Yoh-ichi Koyama

Effects of collagen peptide ingestion on UV-B-induced skin damage.

The effect of daily ingestion of collagen peptide on the skin damage induced by repeated UV-B irradiation was examined. Ingestion of collagen peptide (0.2 g/kg/d) suppressed UV-B-induced decreases in skin hydration, hyperplasia of the epidermis, and decreases in soluble type I collagen. These results suggest that collagen peptide is beneficial as a dietary supplement to suppress UV-B-induced skin damage and photoaging.

Elongated dermatan sulphate in post-inflammatory healing skin distributes among collagen fibrils separated by enlarged interfibrillar gaps.

It has been reported that the disaccharide composition of dermatan sulphate shows transient changes after epicutaneous application of the hapten 2,4-dinitrofluorobenzene to mouse skin, and that these changes are most conspicuous in healing skin on day 15 after chemical insult [Kuwaba, Nomura, Irie and Koyama (1999) J. Dermatol. Sci. 19, 23-30]. In the present study it was found that the molecular size of dermatan sulphate was increased on day 15 after hapten application. The molecular size of decorin increased in healing skin, whereas the size of dermatan-sulphate-depleted core protein did not increase. The length and localization of decorin dermatan sulphate were investigated by electron microscopy. Dermatan sulphate filaments oriented orthogonally to collagen fibrils were longer in healing skin than in control skin. In control skin, dermatan sulphate filaments were found among tightly packed collagen fibrils. In contrast, the interfibrillar gaps between each collagen fibril were enlarged in healing skin; elongated dermatan sulphate filaments extended from the surface of collagen fibrils across the enlarged gap. These results suggest that the increase in molecular size of decorin dermatan sulphate is important in organizing collagen fibrils separated by enlarged interfibrillar gaps in healing skin.

Functional CD40 ligand is expressed on epidermal Langerhans cells.

Epidermal Langerhans cells (LC) are bone‐marrow‐derived major histocompatibility complex (MHC) class II antigen‐expressing antigen‐presenting cells (APC) that comprise 1–3% of total epidermal cells (EC). LC express high levels of MHC class II antigen and augment costimulatory molecules such as B7‐1, B7‐2 during culture. In a previous report, using purified murine LC, we showed that freshly prepared LC (fLC) do not express CD40, whereas cLC express CD40. Tumor necrosis factor α (TNF‐α) enhanced CD40 expression on LC during culture. We examined the expression of CD40L on LC and found that both fLC and cLC expressed mRNA for CD40L. FACS analysis revealed that cLC cultured for 36 h expressed CD40L but fLC did not. When we examined the cytoplasmic CD40L, however, both fLC and cLC expressed cytoplasmic CD40L. TNF‐α, which up‐regulated CD40 expression on LC during culture, did not modulate CD40L. Co‐culture of purified LC with anti‐CD40L markedly inhibited the up‐regulation of B7‐1 expression on LC and caused partial inhibition of B7‐2 expression during culture. These results indicate that CD40L is expressed on cLC, and that CD40L on LC modulates the expression of costimulatory molecules such as B7‐1 and B7‐2 on LC. J. Leukoc. Biol. 66: 281–285; 1999.

Isolation and Culture of Panning Method‐enriched Langerhans Cells from Dispase‐dissociated Epidermal Cells of the Mouse

Langerhans cells (LCs) are bone marrow‐derived, Ia‐positive antigen‐presenting cells in the epidermis which constitute 2–4% of the total epidermal cells. We examined the usefulness of a combination of dispase treatment and the panning method for enriching and culturing mouse LCs. Trunk skin was treated with partially purified dispase (Godo Shusei, type II) to separate epidermal sheets and to dissociate epidermal cells. Suspended cells were treated with ascites or culture supernatant containing anti‐Ia monoclonal antibody, and LCs were enriched by the Ia‐mediated panning method. Per mouse, 3–4 × 105 LCs were recovered with >95% purity and >90% viability. Enriched LCs potently stimulated the allogeneic mixed‐leukocyte reaction. Ultrastructural observations revealed that enriched LCs contained many vesicles but almost no Birbeck granules. A laminal structure, which was apparently adhesive to the surface of LCs, was observed when ascites were employed as the anti‐Ia antibody. These results indicate that a combination of dispase treatment and the Ia‐mediated panning method is very useful for isolating high yields of functionally mature murine Langerhans cells with high purity and viability.

Size control of decorin dermatan sulfate during remodeling of collagen fibrils in healing skin

Recently it has been reported that the molecular size of decorin dermatan sulfate (DS) was increased in healing skin after hapten application and that the elongated DS was distributed in enlarged interfibrillar space among thin collagen fibrils in situ. Here we show that such modulation of the length of decorin DS is temporary. Although the size of decorin DS was evidently increased on day 15, it decreased to almost normal size on day 35 when the altered disaccharide composition of DS was also recovered. Electron microscopic observation revealed that elongated decorin DS was localized among thin collagen fibrils packed loosely in hapten-treated skin on day 15. In contrast, decorin DS of normal size was distributed among thick collagen fibrils packed tightly on day 35. These results suggest that size control of decorin DS plays important roles in organization of collagen fibrils into bundles by regulating interfibrillar space in healing skin, particularly in maturation of collagen fibrils through shortening of decorin DS in later stages of healing.

Collagen peptide and vitamin C additively attenuate age-related skin atrophy in Sod1-deficient mice

Age-related skin thinning is correlated with a decrease in the content of collagen in the skin. Accumulating evidence suggests that collagen peptide (CP) and vitamin C (VC) transcriptionally upregulate type I collagen in vivo. However, the additive effects of CP and VC on age-related skin changes remain unclear. We herein demonstrate that CP and a VC derivative additively corrected age-related skin thinning via reduced oxidative damage in superoxide dismutase 1 (Sod1)-deficient mice. Co-treatment with these compounds significantly normalized the altered gene expression of Col1a1, Has2, and Ci1, a proton-coupled oligopeptide transporter, in Sod1−/− skin. The in vitro analyses further revealed that collagen oligopeptide, a digestive product of ingested CP, significantly promoted the bioactivity of the VC derivative with respect to the migration and proliferation of Sod1−/− fibroblasts. These findings suggest that combined treatment with CP and VC is effective in cases of age-related skin pathology. Graphical Abstract Co-treatment of collagen peptide and vitamin C additively improve aging-like skin atrophy in mice.

Expression of cellular prion-related protein by murine Langerhans cells and keratinocytes.

Transmissible spongiform encephalopathies are characterized by the accumulation of a proteinase-resistant isoform of the cellular prion-related protein (PrP(c)) within the central nervous system (CNS). The accumulation of scrapie-associated PrP (PrP(Sc)) within cells of the lymphoreticular system prior to its accumulation in the CNS is regarded as important for the development of neurological diseases after peripheral inoculation. Little, however, is known as to which cells are the targets for peripheral inoculation. Here, the presence of PrP(c) on murine Langerhans cells (LC), dendritic cells in the skin and mucosa, and keratinocytes (KC) is demonstrated by immunohistochemical staining, Western-blotting and FACS analysis. The expression of PrP(c) mRNA in freshly purified LC and KC was also detected by reverse transcriptase-polymerase chain reaction. The expression of PrP(c) on LC was slightly increased during culture. These data suggest that LC and KC may be the targets for peripheral infection with prions.

Differential expression of tenascin in the skin during hapten-induced dermatitis

Tenascin is a large extracellular matrix glycoprotein which is found in limited regions of normal adult tissues including the skin. We investigated the induction of tenascin expression in mouse skin during hapten-in-duced dermatitis. In the dorsal skin, hapten application first induced a transient expression of tenascin in deeper regions of the skin. Its distribution then spread over the whole dermis corresponding to the infiltration of Mac-2-positive macrophages. In the ear, tenascin was consistently found in the subcutaneous tissue on the inner side, but very little was seen on the outer side. Tenascin did appear transiently, however, on both sides under hapten treatment. In the early phase of allergic contact dermatitis, no apparent induction of tenascin expression was observed in the swollen ear. However, there was an abundant tenascin expression on both sides during healing. Tenascin expressed under normal conditions was mostly the 180-kDa isoform, while the 230-kDa isoform was markedly induced during healing of the dermatitis. These results suggest that tenascin, particularly the larger 230-kDa isoform, may play important roles in the pathogenesis and healing of hapten-induced dermatitis.

Detection of endogenous and food-derived collagen dipeptide prolylhydroxyproline (Pro-Hyp) in allergic contact dermatitis-affected mouse ear

Generation of collagen dipeptides and deposition of orally administered prolylhydroxyproline (Pro-Hyp) in local inflammatory sites were examined in mice with hapten (2,4-dinitrofluorobenzene)-induced dermatitis in the ear. Pro-Hyp content in the hapten-treated ear was significantly higher in the chronic phase of contact dermatitis than the vehicle control. In contrast, hydroxyprolylglycine contents remained at lower levels in all cases compared to Pro-Hyp. Four hours after the ingestion of [13C5,15N]Pro and [13C5,15N]Pro-Hyp, labeled-Pro-Hyp and Pro, respectively, appeared only in the ear with dermatitis. Thus, Pro-Hyp is generated and degraded as part of the rapid synthesis and degradation of collagen in the ear with dermatitis. In addition to the endogenously generated Pro-Hyp, the orally administered Pro-Hyp was deposited in the ears. [1] Pro-Hyp is generated and degraded as part of the rapid synthesis and degradation of collagen in the ear with dermatitis. [2] The orally administered Pro-Hyp was deposited in the ears.

Temporal changes in disaccharide composition of dermatan sulfate in the skin after epicutaneous application of hapten

Glycosaminoglycans change their amount, structure and distribution with age, differentiation and pathologic conditions. In order to investigate temporal changes in dermatan sulfate in the skin during inflammation and subsequent healing, 2,4-dinitrofluorobenzene (DNFB) was applied once to the dorsal skin of female BALB/cA mice, and the disaccharide composition of dermatan sulfate was examined. Application of DNFB induced a transient increase in the thickness of the dermis, which reached a maximum on day 15 and decreased to the control level on day 35. The total amount of unsaturated disaccharides from dermatan sulfate per cm2 in the DNFB-treated skin showed a temporal pattern similar to that of the thickness of the dermis. Mol% of 4,5-unsaturated 2-sulfo-hexuronic acid-4-sulfated N-acetylgalactosamine (deltaDi-diSB), the second most abundant unsaturated disaccharide from dermatan sulfate, decreased rapidly on day 3 in the DNFB-treated skin, remained less than the control on days 7 and 15, and returned to the control level on day 35. These results suggest that a single application of DNFB induces temporal changes not only in the amount but also in the structure of dermatan sulfate.