IA antigen-positive epithelioid cells in experimentally induced granulomatous inflammation

IA antigens on the cell membrane of inflammatory macrophages and epithelioid cells were investigated with immunoelectron microscopic method during development of granulomas induced by subcutaneous inoculation of 10(7) Mycobacterium lepraemurium into mice with and without hypersensitivity. In C57BL/6N (H-2b) immunogenetic high responder mice 6 weeks after infection majority (87%) of infiltrated cells were IA-positive. Two types of the staining reaction, strong and weak reactivity, were recognized among the positive cells. Strongly IA-positive cells showed lower phagocytosis (0.9/cell section) of mycobacteria than the weakly reacted cells (4.9/cell section). The strongly positive cells underwent morphological differentiation into large epithelioid cells during development of the hypersensitivity-type murine lepromas after 10 or more weeks of infection. Types of granulomas and IA-positive cells in C57BL/6N (nu/+) mice were identical to those found in C57BL/6N. In C57BL/6N (nu/nu) athymic nude mice initial infiltrating cells contained 38% of weakly IA-positive macrophages and a small number (7%) of strongly IA-positive macrophages. But the reactivity was lost later and only 4% of IA-positive cells remained in the granulomas without hypersensitivity. CBA/J (H-2k) low responder mice did not show IA-positive cells in either initial or late stage during the development of nonhypersensitivity-type murine lepromas. We suggest that the presence of IA-positive cells, particularly IA-positive epithelioid cells, in the lesions modulates the course of granulomatous tissue reaction in murine lepromas.     

Secretion of plasminogen activator inhibitor by cultured human epidermal cells

A conditioned medium from cultured human epidermal cells was observed to inhibit the activity of exogenous urokinase. By reverse fibrin autography after SDS polyacrylamide gel electrophoresis, a plasminogen activator inhibitor was detected with a molecular weight of 46,000. Using Mr 33,000 [125I]-labelled urokinase we observed the formation of an enzyme-ligand complex. The molecular weight of this complex was 79, 000. These results indicate that cultured human epidermal cells secrete a plasminogen activator inhibitor (urokinase inhibitor) with a molecular weight of 46,000.     

Tissue plasminogen activator inhibitor in the epidermis

The presence of a tissue plasminogen activator inhibitor in the epidermis was investigated. A partially purified tissue plasminogen activator from a Bowes melanoma cell line medium was used as a tissue plasminogen activator. Extracts of epidermis dissolved in a 10 mM phosphate buffer, pH 7.0, were found to contain a tissue plasminogen activator inhibitor. The same extracts also contained a urokinase inhibitor. It is not clear whether these inhibitors are the same. This study is the first to show the existence of a tissue plasminogen activator inhibitor in the epidermis.     

Pemphigus vulgaris and pemphigus foliaceus antibodies are pathogenic in plasminogen activator knockout mice.

Previous studies have suggested that urokinase plasminogen activator is required for blister formation in pemphigus vulgaris and pemphigus foliaceus. Other studies, however, have shown that downregulation of plasminogen activator does not inhibit blisters induced by pemphigus immunoglobulin G. To eliminate the possibility that small amounts of urokinase plasminogen activator might be sufficient for blister formation, we passively transferred pemphigus immunoglobulin G to urokinase plasminogen activator knockout neonatal mice. Pemphigus foliaceus and pemphigus vulgaris immunoglobulin G caused gross blisters and acantholysis in the superficial and suprabasal epidermis, respectively, to the same degree in knockout and control mice, demonstrating that urokinase plasminogen activator is not absolutely required for antibody-induced blisters. Some studies have shown elevated tissue-type plasminogen activator in pemphigus lesions. Tissue-type plasminogen activator, however, is not necessary for blister formation, because pemphigus foliaceus and pemphigus vulgaris immunoglobulin G caused blisters to the same degree in tissue-type plasminogen activator knockout and control mice. To rule out that one plasminogen activator might compensate for the other in the knockout mice, we bred urokinase plasminogen activator, tissue-type plasminogen activator double knockouts. After passive transfer of pemphigus foliaceus and pemphigus vulgaris immunoglobulin G these mice blistered to the same degree as the single knockout and control mice, and histology indicated blisters at the expected level of the epidermis. These data definitively demonstrate that plasminogen activator is not necessary for pemphigus immunoglobulin G to induce acantholysis in the neonatal mouse model of pemphigus.     

新抗凝治疗SLE小鼠的实验研究

观察了新抗凝片对SLE动物模型BXSB小鼠的治疗作用。结果显示：小鼠发病后其纤溶功能低下，主要是血中t－PA活性明显低于正常（P＜0．01），尿蛋白明显增加（P＜0．05）；用新抗凝片（1mg／kg／d）灌胃20天后，t－PA活性恢复正常，尿蛋白量和免疫复合物在肾小球的沉积明显低于未治疗组（P＜0．05，P＜0．01）。实验证明新抗凝片具有恢复病鼠纤溶功能的作用，从而阻止或清除肾小球免疫复合物和纤维蛋白的沉积，使病损得以修复，对SLE具有治疗作用。     

Fibrin deposition and clearance in chronic granulomatous inflammation: correlation with T-cell function and proteinase inhibitor activity in tissue.

Patterns of fibrin deposition were investigated by immunofluorescence microscopy in livers of thymus intact (TI) and athymic (AT) mice infected with Schistosoma mansoni. Thrombin and fibrinolysis inhibitor activity in tissue extracts also were measured. In TI mice fibrin was detected perivascularly by 6 weeks after infection and at 8 weeks it was found over the granulomas as they developed. Fibrin was cleared from the center of granulomas by 10 weeks. Thrombin inhibitor activity increased at 4 to 6 weeks but declined below control levels later as granulomas formed. Fibronolysis inhibitor activity, on the other hand, peaked at 9 to 12 weeks after infection. In AT mice extensive fibrin deposition was detected in the liver throughout the period when smaller and incomplete granulomas developed. Central clearing did not occur. Thrombin inhibitor activity greatly increased by 8 weeks after infection but fibrinolysis inhibitor activity remained unchanged. These findings suggest that fibrin deposition and firbinolysis are orderly events regulated in the lesions by proteinases and their inhibitors and this seems to be a general tissue reaction in the early stage of chronic granuloma formation. Since local clearance of fibrin in vivo and fibrinolysis inhibitor activity from tissue extracts studied in vitro are more evident in TI mice than in AT mice, it appears that T cell fu;ction is important in modulating the tissue response during granulomatous inflammation.     

Plasminogen activators of psoriatic scale extracts

Summary Psoriatic scale extracts were fractioned by using polyacrylamide gel isoelectric focusing (PAGIF) and preparative electrofocusing in granulated gel (PEGG). The largest protein fraction was found with Ip at pH 4.8–5.0, and the main protein bands within pH values 4.0–7.5.PEGG separated three main fractions with plasminogen activator or trypsin-like esterase activity with isoelectric points at pH 6.5–6.6, 5.4–6.2 and 4.9. The enzyme with Ip at pH 6.5–6.6 hydrolyzed trypsin substrates but lacked plasminogen activator capacity. The enzyme with Ip at pH 5.4–6.2 showed both activities but the third enzyme with plasminogen activator capacity with Ip at pH 4.9 was without detectable esterolytic activity towards substituted basic amino acid esters. The thrid enzyme was prominent in KCl-extract and the second in KSCN-extract. The first was equal in both extracts.The enzyme with Ip at pH 4.9 is possibly of bacterial origin while the plasminogen activator with Ip at pH 5.4–6.2 extracted in KSCN probably represents tissue activator of psoriatic scales.     

Plasminogen activator secreted by cultured human melanocytes

A plasminogen activator (PA), Mr 72,000, was detected in conditioned medium from human melanocyte cultures by fibrin autography. The electrophoretic mobility was identical to that of tissue PA produced by Bowes melanoma cells. PA activity in human melanocyte culture medium was inhibited by anti-tissue PA IgG, but not by anti-urokinase IgG. Our results are the first to show that normal human melanocytes in culture secrete tissue plasminogen activator.     

Tissue plasminogen activator in psoriasis

Elevated levels of the serine proteinase plasminogen activator are observed in psoriatic lesions. In contrast to normal epidermis, lesional psoriatic epidermis contains primarily tissue type plasminogen activator (tPA) activity and much lower levels of urokinase type plasminogen activator (uPA) activity. Tissue plasminogen activator is also detectable immunocytochemically in lesional psoriatic but not normal epidermis. Similarly, mRNA for tPA is observed in lesional epidermis only. These results suggest that lesional psoriatic epidermis synthesizes enhanced levels of tPA compared to normal. Additional data support the hypothesis that enhanced tPA may be another marker common to psoriatic epidermis, epidermis during wound repair, and keratinocytes in culture. The significance of elevated tPA in psoriatic lesions is presently unclear, but its possible relationship to epidermal proliferation and cutaneous inflammation is under study.     

An immunohistochemical study of the distribution of plasminogen and plasminogen activators in bullous pemphigoid

Abnormalities of the cutaneous plasminogen/plasminogen activator system have been associated with acantholytic disorders, psoriasis, keratinocytes in culture, and epidermis in healing wounds. The present study was undertaken to investigate the possible role of the plasmin/plasminogen protease system in lesion development in bullous pemphigoid (BP). Using polyclonal antibodies and a fluorescent technique, the immunohistochemical distribution of plasmin/plasminogen, fibrinogen and the plasminogen activators, urokinase (uPA) and tissue plasminogen activator (tPA), were studied in lesional and non-fesional skin from nine BP patients, one with linear IgA disease (LAD) and one with pemphigoid gestationis (PG). The distribution of the proteases was compared with that in normal skin (n=4) and in suction blisters (n=2). In normal skin, fibrinogen, tPA and uPA were absent from the epidermis and plasminogen was confined to the basal layer. Uninvolved BP skin was identical to controls. Focal areas of suprabasal plasminogen expression in the region of a blister was seen in 3/9 BP lesions and in 1/2 suction blisters. In 6/9 BP lesions and both uninvolved and lesional LAD and PG skin were identical to controls, and no suprabasal expression of plasminogen was present. These findings suggest that suprabasal plasminogen expression is unlikely to play a fundamental role in the pathogenesis of blister formation in BP as enhanced expression was not present in every case and the finding was not specific to BP, also occurring in a suction blister. Enhanced plasminogen expression rather may be a reflection of the processes of tissue repair.     

An improved quantitative method for the study of fibrinolysis in the skin as exemplified in patients with leukocytoclastic vasculitis and erysipelas

The activity of plasminogen activator has been measured in tissue sections with the aid of a modified Todd technique. Frozen skin biopsies were sectioned and the tissue covered with fibrin-plasminogen film. After incubation at 37 degrees C fibrinolysis was studied for a period of 30 min and was graded into six exponentially increasing steps. During this period, grades were linear with the llogarithm of time of incubation. The rate of fibrinolysis is a measure of the activity of the plasminogen activator and is expressed by the slope of the linear regression of grades versus the ogarithm of time; the value of the slope is proportional to the common logarithm of the activity of the plasminogen activator present in the section. Using urokinase as a specific plasminogen activator, the same linear expression was shown with time, indicating the validity of our grading and experimental system. Eleven healthy subjects served as controls to 6 patients with leukocytoclastic vasculitis and 4 patients with erysipelas and 4 with necrotizing fascilitis. The controls showed values similar to the non-involved skin in the patients. The activity was higher in the arm than in the thigh sites. The activity in the thighs was lower in women than in men. A decrease in the plasminogen activator activity was found in the three disorders.     

Plasminogen activator in cultured human epidermal cells

Primary cultures of human epidermal cells produce plasminogen activator (PA) as demonstrated by the ability of conditioned medium or cell lysates to hydrolyze fibrin in the presence of plasminogen, and to cleave [125I]plasminogen to characteristic fragments. The major molecular species of PA in human epidermal cells was inhibited by diisopropylfluorophosphate and comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the high molecular weight band of human urokinase (Mr 55,000). Production of PA by human epidermal cells was inhibited by cycloheximide, stimulated by colchicine, and not affected by cytochalasin B or the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Both cholera toxin and epidermal growth factor stimulated PA activity in human epidermal cells, and PA activity was maximal at concentrations that best support in vitro growth of human epidermal cells. Examination of individual cells indicated that at least 15% of cells within a culture produced detectable amounts of PA.     

Increased migration of murine keratinocytes under hypoxia is mediated by induction of urokinase plasminogen activator

One of the key consequences of cutaneous wounding is the development of tissue hypoxia. Recent data have suggested that this is a potent stimulus for increased keratinocyte migration and hence re-epithelialization, although the mechanisms responsible for this remain unclear. In this study we have investigated the relationship between hypoxia, plasminogen activation, and in vitro wound healing. Exposure of keratinocyte cultures to hypoxia resulted in upregulation of urokinase plasminogen activator mRNA and a subsequent increase in urokinase plasminogen activator-mediated plasminogen activation, as determined by indirect chromogenic peptide assay and plasminogen-linked zymography. Analysis of keratinocyte wound healing in vitro confirmed enhanced wound closure in hypoxic cultures compared with normoxic cultures after 16 h. Pretreatment of normoxic and hypoxic cultures with mitomycin C and cytochalasin B indicated that in this system wound closure was due to keratinocyte migration rather than proliferation. Addition of the broad-spectrum serine proteinase inhibitor, p-aminobenzamidine, or the specific urokinase plasminogen activator inhibitors, amiloride and WX-293, significantly reduced wound closure in hypoxic cultures and abrogated the hypoxic enhancement of wound closure. These data indicate a central role for urokinase plasminogen activators in hypoxic keratinocyte migration and suggest a potential mechanism for enhanced re-epithelialization of wounds under low oxygen tensions.     

Epithelial tissue-type plasminogen activator expression, unlike that of urokinase, its receptor, and plasminogen activator inhibitor-1, is increased in chronic venous ulcers

BACKGROUND: The plasminogen activation system represents a potent mechanism of extracellular proteolysis and is an essential component of normal wound healing. It has also been implicated in the pathogenesis of chronic, nonhealing ulcers. Traditionally, urokinase-type plasminogen activator (uPA) has been associated with pericellular proteolytic activity involved in tissue remodelling processes, and tissue-type plasminogen activator (tPA) mainly with intravascular fibrinolysis. OBJECTIVES: The present study was conducted to characterize the spatial distribution of the various plasminogen activation system components in chronic ulcers and acute, well-granulating wounds. METHODS: The expression of uPA, tPA, urokinase receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and vitronectin was investigated by immunohistochemical staining, in addition to uPA, tPA and PAI-1 expression by in-situ hybridization, in samples from eight chronic venous ulcers, five decubitus ulcers, five well-granulating acute wounds and five normal skin samples. RESULTS: In chronic venous leg ulcers tPA mRNA was detected in basal and suprabasal keratinocytes at the leading wound edge, while in well-granulating wounds and in decubitus ulcers tPA mRNA was expressed only in a few keratinocytes. However, tPA was widely expressed in fibroblast- and macrophage-like cells in the stroma of well-granulating wounds, while less tPA was detected in the granulation tissue of chronic ulcers. tPA mRNA and protein were localized in the superficial granular layers in normal skin. Although no qualitative differences in expression of uPA, PAI-1 or uPAR in the wound edge keratinocytes in chronic ulcers vs. normally granulating wounds were found, their expressions were more pronounced in the granulation tissue of well-granulating wounds. CONCLUSIONS: These results suggest that in poorly healing venous leg ulcers, the pattern of tPA expression is altered in keratinocytes at the leading edge of the wound, and the patterns of tPA, uPA and PAI-1 expression are altered in the granulation tissue.     

Epidermal plasminogen activator is abnormal in cutaneous lesions

To investigate the role of plasminogen activator (PA) in cutaneous disease, we have used biochemical and immunocytochemical techniques to examine PA in normal and lesional skin. In normal human dermis, tissue PA is the predominant PA activity; however, in normal epidermis, urokinase type PA is the predominant PA activity. In contrast, PA activity in epidermis from lesions of patients with a variety of cutaneous diseases was predominantly tissue type enzyme with a much smaller contribution from urokinase. Our patient population included individuals with benign chronic pemphigus, pemphigus vulgaris, pemphigus foliaceous, bullous pemphigoid, or psoriasis. Using rabbit antibody specific for tissue type PA, we have immunocytochemically localized this enzyme in lesions from individuals with the above disorders. Our results indicate that tissue PA is consistently elevated in cutaneous lesions with diverse etiology, pathology, and clinical presentation.     

Endothelial cell activation in cutaneous vasculitis

Markers of endothelial cell activation were measured in 28 patients presenting with various forms of limited or focal type cutaneous vasculitis. Plasma levels of tissue plasminogen activator antigen (t-PA:Ag), plasminogen activator inhibitor type 1 antigen(PAI-I; Ag) and PAI-1 activity, fibrin plate, von Willebrand factor antigen (vWF:Ag), tissue factor (TF) and soluble thrombo-modulin (sTM) were measured. In comparison with the control group [n = 20) there was a significant increase in t-PA: Ag vWF:Ag and TF (P < 0.05, Mann Whitney U-test) in the cutaneous vasculitis group This study confirms that measurable degrees ol erido-thelial activation occur in cutaneous vasculitis.     

Co-regulation of p16INK4A and migratory genes in culture conditions that lead to premature senescence in human keratinocytes

Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system.