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

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

Down-regulation of transforming growth factor-β receptors I and II is seen in lesional but not non-lesional psoriatic epidermis

Transforming growth factor-βs (TGF-βs) are a family of growth factors with inhibitory effects on epithelial cell proliferation. Their effects are mediated by two interacting receptors, of which type I (TβR-I) mediates signal transduction after interaction with type II (TβR-II) carrying the TGF-β ligand. We have studied the expression of TβR-I and TβR-II in psoriatic and normal human skin by using polyclonal rabbit antisera and immunohistochemistry. Immunohistochemical analysis revealed an intense immunoreactivity for both receptors in the basal and often also suprabasal layer of normal and non-lesional psoriatic epidermis. In contrast, all psoriatic lesions studied lacked detectable immunoreactivity of either receptor in the epidermis. The results suggest that lack of TGF-β-mediated growth inhibition by down-regulation of TGF-β receptor expression may play an important part in the pathogenesis of psoriasis.     

mRNA for tissue-type plasminogen activator is present in lesional epidermis from patients with psoriasis, pemphigus, or bullous pemphigoid, but is not detected in normal epidermis

Plasminogen activator (PA), which catalyzes the conversion of plasminogen to the proteinase plasmin, has been implicated in a variety of cutaneous disorders. Lesional epidermis from patients with psoriasis, pemphigus, bullous pemphigoid, and Hailey-Hailey disease contains elevated levels of tissue-type PA (tPA) activity compared to non-lesional epidermis or to epidermis from normal individuals. In the present study, we have used Northern blot analysis to demonstrate that mRNA for tPA is detectable in lesions from patients with psoriasis, pemphigus, and bullous pemphigoid, but is not detectable in normal epidermis. These data strongly suggest that the tPA enzymatic activity present in lesional epidermis results from enhanced synthesis of the enzyme in situ, secondary to elevated steady-state levels of tPA mRNA. Cultured keratinocytes likewise are shown to contain tPA mRNA. Previous investigators have suggested that the phenotypes of keratinocytes in culture, psoriatic epidermis, and epidermis in the process of wound reepithelialization are comparable. Our findings, combined with those of other investigators, suggest that elevated tPA expression may be another common feature of epidermis under these circumstances.     

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

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

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

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

Gone but not forgotten: lesional memory in psoriatic skin

One of the most frustrating aspects of treating psoriasis is the tendency of psoriatic skin lesions to recur after therapy has been discontinued. Not only do lesions recur, but they often recur in the same anatomical locations, expanding to the size they were before therapy. This engenders feelings of frustration and futility in both patients and the dermatologists who care for them. In this issue, Suárez-Fari?as and colleagues identified a gene set-the residual disease genomic profile-of psoriasis, suggesting the presence of both immunologic and structural abnormalities within healed psoriatic lesions. By understanding this "invisible lesion," we may be one step closer to curing psoriasis.     

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

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

Lymphocytes and macrophages of the epidermis and dermis in lesional psoriatic skin,but not epidermal Langerhans cells,are depleted by treatment with cyclosporin A

Summary Since cyclosporin A (CsA) is an immuno-suppressive agent, its beneficial effect in psoriasis suggests that immune cells may play a role in the pathogenesis and resolution of psoriasis. To determine early effects of CsA in psoriasis, we quantitated immune cells using double immunofluorescence microscopy on biopsy specimens obtained prior to therapy and after 3,7, and 14 days of CsA therapy. CsA therapy resulted in significant reductions in the absolute number of immune cells (including T cells, monocytes/macrophages, and antigen presenting cells) contained within psoriatic skin. The effect was rapid, with over one-half of the reduction in the density of HLe1⁺ (human leukocyte antigen-1 positive or bone marrow derived) cells, including T cells, activated T cells, monocytes, and Langerhans cells (LCs), occurring within 3 days. Despite the overall reduction in the numbers of immunocytes in the skin, the proportion of T cells, Langerhans cells, and monocytes in relation to the total number of immune cells was unchanged with therapy, reflecting equally proportional losses of each subtype. Dermal CD1⁺DR⁺ cells (putative Langerhans cells), which are not found in normal skin but are present in lesional psoriasis skin, were virtually cleared from the papillary dermis after CsA therapy. Although absolute numbers of epidermal Langerhans cells, defined as cells expressing both CD1 (T6) and DR molecules (CD1⁺DR⁺), were also reduced after CsA, epidermal non-Langerhans CD1^-DR⁺ cells (macrophages, activated T cells, DR^- keratinocytes) demonstrated a proportionally greater decrease, with the ratio of CD1⁺DR⁺ Langerhans cells/non-Langerhans CD1^-DR⁺ epidermal cells changing from a mean of 0.82 at baseline to 1.92 at day 14. Thus, early in the course of therapy, CsA appears to be effective at clearing CD1^-DR⁺ cells while leaving LC relatively intact in the epidermis.This work was supported in part by the Babcock Foundation     

Cyclosporine A metabolism by cytochrome P-450III occurs in microsomes from rat liver but not from normal epidermis or psoriatic lesions.

Cyclosporine A is efficacious in the treatment of psoriasis when taken orally or injected intralesionally but not topically. Lack of penetration to necessary locations or rapid metabolism during passage through the epidermis may account for the ineffectiveness. Cytochromes P-450III in the liver are known to be involved in cyclosporine metabolism and inactivation. This study was undertaken to determine if an epidermal cytochrome P-450III exists that can inactivate topical cyclosporine A. Rats were treated with the macrolide antibiotic erythromycin to induce the cytochrome P-450III family of enzymes. Microsomal fractions were prepared from liver and epidermis of rats and from lesional areas of psoriasis patients. NADPH cytochrome C reductase activity was determined as a positive control for microsomal enzymatic activity. Formation of metabolite 1, the predominant metabolite of cyclosporine A, by liver microsomes was increased 193% after 10 d erythromycin treatment. The cytochrome P-450 dependent activity in microsomes from the epidermis of control and erythromycin-treated rats and in microsomes from psoriatic tissue was at the detection limits of the assay system. Cytochrome P-450III gene family mRNA were detectable by polymerase chain reaction in liver but not in psoriatic or normal epidermis. The lack of detectable P-450III mRNA and the absence or minimal conversion of cyclosporine A to inactive metabolites by epidermal microsomes suggest that the ineffectiveness of topical cyclosporine A in psoriasis may not be due to inactivation of cyclosporine A by cytochrome P-450 in the skin.     

Phorbol ester decreases cytosolic retinoic acid binding protein (CRABP) capacity in normal but not psoriatic epidermis

The retinoic acid binding protein (CRABP) characterized in human epidermal cytosol exhibits a three-fold increased binding capacity in lesional psoriatic epidermis as compared to normal or uninvolved skin. Treatment of epidermal homogenate from normal subjects by phorbol ester + ATP decreases the specific binding capacity of CRABP without affecting its dissociation constant (Kd=10 nM). The same effect was not observed in involved and uninvolved psoriatic epidermis. These results could be related to the previously reported decreased protein kinase C activity in psoriatic skin. They also suggest that posttranslational events could be responsible for pathological and pharmacological variations in CRABP binding capacity.     

LTA4 hydrolase in human skin: decreased activity,but normal concentration in lesional psoriatic skin

Leukotriene A₄ (LTA₄) hydrolase which transforms LTA₄ into the proinflammatory compound LTB₄ has been identified in human epidermis. The purpose of this study was to investigate the potential role of this enzyme in psoriasis, in which LTB₄ is present in biologically active concentrations. The concentration and activity of LTA₄ hydrolase was determined in normal skin and in matched samples of involved and uninvolved psoriatic skin. The enzyme content was determined using an affinity-purified antibody. This antibody was also used for immunohistochemical staining of skin biopsies. Immunohistochemically LTA₄ hydrolase was localized predominantly in the basal and spinous layers in normal skin and in involved and uninvolved psoriatic skin. The LTA₄ hydrolase content varied between 2.8 and 3.1 μg enzyme/mg protein and was found to be similar in normal and psoriatic skin, involved as well as uninvolved. In contrast, the activity of the enzyme was decreased significantly in involved psoriatic skin (9.9±2.1 μg LTB₄/mg enzyme per min) compared with matched uninvolved psoriatic skin (16.4±3.5 μg LTB₄/mg enzyme per min), but was decreased only insignificantly compared with normal skin (12.4±1.8 μg LTB₄/mg enzyme per min). It was found that the conversion of LTA₄ to LTB₄ results in inactivation of LTA₄ hydrolase activity. This finding is compatible with the idea that the decreased LTA₄ hydrolase activity in involved psoriatic skin reflects transcellular LTB₄ formation in vivo. In peripheral lymphocytes the enzyme content was 1.3±0.3 μg enzyme/mg protein in normal lymphocytes and 1.4±0.3 μg enzyme/mg protein in psoriatic lymphocytes, which was significantly lower than in the skin. In contrast, the specific LTA₄ hydrolase activities in normal and psoriatic lymphocytes (23.4±1.3 and 21.3±1.7 μg LTB₄/mg enzyme per min) were significantly higher than in normal skin. These findings may indicate the existence of LTA₄ hydrolase isoforms in human lymphocytes and human skin.     

Interleukin‐33 is expressed in the lesional epidermis in herpes virus infection but not in verruca vulgaris

Interleukin (IL)‐33 is released on cell injury and activates the immune reaction. IL‐33 is involved in antiviral reaction in herpes virus infection, but the source that secretes IL‐33 has not been identified. We speculate that keratinocytes injured in herpes virus infection secrete IL‐33. In order to detect IL‐33 in the lesional epidermis of patients with herpes virus infection, we immunostained several cutaneous herpes virus infection samples with an anti‐IL‐33 antibody, and compared them with cutaneous human papilloma virus (HPV) infection samples. We observed strong nuclear and mild cytoplasmic staining in epidermal keratinocytes of the lesional skin samples with herpes simplex virus and varicella zoster virus infections. However, staining was not observed in the epidermis of verruca vulgaris (VV) samples. We assumed that the strong immune reaction to herpes virus infection may depend on strong IL‐33 expression in the epidermis, while very weak immune reaction in samples from patients with VV may be due to low or no expression of IL‐33 in the lesional epidermis.     

Evidence for an alternative pathway of keratinocyte maturation in psoriasis from an antigen found in psoriatic but not normal epidermis

We have isolated a murine monoclonal antibody, called psi-3, which immunolabels maturing keratinocytes in psoriatic skin but not in normal epidermis. The staining is cytoplasmic and is not extractable with 1% Triton X-100, which suggests that the psi-3 antigen is a structural component of the keratinocyte. Neither basal cells nor invading inflammatory cells are stained in psoriatic skin and the antigen appears to be associated specifically with maturing and not proliferating keratinocytes. Keratinocytes cultured in vitro from skin from nonpsoriatic individuals display the antigen in a granular pattern in differentiated cells. The antigen is also expressed after tape-stripping of normal skin and, therefore, represents an inducible product of normal keratinocytes. The antigen is destroyed by proteinase K and appears to be a protein. On discontinuous sodium dodecyl sulfate-gel electrophoresis, the antigen has been found to have a molecular weight of 135,000. The psi-3 antigen is interpreted as a new keratinocyte product expressed in psoriasis, culture, wound healing, and certain other pathologic skin conditions. The synthesis of such a new antigen would not be expected if keratinocyte maturation in psoriasis is a truncated version of the normal system and supports the hypothesis that psoriatic keratinocytes are following an alternative pathway. Results using experimental injury suggest that the psoriatic pathway is normally expressed during wound healing.     

Key component of inflammasome,NLRC4, was identified in the lesional epidermis of psoriatic patients

Inflammasomes are multimolecular complexes that control the inflammatory response. The function of inflammasomes in the pathogenesis of psoriasis is still unclear. To clarify the relationship between inflammasomes and the pathophysiology of psoriasis, and in particular, to identify molecules interacting with caspase‐1, a crucial component of inflammasomes, scale extracts obtained from patients with psoriasis were immunoprecipitated with anti‐caspase‐1 antibody and analyzed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC‐MS/MS). The expression of the inflammasome component was assessed by immunohistochemical analysis and an in vitro assay. We identified several candidates for caspase‐1‐interacting proteins from the psoriatic scale extracts by immunoprecipitation and LC‐MS/MS. Nucleotide‐binding oligomerization domain‐containing protein‐like receptor family CARD domain‐containing protein 4 (NLRC4) was the only inflammasome component among the candidates; thus, the protein is considered to be a key factor of inflammasomes in psoriasis. No inflammasome component was found in the extracts of atopic dermatitis or normal skin by LC‐MS/MS. Immunohistochemical analysis demonstrated upregulation of NLRC4 in the lesional epidermis of some psoriatic patients whereas weak expression of NLRC4 was detected in the normal and non‐lesional epidermis. The mRNA expression of the NLRC4 gene increased in keratinocytes at confluency, 48 h after air exposure and after the addition of 1.5 mmol/L calcium chloride. Our findings suggest that NLRC4 may be involved in the exacerbation or modification of psoriatic lesions.