Superoxide dismutase (SOD), which catalytically scavenges superoxide anion (O–2), constitutes an essential defense against the toxicity of oxygen. We investigated the enzyme activity of pig skin epidermis. SOD activity was determined by monitoring the inhibitory effect of SOD on red formazan formation from neotetrazolium, which depends on (O–2) generation. (O–2) was generated by the hypoxanthine‐xanthine oxidase reaction. Pig epidermis contained significant amounts of heat‐labile SOD activity which was proportional to the added epidermal homogenate. The optimal pH of the reaction was between pH 8.2 and 8.5. Metallochelating agents such as cyanide, sodium azide, and diethyldithiocarbamate (DDC) inhibited the epidermal SOD activity in a dose‐dependent manner. It has been known that two types of SOD, a Cu, Zn‐type and a Mn‐type, are present in eukaryotes; that the latter is insensitive to cyanide inhibition. Using this property, the main SOD present in the epidermis was hypothesized to be the Cu, Zn‐type (8.6 ±1.1 unit/mg protein; around 75%); the Mn‐type was a minor component (2.8 ± 0.2 unit/mg protein; around 25%). SOD staining following acrylamide disc gel electrophoresis revealed two epidermal SOD bands, one of which was abolished by the addition of cyanide. These results are consistent with the view that pig epidermis contains two types of SOD, a Cu, Zn‐type and a Mn‐type; the former appears to be predominant.

Epidermal keratinocytes form cornified cell envelopes during terminal differentiation. These envelopes are composed of several cross-linked molecules, including involucrin, loricrin, and SPRR. We have previously reported that involucrin is synthesized earlier in terminal differentiation than loricrin. To further elucidate the mechanisms of terminal differentiation, we have now examined the expression of the two differentiation markers, involucrin and SPRR, in cultured human epidermal keratinocytes. In confluent nonstratified cultures, many involucrin-immunoreactive cells were detected, but few SPRR1/3-positive cells. Double staining demonstrated that cells containing SPRR1/3 almost always contained involucrin, but involucrin was present in many cells that did not contain SPRR. Light and electron microscopic immunohistochemistry of a stratified culture demonstrated that lower cells (close to the basal layer) were occasionally involucrin-positive, but lacked SPRR1/3, whereas more superficial cells contained both involucrin and SPRR. We conclude that involucrin and SPRR are sequentially induced in this order during keratinocyte differentiation.

A 49-year-old man presented a progressive swelling and induration of the skin resulting in flexion contracture. He had a history of two tick bites at the age of 17 and 47 years. Serum anti-Borrelia-burgdorferi antibody was positive; isolation of B. burgdorferi from the skin lesion was unsuccessful. He had eosinophilia (white blood cells 8,300/μl, 33% eosinophils) and hypergammaglobulinemia. The diagnosis of Shulman syndrome (eosinophilic fasciitis) from clinical and histological findings was established. A part of the flagellin gene of B. burgdorferi was detected in a skin biopsy sample by using the polymerase chain reaction method. To the best of our knowledge, this is the first report of detection of B.-burgdorferi-specific DNA from a skin sample of Shulman syndrome.

Transforming growth factor beta 1 (TGF beta 1) is a regulator of cell proliferation and differentiation. Using a mouse peritoneal cell-derived mast cell culture system, we investigated the effects of TGF beta 1 on mast cell proliferation. TGF beta 1 inhibited IL-3- and IL-4-dependent connective tissue-type mast cell proliferation. The effect was concentration dependent: 50% inhibition was observed with 1.0 ng/ml TGF beta 1 and the maximal inhibitory effect (no proliferation), was observed with 10 ng/ml. Flow cytometric analysis suggested that the inhibitory effect of TGF beta 1 was due to blocking of both G1 and G2 phases. Both control and TGF beta 1-treated mast cells showed similar histamine release induced by the calcium ionophore, A23187. TGF beta 1 seems to be an important negative regulator of connective tissue-type mast cell proliferation with apparently no appreciable effect on mast cell function.

Fas antigen is a cell membrane protein that has been suggested to mediate apoptosis. Using SV40-transformed human keratinocytes, we investigated the Fas-antigen-dependent apoptotic process. The expression of Fas antigen mRNA was markedly induced by interferon-gamma (IFN-gamma) treatment (500 U/ml). After IFN-gamma treatment in the presence of anti-Fas monoclonal antibody, apoptosis was induced, as detected by the formation of nucleosome-sized fragments of DNA and morphologically by apoptotic cells with round homogeneous nuclear beads detected by acridine orange staining. The apoptotic SV40-transformed keratinocytes were analyzed quantitatively by enzyme-linked immunosorbent assay using antihistone and peroxidase-conjugated anti-DNA antibodies to detect cell death. The IFN-gamma- and anti-Fas antibody-dependent apoptotis was observed by 3 h, and the maximal response was observed by 12 h. The induction of apoptosis was significantly augmented by treatment with 10 ng/ml 12-o-tetradecanoyl-phorbol-13-acetate (TPA). TPA alone had no effect on either Fas antigen expression or on the apoptotic process. Other protein kinase C activators (1-oleoyl-2-acetylglycerol and mezerein) also stimulated IFN-gamma-dependent apoptosis, whereas 4-o-methyl phorbol myristate acetate, a very weak protein kinase C activator, had only a slight effect. The TPA-induced augmentation of apoptosis was inhibited by the protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7). However, H-7 inhibited only the TPA-induced augmentation of apoptosis; the basal IFN-gamma- and anti-Fas-dependent apoptosis remained in the presence of H-7. Northern blot analysis revealed that c-jun mRNA was induced by IFN-gamma plus anti-Fas antibody treatment as well as by TPA treatment; the addition of IFN-gamma alone to the incubation medium had no effect on the expression of c-jun mRNA. These results indicate that IFN-gamma induces a Fas-antigen-dependent apoptotic process in SV40-transformed keratinocytes and that TPA augments the process through the activation of protein kinase C.