Immunohistochemical localization of SKALP/elafin in psoriatic epidermis.

Recently we have reported the purification and biochemical characterization of a new, inducible elastase inhibitor [skin-derived antileukoproteinase (SKALP)], which could be extracted in high amounts from psoriatic skin but not from normal human skin. Here we demonstrate the immunohistochemical localization of SKALP in psoriatic epidermis. SKALP was found exclusively in the upper layers of the suprabasal compartment and stratum corneum of lesional psoriatic epidermis.

Normal human skin demonstrates marked site-variation of tenascin expression, not correlated to epidermal proliferation (Ki-67 binding).

In disorders of the skin characterized by epidermal hyperproliferation, it has been demonstrated that the expression of tenascin in the dermis is markedly increased. In normal dermis, however, tenascin is slightly expressed in the upper dermis beneath the basal membrane. Using an immunohistochemical approach, tenascin expression (T2H5 binding) and recruitment of cycling epidermal cells (nuclear binding to Ki-67) were studied in normal skin at various localizations of the body surface.

Tenascin expression in basal cell carcinoma.

Tenascin (hexabrachion, cytotactin) is an extracellular matrix glycoprotein whose expression is strongly increased in hyperproliferative skin diseases, as shown by immunohistochemistry with polyclonal sera. In this study we describe a new monoclonal antibody (T2H5) against human tenascin. The specificity of T2H5 was validated by sequential immunoprecipitation of tenascin with polyclonal sera.

Demonstration of skin-derived antileukoproteinase (SKALP) in urine of psoriatic patients.

Recently we described a new elastase inhibitor (skin-derived antileukoproteinase, SKALP) that is expressed in psoriatic epidermis and cultured keratinocytes, but is virtually absent in normal skin. In this study we investigated whether SKALP activity could be measured in urine of psoriatic patients and healthy controls. We found that urine of psoriatic patients contained considerable amounts of anti-elastase activity, whereas this activity in urine from normals was significantly lower.

MON-150, a versatile monoclonal antibody against involucrin: characterization and applications.

A monoclonal antibody, designated MON-150, was found serendipitously to react strongly with the granular layer of normal human epidermis and with the upper spinous layers of psoriatic epidermis. From analysis by flow cytometry of cultured human keratinocytes, it appeared that the percentage of MON-150-positive cells strongly increased when the cells reached confluence and the growth fraction declined. To identify the antigen recognized by MON-150, a lysate of human keratinocytes was subjected to affinity chromatography using a MON-150 Sepharose column.

Abnormal expression of Ki-67 antigen in hair follicle of alopecia areata.

The monoclonal antibody Ki-67 was used to determine the numbers of cycling cells in hair follicles both in alopecia areata and in normal scalp skin. Pronounced nuclear staining was limited to the area below the critical line of Auber and the exterior part of the outer root sheath. In alopecia areata there is reduced nuclear Ki-67 binding in the bulb of anagen hair follicles.

Tenascin expression in human dermis is related to epidermal proliferation.

The extracellular matrix glycoprotein tenascin is sparsely distributed in normal human dermis. The authors have shown that in a number of skin diseases (psoriasis, skin tumors), tenascin expression is strongly increased. In this immunohistochemical study, using polyclonal and monoclonal antisera, we have tested the hypothesis that tenascin expression in vivo is linked to epidermal proliferation.

Skin-derived antileukoproteinase (SKALP), an elastase inhibitor from human keratinocytes. Purification and biochemical properties.

Recently we reported a preliminary characterization of anti-elastase activity which is found in cultured keratinocytes and in epidermis from psoriasis patients, but not in normal human epidermis. Here we present evidence that this inhibitory activity is derived from a cationic protein with a molecular mass of 18 kDa. In psoriatic scales the inhibitor is mainly present as a biologically active 11 kDa fragment.

Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry.

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH.

Distribution of skin-derived antileucoproteases (SKALP) in the marginal zone of the spreading psoriatic lesion.

Two new elastase inhibitors (SKALP, skin-derived antileucoproteases) were recently described in the lesional skin in psoriasis. The present study investigated the distribution of SKALP activity in the marginal zone of spreading psoriatic plaques. In a 4-mm zone immediately adjacent to the erythemato-squamous plaques, SKALP activity was slightly increased compared to distant uninvolved skin.

Expression of tenascin in perifollicular connective tissue: comparison of normal scalp and alopecia areata.

Tenascin, a recently discovered extracellular matrix protein, was demonstrated in perifollicular connective tissue of normal human scalp using immunohistochemistry. Its localization was different from other well-known extracellular matrix components, like fibronectin, laminin and heparan sulphate proteoglycan. A comparison between alopecia areata and normal scalps did not reveal major qualitative differences, except for an increased expression near heavily infiltrated follicles.

Tenascin expression in hyperproliferative skin diseases.

The expression of tenascin, a recently discovered extracellular matrix glycoprotein, was studied by immunohistochemistry in normal human skin and in a number of skin diseases with epidermal hyperproliferation such as psoriasis, basal cell carcinoma, Bowen's disease and solar keratosis. Tenascin expression in the upper dermis of normal skin was found to vary from almost absent to patchy along the basal membrane. Staining was continuous and intense around blood vessels, hair follicles and eccrine sweat ducts.

Antigen induced arthritis in beige (Chediak-Higashi) mice.

Mice with the beige mutation, which are known to be deficient for leucocyte elastase and cathepsin G, were used to investigate the role of neutral proteases in a model for antigen induced arthritis. Surprisingly, it was shown that in this model of arthritis, using methylated bovine serum albumin as an antigen, C57/black/6 'beige' mice (deficient for leucocyte neutral proteases) developed a more severe form of arthritis than the control mice ('black' mice), resulting in a higher degree of tissue damage. The incidence and degree of bone apposition and destruction of articular cartilage at day 21 after induction of arthritis were significantly higher in the beige mice.

Skin-derived antileucoproteases (SKALPs): characterization of two new elastase inhibitors from psoriatic epidermis.

Elastase inhibiting activity (EIA) was demonstrated in the epidermis from lesions and in psoriatic scale, whereas normal epidermis did not contain significant EIA. Two new elastase inhibitors were partially purified and characterized using psoriatic scale as a source. The two species (approximate molecular weights 10 and 20 kDa) were shown to be stable, and high-affinity inhibitors of human leucocyte elastase (Ki less than 10(-10) M).

Cryosectioning of hair follicles. An improved method using liquid nitrogen conduction freezing.

Horizontal sectioning of scalp biopsies is especially useful in hair diseases characterized by a reduction of follicles in size or number. Horizontal sectioning using standard cryo-microtomes is hampered by the differences in cutting properties between hair follicles and fatty tissue, resulting in loss of topography. We report a simple, inexpensive method to temporarily cool the specimen to an appropriate temperature by means of a conduction system using liquid nitrogen.

Elastase-inhibiting activity in scaling skin disorders.

Elastase inhibiting activity (EIA) has been observed in normal skin as a response to surface trauma, immediately following the intra-epidermal accumulation of polymorphonuclear leukocytes (PMN). In order to elucidate the relation between EIA and inflammation, the inhibiting activity was assessed in skin samples of scaling dermatoses (a) without significant inflammation: erythrodermic autosomal recessive lamellar ichthyosis (EARLI), non-erythrodermic autosomal recessive lamellar ichthyosis (NEARLI), X-linked recessive ichthyosis (XLRI) and X-linked dominant chondrodysplasia punctata (XLD-CDP); (b) with predominantly mononuclear cell infiltration: atopic dermatitis; (c) with mixed infiltration of PMN and mononuclear cells: psoriasis and Netherton syndrome. All skin disorders investigated showed an increased EIA as compared with normal skin.

Flow cytometric analysis of epidermal subpopulations from normal and psoriatic skin using monoclonal antibodies against intermediate filaments.

Keratin-type intermediate filament proteins show characteristic expression in normal and pathologic epidermis. Some keratins are restricted to the basal cell layers, and others occur exclusively in the suprabasal compartment. SDS-gel-electrophoresis and immunohistochemistry are generally used for the assessment of keratin profiles and their localizations.

Chondrocyte nonresponsiveness to insulin-like growth factor 1 in experimental arthritis.

Inhibition of chondrocyte proteoglycan (PG) synthesis is one of the mechanisms leading to cartilage destruction in joint inflammation. Using murine cartilage from normal and arthritic knee joints, we examined this process. We found that for normal, anatomically intact murine articular cartilage, insulin-like growth factor 1 (IGF-1) is a potent anabolic factor.

Age- and sex-related differences in antigen-induced arthritis in C57Bl/10 mice.

The influence of both age and sex on antigen-induced arthritis in C57Bl/10 mice was studied. Methylated bovine serum albumin was used to induce arthritis in young adult (3 months) and old (18 months) male and female mice. Arthritis became chronic and led to severe joint damage more often in 18-month-old female mice, compared with both young adult female mice and with male mice of both ages.

Antiglomerular basement membrane nephritis in beige mice. Deficiency of leukocytic neutral proteinases prevents the induction of albuminuria in the heterologous phase.

Antiglomerular basement membrane (GBM) nephritis with massive albuminuria can be induced in mice by injection of heterologous antibodies against mouse GBM. The albuminuria and the glomerular lesions in this model are not mediated by complement, but are dependent on the presence of polymorphonuclear granulocytes (PMN) in the glomeruli. Neutral serine proteinases and reactive oxygen metabolites produced by activated PMN have been implicated as agents contributing to tissue damage.

Insulin-like growth factor stimulation of chondrocyte proteoglycan synthesis by human synovial fluid.

We investigated the role of insulin-like growth factors (IGFs) as regulating factors of cartilage metabolism in human synovial fluid (SF), using a bovine explant culture system that was shown to respond to recombinant IGF-1 in vitro. SF from rheumatoid arthritis (RA) patients and from control patients was found to stimulate chondrocyte proteoglycan synthesis in bovine articular cartilage. A monoclonal antibody directed primarily against IGF-1 (and to some extent, IGF-2) partially blocked the stimulatory action of serum and totally blocked the stimulation by SF.

Experimental arthritis in C57black/6 normal and beige (Chediak-Higashi) mice: in vivo and in vitro observations on cartilage degradation.

Mice with the beige mutation are known to be deficient for polymorphonuclear leucocyte (PMN) elastase and cathepsin G and can therefore be used as a model for protease dependence of tissue destruction in inflammatory conditions. The in vitro and in vivo effect of PMN activation on cartilage damage in C57black/6 normal and beige mice was measured. In vitro it was found that stimulation of normal PMNs with chemotactic peptide caused degradation of articular cartilage matrix owing to an elastase dependent mechanism; PMNs of beige mice did not induce degradation of cartilage.

Effects of synovial fluid and synovial fluid cells on chondrocyte metabolism in short term tissue culture.

Freshly isolated synovial fluid (SF) and plasma samples from 20 patients with rheumatoid arthritis (RA) and 9 patients with joint effusions of other diagnoses (non-RA) were immediately (without fractionation or dilution) used as a culture medium for murine articular cartilage. Both intact SF, and cell depleted SF were tested for the effect on proteoglycan synthesis (35S-incorporation) compared to the patients' plasma or standard tissue culture medium. In addition, the effect on proteoglycan degradation was measured using 35S-prelabeled cartilage.