I. Joronen

Cysteine proteinase inhibitors in psoriatic epidermis

SummaryHuman psoriatic epidermis and scales were demostrated to contain two antigenically separate cysteine proteinase inhibitors, one acidic with an isoelectric point of 4.7–5.0 (ACPI) and one neutral with an isoelectric point of 6.0–6.5 (NCPI), while normal epidermis contains only ACPI. The total papain (cysteine proteinase) inhibiting activity of the psoriatic epidermis as calculated per mg protein was higher than that in normal epidermis. Both ACPI and NCPI were localized immunocytochemically, mainly in the highest spinous cell layers with less activity in the parakeratotic cells and lower layers of spinous cells. Basal cells were essentially negative.

Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoroimmunoassay

A time-resolved fluoroimmunoassay was developed for the detection of 3 human low molecular weight cysteine proteinase inhibitors, ACPI (cystatin A), NCPI (cystatin B), and gamma-trace (cystatin C). Polystyrene tubes or polystyrene microtitration strips were used as solid phase. The rabbit anti-inhibitor immunoglobulins were used as the capture antibody, and, when labelled with europium, also as the detector antibody. The threshold sensitivity of the tests was 0.1 ng/ml for NCPI and 1 ng/ml for the others. All the 3 cysteine proteinase inhibitors, ACPI, NCPI, and gamma-trace, were detected in pooled serum samples of patients with kidney failure. gamma-Trace seemed to be quantitatively the major and ACPI the minor inhibitor. No other low molecular mass cysteine proteinase inhibitor was detected after isoelectric focusing of the 12 kDa area of gel filtered human serum.

Serum cysteine proteinase inhibitors with special reference to kidney failure

Serum levels of proteins reactive in radioimmunoassay with an antiserum prepared in rabbits against purified human spleen neutral cysteine proteinase inhibitor (NCPI) was determined in 70 healthy controls and from 80 patients suffering from suspected or proven kidney failure. The values varied from less than 0.2 mg/l in normal sera to levels over 2 mg/l in some patient sera. Serum level of NCPI was found to roughly correlate with serum creatinine values. However, there were sera with high NCPI levels which did not have increased serum creatinine values. In sera with high NCPI levels subjected to double radial immunodiffusion two precipitin lines, one completely and the other partially identical to NCPI were registered. After fractionating of serum proteins with gel chromatography on Sephadex G 100, two protein peaks of immunological similarity to purified NCPI were found: one low molecular weight (MW around 12,000) and one high molecular weight (MW around 100,000). The low molecular weight NCPI-like material appeared to inhibit human cathepsin B and papain and is thus free serum NCPI. alpha-Cysteine proteinase inhibitor did not increase with serum creatinine as did NCPI.

Cysteine proteinase inhibitors produced by mononuclear phagocytes

SummaryMonocytes were separated from human peripheral blood and allowed to attach to culture flasks, after which the content and production of a number of cysteine proteinase inhibitors was assayed. These were: a low molecular weight (MW 12000) acid cysteine proteinase inhibitor (ACPI); a low-molecular weight inhibitor of the same size with neutral pH (NCPI), and α-cysteine proteinase inhibitor with a molecular weight around 90000 (α-CPI). Only NCPI was detectable in the cultures at the beginning of the incubation, and it was synthesized and released into the incubation mixture during the incubation, especially if the cells were stimulated with silica. The amount of NCPI contained in and released from the cells was drastically decreased by puromycin. Immunoblots after cell electrophoresis in polyacrylamide gel revealed only one molecular form of NCPI with a molecular weight of 12000 both in the cells and in the culture medium. No ACPI or α-CPI could be detected.

A 43-kDa papain inhibitor produced by a cultured human epidermal cell line

SummaryEpidermis-derived cells (NCTC 2544) were cultured and the proteins of the culture medium, as well as of the cells, were fractionated by gel-chromatography. The fractions were analyzed for their papain-inhibitory capacity and for the presence of socalled 43-kDa papain inhibitor. A papain inhibitor was identified with molecular weight and immunological characteristics similar to the original 43-kDa inhibitor that was isolated from psoriatic scales. The result proves that NCTC-2544 cells can produce the so-called psoriasis inhibitor under culture conditions.

Production of acid and neutral cysteine-proteinase inhibitors by a cultured human skin epithelium cell line

SummaryHuman skin epithelial-like cells (NCTC-strain 2544) were grown in RPMI-1640 medium supplemented with foetal calf serum for up to 2 weeks. The culture medium and extracts made from the cells were subjected to gel-filtration chromatography in a Sephacryl S-200 column for fractionation of the proteins. The fractions were assayed for acid and neutral cysteine-proteinase inhibitor (ACPI, NCPI) using time-resolved fluoroimmunoassay and radioimmunoassay, and the cysteine-proteinase-inhibiting activities were assayed using papain. Free NCPI, i.e. a molecule with isoelectric variants at pHs 6.0 and 6.5, which has an Mr of around 12,000 and is capable of inhibiting papain, was detected both in the culture medium and in the cells. Immunodiffusion studies revealed its immunological identity with human spleen-derived NCPI. The amount of NCPI increased during the incubation period. ACPI — characterized as a molecule having an isoelectric point of 4.9, an Mr of about 12,000, papain-inhibiting capacity and antigenic reactivity with spleen-derived ACPI — was not detected in the culture medium. It was, however, detected in the cells after 2 weeks in culture. These data prove that ACPI and NCPI are synthesized by the NCTC-2544 cells under the present culture conditions.

The 43 kDa papain inhibitor in normal and diseased skin.

The presence of 43 kDa papain inhibitor in 43 different skin diseases was immunohistochemically studied by using both poly‐and monoclonal antibodies. Psoriasis and various eczematoid reactions as well as viral infections showed the most pronounced staining in the squamous cells of the epidermis. The antigen was also present in benign tumours or precancerous lesions which showed keratinization. Cells of poorly differentiated squamous cell carcinomas, basal cell carcinomas and melanocytic tumours were negative. The antigen seems to be related to disturbed keratinization and benign proliferation in non‐neoplastic derma‐toses and it is also present in differentiating squamous neoplasms.

Separation and partial characterization of four cysteine proteinases from a human epidermal cell line.

SummaryFour different cysteine proteinases from a cultured human epidermal cell line (NCTC 2544) were partially purified and characterized. The biggest hydrolase was an endoaminopeptidase with the molecular weight of several hundred kilodaltons. It was a glycoprotein and had an almost neutral pH optimum. The three other hydrolases resembled lysosomal cathepsins B, H, and L in various respects except for somewhat higher molecular weight for cathepsin B (29 kDa) and the cathepsin H-like (70 kDa) hydrolase than those reported from most other tissues. Low molecular weight cysteine proteinase inhibitors ACPI (cystatin A) and NCPI (cystatin B) inhibited the cathepsins, but not the high molecular weight proteinase.

The 43 KDa inhibitor of human skin and other epithelia

We have used immunohistochemical and quantitative immunochemical techniques to study the tissue distribution of the 43 kDa papain inhibiting protein, originally isolated from psoriatic scale. High amounts of the inhibitor were found in the epidermis only in several skin diseases where the epidermal cell proliferation was increased (psoriasis, various eczemas). The inhibitor was also found in the basal cells of the bronchial epithelium and in the squamous epithelia of mouth and oesophagus, and the Hassals corpuscles of the thymus. Sera of patients suffering from several skin diseases did not contain the inhibitor, but antibodies against it were found in the sera of two patients suffering from leg ulceration. The inhibitor was immunologically unrelated to filaggrin, involucrin, or keratins.

Natural cysteine proteinase inhibitors reduce lectin induced lymphocyte stimulation.

Lymphocyte stimulation by lectins can be inhibited by several synthetic inhibitors of proteolytic enzymes, notably those of cysteine proteinases. The effects of naturally occurring enzyme inhibitors are less well known. The effect of the neutral low-molecular weight cysteine proteinase inhibitor (NCPI) recently purified from lymph nodes and spleen was therefore investigated. Cultured peripheral blood lymphocytes were stimulated by PHA or ConA in the presence or absence of NCPI and the incorporation of 3H-thymidine was measured. NCPI was found to inhibit these lymphocyte responses in these circumstances.