Search

Toll-like receptors (TLRs) on keratinocytes are important cell surface receptors involved in the innate and acquired immune response to invading microorganisms. In acne vulgaris, TLR2 activation by Propionibacterium acnes (P. acnes) may induce skin inflammation via induction of various proinflammatory molecules that stimulate the invasion of inflammatory cells. Although corticosteroids themselves exert immunosuppressive or anti-inflammatory effects, it is well known clinically that systemic or topical glucocorticoid treatment provokes an acneiform reaction. Nevertheless, the effect of steroids on TLR2 expression in human keratinocytes remains unknown. Here, we found that the addition of glucocorticoids, such as dexamethasone and cortisol, to cultured human keratinocytes increased their TLR2 gene expression. Moreover, these glucocorticoids markedly enhanced TLR2 gene expression, which was further stimulated by P. acnes, tumor necrosis factor-alpha, and IL-1alpha. Gene expression of mitogen-activated protein kinase (MAPK) phosphatase-1 was also increased by the addition of dexamethasone. By using several inhibitors and activators, we found that TLR2 gene induction by glucocorticoids was mediated by the suppression of p38 MAPK activity following induction of MAPK phosphatase-1. These findings strongly suggest that steroid-induced TLR2 together with P. acnes existing as normal resident flora plays an important role in the exacerbation of acne vulgaris as well as in possible induction of corticosteroid-induced acne or in that of rosacea-like dermatitis.

Although ultraviolet B (UVB) induces apoptosis and functional perturbations in dendritic cells (DC), for example, Langerhans cells (LC), it also stimulates some LC into maturation after irradiation in vivo. To analyze its reciprocal effects on DC, we elucidated the direct effect of UVB on DC in vitro using human monocyte-derived DC (MoDC). UVB from 50 to 200 J per m2 stimulated the maturation of MoDC with (1) augmented expression of CD86 and HLA-DR, (2) enhanced production of IL-1beta, IL-6, IL-8, and TNF-alpha at both the mRNA and protein levels, and (3) enhanced allostimulatory capacity on a per-cell basis, whereas the exceeded doses induced apoptotic cell death. Western-blot analysis of MoDC after UVB demonstrated a concentration-dependent phosphorylation of p38- and c-JUN N-terminal kinase (JNK)-mitogen-activated protein kinases (MAPK), but not that of extracellular signal-regulated kinases. p38 MAPK-inhibitor, SB203580, inhibited both UVB-induced maturation and apoptosis of MoDC. Interestingly, MoDC that had undergone apoptosis exhibited an augmented expression of HLA-DR without upregulation of CD86 antigen, suggesting their tolerogenic phenotype. Thus, our study revealed a dual effect of UVB, to stimulate maturation or to induce apoptosis in MoDC, depending on the dosage, via p38 MAPK pathway.

To elucidate the biological significance of activating mutations of BRAF in human malignant tumors, we performed a mutation analysis using 43 cell lines established from tumors that had developed in several kinds of human organs. Because the same V599E point mutation was observed in three of six melanoma cell lines and no such mutations were observed in other types of cancers, we focused further on melanoma, performed mutation analyses of NRAS, KRAS, CTNNB1, and p16/p14(ARF) in these cell lines, and found one NRAS mutation and three p16/p14(ARF) mutations. We further searched for mutations of BRAF and NRAS in 35 primary sporadic melanomas from 35 Japanese patients and detected the V599E BRAF point mutation in only nine (26%) of them. Significant differences in mutation frequency were observed among four histological subtypes; four (50%) of eight superficially spreading melanoma and five (33%) of 15 acral lentiginous melanoma had the mutation, whereas none of 12 other types (six nodular melanoma, five lentigo melanoma, and one mucosal melanoma) had it. The BRAF mutation was observed frequently even in small lesions, indicating that activation of this gene may be one of the early events in the pathogenesis of some melanomas.

Human monocyte-derived dendritic cells (MoDC) have both histamine H1 and H2 receptors and can induce CD86 expression by histamine. Nevertheless, it has not been reported whether human epidermal Langerhans cells (LC) have histamine receptors or not. In this study, using RT-PCR, we investigated the expression of H1 and H2 receptor mRNA on DC with the features of LC (LC-like DC) that were generated in vitro from peripheral blood monocytes, LC derived from CD34+ hematopoietic progenitor cells, and LC obtained from human epidermis. We compared the histamine-induced CD86 expression among these cells. In contrast to MoDC, LC and LC-like DC did not express H1 or H2 receptors. In addition, they could not augment the CD86 expression by histamine. Interestingly, when transforming growth factor-beta1 (TGF-beta1) was added to the culture of MoDC, the expression of H1 and H2 receptors and the histamine-induced CD86 expression were abrogated in a concentration-dependent fashion. Finally, in the assessment of the cell surface expression of histamine receptors using fluorescence-labeled histamine, histamine could bind to MoDC and dermal dendritic cells obtained from the skin, whereas there was no specific binding of histamine to LC-like DC or LC obtained from the skin. These data suggest that LC do not express either H1 or H2 receptors, mainly because of the effect of TGF-beta1. This made a striking contrast with the expression of the functional H1 and H2 receptors on MoDC and dermal dendritic cells.

Diabetes mellitus induces many pathophysiologic changes in the skin. Even so, dermatologists still lack an animal model of diabetes that enables the direct evaluation of the various functional properties of the skin. Our group induced two types of an experimental type 1 diabetes model in hairless mice by administering either streptozotocin or alloxan, in order to examine the properties of the stratum corneum and epidermis of these animals. The plasma glucose concentrations of the mice at 3 wk after their i.v. injection were significantly higher than those of control mice (streptozotocin, 3.2-fold; alloxan, 3.7-fold). The stratum corneum water content was significantly reduced in both types of diabetic mice, whereas the transepidermal water loss remained unchanged. The amino acid content with normal epidermal profilaggrin processing was either normal or elevated in the stratum corneum of the streptozotocin-treated mice. In contrast, the stratum corneum triglyceride content in the streptozotocin-treated mice was significantly lower than the control level, even though the levels of ceramides, cholesterols, and fatty acids in the stratum corneum were all higher than the control levels. The streptozotocin-treated group also exhibited decreases in basal cell proliferation and epidermal DNA content linked with an increase in the number of corneocyte layers in the stratum corneum, suggesting that the rates of epidermal and stratum corneum turnover were slower in the streptozotocin-treated animals than in the normal controls. In contrast, there were no remarkable changes in any of the epidermal differentiation marker proteins examined. This finding in diabetic mice, namely, reduction in both the epidermal proliferation and stratum corneum water content without any accompanying impairment in the stratum corneum barrier function, is similar to that found in aged human skin. Our new animal model of diabetes will be useful for the study of diabetic dermopathy as well as the mechanisms of stratum corneum moisturization.

We have generated mutant mice for epidermal-type fatty acid binding protein by the gene targeting technique and examined the phenotype in detail. Despite a lack in the expression of epidermal-type fatty acid binding protein mRNA and its protein in the skin and other tissues of the mutant mice, the animals appeared normal in gross and histologic examination. Northern blot analysis of other fatty acid binding proteins revealed a distinct elevated gene expression of heart-type fatty acid binding protein in the skin of the homozygous mice. In analyses of the skin, no differences were observed in contents of major fatty acids, electron microscopic appearance as well as inflammatory responses in ear skin between the mutant and wild-type mice. Basal transepidermal water loss of homozygous mice was lower than that of the wild mice. When acetone was applied to the skin for disruption of the water permeability barrier, recovery in transepidermal water loss was delayed, although maximum transepidermal water loss upon acetone treatment was similar between homozygous and wild-type mice in terms of size and time course. The molecular mechanism by which epidermal-type fatty acid binding protein contributes to the water barrier function of the skin remains to be elucidated.

Because interferon-gamma, interleukin-4, and interleukin-5 have been identified at the mRNA and protein levels in the lesional skin of patients with atopic dermatitis, we investigated the roles played by granulocytes as effector cells in allergic inflammation by using two unique murine skin models. In vitro generated Th1 and Th2 cells from naïve splenocytes of antiovalbumin T cell receptor transgenic BALB/C mice were adoptively transferred with ovalbumin into the ear pinnae or air-pouches produced in the back skin of naïve, nontransgenic BALB/C mice. The injection of Th1 cells with ovalbumin induced delayed type ear swelling that peaked at 48 h, whereas that of Th2 resulted in ear swelling that peaked at a much earlier time, 24 h. Histologic study of the swollen ear skin and granulocytes recruited into the air-pouch demonstrated that, although the Th1-induced inflammation caused a neutrophil-predominant infiltrate with few eosinophils, larger numbers of eosinophils accumulated in the Th2-induced inflammation. Using these murine models, we further evaluated the effects of drugs used for the treatment of atopic diseases. The results showed that FK506 administration could effectively reduce skin inflammation induced by either Th cells. Interestingly, the neutrophil elastase inhibitor ONO-6818 efficiently inhibited Th1-induced inflammation. In contrast, a leukotriene receptor antagonist, ONO-1078, specifically suppressed Th2-induced inflammation. We also found that each ONO drug exerted direct influence on specified granulocytes, as neither affected in vitro production of relevant Th cytokines. Thus, we succeeded in developing animal skin inflammation models in which we can evaluate the contribution of protein antigen-specific Th1 or Th2 cells through the action of granulocytic effector cells.

We investigated the regulation of C3 production by human cultured epidermal keratinocytes by enzyme-linked immunosorbent assay. The results showed that IFNgamma and TNFalpha enhanced the synthesis of C3 by epidermal keratinocytes in a concentration-dependent manner. Moreover, a protein kinase C (PKC) inhibitor blocked C3 production, whereas PMA enhanced it. There was a synergistic effect between IFNgamma and TNFalpha. In experiments to investigate the role of protein tyrosine kinase (PTK) in C3 production, we found that treatment with herbimycin A, a specific inhibitor for the c-Src-related PTK, caused significant enhancement of the C3 production induced by IFNgamma or TNFalpha, suggesting that c-Src-type PTK(s) provides a negative signal to C3 production. Each competitive inhibitor of PTK, genistein or tyrphostin, substantially increased the C3 production by IFNgamma at lower concentrations, although each agent had little effect on TNFalpha-associated production of C3 at the same concentrations. The data show that pro-inflammatory cytokines IFNgamma and TNFalpha synergistically augment C3 production by epidermal keratinocytes by different pathways.

It has been reported that the in vivo maturation of Langerhans cells after hapten painting is mediated by IL-1 beta while Langerhans cell maturation after in vitro culture is mediated by granulocyte-macrophage colony-stimulating factor (GM-CSF). To clarify the reason for this discrepancy, we examine the expression of Ia antigen and several co-stimulatory molecules on Langerhans cells that were activated by in vitro culture, by hapten painting, or by an intradermal injection of several cytokines. Both cultured Langerhans cells and those activated by hapten painting increased the expression of Ia antigen and all the co-stimulatory molecules (i.e., intercellular adhesion molecule-1 [ICAM-1], B7-1, B7-2, and CD40). In contrast, an intradermal injection of interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) increased the expression of Ia antigen, ICAM-1, B7-2, and CD40, but not that of B7-1. These data indicate that IL-1 beta or TNF-alpha is not sufficient to induce B7-1 expression on Langerhans cells in vivo. Subsequently we examined the effect of anti-cytokine antibodies (Abs) on the expression of those molecules on cultured Langerhans cells. While none of the Abs to IL-1 beta, TNF-alpha, or GM-CSF changed the upregulation of Ia antigen, ICAM-1, or CD40 on cultured Langerhans cells, anti-GM-CSF Ab suppressed that of B7-1 and B7-2. Taken together, our present results suggest that IL-1 beta is required for the upregulation of Ia, ICAM-1, B7-2, and CD40, while GM-CSF is required for the upregulation of B7-1 and B7-2, although it still remains unclear why the injected GM-CSF could not augment B7-1 expression on Langerhans cells in vivo and why anti-IL-1 beta Ab did not suppress the upregulation of Ia, ICAM-1, or CD40 on cultured Langerhans cells.

Recently we have demonstrated that, besides Vγ5 + γ/δ TcR + T cells (Vγ5 + γ/δ T cells), Vγ5 - γ/δslash; TcR + T cells (Vγ5 + γ/δ T cells) are also present in murine skin. In the present study, to characterize the functional differences between these two different cutaneous γ/δ T cells we examined the expression pattern of E-cadherin and its two integrins. After co-culturing of Ly-5 + epidermal cells and migrating cells from organ-cultured murine skin with cutaneous stromal cells, we could expand Vγ5 + γ/δ T cells and Vγ5 - γ/δ T cells, respectively. Flow cytometry demonstrated that cultured Vγ5 + γ/δ T cells expressed E-cadherin, but Vγ5 - γ/δ T cells did not. This difference in E-cadherin expression was also observed in freshly isolated Vγ 5+ and Vγ5 - γ/δ T cells. On the other hand, both Vγ5 + and Vγ5 - γ/δ T cells expressed the a chain of the vitronectin receptor, but did not express the α4 integrin. Of these two cutaneous &/δ T cells, only Vγ5 + γ/δ T cells adhered to murine keratinocyte cell line, PAM 212 cells. Unexpectedly, however, the adhesion of E-cadherin-expressing Vγ5 + γ/δ T cells to PAM 212 cells was not inhibited by anti-E-cadherin antibody, which effectively abrogated the adhesion of Langerhans cells to PAM 212 cells. These distinct phenotypic and functional characteristics in the subsets of cutaneous γ/δ T cells may suggest that they reside in different locations in the skin to play different functional roles in skin immunophysiology.

We have established a dermal fibroblast-like stromal cell line, DFB-1, and a clone, 12E2, from epidermal sheets prepared from the skin of BALB/c mouse ears by trypsin digestion. They were suggested to be fibroblasts or myofibroblasts, as 1) they were polygonal or spindle-shaped under the phasecontrast microscope, 2) they did not possess any tonofilaments or desmosomes, and 3) they did not express any marker for bone marrow-derived cells or macrophages. Interestingly, these cells showed a unique phenomenon of "pseudoemperiporesis," which was first recognized in the interaction between thymic nurse cells and thymocytes. Namely, two T-cell clones and one T-cell hybridoma migrated beneath the cytoplasmic projections of the fibroblastlike cutaneous stromal cells in culture. Furthermore, secretion of interleukin 7 by these cells was confirmed by bioassay using an IL-7-dependent cell line and by inhibition with anti-interleukin 7 antibody, and the expression of interleukin 7 mRNA was also demonstrated in these cells by a combination of reverse-transcriptase polymerase chain reaction and Southern blot analysis. These data strongly suggest the presence of unique stromal cells even in the skin, probably at the upper dermis, which can function like the nurse cells in the thymus. These stromal cells may play a crucial role in cutaneous immunophysiology.

Recently, it has been demonstrated that the skin-infiltrating T cells express cutaneous lymphocyte-associated antigen, which is the ligand of E-selectin or endothelial-leukocyte adhesion molecule, suggesting that cutaneous lymphocyte- associated antigen functions as the homing receptor of the skin infiltrating T cells. In contrast, the mechanism for the migration of Langerhans cells from the bone marrow to the skin has not been clarified. Sialyl LewisX acts as a ligand for endothelial-leukocyte adhesion molecule and granule membrane protein 140. We examined the expression of sialyl LewisX in epidermal dendritic cells in human skin. Two color immunofluorescence study on an epidermal sheet revealed that human leukocyte antigen DR+ or CD1a+ epidermal dendritic cells were partially sialyl LewisX+, although all of the sialyl LewisX+ dendritic cells were human leukocyte antigen DR+ and CD1a+. Further analysis of these dendritic cells by flow cytometry demonstrated that most of the human leukocyte antigen DR+ and CD1a+ epidermal cells expressed sialyl LewisX, although the magnitude of its expression was more variable than that of CD1a expression, and that some of human leukocyte antigen DR+ cells were clearly sialyl LewisX-. Immunoperoxidase study of normal skin showed the presence of sialyl LewisX+ dendritic cells not only in the epidermis but also in the upper dermis. These data demonstrating the heterogeneity of the expression of sialyl LewisX by epidermal Langerhans cells suggest their possible relationship to the stage of maturation as well as to the migration of Langerhans cells from the bone marrow to the skin.

After hapten application, epidermal Langerhans cells migrate into the regional lymph nodes through dermal lymphatics. Recently, we have demonstrated that some of them take the phenotypic and functional characteristics similar to those of in vitro cultured Langerhans cells, before disappearing from the epidermis. To analyze the mechanisms underlying the migration of Langerhans cells, we studied the expression of several adhesion molecules on freshly isolated LC and cultured LC. Pgp-1 (CD44), intercellular adhesion molecule 1, and alpha 4 integrin were strongly expressed on cultured Langerhans cells. Among them, only alpha 4 integrin was strongly up-regulated by cultured Langerhans cells, because its expression by freshly isolated Langerhans cells was very weak. This up-regulation of alpha 4 integrin was also observed on in vivo activated Langerhans cells in the epidermis and draining lymph nodes after hapten application. These data suggest a possible role played by VLA-4 in the migration of Langerhans cells from the epidermis into the regional lymph nodes after hapten application.

Anti-DNA autoantibodies are known to cross-react with a wide variety of substances including cell-surface molecules. Thus, we examined cross-reactivities of 20 murine monoclonal anti-DNA antibodies with normal human and mouse skin tissues. Hybridomas producing these monoclonal antibodies were established from non-immunized spleen cells from autoimmune MRL-1pr/1pr mice, a strain characterized by spontaneous development of SLE-like disorders including skin changes. They were selected based on their reactivity to DNA in a typical enzyme-linked immunosorbent assay, in which nine monoclonal antibodies were reactive with both double-stranded DNA and single-stranded DNA, whereas nine monoclonals were reactive only with single-stranded DNA. Even though only seven of them were observed to stain nuclei, most of the monoclonal antibodies revealed strong and distinct cross-reactivities to various components of the skin tissues including the epidermal basement membrane, keratinocytes at different locations of the epidermis, melanocytes, Langerhans cells, Thy-1+ dendritic cells in the case of murine skin, and mast cells. Our results suggest a possible role of so-called anti-DNA antibodies with high or low affinities to DNA in the pathogenesis of cutaneous lesions of lupus erythematosus.

We provide evidence that stratum corneum (SC) activates complement through the alternative pathway to generate C5a anaphylatoxin. By immunofluorescence studies it was shown that in addition to circulating IgG autoantibody, there were anti-SC antibodies of IgM and IgA classes in the sera from normal individuals. However, all the titers were significantly lower than the level of C3 deposition between corneocytes. By contrast, no C1q deposition occurred. Immunoelectrophoretically the orthokeratotic SC homogenates were found to induce the conversion of C3 from native C3 to C3B in fresh human serum even when the classic pathway was blocked by Ca2+-chelation, Enzyme immunoassay showed that factor B split product, Bb, was generated by the SC homogenates in the Ca2+-chelated serum. Radioimmunoassay for C5a also demonstrated that the SC homogenates could generate C5a also demonstrated that the SC homogenates could generate C5a anaphylatoxin in serum to an extent similar to that in non-treated serum when restricted to the alternative pathway activation; neutrophil chemotactic activity was generated in Ca2+-chelated serum at levels comparable to that generated to non-treated fresh serum. We separated the SC samples into a cornified envelope and soluble and keratin fractions. The cornified envelope was more effective in activating complement. This activity resided in heat-stable and non-lipid substances of corneocytes. Our hypothesis is that when the SC comes in contact with serum, it activates complement mainly through the alternative pathway to induce chemotactic C5a anaphylatoxin. Hence, inflammation in normal individuals after a traumatic injury to the skin or rupture of acne comedones, or epidermal cyst and possibly the formation of subcorneal sterile pustules noted in several dermatoses are explainable through this mechanism.

Transepidermal migration of leukocytes, with resultant formation of microscopic or macroscopic sterile subcorneal pustules is a phenomenon characteristically noted in psoriasis and related sterile pustular dermatoses. It is natural to assume the presence of potent neutrophil chemotactic substances in the subcorneal portion of the lesional epidermis, because this location is the target of the in vivo leukocyte chemotaxis. In fact, crude psoriasis scale extracts show remarkably high neutrophil chemotactic and activating properties as compared with those of other non-psoriatic inflammatory dermatoses. We isolated a psoriatic leukotactic factor (PLF) having a molecular mass of around 12 kD, distinct from those common to other inflammatory changes involving the skin or those released by bacteria. Further analysis of PLF identified C5 cleavage fragments, together with other chemotactic peptides, such as those derived from monocytes. Likewise, potent low-molecular-mass chemotactic factors, including cell membrane lipid derived chemotactic factor, e.g. leukotriene B 4 , are also increased in psoriatic lesions, as in other nonpsoriatic inflammatory dermatoses. However, their activity to stimulate the generation of oxygen radicals in neutrophils was found to be much weaker than that of PLF. The peripheral blood leukocytes from active psoriatic patients show enhanced function in chemotaxis, phagocytosis, active oxygen production, and enzyme release; patients' sera contain substances such as anaphylatoxins that activate leukocyte function. Further research is required for clearer understanding of the scries of events resulting in the leukocyte chemotaxis, as well as for the elucidation of the background immunoregulatory mechanisms. J Invest Dermatol 88:18s—23s, 1987

Based on the evidence from our previous studies that we can evaluate the hydration state of the skin surface quickly and quantitatively in terms of conductance to the high frequency electric current of 3.5 MHz, we have established a simple in vivo function test that furnishes information on the hygroscopic property and water-holding capacity of the stratum corneum in a few minutes. The test procedure consists of electromeasurements before and after application of a droplet of water on the skin for 10 seconds to obtain data on the hygroscopic property of the skin surface and later serial measurements at an interval of 30 seconds for 2 min to evaluate the water-holding capacity. Under usual ambient conditions normal skin surface showed a high rise in conductance just after application of water, which was followed by a rapid fall-off within 30 seconds, thereafter by gradual return to the prehydration levels by 2 min. By this method we have demonstrated that (i) the superficial horny layer of normal skin is much less hygroscopic and less capable of holding water than the corresponding deeper portions and that (ii) scaly skin shows functional defects in both hygroscopicity and water-holding capacity, between which the former normalizes much faster than the latter.

Ia antigen-bearing (Ia+) Langerhans cells have attained an important position as immunocompetent cells in the epidermis. Recently there have been successive reports on other new possible candidates for immunocompetent cells in the epidermis, i.e., Ia+ keratinocytes and dendritic Thy-1 antigen-bearing (Thy-1+) epidermal cells which also express Ly-5 antigen and asialo-GM1. Based on our previous findings that in allergic contact sensitivity reactions, keratinocytes express Ia antigen 3-9 days postchallenge, in this report, we have attempted to define more clearly the dynamic changes of Ia+ keratinocytes and dendritic Thy-1+ epidermal cells by enumeration of the precise percentages of Ia+, Thy-1+, and Ly-5 antigen-bearing (Ly-5+) cells in epidermal cells at various times of the challenge phase in allergic contact sensitivity reactions by use of a fluorescence-activated cell sorter. By 24 h postchallenge, the percentages of Ia+, Thy-1+, and Ly-5+ cells showed hardly any change. There were approximately 2% Ia+ cells, 50% Thy-1+ cells which consist of 2 populations (i.e., 45% weakly Thy-1 antigen-positive cells and 4% strongly Thy-1 antigen-positive cells), and 3.5% Ly-5+ cells. From 48 h postchallenge, however, the percentage of Ia+ cells and that of Thy-1+ cells began to increase and reached a plateau, with approximately 20% Ia+ cells and 70% Thy-1+ cells, respectively, at 120 h postchallenge. The change of the percentages of Ly-5+ cells appears to correspond to that of strongly Thy-1 antigen-positive cells. Only at 48 h postchallenge, Ly-5+ cells and strongly Thy-1 antigen-positive cells showed a small increase in number, comprising approximately 10% of the epidermal cells. These data suggest that among Thy-1+ epidermal cells, strongly Thy-1 antigen-positive cells correspond to dendritic Thy-1+ epidermal cells, and in contact sensitivity reactions in mice, dendritic Thy-1+ epidermal cells show only a minor dynamic change in contrast to Ia+ cells, in which more than 15% of keratinocytes express Ia antigen from 48 h postchallenge.

Nobody denies the fact that cracking and scaling of the skin surface develop because of decreased water content in the horny layer. However, functional research of such stratum corneum has been greatly hampered by the lack of an adequate, noninvasive in vivo method to assess the hydration state of the skin surface. While studing the electrical properties of the skin using a newly developed instrument that can be measure the resistance and capacitance to the high frequency current of 3.5 MHz, we have found that by employing dry electrodes we can evaluate the hydration state of the skin surface quickly and quantitatively in terms of output voltage of the loss resistance detector(Vr), which is in a reciprocal relation to resistance. The sensitivity is very high since it can detect a high Vr value at the site where a water droplet is applied and blotted out instantaneously and where no special change can be found with the naked eye. The principal hydration detected by this method seems to be that in the outermost portion of the stratum corundum on the basis of the following finding: (i) contact of the skin with water even for a second caused a great increase in Vr: (ii) Vr progressively increased as deeper layers of the stratum corundum was serially exposed by cellophane tape-stripping until reaching a certain level of the hydrated, viable epidermis, while the Vr on the surface of the intact stratum corundum was not influenced by the amount of tissue fluid contained in the underlying epidermis; and (iii) in vitro the Vr of pieces of fully hydrated stratum corundum rapidly decreased in a dry atmosphere with evaporation of the water contained in their superficial portion. Furthermore, we have been able to demonstrate the highly hygroscopic nature of the stratum corneum, (b) lesser hydration on the extremities that on the trunk, (c) the decreased water-holding capacity of scaly lesions despite the increased transepidermal water loss, and (d) the water supplying ability of skin emollients.

Trichophytin contact sensitivity was demonstrated for the first time in guinea pigs infected with Trichophyton mentagrophytes using a new inoculation technique arid a provocative patch test. The inoculation method utilizes an occlusive dressing to keep a high humidity in the inoculation site. With a zoophilic strain of T. mentagrophytes , secure occlusion of the inoculated site for 24hr produced definite infection for several weeks. Trichophytin contact sensitivity was detected by means of a provocative patch test with undiluted crude trichophytin from filtrates of broth medium cultured with the organism. The sensitivity was confirmed by a patch test with purified trichophytin consisting of polysaccharide with attached peptides. The incidence of contact sensitivity increased with the number of infections-11 out of 20 animals (55%) after the first infection. 12 of 15(80%) alter the second. and 11 of 12 (91.5%) after the third.

The present study was undertaken in order to clarify the exact mode of the Langerhans cell (LC) depleting process caused by UV irradiation. Following irradiation with a single dose of various wavelengths of monochromatic UV radiation (UVR), we studied the number of Ia-positive cells in mouse epidermal sheets quantitatively, particularly with regard to dose-response relationship, action spectrum, and time course change. In addition, we studied morphologic alterations of these cells using electron- and immunoelectron microscopy (EM and IEM). We obtained the following results after a single dose of UVB radiation (200 mJ/cm2 of 300 nm) or PUVA (1% of 8-methoxypsoralen (8-MOP) 20 microliter and 1 J/cm2 of 360 nm): (1) EM and IEM showed that while some LCs simply lost their Ia marker without any structural alterations, the majority of the LCs disappeared due to actual cell damage. (2) During an "injury phase," the initial 48 h, and a "recovery phase," lasting from 4-14 days after irradiation, enlargement of the size of remaining Ia-positive LCs occurred. The degree of enlargement was closely related to the degree of reduction in number, suggesting a process compensating for the loss of the LC population. (3) It was found that the recovery rate of LCs after irradiation damage was slower than that of keratinocytes, indicating different cell kinetics between these distinct cell populations in the epidermis, i.e., restoration of LCs after irradiation seems to be achieved at least partially through a repopulation process originating in the bone marrow. Studies with irradiation of various monochromatic wavebands, with or without topical 8-MOP, showed that the action spectrum for Ia-positive cell depletion activity lay within the spectrum shorter than 300 nm for UVR alone, and between 320-380 nm for 8-MOP plus UVR. Since the action spectra were similar to those for keratinocyte damage, i.e., sunburn cell formation, induction of unscheduled DNA synthesis, and to those for UVR-induced erythema, we conclude that common mechanisms underlie these types of tissue damage.

The accumulation of polymorphonuclear leukocytes (PMN) beneath the stratum corneum is a characteristic histopathologic finding in various aseptic pustular dermatoses. To elucidate the pathomechanism involved in this phenomenon, we investigated whether PMN also attach to a sheet of corneocytes in vitro. A 1-cm2 corneocyte sheet was attached to a sterile glass slide with double adhesive tape used for skin graft surgery before incubating with human serum. The PMN suspension then was applied to the sheet. Attached cells were stained with May-Grunwald-Giemsa and counted with a computer image analyzer. We quantitatively assessed PMN adhesion to the serum-treated corneocyte sheets, which was mediated by activation of the alternative complement pathway. Addition of either anti-CD18 or anti-CD11b antibody to the assay system resulted in a marked reduction of PMN adhesion. We also demonstrated immunohistochemically that iC3b was formed on the serum-treated corneocytes. These findings suggest that PMN attach to serum-treated corneocytes through an interaction of CR3 expressed on PMN with iC3b-coated corneocytes. In addition, we found that this adhesion was enhanced by activation of PMN with phorbol myristate acetate. From these results, we speculate that complement activation by corneocytes occurs in the cutaneous lesions of aseptic pustular dermatoses and that PMN can be stimulated by the interaction with iC3b-opsonized corneocytes as well as by chemotaxins, leading to damage of the surrounding epidermal keratinocytes.

Leptin, a satiety-regulating cytokine, is predominantly expressed by adipocytes, although recently the nonadipose tissue production of leptin has been reported. To investigate the possibility of leptin production by human scalp hair follicles, we examined leptin production and its mRNA expression by cultured human follicular papilla cells. We isolated 12 human follicular papilla cell lines from different individuals. They were identified by their morphology, their high alpha-smooth-muscle actin expression, their inability to differentiate into adipocytes, and by the lack of mRNA for adipose-specific fatty acid binding protein. All the human follicular papilla cell lines, but not neonatal human dermal fibroblasts, produced significant amounts of leptin demonstrable by enzyme-linked immunosorbent assay. We demonstrated leptin mRNA expression by human follicular papilla cell lines, but not by neonatal human dermal fibroblasts, by reverse transcription polymerase chain reaction. By immunohistochemistry and in situ hybridization, we detected both leptin protein and mRNA at the lower portion of the hair follicle, i.e., hair matrix, inner root sheath of the hair bulb, and human follicular papilla cells. In contrast, the leptin receptor with intracytoplasmic signal sequence was detected in the follicular papilla cells immunohistochemically, and the long isoform of the leptin receptor mRNA was demonstrated in the human follicular papilla cell lines by reverse transcription polymerase chain reaction. Finally, by using these human follicular papilla cell lines, we showed that cytokines such as interleukin-1 beta, tumor necrosis factor alpha, interferon-gamma, and interleukin-4, and growth factors such as epidermal growth factor, basic fibroblast growth factor, and transforming growth factor beta1, but not vascular endothelial growth factor, hepatocyte growth factor, keratinocyte growth factor, and insulin-like growth factor 1, significantly downregulated the production of leptin. These data demonstrated that human follicular papilla cells produce leptin and express the functional leptin receptor in vivo and in vitro, suggesting its autocrine function. Moreover, the regulation pattern of its production by various factors suggests a pivotal role of leptin in hair biology.

Macrophage colony-stimulating factor has not been considered as a factor responsible for dendritic cell or Langerhans cell development from hematopoietic progenitor cells. In this study, we examined whether macrophage colony-stimulating factor could be used instead of granulocyte-macrophage colony-stimulating factor for the in vitro development of Langerhans cells from hematopoietic progenitor cells. We replaced granulocyte-macrophage colony-stimulating factor with macrophage colony-stimulating factor from a serum-free culture containing granulocyte-macrophage colony-stimulating factor, stem cell factor, Flt3 ligand, tumor necrosis factor-alpha, and transforming growth factor-beta1. This serum-free culture medium containing macrophage colony-stimulating factor, but not granulocyte-macrophage colony-stimulating factor (macrophage colony-stimulating factor culture), could induce CD1a+ Birbeck granule+ Langerin+ E-cadherin+ factor-like XIIIa Langerhans cells. As a control, the culture of hematopoietic progenitor cells in this culture medium depleted of macrophage colony-stimulating factor or transforming growth factor-beta1 resulted in far fewer or null CD1a+ cells, respectively. Macrophage colony-stimulating factor increased the number of CD1a+ cells in a concentration-dependent fashion. These macrophage colony-stimulating factor-induced Langerhans cells were different from granulocyte-macrophage colony-stimulating factor-induced Langerhans cells in their decreased expression of CD11c and their immature phenotype. The decreased expression of CD11c, however, was recovered by culturing them with granulocyte-macrophage colony-stimulating factor, while they acquired a mature phenotype qby granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, interleukin-1alpha, or lipo-polysaccharide. Macrophage colony-stimulating factor-induced Langerhans cells could stimulate allogeneic T cells. Interestingly, we could keep the growth and immature phenotypes of macrophage colony-stimulating factor-induced Langerhans cells for at least 28 d of culture. These studies demonstrated that macrophage colony-stimulating factor in cooperation with transforming growth factor-beta1 could induce Langerhans cell development from hematopoietic progenitor cells in vitro without granulocyte-macrophage colony-stimulating factor, which suggests the possibility that macrophage colony-stimulating factor plays a part in the Langerhans cell development in vivo. In addition, the culture using macrophage colony-stimulating factor presents a novel culture system to enable a large-scale and long-term culture of immature Langerhans cells.

Although melanoma mostly affects the skin, it is notorious for its propensity to easily develop metastasis. Metastatic melanoma is highly resistant to a variety of therapies. We examined the anti-metastatic potential of peritumoral monotherapy against murine cutaneous B16F10 melanoma with synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs. We demonstrated that repeated peritumoral injections of CpG ODN significantly reduced skin tumor size. Peritumoral CpG ODN-treatment of skin tumors prevented the development of pulmonary B16F10 colonies. Adoptive transfer of splenocytes obtained from CpG ODN-treated mice markedly reduced the number of previously established pulmonary colonies in recipient naïve mice. T-lymphocyte depletion studies indicated that the anti-metastatic effect was dependent on both CD4+ and CD8+ T cells. These results suggest that CpG ODN are promising as a preventive and therapeutic anti-metastatic measure against melanoma.

Concentrations of C3a and C4a anaphylatoxins in suction blister fluids were determined by radioimmunoassay in patients with psoriasis and normal controls. Comparison of anaphylatoxin levels between serum samples and blister contents in the same subjects revealed that the levels of both C3a and C4a anaphylatoxins were significantly higher in the former than the latter even in those raised on normal skin, suggesting that the classic complement pathway is activated during suction procedure. Therefore we cannot regard suction blister fluid to be simply representative of undisturbed interstitial tissue fluid as far as the complement system is concerned. There was no difference in anaphylatoxin levels between those from uninvolved skin of psoriatic patients and those from normal controls. However, significantly high anaphylatoxin levels were noted in fluids of suction blisters raised on lesional skin as compared with those produced on uninvolved skin in psoriatic patients.

We found an increased amount of immunoreactive tyrosinase in human melanocytes after 6-d culturing with vitamin D3 (cholecalciferol). Most of these melanocytes became more dendritic and swollen in a fashion similar to that noted in the skin after ultraviolet irradiation. However, 7-dehydrocholesterol (pro-vitamin D3) or 1 alpha,25-dihydroxy-vitamin D3 (activated vitamin D3) were found to have little effect on the same system. Because vitamin D3 is known to be photochemically converted from pro-vitamin D3 in the skin by ultraviolet irradiation, the mechanism of human skin pigmentation after ultraviolet irradiation, thus far unknown, may be at least partly explained by this stimulating effect of vitamin D3 on melanocytes.

Human sera were examined for evidence of complement activation by Fonsecaea pedrosoi. The serum that had been incubated with F. pedrosoi showed immunoelectrophoretic C3 conversion and generation of C5a, as measured by radioimmunoassay. C3 conversion and C5a generation did not occur in serum chelated with EDTA, or in serum heated to 56 degrees C for 30 min. Serum depleted of the heat (50 degrees C X 30 min)-labile factor B was also deficient in C3 conversion and C5a generation. Complement activation was not affected when the classical pathway was blocked by Mg++-EGTA. These studies indicate that F. pedrosoi can activate the complement system via the alternative pathway, with the resultant development of C5a.

Normal human epidermal melanocytes became swollen and more dendritic when they were cultured for 6 days with prostaglandin (PG) E2, but not with PGE1, although the amount of immunoreactive tyrosinase in the melanocytes did not appear to be increased markedly after these treatments. From these data we suggest that PGE2 may be one of the factors responsible for the induction of postinflammatory hyperpigmentation of the skin.

We measured in vitro the hygroscopicity and bound (non-freezing) water of various samples of pathologic horny layer obtained from the lesions of senile xerotic skin and psoriasis vulgaris and the normal horny layer from glabrous skin and plantar horny layer. The amount of water taken up by pathologic stratum corneum was much smaller than that by normal horny layer in an environment at a high relative humidity (RH). Tightly bound primary water to stratum corneum measured by Karl Fischer's method was about 5 mg/100 mg of dry stratum corneum in all the samples studied, while less tightly bound secondary water was much smaller in amount in pathologic stratum corneum than in the controls, i.e., 31.7 mg/100 mg dry scale from senile xerosis and 27.2 mg/100 mg dry psoriatic scale as compared with 38.2 mg/100 mg dry normal stratum corneum from glabrous skin and 37.3 mg/100 mg dry normal plantar stratum corneum. We believe that the low hygroscopicity of the pathologic stratum corneum is due to this smaller capacity for secondary bound water, which is responsible for the development of a dry scaly appearance even at high RH.

To evaluate the role of the complement system in inflammation induced by chlorpromazine (CPZ) and ultraviolet-A (UVA) irradiation, the phototoxic response in guinea pigs decomplemented by cobra venom factor was compared with that in saline-treated animals. Phototoxic lesions were induced in animals by intradermal injections of CPZ solution, followed by UVA irradiation. Clinically, the normal animals developed erythema and induration which showed a maximal response at 10 h with a mean value of 1.6 on a scale of 0 to 3+. The complement-depleted animals showed a weaker clinical response than the normal animals 6-24 h postirradiation (p less than 0.05). These clinical changes were associated with increased vascular permeability, as demonstrated by extravasation of i.v. injected Evans blue in saline-treated animals. In vitro UVA irradiation of serum containing CPZ resulted in a dose-dependent diminution of total complement activity. Such irradiated serum showed immunoelectrophoretic C3 conversion. These results suggest that the complement system is involved in the development of CPZ-induced phototoxic lesions.