Hiroko Koizumi

Distribution of IL-18 and IL-18 receptor in human skin: various forms of IL-18 are produced in keratinocytes.

Abstract Human interleukin-18 (IL-18) enhances IL-12-mediated IFN-γ production by lymphocytes and Fas/ perforin-mediated cytolysis by NK cells. IL-18 is synthesized as a 24 kDa proform, and the proform is processed in the cytoplasm into an 18 kDa mature form. Active and precursor forms of IL-18 have been detected in immunocompetent cells, and active IL-18 exerts its functions through its receptor. We sought to determine which human skin cells are responsible for production of IL-18 and which express its receptor. Monoclonal antibodies against human IL-18 and polyclonal antibody against IL-18 receptor were provided for this analysis. Formalin-embedded and frozen sections of human epidermis were analyzed by immunoperoxidase and immunofluorescence staining. IL-18 was detected in all living cell layers of the epidermis, hair follicles, arrectores pilorum, eccrine ducts and endothelial cells. IL-18 was localized in the cytoplasm of cells in living epidermal cell layers. In contrast, IL-18 receptor was mainly detected in keratinocytes and expressed in the cell periphery in living cell layers. Since keratinocytes were the main source of IL-18 and its receptor, cultured human keratinocytes were further analyzed by immunoblotting. IL-18 receptor was identified as an 80 kDa single band. The mature 18 kDa and precursor 24 kDa forms of IL-18 were detected by our monoclonal antibody (mAb) 21 and mAb 132, respectively, while only the 18 kDa form was detected by a commercial mAb, 125-2H. Cultured keratinocytes showed positive granular staining for IL-18 in the cytoplasm and positive staining for IL-18 receptor mainly in the cell periphery. Our findings indicate that mature IL-18, precursor IL-18 and IL-18 receptor are simultaneously expressed with different localizations by human epidermal keratinocytes. Keratinocytes might be activated by their own IL-18 in an autocrine or paracrine fashion.

Differentiation-associated localization of small proline-rich protein in normal and diseased human skin

Summary The expression of SPRR (small proline‐rich protein) was investigated in normal human skin and in diseased skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma. Naevus pigmentosus, ichthyosis vulgaris and several inflammatory skin diseases, by immunohistochemical staining. A polyclonal antibody was raised against a synthetic peptide for a C‐terminal common region for SPRR l and SPRR 3. In immunoblot analysis, a positive band of 18kDa was detected, which showed the presence of SPRR l in human epidermal keratinocytes. In normal epidermis, positive staining for SPRK was observed in keratinocytes in the granular layer and the uppermost or two spinous cell layers, with no staining of the other spinous or basal layers. The staining was obvious at the cell periphery, weak at the cytoplasm, and absent in the nucleus. Staining was observed in several outer layers of the follicular infundibulum to the isthmus. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, eccrine duct, melanocytes. Langerhans cells or fibroblasts. The arrectores pilorum, striated muscles, muscle layers of vessels, and myoepithelia of eccrine gland, were weakly stained. In psoriatic skin, stained keratinocytes were distributed in the spinous cell layers except for the basal layer, in ichthyosis vulgaris. SPRR was barely expressed in the uppermost living cell layers of the epidermis in epidermolytic hyperkeratosis. degenerated squamous cells widely expressed SPRR. In Dariers disease, dyskeratolic cells were clearly stained. In squamous cell carcinoma, staining was observed in keratotic cells around horny pearls. In basal cell epithelioma, naevus pigmentosus, and malignant melanoma, the tumour cells or naevus cells were not stained. The distribution of SPRR was similar to that of involucrin in normal and several diseased skin, except for ichthyosis vulgaris. We conclude that SPRR is expressed in close association with epidermal differentiation in normal skin and skin diseases. The alteration of the expression of the proteins correlated to terminal differentiation, and differs from disease to disease.

Leukemia cutis in acute myelomonocytic leukemia: infiltration to minor traumas and scars.

We describe a patient with acute myelomonocytic leukemia who demonstrated leukemic cell infiltration to scratched wounds and scars from trauma. A 65‐year‐old Japanese woman developed low grade fever, headache and exanthema. Hematology testing disclosed leukocytosis of 95,600/mm3 with 65% monocytes and 9% blast cells. Infiltrated erythema and nodules were disseminated over most of her body. Moreover, linear scratched wounds and traumatic scars were indurated. Skin biopsy showed dense atypical mononuclear cell infiltration with monocytic characteristics.

Use of dihydrorhodamine 123 for detecting intracellular generation of peroxides upon UV irradiation in epidermal keratinocytes.

Ultraviolet (UV) radiation from the sun and artificial UV sources is an environmental hazard that produces inflammation, pigmentation and carcinogenesis in human skin [15]. Recently, peroxides such as superoxide anion, hydroxyradical and hydrogen peroxide have been suggested as primary hazardous substances [7, 11, 14]. However, a direct method to detect peroxide in living keratinocytes after UV irradiation has not yet been developed. At present, peroxides are detected either by ESR spectroscopy using spin trapping reagents [6, 14], or by flow cytometry using fluorescent dyes such as dichlorofluorescein (DCF) [1, 3, 8, 10, 20]. When these methods were applied to irradiation studies, we encountered serious problems. The reagents used absorb UV, and so complicate the interpretation of the results. It is preferable to measure the generation of peroxides under physiological conditions, but spin trapping reagents can be used only after homogenizing the cells, and some dyes such as DCF are not stable in the cell. Recently a new dye, dihydrorhodamine 123 (DHR 123), has been developed, and applied successfully to the study of peroxide generation in leucocytes upon chemical stimulation [16, 17]. Here we extend the use of this dye for radiation studies, and report for the first time that peroxides are generated in intact epidermal keratinocytes upon UV irradiation.

H2 histamine receptor-mediated increase in intracellular Ca2+ in cultured human keratinocytes

Histamine is present in the epidermis in intracellular and extracellular area and is released from mast cells and keratinocytes in the early stage of inflammation of the skin. Such release may contribute to common itching or intensify the inflammatory responses. Histamine binds to its receptors and participate in regulation of the inflammatory responses by acting on endothelial cells, nerve endings, lymphocytes, monocytes, and leukocytes. Histamine has direct effects on keratinocytes as well. Histamine modulates the proliferation of keratinocytes. The binding of histamine to the receptor on keratinocyte membrane induces activation of adenylate cyclase and phospholipase C through GTP binding protein. We previously reported that histamine induces transient increase in intracellular Ca2+ in cultured normal human epidermal keratinocytes (NHEK) and normal epidermis. H1 and H2 histamine receptors are widely distributed in many tissues and cells. In this study, we investigated which types of histamine receptors are related to the increase in intracellular Ca2+ by histamine stimulation in cultured human epidermal keratinocytes. NHEK were cultured in serum-free KGM medium. With H1 antihistamines, mepyramine and diphenhydramine, histamine responses were moderately but not statistically significantly inhibited. With H2 antihistamine, cimetidine, histamine response was significantly inhibited. Epinephrine response was not affected by these antihistamines. Thus, it is considered that H2 antihistamines specifically block histamine-mediated increase in intracellular Ca2+ of cultured normal human keratinocytes.

1,25-Dihydroxyvitamin D3 and a new analogue, 22-oxacalcitriol, modulate proliferation and interleukin-8 secretion of normal human keratinocytes.

Vitamin D3 is a new therapeutic agent for psoriasis. Hyperproliferation of epidermis and over-secretion of IL-8 by keratinocytes are the characteristic features of psoriasis. The present study was conducted to determine whether a new vitamin D3 analogue, 22-oxacalcitriol, could be effective in inhibiting the proliferation and IL-8 secretion of normal human epidermal keratinocytes. Cell proliferation was measured colorimetrically by the MTS assay. IL-8 secretion was measured with ELISA. Proliferation of cultured normal human epidermal keratinocytes was inhibited in the presence of 1,25-dihydroxyvitamin D3 at the concentrations of 1 x 10(-8) to 1 x 10(-6) M and 22-oxacalcitriol at concentrations of more than 1 x 10(-9) M at 48 h. IL-8 secretion from normal human epidermal keratinocytes was augmented by TNF-alpha, or synergistically by TNF-alpha and IFN-gamma. 1,25-Dihydroxyvitamin D3 and 22-oxacalcitriol inhibited cytokine-stimulated IL-8 production dose dependently after 24 h incubation without inhibition of cell proliferation. 1,25-Dihydroxyvitamin D3 and 22-oxacalcitriol are considered to inhibit the proliferation of keratinocytes directly and indirectly with inhibition of the secretion of IL-8 from keratinocytes. The inhibition of IL-8 secretion from keratinocytes by vitamin D3 could modulate the behaviour of immunocompetent cells infiltrating in the skin.

TWO FORMS OF ADENOSINE DEAMINASE IN PIG EPIDERMIS (II)

Molecular heterogeneity of adenosine deaminase in pig epidermis is reported. Gel filtration of pig skin extracts with Sephadex G‐150 revealed the existence of two forms of adenosine deaminase. Molecular weights of the enzymes were around 30,000–40,000 and 300,000–350,000 respectively. Several catalytic properties of these two adenosine deaminases were quite similar: Km value, substrate specificity, pH activity profile, and the effect of coformycin, a strong inhibitor of adenosine deaminase.

Dermal dendrocyte hamartoma with stubby white hair: A novel connective tissue hamartoma of infancy

A previously undescribed hamartoma with stubby white hair was observed in a 1-week-old girl. A deep red, soft nodule with fine wrinkles was present on the back. White bizarre short thick hairs with irregular exterior cuticular squamae were noted. The main cells proliferating in the nodule were fibroblast-like spindle cells that had dendrites and showed positive staining for CD34 antigen. These cells surrounded vessels and nerves. In addition, there were immature hair follicles, relatively thick-walled small vessels, and small adipose cells with fine connective tissue. This hamartoma was considered to be a dermal dendritic cell hamartoma originating from CD34-positive cells.

Lichenoid skin lesions as a sign of β2‐microglobulin‐induced amyloidosis in a long‐term haemodialysis patient

We report a case of β2‐microglobulin‐induced amyloidosis. The patient was a 40‐year‐old man suffering from non‐amyloid nephropathy, who had been treated by haemodialysis for 20 years. Lichenoid skin lesions, consisting of groups of pin‐head‐sized shiny papules, were present on the arms and trunk. On histological examination, amyloid deposits were present, principally in the dermal papillae, hut also around the sweat ducts and hair follicles. The amyloid displayed potassium‐permanganate‐resistant Congo red affinity, and green birefringence under polarized light, Immuno‐histochemically, β2‐microglobulin was demonstrated in the lesions, confirming that they were a manifestation of β2‐microglobulin‐associated amyloidosis. Skin lesions of this type have not been reported previously in β2‐microglobulin‐associated amyloidosis.

Substance P induces intracellular calcium increase and translocation of protein kinase C in epidermis

Substance P is a neuropeptide present in, and released from, peripheral C nerve endings. The presence of substance P‐positive nerve fibres in the epidermis has been reported. We investigated the effect of substance P on the transmembrane signalling system of pig epidermal keratinocytes. Treatment of pig epidermis with substance P resulted in an increase in inositol 1,4,5‐trisphosphate (IP3), and in intracellular free calcium. The treatment also resulted in translocation of protein kinase C from a cytosol to a membrane fraction. Substance P, however, did not affect the β‐adrenergic‐ or histamine (H2)‐ adenylate cyclase responses of the epidermis. Neither forskolin‐induced, nor cholera toxin‐induced cyclic AMP accumulation were affected by substance P treatment. These results are consistent with the view that substance P stimulates phosphatidylinositol‐4,5‐bisphosphate (PIP2) hydrolysis of keratinocytes, resulting in IP3‐Ca2+ and diacylglycerol‐protein kinase C signal activation. Although protein kinase C is known to affect the epidermal adenylate cyclase system, no evidence for such ‘cross‐talk regulation’ was detected in keratinocytes by substance P treatment.