Location of HLA-A,B,C antigens in dendritic cells of normal human skin: an immunoelectron microscopic study

The distribution of the major histocompatibility antigens HLA-A,B,C, (HLA) on dendritic cells of normal human skin was studied by immunoelectron microscopy and a 4-step immunoperoxidase technique utilizing monoclonal antibodies. Light microscopy revealed peripheral staining for HLA of epidermal and pilar infundibular keratinocytes. In the epidermis, the staining was present from the basal layer to the upper stratum spinosum. In the follicles below the level of the infundibulum, HLA was detected only on rare intraepithelial dendritic cells. These dendritic cells could not be identified in the epidermis due to the HLA staining of the surrounding keratinocytes. Similar cells stained diffusely with anti-T6 antibody; the keratinocytes did not. Immunoelectron microscopy demonstrated: (1) the presence of HLA staining of keratinocyte membranes from the stratum basalis to the level of the upper stratum spinosum and in the pilar infundibulum, (2) the possible absence of HLA on melanocytes, (3) the presence of focal HLA staining of the membranes of epidermal and follicular dendritic cells that contained Birbeck granules and were, therefore, Langerhans cells, (4) dendritic mononuclear cells within the follicular epithelium, which although devoid of Birbeck granules, exhibited similar reactivity with anti-HLA antibody. These findings suggest that HLA antigens are present on the membranes of Langerhans cells, but are not demonstrable on melanocytes in normal human skin.     

Ultrastructural changes in epidermal Langerhans cells and melanocytes in response to ultraviolet irradiation, in Australians of Aboriginal and Celtic descent

The effects of exposure to small doses of artificial ultraviolet radiation (UVR) on the ultrastructure of epidermal Langerhans cells (LC) and melanocytes were studied in two groups of Australian subjects, one of Aboriginal and the other of Celtic descent. UV exposure induced an apparent depletion of LC in the epidermis of both groups. However, LC depletion in the Aboriginal subjects was associated with apoptosis, whereas organelle and membrane disruption in the LC of Celtic subjects suggested a reduction by direct cellular damage. LC in Aboriginal epidermis tended to become relocated at more superficial levels following UV exposure, and their Birbeck granules became more numerous. LC in Celtic epidermis appeared to become relocated in a basal location and contained fewer Birbeck granules. The central lamina of the Birbeck granules in Aboriginal LC, which was more electron-dense than that in Celtic subjects prior to UV treatment, was temporarily lost following treatment, while the ultrastructure of Birbeck granules in Celtic LC was unchanged. LC and 'indeterminate cells' in intimate association with lymphocyte-like cells occurred in the basal layer of Celtic epidermis 5 days after exposure. These complexes were not observed in Aboriginal epidermis although isolated lymphocyte-like cells were observed in the same location. Melanocytes in Aboriginal epidermis contained greater numbers of melanosomes than those in Celtic epidermis throughout the experiment. Inactive epidermal melanocytes in Celtic subjects initially responded to UV exposure with a slight increase in melanosome content followed by a substantial further increase, whereas active melanocytes in the Aboriginal subjects showed the opposite response. The implications of the different responses of LC and melanocytes in the two groups, in relation to immunological function of the epidermis and the marked racial difference in the incidence of skin cancer, are discussed. Cancer of the skin, particularly basal and squamous cell carcinoma, occurs primarily in people with fair skin who burn easily following exposure to ultraviolet radiation (UVR). In contrast, the incidence of skin cancer in inherently dark-skinned people is low. Melanin is synthesized by melanocytes in response to UVR and is thought to protect epidermal cells against damage to their genetic material by absorbing UVR and thereby reducing its penetration into the skin. Thus darkly pigmented skin is more resistant to the effects of UVR.(ABSTRACT TRUNCATED AT 400 WORDS)     

Circular and branched Birbeck granules and cytomembrane blebbing in Langerhans' cells after dithranol irritation

Mild dithranol irritation of healthy human skin has a stronger effect on the fine structure of Langerhans' cells (LC) than on that of other epidermal cells, causing mitochondrial enlargement and disruption of the cristae of LCs. With a stronger dithranol irritation, LCs were even more affected resulting in circular and branched Birbeck granules (BG) and blebbing of the LC cytomembrane. More often than is normal, BGs were contiguous with the LC cytomembrane. Electron microscopic observations indicated that blebbing and the abnormal BG formation were associated phenomena, in accordance with the hypothesis that BGs are endocytotic organelles formed from the cytomembrane.     

Alteration of the expression of Bcl-2, Bcl-x,Bax, Fas,and Fas ligand in the involved skin of psoriasis vulgaris following topical anthralin therapy

Anthralin (dithranol) is frequently used for the treatment of psoriasis. However, the mode of action of anthralin has not been completely elucidated as yet. Recent findings suggest that psoriatic keratinocytes are resistant to the apoptotic process. In this study, we examined the immunohistochemical expression of apoptosis-regulated protein in the involved psoriatic skin following topical anthralin therapy. Biopsy specimens were obtained from back skins treated with topical anthralin or white petrolatum (control) in 4 patients with psoriasis vulgaris. Immunohistochemical examination revealed that psoriatic keratinocytes expressed high levels of Bcl-x, which was significantly reduced after anthralin treatment. Bax was not detected in the epidermal keratinocytes in the petrolatum-treated skin, while it was present in the upper keratinocytes after anthralin therapy. Bcl-2 was detected only in basal layers of psoriatic epidermis following both petrolatum and anthralin application. Psoriatic keratinocytes expressed higher levels of Fas in the lower epidermis, while only weak expression was detected in anthralin-treated plaques. On the other hand, hyperproliferative keratinocytes strongly expressed Fas ligand (FasL) on their plasma membranes as well as infiltrating lymphocytes in the upper dermis. Furthermore, anthralin-treated psoriatic epidermis did not express FasL. In normal skin, keratinocytes expressed low to absent levels of Bcl-x and Bax, while Bcl-2 was detected only in melanocytes in basal layers. Neither Fas nor FasL were detected in the epidermis of normal skin. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining revealed positive labeling on the majority of psoriatic keratinocytes through the epidermis in petrolatum-treated skin, whereas anthralin treatment markedly reduced TUNEL-positive keratinocytes. These in vivo results may reflect improvement of the psoriatic skin following effective anthralin therapy.     

Lymphocytes and Langerhans cells in patch tests

The distribution of immunocompetent cells was analysed in allergic (nickel) and irritant (dithranol) patch tests using conventional transmission electron microscopy and labelling with monoclonal antibodies in an avidin-biotin immunoperoxidase study. The biopsies were taken 24 or 48 h after the allergen/irritant application. In allergic and irritant reactions, most inflammatory cells were OKT11 positive (pan T lymphocytes). The majority of these cells were also OKT4 positive (helper/inducer T lymphocytes), while the minority were OKT8 positive (suppressor/cytotoxic T lymphocytes). NK9 positive cells (natural killer cells) were observed in small numbers. The number of dendritic OKT6 and OKIal positive cells (Langerhans cells) in the epidermis was unaffected in allergic reactions. In irritant reactions, a normal number of OKT6 positive Langerhans cells was observed, while the number of OKIal positive cells had increased in the epidermis. Dithranol caused prominent fine structural changes in the mitochondria of the Langerhans cells, while the keratinocytes appeared largely unaffected. The present study indicates that allergic and irritant patch tests cannot be differentiated reliably using current immunohistopathological or electron microscopic techniques, in spite of the small differences observed.     

Electron microscopic observations of dyskeratosis, apoptosis, colloid bodies and fibrillar degeneration after skin irritation with dithranol

Dying cells undergo coagulative necrosis or apoptosis. In the skin, apoptosis is known to occur in graft-versus-host reactions, in lichen planus, during regression of plane warts and neoplasms, and after physical injury caused by ultraviolet light resulting in sunburn cells. The present study shows that primary skin irritation also causes apoptosis. Mild, or moderate-to-considerable, dithranol irritation of healthy uninvolved human skin caused focally coagulative necrosis of keratinocytes and also apoptosis of scattered keratinocytes, i.e. condensation of chromatin and cytosol, clumping of tonofilaments and budding of membrane-bound cell fragments. These apoptotic cell fragments were engulfed in the epidermis by macrophages. Colloid bodies were detected in the upper dermis and apparently represented nonphagocytosed apoptotic cell fragments that had dropped down from the epidermis. Dithranol also caused fibrillar degeneration of melanocytes and in some cases of Langerhans' cells, indicating that colloid bodies in the upper dermis could partly derive from these cell types. The significance of apoptosis in irritant contact dermatitis could be to maintain homeostasis of epidermis and counteract the hyperplastic response caused by irritant stimuli. Another possibility is that apoptosis was the response to an injury less severe than that causing necrosis.     

Distributional changes of Langerhans cells in human skin during irritant contact dermatitis

We used light and electron microscopic immunocytochemistry to study distributional changes in the human Langerhans cell (LC) system during the first 14 days of a mild irritancy caused by sodium lauryl sulphate (SLS). A marked initial decrease in epidermal LC was noted possibly resulting from migration from the epidermis to the dermis and from irreversible cell damage. Several studies have previously found an unchanged number of LC in SLS-induced contact irritant dermatitis, but these studies may not have taken into account the fact that SLS is effectively absorbed from the test chamber. Unless certain precautions are taken the SLS concentration rapidly falls to topical levels that have no effect on the LC system. Simultaneously with the decrease in the epidermis we observed an increase in dermal CD1a+ cells, confirming an often reported finding. There is, however, no consensus as to the identity of these cells, and several authors have reported that such cells lack LC granules and thus these cells have often been classed as indeterminate cells. We found that, during irritant contact dermatitis, provided an adequate number of sections were scrutinized in the electron microscope, all dermal CD1+ cells contained Birbeck granules.     

Immunohistochemical localization of lipocortins in normal and psoriatic human skin

The distribution of lipocortin I, a steroid-induced inhibitory protein of phospholipase A₂, was examined in normal and psoriatic human skin. Using immunoblotting analysis with specific antibody against human lipocortin I purified from human placenta, lipocortin I was detected as a 37 kDa protein in cultured epidermal cells, whole skin and epidermis. In the dermis and stratum corneum, lipocortin I was only weakly detectable by Western blotting. In contrast to normal skin, much less lipocortin I was detected by Western blotting analysis in psoriatic skin. Using immunoperoxidase immunohistochemical analysis, lipocortin I was demonstrated in the cytoplasm of keratinocytes in the upper and middle layers of the epidermis and in some infiltrating cells in the dermis in normal skin. In involved psoriatic skin, by contrast, lipocortin I was almost undetectable in the epidermis, although it was demonstrated in some infiltrating cells in the dermis. No immunostaining of lipocortin I was observed in the stratum corneum of normal or psoriatic skin. These results, together with the finding that phospholipase A₂ activity is higher in psoriatic epidermis than in normal epidermis, suggest that lipocortin I plays an important role in the regulation of differentiation and proliferation of epidermal keratinocytes.     

Abnormal distribution of epidermal protein antigens in psoriatic epidermis.

The immunohistochemical distribution of the epidermal proteins filaggrin, involucrin, and cytokeratins is characteristic in normal epidermis. This distribution may change as a result of malignant transformation or abnormal differentiation. The present study was conducted to determine the patterns of reactivity of psoriatic epidermis to antibodies against various epidermal proteins and to clarify abnormal differentiation or maturation of the keratinocytes in psoriatic epidermis. Anti-human filaggrin, anti-human involucrin, and twelve kinds of anti-cytokeratin antibodies were used in this study. Cryostat or paraffin-embedded sections were stained with these antibodies by the avidin-biotin peroxidase technique. The epidermis of the noninvolved skin of patients with psoriasis vulgaris showed the distribution seen in normal skin. However, involved psoriatic skin revealed little or no reaction in the stratum corneum or in the granular layer with the anti-filaggrin antibody. Cells positively staining with anti-involucrin antibody paradoxically appeared in the lower cell layers of involved psoriatic epidermis. An anti-keratin antibody, AE1, stained suprabasal cells in involved psoriatic epidermis, although this antibody selectively stained epidermal basal cells in normal skin. The other anti-keratin antibodies, especially KL1, PKK1, and a polyclonal anti-keratin antibody, were less reactive with involved psoriatic skin than with normal skin. These observations suggest that the maturation pathway of keratinocytes in active psoriatic lesions differs qualitatively from that in normal epidermis.     

Migration of Langerhans cells into the epidermis of human skin grafted into nude mice.

In a previous study, it was demonstrated that human Langerhans cells (LC) are preserved in human skin grafted onto a nude mouse. Moreover, although it was observed that mouse LC of the host invade skin grafts from allogeneic mouse or rat, they do not penetrate in human skin grafts. In most of the human skin equivalent systems produced in vitro, LC appear to be lost. The present study was designed to investigate whether the mouse LC will repopulate a human skin equivalent. For this purpose, two different systems of skin equivalent have been grafted into the nude mouse. They were composed of human keratinocytes deposited on dead human dermis, or on lattice composed of human fibroblasts embedded in type I collagen. At different times after grafting, the presence of LC in the transplants was assayed either by indirect immunofluorescence or by electron microscopy. Indirect immunofluorescence was performed on frozen sections or on epidermal sheets with anti-Ia, anti-HLA-DR, or OKT6 antibodies. It was observed that, at 2 months after grafting, Ia(+) HLA-DR(-) OKT6(-) cells are present in grafted human epidermis. Moreover, LC with typical Birbeck granules are also detected by electron microscopy. It could be concluded, from this study, that mouse LC can repopulate human epidermis devoid of human LC.     

银屑病患者表皮及真皮中Bcl-X、Bcl-2、Bax表达的免疫组化研究

目的 探讨银屑病患者细胞增殖与凋亡的调节。方法 用免疫组化ＳＰ法检测银屑病皮损及非皮损中Ｂｃｌ－Ｘ、Ｂｃｌ－２、Ｂａｘ的表达情况。结果 银屑病皮损中,Ｂｃｌ－Ｘ在表皮各层、真皮炎症细胞及血管内皮的表达均较正常明显增高;Ｂａｘ在颗粒层与棘层的表达轻度增高。上述异常在非皮损中已经部分存在,且隍未完全恢复正常。而Ｂｃｌ－２表达与正常相似,局限于基底层黑素细胞,角质形成细胞未是性。结论 银屑病中角质形成细     

Expression of interferon-gamma receptors in normal and psoriatic skin.

Psoriatic keratinocytes have a reduced antiproliferative response to interferon (IFN)-gamma, and HLA-DR expression is usually not observed on keratinocytes in psoriatic plaques despite the presence of activated T cells. We have therefore compared the expression of IFN-gamma receptors in psoriatic skin with that of normal human skin. Using mouse monoclonal antibodies and immunoperoxidase staining on cryostat cut sections, we detected IFN-gamma receptors on keratinocytes throughout the epidermal layers except stratum corneum in normal skin (n = 11). Biopsy specimens from involved psoriatic skin (n = 17) consistently showed a staining pattern that differed from that of normal skin in that only the lower part of epidermis reacted with the antibodies to IFN-gamma receptors, whereas the upper layers showed no or minimal staining. Expression of IFN-gamma receptors in uninvolved psoriatic skin (n = 16) did not differ from that of healthy controls. Forty-five percent of the biopsies from lesional psoriatic skin displayed ICAM-1 positive keratinocytes, and only two specimens had a limited expression of HLA-DR reactive keratinocytes. The decreased binding of antibodies against the IFN-gamma receptors in the upper part of psoriatic epidermis might be secondary to abnormal maturation of psoriatic keratinocytes or a primary defect involving abnormal modulation of IFN-gamma receptors.     

C-5 propyne-modified oligonucleotides penetrate the epidermis in psoriatic and not normal human skin after topical application

We have previously shown that antisense oligonucleotides effectively reduced insulin-like growth factor I receptor expression in human psoriatic skin grafted on to nude mice when injected intradermally. We therefore investigated the penetration of C-5 propyne modified antisense oligonucleotides into human normal and psoriatic skin after topical administration. Oligonucleotide (37.5 microg; 250 microM) was applied in aqueous solution or 5% methylcellulose gel for 24 h, prior to live confocal microscopy and fluorescence microscopy of fixed sections. We found that oligonucleotide could penetrate through the stratum corneum of psoriatic but not normal human skin over large regions of the epidermis. The oligonucleotide was localized to the nucleus of large parakeratotic cells in the psoriatic skin as well as smaller basal and suprabasal keratinocytes. In normal human skin, oligonucleotide was confined to the stratum corneum, with little or no oligonucleotide apparent in the viable epidermis. Electrophoresis of oligonucleotide recovered from treated psoriatic and normal skin revealed that the oligonucleotide remained intact over the 24 h period. In summary, we found that C-5 propyne modified antisense oligonucleotides could reach the target cells (in this case basal keratinocytes) after topical administration to psoriatic but not normal skin.     

Reappraisal of in situ immunophenotypic analysis of psoriasis skin: interaction of activated HLA-DR+ immunocompetent cells and endothelial cells is a major feature of psoriatic lesions

Psoriasis is an inflammatory skin disease of unknown aetiology. Many observations indicate that T cells play an important role in the pathogenesis of the disease. Upregulation of MHC class-II molecules on immunocompetent cells, endothelial cells and keratinocytes on lesional psoriatic skin has been regarded as a hallmark of the disease. However, there is some controversy in the literature regarding the cell types expressing class-II molecules and there is limited information about the presence of immune cells other than T cells and antigen presenting cells in the cellular infiltrates of psoriatic skin. We therefore reinvestigated the subject using immunocytochemical single and multiple staining techniques. In agreement with earlier reports, our studies showed that the cellular infiltrates in lesional skin consist largely of HLA-DR⁺/IL-2R⁺ T cells, HLA-DR⁺/CD1a⁺ Langerhans cells, and HLA-DR⁺/CD68⁺ macrophages. We found increased HLA-DR expression mostly on immunocompetent cells and endothelial cells, but no prominent HLA-DR expression on keratinocytes in lesional psoriatic skin. Upregulation of HLA-DR on endothelial cells and in mononuclear infiltrates was also evident in the non-lesional skin of psoriatic patients as compared with normal controls. B cells and natural killer cells were also found in the cellular infiltrates in lesional psoriatic skin. In spite of the presence of a large amount of activated T cells in the epidermis, we found that HLA-DR expression on keratinocytes was not a major feature of psoriatic skin.     

Azelaic acid vs. placebo: effects on normal human keratinocytes and melanocytes. Electron microscopic evaluation after long-term application in vivo

The effects of topically applied 20% azelaic acid (AA) on normal human epidermis were investigated vs. placebo in a double blind study by electron microscopy in 15 volunteers. After 3 months of local application twice daily, the pattern of epidermal keratinization was found altered in skin treated with AA. In particular, the number and thickness of tonofilament bundles and the number of keratohyaline granules seemed decreased; the remaining granules were smaller, occasionally showing irregular electron densities. The perinuclear endoplasmic reticulum and the cytoplasmic cisternae were enlarged and swollen mitochondria were regularly observed in most malpighian keratinocytes. Thorough quantitative evaluation of the number and distribution of melanocytes by a MOP-videoplan computer system showed no differences between verum and placebo sites, although, the mean number of melanocytes had increased in both, as compared to the untreated controls taken before onset of therapy. No significant qualitative changes of the normal melanocytes were found. These findings indicate that azelaic acid may influence the differentiation of normal human keratinocytes by reducing the synthesis of keratin precursors and may, therefore, act as a mild antikeratinizing agent, whereas, the pigmentary system in normal human epidermis does not show any specific change after 3 months of treatment with AA.     

Alterations of Clinically Normal Skin in Early Eruptive Guttate Psoriasis

In clinically normal skin of early eruptive guttate psoriasis, in patients with psoriasis for the first time, the epidermal changes were restricted to small tissue areas showing a slight hyperplasia and a condensed stratum corneum but no parakeratosis. The center (zone 1) of the small tissue areas was characterized by (1) exoserosis with spongiotic dilatation of the intercellular space in the non-cornified epidermis, (2) derangement of keratinocytes with respect to shape, orientation, and number of desmosomes and fibrils, (3) dyskeratotic keratinocytes of two types, A and B, (4) agranulosis restricted to the dyskeratotic keratinocytes of type A, (5) exocytosis of mononuclear cells and Langerhans' cells into the entire non-cornified epidermis, (6) large gaps in the basement membrane between the dermis and epidermis. No polymorphonuclear leukocytes were seen. The periphery (zones 2) of the small tissue areas showed slight changes. The alterations of the upper dermis were slight: besides a very mild general inflammatory reaction with capillary dilatation and perivascular cell infiltrate there was also a focal accumulation of inflammatory cells immediately beneath the epidermis, with cells seemingly infiltrating the stratum basale. It is postulated that the epidermal and dermal changes as shown here represent primary psoriatic changes.     

Embryology of the epidermis: ultrastructural aspects. III. Maturation and primary appearance of dendritic cells in the mouse with mammalian comparisons.

The epidermis of mice ranging in age from prenatal day 17 through postnatal day 4 and, in addition, postnatal day 18, was studied with the electron microscope. In the 17 day fetus, the periderm may or may not be present and a stratum corneum is developing in the latter case. The cells of the strata spinosum and granulosum contain much glycogen and many keratinosomes and homogeneous keratohyalin granules which appear to line up in a row near the distal cell membranes, fuse and form keratinized cells which contain material similar in density to that of the individual keratohyalin granules. In the 18 to 20 day fetuses, no periderm is present. The stratum corneum becomes well-developed. The quantity of glycogen decreases but the number of keratinosomes and keratohyalin granules increases. Some cells in the basal region contain necrotic phagocytosed cells. Few changes occur in the epidermis of the neonate, although the stratum corneum increases in thickness. In the early postnatal period, the mouse epidermis is mature in appearance and resembles that of man. By postnatal day 18, the whole epidermis is much thinner, although all classical strata can usually be identified. The primary appearance and subsequent maturation of epidermal dendritic cells was also studied in the mouse during the embryonic, fetal, early postnatal and 18 day postnatal periods. Melanocytes which lacked cell processes were first identified in the 15 day fetus. Melanocyte cell processes and an increasing number of melanosomes, as well as melanocytes, are present between prenatal day 18 and postnatal day 3. On postnatal day 4, fewer melanocytes are present and they contain vacuoles. Phagocytosed melanocytes are also present within basal keratinocytes. Undifferentiated dendritic cells or indeterminate cells are observed beginning on prenatal day 16. These cells do not appear to increase significantly in number subsequently. On postnatal day 18, Langerhans cells and indeterminate cells are present but no melanocytes can be identified. No Merkel cells were observed. Mitotic dendritic cells or dendritic cells traversing the basal lamina were not observed.     

Ultrastructural effects of UVB radiation and subsequent retinoic acid treatment on the skin of hairless mice

The ultrastructure of hairless mouse skin exposed to UVB radiation and followed by retinoic acid treatment was studied to identify alterations induced in both epidermis and dermis. Female mice were irradiated 3 times weekly for 5-6 months; a group of these mice was then treated topically 3 times weekly for 10 weeks with either 25 micrograms all-trans-retinoic acid dissolved in acetone or with acetone alone. Age-matched, unexposed, untreated mice served as controls. Cutaneous changes induced by UVB radiation included keratinocyte mitochondrial inclusions often accompanied by damaged cristae, duplication of basement membrane, increased number of dermal fibroblasts, inflammatory cells and elastic fibers, and abnormal elastic fibers. Subsequent retinoic acid treatment resulted in more prominent mitochondrial inclusions which sometimes coalesced to form irregular contoured bodies. Also observed were lipid droplets in the stratum corneum, glycogen deposits in keratinocytes and granular material in dilated keratinocyte endoplasmic reticulum. Poorly differentiated epidermis with necrotic or apoptotic cells was present in some specimens. Elastic fibers were fewer and usually morphologically normal. Skin exposed to UVB and treated with vehicle appeared similar to control except for the presence of excess basement membrane and occasional small mitochondrial inclusions. Because of the heightened concern regarding UV radiation-induced damage to the human skin and the current topical use of retinoids, the cutaneous changes described are considered worthy of attention.     

Nucleolar ultrastructure in psoriatic skin lesions

Keratinocytes in psoriatic skin lesions were studied to provide more information on the nucleolar ultrastructure in these cells. In contrast to normal keratinocytes, nucleoli in the keratinocytes of the uppermost layer of stratum granulosum (intermedium) of psoriatic lesions were characterized by the absence of the segregation of nucleolar components. In addition, the keratinocytes without keratohyalin of the uppermost layer of stratum intermedium of psoriatic lesions did not contain degranulated nucleoli. Such observations indicate an alteration of the maturation processes in the keratinocytes of the psoriatic skin lesions and the characteristic nucleolar changes apparently represent the morphological expression of the altered inhibition of the nucleolar (preribosomal) RNA synthesis.