerve growth factor and keratinocytes: a role in psoriasis

Nerve growth factor (NGF) is synthesized and released by human keratinocytes. NGF acts as a neurotrophic molecule at the skin level, as it stimulates the sprouting of nerve fibers and regulates the synthesis and expression of neuropeptides. NGF can thus take part in neurogenic inflammation which in turn is involved in the pathogenesis of several inflammatory dermatoses. Human keratinocytes also synthesize and express the low (p75)-and the high-affinity (trk) NGF-receptor (NGF-R). NGF stimulates keratinocyte proliferation which is blocked by the natural alcaloid K252, a specific inhibitor of trk phosphorylation. K252 inhibits keratinocyte proliferation and induces keratinocyte apoptosis, in the absence of exogenous NGF, indicating the existence of an autocrine loop where NGF and trk act as key players. Finally, NGF protein levels are increased in psoriatic as compared to non-lesional and normal skin, and psoriatic keratinocytes express higher amounts of NGF than normal keratinocytes. This review will discuss the above findings in view of a possible involvement of NGF in the pathomechanisms associated with the development of the psoriatic lesion.     

IL-23在寻常型银屑病皮损角质形成细胞中的表达

目的：探讨IL-23在寻常型银屑病患者角质形成细胞中的表达。方法：分离培养正常人表皮组、银屑病皮损组和银屑病非皮损组中角质形成细胞,给予混合刺激物处理。用RT-PCR方法比较培养的上述各组角质形成细胞在刺激后IL-23p19亚单位的mRNA水平,并用ELISA方法检测刺激前后培养液上清中的IL-23 p19浓度。结果：刺激前,银屑病皮损组角质形成细胞中IL-23水平均高于正常人表皮组和银屑病非皮损组;刺激后,银屑病皮损组角质形成细胞中IL-23 p19 mRNA和IL-23水平均明显高于正常人表皮组和银屑病非皮损组。结论：IL-23在银屑病皮损角质形成细胞中刺激前后的表达均增加,IL-23可能在银屑病的发生中发挥重要作用。     

Differential expression of D-type cyclins in HaCaT keratinocytes and in psoriasis

In this study, we show that the G0-G1/S phase of HaCaT keratinocyte cell cycle is characterized by D1-type cyclin expression, whereas during the repeated rapid turnover of highly proliferating cells, the expression of cyclins D2 and D3 dominates. Knocking down cyclin D1 mRNA resulted in no change of cell proliferation and morphology, indicating that D2 and D3 cyclins could substitute for D1 in driving the cell cycle. Increased numbers of cyclin D1-expressing keratinocytes were found in the basal layers of the lesional psoriatic epidermis compared to both normal and non-lesional epidermis without increased expression of cyclin D1 mRNA, suggesting a possible dysfunction in the degradation of cyclin D1 protein. We also detected a significant increase in cyclin D2 and D3 mRNA expressions in psoriatic epidermis compared to normal epidermis with no difference in protein expressions. Blocking alpha5-integrin function by a neutralizing antibody in HaCaT keratinocytes downregulated the expression of cyclin D1 mRNA without affecting the expressions of cyclin D2 and D3 indicating a regulatory role for alpha5-integrin in the expression of cyclin D1. Our data suggest a possible role for D-type cyclins in the excessive basal-cell proliferation and perturbed keratinocyte differentiation in the psoriatic epidermis.     

角质形成细胞异常诱发银屑病：基因工程动物模型的启示

银屑病的发病机制目前尚未完全阐明［1］。30年来,学术界一直对角质形成细胞和免疫细胞的功能异常在银屑病发病中孰为因果存在争论［2］。起初认为银屑病是由于角质形成细胞异常增殖引起的,但随着研究的深入,在皮损区发现Th1和Th17细胞浸润,遂将注意力转向T细胞介导的免疫反应,并逐渐将其作为银屑病发病机制的核心［1］。近年来,基因工程银屑病动物模型研究发现,角质形成细胞在银屑病皮肤炎症的启动和维持中均发挥关键作用。根据基因工程技术的特点,银屑病小鼠模型主要分为条件性基因敲除和先天性基因表达异常小鼠模型。前者是在小鼠成年以后,通过注射枸橼酸他莫昔芬特异性敲除角质形成细胞内的某些蛋白质而诱发银屑病,更接近于人类银屑病的发生过程。后者是通过特异性细胞内基因敲除或插入技术,使小鼠出生时即有角质形成细胞内蛋白质表达增多或缺失,更接近于先天性疾病的发病模式。本文简述几种银屑病基因工程动物模型以及相关研究,并结合近年我们团队取得的一些研究成果,分析并深入理解角质形成细胞在银屑病发病机制中的作用……     

Induction of new proteins by gamma interferon in cultured human keratinocytes

Human epidermal keratinocytes incubated with recombinant gamma interferon (r-IFN-gamma) show, on two-dimensional gel electrophoresis, both the appearance of new proteins and the loss of others. Among [35S]methionine-labeled proteins, which are induced in an actinomycin- or alpha-amanitin-sensitive manner, is a prominent group with an apparent relative molecular mass of 53,000 and pI of 5.3-5.8. The synthesis of these proteins continues for at least 4 days in the presence of gamma interferon (IFN-gamma). Over the concentration range tested, up to 670 pM, there is no inhibition of protein synthesis, so the appearance of these proteins cannot be explained by overall inhibition of protein synthesis. Furthermore, at 4 pM we found only minor inhibition of DNA (21%) and RNA (29%) synthesis. Half-maximal induction of the prominent 53 kD proteins occurs at an interferon concentration of 0.8-3.5 pM which may be compared with a range of 1.5-30 pM for HLA-DR induction. The same prominent proteins are also induced by type I interferons. The 53 kD protein complex appears to consist of at least 4 different proteins, one of which is phosphorylated and another one of which is not induced in fibroblasts treated with IFN-gamma. We could obtain no evidence that the proteins were related by glycosylation. The presence of these proteins provides a sensitive means of identifying keratinocytes responding to interferons. Lack of these proteins in normal epidermis indicates that interferon does not play a major role in the control of keratinocyte behavior in sound skin.     

Increased IL-6 production by monocytes and keratinocytes in patients with psoriasis

Interleukin-6 (IL-6) is a multifunctional inflammatory cytokine that is produced by monocytes and keratinocytes upon stimulation. Because psoriasis is a skin disease characterized by a hyperproliferative activity of keratinocytes and an inflammatory infiltrate, in the present study IL-6 production of monocytes and keratinocytes of patients with psoriasis was investigated. Peripheral blood mononuclear cells (PBMC) derived from psoriatics, atopics, and healthy controls were incubated for 24 h and, subsequently, supernatant IL-6 activity was measured using an IL-6-dependent hybridoma cell line (B9). Compared to controls and atopics, PBMC of psoriatics produced significantly increased amounts of biologically active IL-6. These findings were also confirmed by Western blot analysis using a specific antiserum directed against IL-6. Moreover, when the sera of the same patients were tested for IL-6 activity, sera of psoriatics contained significantly elevated amounts of circulating IL-6 in comparison to samples from atopics and healthy controls. In contrast to normal or uninvolved skin, keratinocytes in psoriatic lesions were remarkably positive for IL-6 as detected by immunohistochemistry and in situ hybridization. In addition, IL-6 also was found to induce its own synthesis and release by monocytes. These findings indicate that keratinocytes and monocytes in psoriasis are activated to produce increased amounts of IL-6, which may be one of the mediators involved in the regulation of both local and systemic inflammatory reactions occurring in skin diseases such as psoriasis.     

Differential proliferation of endothelial cells and keratinocytes in psoriasis and spongiotic dermatitis

This study used MIB-1 monoclonal antibody to quantify the proliferating keratinocytes and endothelial cells and their proliferation fractions in cases of normal skin, acute and established plaque psoriasis, and acute and chronic spongiotic dermatitis. The number and the proliferation fraction of MIB-1 positive cells were higher in psoriatic and chronic spongiotic lesions than in normal skin (p < 0.05). Established plaque psoriasis had a higher number of proliferating keratinocytes and a higher keratinocytic proliferation fraction than did acute psoriasis (p < 0.05). The number of proliferating endothelial cells decreased as acute psoriatic lesion became chronic, but the number in acute spongiotic lesion increased as it became chronic. The endothelial proliferation fraction was higher in acute psoriasis than in established plaque psoriasis (p < 0.05). The ratio of keratinocytic proliferation fraction to endothelial cell proliferation fraction of the psoriatic and spongiotic lesions suggested the presence of different reaction patterns to inflammation in psoriasis and spongiotic dermatitis.     

Interferon gamma is a marker for histiocytosis X cells in the skin

In histiocytosis X (HX), which is regarded as a proliferative disease of Langerhans cells (LCs), the tumor cells share characteristic membrane antigens and ultrastructural features with normal LCs. To the present no markers have been described which distinguish HX cells from normal epidermal LCs. Here we report on the selective reactivity of HX cells with a monoclonal antibody against interferon gamma (IFNg). Our results show that HX cells share an epitope with human IFNg while normal LCs do not. It remains to be established whether the expression of IFNg is specific for HX cells or rather characterizes a certain activation state of LCs.     

Recombinant gamma interferon induces HLA-DR expression on cultured human keratinocytes

In normal human epidermis, expression of HLA-DR antigen is restricted to Langerhans cells (LC) and acrosyringial epithelium. However, in diseases such as lichen planus and graft-vs.-host, HLA-DR antigen appears to be expressed by keratinocytes, although the exact source of the HLA-DR is unclear. Two possibilities are that (1) the HLA-DR is shed by neighboring immunocompetent cells, or (2) that the keratinocytes are synthesizing the antigen themselves. Recently, gamma interferon has been shown to induce HLA-DR biosynthesis and expression on human malignant melanoma cells lines and on normal vascular endothelium. We report here that pure recombinant human gamma interferon (100 units/ml) induces HLA-DR expression on 60-70% of cultured human adult keratinocytes depleted of LC within 2-4 days of culture as determined by fluorescence-activated cell sorter (FACS) analysis using monoclonal antibodies. No residual LC or lymphocytes could be detected in these cultures. This is the first demonstration of HLA-DR expression by cultured human keratinocytes. This expression may be of functional significance in antigen presentation and cell-mediated cytotoxicity involving the epidermis.     

Modulation of bullous pemphigoid antigens by gamma interferon in cultured human keratinocytes

We report the effects of human recombinant gamma interferon (gamma-IFN) on the expression of bullous pemphigoid (BP) antigens by human cultured keratinocytes. Secondary epidermal cell cultures were grown on 3T3 mouse fibroblasts; when confluent, some cultures were maintained in control medium while others were exposed to various concentrations of gamma-IFN (100, 200, 400 U/ml) for 14 days. The expression of BP antigens was analyzed by indirect immunofluorescence on epithelial sheets and immunoblotting of Tris, SDS, beta-mercaptoethanol culture extracts using different BP sera. Our results show that gamma-IFN alters the expression of BP antigens in a way varying according to the skin donor: we observed results ranging from complete loss and decreased expression to unchanged reactivity patterns. Thus, gamma-IFN modifies BP antigen expression; this behavior has been previously shown for other adhesion molecules such as fibronectin and thrombospondin. However, the observed variability of the expression of BP antigens according to the skin donor suggests an unexpected variability in keratinocyte sensitivity to gamma interferon, which remains to be explored both in vitro and in vivo.     

Upregulation of RANTES in psoriatic keratinocytes: a possible pathogenic mechanism for psoriasis

Intraepidermal collections of neutrophils and lymphocytes are unique features of the inflammatory reaction of psoriasis. Migration of leukocytes from dermis to the epidermis suggests a role for chemotactic agent(s). In recent years, increased levels of chemokines such as IL-8 , GRO-a and MCP-1 have been reported in the keratinocytes of psoriatic tissue. IL-8 and GRO-alpha belong to a subfamily (C x C) class and MCP-1 is a beta chemokine. In this study, we investigated RANTES, which is a beta chemokine (C-C class); RANTES has been found to be associated with various cell-mediated hypersensitive disorders. We obtained eight skin biopsies from chronic psoriatic plaques, and five biopsies each from non-lesional psoriatic skin, lichen planus, eczematous dermatitis and skin from healthy controls. Snap-frozen samples were cut into 7 microm cryosections and stained with 6 mg/ml of monoclonal anti-RANTES mouse IgG (DNAX, Palo Alto, CA). Standard immunohistochemistry techniques were applied. RANTES was detected only in the keratinocytes. The number of keratinocytes in per mm2 of epidermis stained for RANTES were 116.79+/-98.42 in psoriatic tissues compared to 32.00+/-46.05 (p<0.05), 6.39+/-3.59 (p<0.01), 2.64 +/-1.15 (p<0.01) and 3.53+/-5.26 (p<0.01), respectively, in the non-lesional, lichen planus, eczematous lesions and normal skin. This is the first study to report that the keratinocytes of psoriatic tissue express high levels of RANTES compared to the controls. IL-8 and related molecules (C x C class) are predominantly chemotactic for neutrophils and MCP-1 is a strong chemotactic factor for monocytes. In contrast, RANTES is chemotactic for memory T cells and activated naive T cells. Increased amounts of RANTES as reported here provide an explanation for migration of the activated T cells to the epidermis of the psoriatic lesions. In addition, RANTES activates T cells. These results suggest that RANTES may have a significant role in the inflammatory process of psoriasis. Our findings further substantiate a regulatory role for keratinocytes in the inflammatory process of psoriasis.     

The distribution of Fc gamma RI, Fc gamma RII and Fc gamma R III on Langerhans' cells and keratinocytes in normal skin

The distribution of Fc-receptors for IgG (FcR) on human epidermal cells (EC) was characterized in situ using monoclonal antibodies (MoAbs) by indirect immunofluorescence staining of cryosections. The results showed heterogeneity of FcR expression on Langerhans' cells (LC) and keratinocytes (KC). The MoAb IV.3 against FcR II (CDw32) gave granular staining of most LC whereas the MoAb 32.2 against FcR I (CD64) occasionally stained a few dendritic cells. 32.2 demonstrated weak granular staining along the outer aspect of KC in stratum spinosum and stratum basale and intense staining of stratum corneum and stratum granulosum. The MoAbs Leu 11b against FcR III (CD16) and B1D6 reacting with a placental FcR with low affinity for IgG gave intense linear membrane staining of KC. Leu 11b produced strongest staining of stratum granulosum and B1D6 the strongest staining of stratum spinosum and basale. The results confirm our previous observations of FcR in situ on LC and KC in normal skin using functional assays and demonstrate that these EC possess different types of low affinity FcR. The data support the contention of an immune function of KC. FcR may be mediators for interaction between KC and LC. The FcR activity in stratum granulosum may have an immune function as a barrier against microorganisms and other antigens.     

Normal response to tumor necrosis factor-alpha and transforming growth factor-beta by keratinocytes in psoriasis

Abstract Normal and chronic plaque psoriatic keralinocyte cultures were tested for their in vitro response to 2–200 ng/nil TNF-α and 0.1–10 ng/ml TGF-β in a serum-free culture system. All normal and lesional psoriatic epidermal cell cultures showed a dose- and lime-dependent inhibition of growth in response to TNF-α and TGF-β. Inhibition in individual cultures was first seen at a concentration of 2 ng/ml for TNF-α and 0.1 ng/ml for TGF-β at day 2, but became significant at 20 ng/ml and 1 ng/ ml for TNF-α and TGF-β respectively at days 2-6. This effect was statistically significant at days 3–4 for the group of normal (TNF-α and TGF-β, n = 10, p<0.01 and psoriatic cultures (TNF-α. n = 9, p<0.0l; TGF-β, n = 7, p<0.05). Epidermal cells from normal and psoriatic skin were inhibited to the same extent at the same optimal concentrations by each cytokine. Inhibition was abolished by the addition of specific antibody to each cytokine, whilst antibody to a different cytokine had no effect. Nuclear and/or nuclear membrane staining was observed with antibody to the p55 TNF receptor both in cultured keralinocyles and in the tipper epidermal layers of both normal and psoriatic skin. In contrast, plasma membrane and cytoplasmic expression of the p55 TNF receptor was observed on macrophages and lymphocytes infiltrating psoriatic der-mis. This study has shown that the growth of normal and psoriatic keratinocytes was equally inhibited by TNF-α and TGF-βin vitro. The expression of TNF receptor by psoriatic keratinocytes in vivo may permit regulation of epidermal growth by administration of TNF-α in this disease.     

银屑病患者淋巴细胞与角质形成细胞体外培养NF-κB的表达

目的：探讨银屑病发病中淋巴细胞和角质形成细胞的相互作用及核因子调控机制。方法：从银屑病患者外周血分离单一核细胞（PBMC）．正常人皮肤体外培养获角质形成细胞（KC），两者混合培养，利用流式细胞仪分析培养体系及PBMC、KC各自的转录因子NF-κB表达，ELISA法测上清液中IL-8、ICAM—I含量。结果：银屑病患者培养体系及PBMC、KC中NF—κB和细胞因子的表达水平均高于健康对照组（P〈0．01）。结论：银屑病患者中NF—κB的活化增强是其发病的重要因素，同时提示抑制NF—κB活性的药物可能有助于银屑病的治疗。