Keratinocytes constitutively express the CD95 ligand molecule on the plasma membrane: an in situ immunoelectron microscopy study on ultracryosections of normal human skin.

BACKGROUND: Tissue homeostasis is mainly preserved by cytolytic functions. Cytolytic cells, when expressing the CD95 ligand (Fas-L) molecule on the cell membrane, are able to kill CD95 (Fas)-expressing target cells. Although cultured epidermal keratinocytes (KC) have been shown to express Fas-L, and normal skin has been shown to bear Fas-L mRNA, efforts so far to find possible constitutive Fas-L expression on the cell membrane by resting KC in normal human epidermis (i.e. in a functionally active location) have been inconclusive. OBJECTIVES: The aim of the present study was therefore to show the constitutive expression of Fas-L on the plasma membrane of KC. METHODS: Gold immunoelectronmicroscopy, a highly specific and sensitive immunodetection system, was performed in situ on skin sections obtained by ultracryomicrotomy, without previous embedding (i.e. in conditions strictly similar to the in vivo situation). RESULTS: Relatively few (51.55 +/- 28.61), 10-nm colloidal gold particles were observed at the cell surface of KC in the basal layer of the epidermis and an even smaller (P < 0.005) number of gold granules was detected in the KC of the spinous layer. CONCLUSIONS: Although scanty, the constitutive Fas-L expressed on the surface of KC can bind Fas expressed by possible occasional inflammatory cells entering the epidermis, and kill them, so preventing inflammation. Fas-L-expressing KC could moreover induce apoptosis of epidermal cells bearing viral or neoplastic antigens. Thus, the expression of Fas-L by KC may contribute to the preservation of epidermal homeostasis in vivo.

Ultraviolet-B radiation induces modulation of antigen presentation of herpes simplex virus by human epidermal cells.

Although ultraviolet (UV) B radiation is known to be immunosuppressive, there is little information regarding a relevant immunological endpoint to assess human subjects in vivo. Therefore, we have examined the effect of in vivo UV radiation on the ability of human epidermal cells (EC) to present herpes simplex virus (HSV) antigens to memory T cells. Human volunteers, who were seropositive for HSV, were exposed to one minimal erythemal dose (MED) for four consecutive days. EC, prepared from suction blister roofs, were co-cultured with autologous T cells in the presence of HSV. HSV antigen presentation by UV-exposed EC was increased compared with control, nonexposed EC. This up-regulation correlated with an influx of macrophages into the epidermis, which are considered to be associated with UV-induced tolerance. Altering the UV protocol to a sub-erythemal UV dose for four consecutive days or to a single high dose of 2 MED, resulted in suppressed HSV antigen presentation, without the influx of the UV-macrophages. One of the goals of the present study was to eventually use this HSV system to investigate sunscreen immunoprotection. A pilot study with a TiO2-containing sunscreen suggested that the endpoint for UV-induced immunosuppression presented here is promising to be used for human in vivo sunscreen immunoprotection studies.

CD1a molecules traffic through the early recycling endosomal pathway in human Langerhans cells.

In this work, we studied the localization and traffic of CD1a molecules in human epidermal Langerhans cells and the ability of these cells to stimulate CD1a-restricted T cell clones. We found that CD1a was spontaneously internalized into freshly isolated Langerhans cells, where it was rapidly distributed to the early/sorting endosomes and then to the early/recycling endosomes. In the latter compartments, CD1a colocalized with Rab11, a small GTPase known to be involved in the recycling of transmembrane proteins from early endosomes to the cell surface. In the steady state, intracellular CD1a was mainly located in Rab11+ recycling endosomal compartments. When endocytosis was blocked, intracellular CD1a moved rapidly from the early/recycling endosomes to the cell surface where it accumulated. The resultant increase in the cell surface expression of CD1a enhanced the capacity of Langerhans cells to stimulate a CD1a-restricted T cell clone. These findings are consistent with a dynamic exchange of CD1a between recycling compartments and the plasma membrane and suggest that the antigen-presenting function of CD1a depends on its traffic through the early/recycling endosomal pathway.

Major histocompatibility complex class II- fetal skin dendritic cells are potent accessory cells of polyclonal T-cell responses.

Whereas dendritic cells (DC) and Langerhans cells (LC) isolated from organs of adult individuals express surface major histocompatibility complex (MHC) class II antigens, DC lines generated from fetal murine skin, while capable of activating naive, allogeneic CD8+ T cells in a MHC class I-restricted fashion, do not exhibit anti-MHC class II surface reactivity and fail to stimulate the proliferation of naive, allogeneic CD4+ T cells. To test whether the CD45+ MHC class I+ CD80+ DC line 80/1 expresses incompetent, or fails to transcribe, MHC class II molecules, we performed biochemical and molecular studies using Western blot and polymerase chain reaction analysis. We found that 80/1 DC express MHC class II molecules neither at the protein nor at the transcriptional level. Ultrastructural examination of these cells revealed the presence of a LC-like morphology with indented nuclei, active cytoplasm, intermediate filaments and dendritic processes. In contrast to adult LC, no LC-specific cytoplasmic organelles (Birbeck granules) were present. Functionally, 80/1 DC in the presence, but not in the absence, of concanavalin A and anti-T-cell receptor monoclonal antibodies stimulated a vigorous proliferative response of naive CD4+ and CD8+ T cells. Furthermore, we found that the anti-CD3-induced stimulation of naive CD4+ and CD8+ T cells was critically dependent on the expression of FcgammaR on 80/1 DC and that the requirement for co-stimulation depends on the intensity of T-cell receptor signalling.

Cytoplasmic dynein colocalizes with melanosomes in normal human melanocytes.

BACKGROUND: Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. Several studies provide arguments supporting the hypothesis that actin-based and microtubule-based motor proteins co-operate in melanosome transport towards the dendrite tips. Melanosomes can move bidirectionally along microtubules in vitro, and in murine melanocytes, they move towards the cell periphery and back again. Microtubules have a fast-growing plus end and a slow-growing minus end. Microtubule-associated motor proteins move unidirectionally either towards the plus or towards the minus end. However, it is not known which motor protein is responsible for minus end-directed movement of melanosomes. OBJECTIVES: We aimed to investigate the in vitro expression of the minus end-directed motor protein cytoplasmic dynein in normal human epidermal melanocytes, keratinocytes and dermal fibroblasts. METHODS: Reverse transcription-polymerase chain reaction and Northern blot analysis were used. In addition, an attempt to obtain insight into the subcellular localization of cytoplasmic dynein, immunofluorescence studies and immunogold electron microscopic studies were performed. RESULTS: The three different forms of cytoplasmic dynein heavy chain were expressed in all studied skin cells. Immunofluorescence staining showed similar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tips. Double labelling with a melanosome marker showed apparent co-localization of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear area and dendrite tips. For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A close association of dynein intermediate chain 74 and alpha-tubulin with the melanosome surface was detected using immunogold electron microscopy. CONCLUSIONS: The colocalization of different subunits of the cytoplasmic dynein complex with melanosomes is consistent with the hypothesis that this motor protein supports minus end-directed melanosome movement along microtubules.

Broad-spectrum sunscreens offer protection against urocanic acid photoisomerization by artificial ultraviolet radiation in human skin.

Cis-urocanic acid (UCA) has been indicated as an important mediator of ultraviolet (UV)-induced immunosuppression. In this study we describe a rapid, noninvasive method for the determination of the protective capacity of various sunscreens against the UV-induced isomerization of trans-UCA into its cis form. For this purpose we applied sunscreens prior to in vivo exposure of human volunteers with single or repeated broadband UVB irradiations of 100 mJ per cm2. We found significant but different levels of protection against UCA photoisomerization by all sunscreens that correlated with the sun protection factor. A comparison of various sunscreens with a sun protection factor of 10, showed that the best protection was offered by the sunscreens (containing organic UV filters or TiO2) with broad absorption spectra. The ability to inhibit cis-UCA formation was not influenced by the penetration characteristics of sunscreens, as determined by application of the sunscreen on quartz glass that was placed on the skin, preventing penetration of sunscreen in the skin. In addition ex vivo UV exposure of human skin was employed to permit other tests of immunomodulation, in this case the mixed epidermal cell lymphocyte reaction. The advantage of this ex vivo method is that there is no need to take biopsies from volunteers. Ex vivo irradiation of human skin with a single dose of 200 mJ per cm2 resulted in similar protection by the sunscreens against cis-UCA formation as in the in vivo system. Furthermore, the mixed epidermal cell lymphocyte reaction data correlated with the cis-UCA findings. We conclude that UCA isomerization is an excellent method to determine sunscreen efficacy and that broad-spectrum sunscreens offer good immunoprotection.

Infection of a canine macrophage cell line with leishmania infantum: determination of nitric oxide production and anti-leishmanial activity.

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-gamma, TNF-alpha and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N(G)-nitroargininemethyl ester (L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.

Kinesin and kinectin can associate with the melanosomal surface and form a link with microtubules in normal human melanocytes.

Microtubuli play an important role in the organization of organelles and membrane traffic. They are present in melanocytic dendrites through which melanosomes are transported towards keratinocytes. Besides the actin-based motility systems, microtubuli-associated motor proteins also play a critical role in melanosome movement, as has recently been confirmed in mouse melanocytes. We investigated the in vitro expression of two forms of human conventional kinesin and its receptor kinectin in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription polymerase chain reaction and northern blot analysis. In an attempt to gain insight into the subcellular distribution of kinesin and kinectin in melanocytes, double immunofluorescent staining and immunogold electron microscopy were performed. In all studied skin cells ubiquitous and neuronal kinesin are expressed, as well as the kinectin receptor. Immunofluorescent staining shows distinct but partially overlapping distributions for kinesin heavy chain and melanosomes, suggesting that kinesin is associated with some but not all of the melanosomes. Similar observations for kinectin indicate that this receptor can colocalize with melanosomes, which was confirmed by immunoelectron microscopy. The latter technique allowed us to demonstrate a close association between kinesin heavy chain, microtubuli, and melanosomes. The combined data from reverse transcription polymerase chain reaction, northern blot analysis, double immunofluorescent staining, and immunogold electron microscopy suggest that kinesins and kinectin have an important role in microtubuli-based melanosome transport in human melanocytes.

Melanosome capping of keratinocytes in pigmented reconstructed epidermis--effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis.

Reconstructed pigmented epidermis was established by co-seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air-liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3-isobutyl-1-methyl-xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.