Activated immunocompetent cells in human skin lymph derived from irritant contact dermatitis: an immunomorphological study

By means of microsurgical lymph cannulation, skin lymph was sampled in the course of a sodium lauryl sulphate (SLS)-induced irritant contact dermatitis in human volunteers. The lymph cells were isolated by centrifugation, and then characterized immunocytochemically using different monoclonal antibodies, and in the late phase of the skin reaction also by electron microscopy. Analyses of lymph cells before the induction of the contact dermatitis revealed median values of about 60% T cells (CD4/CD8 ratio about 2:1), 4% Langerhans cells (LCs), and 1% B cells. The remainder were varying proportions of erythrocytes and uncharacterized cells. During the skin reaction, and even after resolution of the clinical signs of dermatitis, a relative and absolute increase of T and B cells, as well as of HLA-DR positive cells, paralleled the previously reported increase of LCs; a high percentage of the T cells were CD4 and CD8 negative. In addition, surface markers such as CD11a, CD25, CD54 and CD58 were detected on lymph cells sampled during the irritant skin reaction. Cell rosettes observed in the lymph throughout the experiment were analysed in the late phase of the skin reaction, and showed a central LC with three to five peripheral, in part activated, T cells, ultrastructurally revealing gap junction-like structures between the two cell types. These data indicate that immunocompetent cells in the skin are activated by a variety of non-immunological stimuli such as operative trauma and irritant contact dermatitis.     

Down-regulation of Langerhans cell protein kinase C-β isoenzyme expression in inflammatory and hyperplastic dermatoses

The family of protein kinase C (PKC) isoenzymes plays a fundamental part in signal transduction, and thereby regulates important cellular functions, including growth, differentiation, cytokine production and adhesion molecule expression. In lesional psoriatic skin. Ca²⁺-dependent PKC activity, PKC-β protein and epidermal Langerhans cell (LC) PKC-β immunostaining are significantly decreased, indicating activation and subsequent down-regulation of PKC. Whether these changes occur in other inflammatory/hyperplastic dermatoses is, however, unknown. We examined PKC-α and PKC-β expression in normal skin, psoriasis, cutaneous T-cell lymphoma (CTCL), lamellar ichthyosis, non-bullous ichthyosiform erythroderma, atopic dermatitis, urushiol-induced allergic contact dermatitis, and sodium lauryl sulphate (SLS)-induced irritant contact dermatitis. Cryostat sections were stained for PKC-α and PKC-β, and the LC marker CDla, using an immunoperoxidase technique and specific monoclonal antibodies. Double-labelling studies, in normal skin, revealed co-expression of PKC-β and CDla by epidermal LCs. Analysis of the number of PKC-^β+ and CDl^a+ epidermal LCs, in diseased compared with normal skin, revealed three categories: (i) in psoriasis and CTCL. the PKC-^β+ epidermal LC number was significantly reduced, whereas the CDl^a+ epidermal LC number was unchanged; (ii) in allergic and irritant contact dermatitis, both PKC-β+ and CDla⁺ epidermal LCs were significantly reduced in number; and (iii) in atopic dermatitis, the PKC-β⁺ epidermal LC number was normal, and CDla⁺ epidermal LCs were significantly increased in number. Moreover, the ratio of epidermal LC PKC⁺/CDla⁺ was reduced in all the dermatoses studied, suggesting activation of PKC-β, with subsequent down-regulation. Within the dermis, increased PKC-β staining of infiltrating cells was observed in all the conditions studied except lamellar ichthyosis and non-bullous ichthyosiform erythroderma. These data indicate that: (i) down-regulation of LC FKC-β occurs in a variety of inflammatory and hyperplastic skin disorders, and is not unique to psoriasis, and (iii the pattern of epidermal LC PKC-β and CDla expression varies among the diseases studied. In mice, PKC activation induces LC migration. Thus, down-regulation of epidermal LC PKC-β associated with reduced CDla⁺ epidermal LCs in allergic and irritant contact dermatitis suggests that PK.C-β may transduce the signal for migration of LCs from human epidermis.     

Sodium lauryl sulfate effect on the density of epidermal Langerhans cells.

The effect of different test models for sodium lauryl sulfate (SLS)-induced irritant contact dermatitis on epidermal Langerhans cells (LC) numbers was examined. Finn Chambers, 8 and 12 mm, containing 15 and 34 or 50 μl, respectively. of l% aq. solution of SLS were applied to human forearm skin for 48h as single or repeated application. The results showed a clear difference between the effects with the 2 chamber Sizes. The effect of the 8-mm chambers could result in increased, unchanged or decreased LC numbers, while 12-mm chambers always produced a decrease. These results seem to explain, at least partly, the discrepant results reported from various laboratories.     

Electron microscopy of the effects of dithranol on healthy and on psoriatic skin

The fine structure of healthy and of psoriatic skin was studied after dithranol irritation. Following a minimum erythematogenic or a moderate-to-considerable irritant reaction of healthy skin with dithranol, all epidermal cell types were focally affected. The Langerhans' cells (LC) were most sensitive, reacting with strongly swollen mitochondria with broken cristae, and sometimes by forming branched and circular Birbeck granules. More often than is normal, the Birbeck granules showed continuity with the LC cytomembrane. Also, melanocytes were more sensitive than keratinocytes. Most of the more strongly affected keratinocytes became cytolytic with edemic cytoplasm, but scattered cells underwent dyskeratosis (apoptosis) resulting in colloid bodies in the upper dermis. High amounts of lipid droplets developed in the basal keratinocytes, LCs, melanocytes, and dermal cells. Lipid droplets also developed in the keratinocytes at the stratum granulosum/stratum corneum interface concomitantly with a decrease in keratohyalin. Ten days after challenge, keratohyalin granules were normal again, but otherwise many changes in epidermis persisted, with increased numbers of exocytic cells and LC-mononuclear cell contacts in the epidermis and immunocompetent cells crossing the dermal-epidermal junction. These findings indicate that an irritant reaction could predispose to sensitization via nonspecifically activated immunocompetent cells. A single half-hour contact treatment with dithranol caused negligible changes in the psoriatic skin, while 24 hours' occlusion caused moderate-to-massive changes in mitochondria of keratinocytes, resulting in giant perinuclear mitochondria with broken cristae. Dithranol affects all cell types in the skin and morphologically it cannot be concluded which effect is the important one in clearing the psoriatic lesions.     

A comparative study of allergic and primary irritant contact dermatitis with dinitrochlorobenzene (DNCB) in dogs.

Attempts were made to induce allergic contact dermatitis in dogs, a species generally considered poorly responsive to experimental allergic contact dermatitis. Yound Beagles were sensitized to 2,4 dinitrochlorobenzene (DNCB) by multiple intradermal injections. Two weeks after sensitization, these dogs were challenged topically with 0.1% DNCB by a standard closed-patch technique. Sensitization evidenced by various degrees of reaction following challenge was established in all of 14 pups used, while 7 nonsensitized control pups did not react to challenge. Primary irritant contact dermatitis was induced in the skin of nonsensitized Beagle pups by 1%, 5%, and 10% solutions of DNCB. In allergic contact dermatitis the sites of challenge were grossly indurated, erythematous, and edematous. Histologically at these sites there was an infiltration of mononuclear cells which reached maximum intensity at 3 to 4 days. Accumulations of lymphoid cells were marked around sweat galnds and hair follicles. Penetration of leukocytes into these cutaneous adnexa was associated with degenerative processes in their cellular structures. Mononuclear cell infiltration into the epidermis was mild. Spongiosis was observed in the epidermis, but vesicle formation was rare. In primary irritant contact dermatitis gross lesions were characterized by severe erythema, edema, and gangreen of the skin. Microscopically, the main lesions were necrosis of the epidermal cells, separation of the epidermis from the dermis, dermal edema, and massive infiltration of the dermis with polymorphonuclear cells.     

Studies on human skin lymph containing Langerhans cells from sodium lauryl sulphate contact dermatitis.

Immunologic processes in diseased human skin have been extensively investigated, but little is known about the effect of skin diseases on human afferent skin lymph. Starting in the papillary dermis, the skin lymphatics drain the adjacent tissue in a one-way flow toward the regional lymph nodes. The composition of the afferent lymph, therefore, reflects the immunologic inflammatory processes in the drained tissue. To obtain afferent lymph to investigate its content, we inserted a cannula, by means of microsurgery, into a superficial peripheral lymph vessel draining a defined skin area. By manipulating the drained skin area and subsequent examination of the lymph we established an in vivo system for investigating the kinetics of lymph changes during the course of skin reactions. In lymph derived from a mild sodium lauryl sulphate (SLS)--induced contact dermatitis we could demonstrate an increase of both flow and cells. In particular, the number of Langerhans cells (LC) increased enormously during the course of the skin reaction. It, therefore, seems that a large increase in the migration of LC from the skin to the regional lymph nodes is a major feature of SLS-induced contact dermatitis, suggesting that LC may play a major role in the irritant contact dermatitis reaction.     

Turnover and kinetics of epidermal langerhans cells and their dendritic precursor cells in experimental contact dermatitis

The numerical density of epidermal Langerhans cells (LCs) in contact sensitivity and toxic contact dermatitis is still a matter of controvery, mainly due to changes in the phenotypic markers of this antigen-presenting cell during the skin reactions. Since the electron microscopic detection of Birbeck granules is the most reliable marker for the identification of normal and pathologically altered LCs, we performed an ultrastructural-morphometric time-course analysis to evaluate their epidermal turnover in the earskin of BALB/c mice after painting the ears with the hapten 2,4-dinitrofluorobenzene and the irritant croton oil. The counts revealed degeneration and depletion of epidermal LCs in both allergic and toxic dermatitis. In contrast, a slightly increased number of activated epidermal LCs was found during contact sensitization. All experimental procedures resulted in an enhanced immigration of so-called indeterminate dendritic cells which also became ultrastructurally activated and often showed Birbeck granule-like formations at their cell membrane. Immunohistochemistry with the monoclonal antibody 4F7, a new marker for dendritic precursor cells of LCs, demonstrated a significant increase in these accessory cells in the epidermis. Our results indicate that contact sensitivity and toxic skin reactions are characterized by complex but distinct changes in the turnover, kinetics and cellular properties of epidermal LCs and their dendritic precursor cells. Received: 16 March 1995     

Irritant-induced migration of Langerhans cells coincides with an IL-10-dependent switch to a macrophage-like phenotype

Langerhans cells (LCs) migrate after topical exposure of the skin to irritants, despite the supposed independence of irritant contact dermatitis from adaptive immunity. Whereas allergen-activated LCs are known to migrate to the draining lymph nodes (LNs), the fate of migrated LCs upon topical irritant exposure is unknown. Here, we identified a phenotypic switch of LCs after their migration into the dermis upon irritant exposure. With the aid of ex vivo intact human skin and epidermal sheets, we show that dermal fibroblasts are necessary for an IL-10-dependent postmigrational phenotypic switch of LCs into macrophage-like cells. Exposure of ex vivo skin to a panel of seven irritants resulted in a decrease in the number of CD1a(+) cells and an increase in CD14(+)/CD68(+) cells in the dermis. Neutralizing antibodies against IL-10 totally inhibited the phenotypic LC-to-macrophage transition, but did not influence the migration of CD1a(+) cells. Exposure of epidermal sheets to irritants resulted in a fibroblast-dependent LC-to-CD14(+)/CD68(+) switch coinciding with migration, which could be totally inhibited by neutralizing antibodies against either IL-10 or CCL2/CCL5 (two chemokines responsible for epidermal-to-dermal migration). We have thus identified an IL-10-dependent phenotypic switch of LCs into macrophage-like cells upon irritant exposure and emigration from the epidermis.     

Elemental changes in guinea pig epidermis at repeated exposure to sodium lauryl sulfate.

Epidermal hyperplasia is the response of the epidermis to external harmful stimuli. The control and regulation of this hyperplasia is not completely understood. It has been proposed that changes in the cellular sodium/potassium ratio are of importance in the regulation of cell proliferation. To evaluate if such a change in the elemental content of epidermal cells can be one factor to consider at irritant contact dermatitis, we performed a quantitative assessment of sodium lauryl sulfate (SLS)-induced contact reactions in the guinea pig. SLS was applied 1, 2 or 3 times and biopsies were obtained at 24 and 84 h after the last application. It was found that repeated exposures to SLS induced a hyperplasia of epidermis at 24 h persisting at 84 h. At 24 h there were significant changes in the sodium and potassium content of the keratinocytes. At 84 h there was still an increased potassium level in the cells and the sodium/potassium ratio was significantly decreased in epidermis exposed three times to SLS. This implies that changes in cellular sodium/potassium ratios occur in epidermal hyperplasia following irritant stimuli.     

Epidermal Langerhans cell apoptosis is induced in vivo by nonanoic acid but not by sodium lauryl sulphate

Exposure to irritants may cause chronic irritant contact dermatitis (ICD), characterized by irregular epidermal thickening and a predominantly dermal mononuclear cell infiltrate. The mechanisms involved, and why only certain individuals are affected, are not clearly understood. Different irritants may trigger different cellular and molecular interactions between resident skin cells and recruited inflammatory cells. In some individuals these interactions may become self-perpetuating resulting in persistent inflammation in the absence of continued exposure. This study examined Langerhans cell (LC) density in clinically normal skin of 46 patients with chronic ICD and 10 healthy individuals, and compared the action of the two irritants nonanoic acid (NA) and sodium lauryl sulphate (SLS) on the LCs and keratinocytes of clinically normal skin in patients with chronic ICD. There was a higher number of LCs/mm basement membrane in patients compared with controls, although there was no difference in the number of dendrites/LC nor in dendrite length. SLS induced keratinocyte proliferation after 48 h exposure, had no effect on LC number or distribution, and induced keratinocyte apoptosis after 24 and 48 h exposure. In contrast, NA decreased keratinocyte proliferation after 24 h exposure but this returned to basal levels after 48 h, and induced epidermal cell apoptosis after only 6 h exposure. NA dramatically decreased LC number after 24 and 48 h exposure, which was accompanied by basal redistribution and decreased dendrite length. Most significantly, NA induced apoptosis in over half of the LCs present after 24 and 48 h exposure.     

Differential effects of structurally unrelated chemical irritants on the density and morphology of epidermal CD1+ cells.

In order to gain a greater insight into the complex mechanisms of action of different irritant chemicals on the skin, we have studied the behavior of epidermal CD1+ cells in experimentally induced irritant contact dermatitis. Healthy, human volunteers were patch tested for 48 h with the following six chemically unrelated irritants and their appropriate vehicle controls; benzalkonium chloride, sodium lauryl sulphate, dithranol, nonanoic acid, croton oil, and propylene glycol. After visually assessing and grading the resulting inflammatory reactions, punch biopsies were taken and the morphology and density of CD1+ cells in the epidermis studied using immunocytochemical techniques in combination with image analysis and electron microscopy. Statistically significant decreases in the epidermal density of CD1+ cells occurred in the responses to dithranol (p less than 0.05) and nonanoic acid (p less than 0.01). Importantly, these changes in density were not simply due to variations in the intensity of inflammatory response (r = 0.1157). Alterations in the length of the dendritic processes of CD1+ cells were also induced, and semi-quantitative analysis revealed significant decreases in dendrite length in the reactions to sodium lauryl sulphate (p less than 0.05), nonanoic acid (p less than 0.001), croton oil (p less than 0.05), and dithranol (p less than 0.005). Unlike epidermal density, however, this effect on cell morphology was directly related to the severity of inflammation (r = -0.74, p less than 0.01). Morphologic evidence of cellular injury to Langerhans cells was seen by electron microscopy in the majority of biopsies, although relatively few cells were affected in sodium lauryl sulphate and propylene glycol reactions. Benzalkonium chloride, unlike the other irritants, also induced a state of metabolic activation in a high proportion of epidermal Langerhans cells. Lymphocyte/Langerhans cell apposition was observed in most samples, but was particularly prevalent in the reactions to dithranol. The results of this study demonstrate that significant changes in the morphology and density of Langerhans cells occur in irritant contact dermatitis, some of which are directly influenced by the chemical nature of the irritant.     

Skin irritants and contact sensitizers induce Langerhans cell migration and maturation at irritant concentration

Skin irritants and contact allergens reduce the number of Langerhans cells (LCs). It has been assumed that this reduction is due their migration to the draining lymph node (LN) for initiating immune sensitization in a host. Skin irritation, however, as opposed to contact allergy is not considered to be an immunological disease. Nevertheless, skin irritants are also known for their adjuvant-like effects on contact allergy, resulting in skin hypersensitivity reactions like toxic dermatitis. The human organotypic skin explant culture (hOSEC) model is used to study the characteristics of chemical-induced migration of CD1a(+) LCs out of the epidermis in relation to irritancy or toxicity. We analysed cells emigrating out of hOSEC for CD1a(+) LCs, CD83(+) mature dendritic cells (DCs) and CCR7(+) LN homing cells. After exposure to a toxic concentration of a non-immunogenic irritant, an increase of CD1a(+)CD83(+) LCs was found in the culture medium. A non-toxic concentration of an sensitizer induced an increase of CD1a(+) cells. About 50% of skin emigrating CD1a(+) LCs were CD83(-) (immature) but all were CCR7(+). Skin irritation by both non-allergenic and allergenic compounds induces LC migration and maturation. In contrast, only allergenic compounds induced LC migration with partial maturation at subtoxic concentration. This effectively demonstrates that irritation is physiologically needed stimuli for inducing LC maturation.     

Immunocytochemical detection of the carbohydrate antigen, Sialyl Lewisx, in normal human skin and during irritant contact dermatitis

Abstract Sialys Lewis^x (SLex) is a ligand for the E-selectin and the interaction of E-selectin on the endothelium and SLe^x on T cells may be important for T-cell migration into the skin. We investigated the expression of SLe^x on Langerhans cells (LC) in normal skin and on LC repopulating epidermis deprived of LC due to a preceding irritant contact dermatitis. SLe^x was visualized by fluorescence and light microscopic immunocytochemistry using the monoclonal antibody. CSLEX-1. The results showed that about 40% of LC in normal epidermis express SLe^x. In the repopulation phase, most of the epidermal cells were CDla⁺/SLe^x+. We suggest that SLe^x is present on epidermal LC that have recently immigrated from the dermis.     

Epidermal Langerhans' cells in chronic eczematous dermatitis of the palms treated with PUVA and UVB

Epidermal Langerhans' cells (LC) were studied in patients with chronic eczematous dermatitis of the palms. The monoclonal antibodies anti-Leu 6 and anti-HLA-DR were used, and the cells visualized with an immunoperoxidase technique. Increased numbers of LC were found in allergic contact dermatitis as well as in irritant contact dermatitis and hyperkeratotic dermatitis of the palms. The increased number of epidermal LC in lesional skin may facilitate presentation of exogenous or endogenous antigens to activated T-cells. One hand was treated with PUVA or UVB and the other hand served as a non-exposed control. PUVA treatment cleared the dermatitis and the LC number decreased markedly. With UVB treatment clinical improvement was achieved, and a less pronounced decrease in epidermal LC was noticed. There seems to be a crude relationship between the extent of clinical improvement and the reduction in epidermal LC numbers. The change in LC might be a primary event or secondary to a reduction of the inflammatory process.     

模拟日光照射后皮肤CD1a、CD68阳性细胞变化的研究

目的 观察正常人皮肤经日光模拟器照射（solar-simulated ultraviolet radiation,ssUVR)后,朗格汉斯细胞（Langerhans cells, LC）和CD68阳性的巨噬细胞的变化。方法14名健康汉族女性志愿者于背部非曝光部位接受ssUVR照射。选择2个正方形部位,一处为正常对照,另一处为每日一次ssUVR照射。第4天照射后的72小时,进行活检取材。对所有标本进行CD1a和CD68免疫组化染色。结果 未照射部位正常表皮内的LC密度为258±61个/mm2,ssUVR照射部位的LC密度明显降低为96±53个/mm2,LC的形态不完整,树突变短而不明显。真皮浅层CD68阳性的巨噬细胞,未照射部位密度为290±22个/mm2,ssUVR照射部位的密度升高为399±65个/mm2。经过照射后真皮这些巨噬细胞数目明显增多位置上移,形态上树突变长并且大多数互相连接紧密。结论ssUVR照射可使LC数目减少,形态破损。真皮内的巨噬细胞则增高,这似有助于弥补紫外线照射对局部免疫的抑制作用。     

Cytochemical and Ultrastructural Studies of the Langerhans'' cells

In the guinea pig, experimental allergic contact dermatitis (ACD) and primary irritant contact dermatitis (PICD) were induced with different concentrations of dinitrochlorobenzene (DNCB). The epidermal Langerhans' cells (LCs) were observed sequentially by both adenosine triphosphatase (ATPase) and electron microscopy. Light microscopically, in ACD, the density and dendritic processes of LC decreased markedly within 12 h after antigen challenge. Almost no recognization LCs could be seen within 2 to 5 days. Later, LCs began to repopulate in the epidermis. Within 14 days, the density and shape of the LCs returned to normal. On the contrary, LCs changed more rapidly in PICD. The dendritic processes of LC decreased within 2 h and cell density decreased dramatically within 6 h after DNCB application. LCs also repopulated more rapidly in the epidermis. Electron microscopically, in ACD, we observed that lymphocyte-like cells apposed to LCs; LCs were activated and damaged; however, in PICD, we found neither the apposition of lymphocyte-like cells to LCs, nor the activation of LCs. LCs play an important role in the convalescence phase as well as in the early and later phases of contact allergic reaction.     

Cytochemical and Ultrastructural Studies of the Langerhans' Cells

Abstract: In the guinea pig, experimental allergic contact dermatitis (ACD) And primary irritant contact dermatitis (PICD) were induced with different concentrations of dinitrochlorobenzene (DNCB). The epidermal Langerhans' cells (LCs) were observed sequentially by both adenosine triphosphatase (ATPase) and electron microscopy. Light microscopically, in ACD, the density and dendritic processes of LC decreased markedly within 12 h after antigen challenge. Almost no recognization LCs could be seen within 2 to 5 days. Later, LCs began to repopulale in the epidermis. Within 14 days, the density and shape of the LCs returned to normal. On the contrary, LCs changed more rapidly in PICD. The dendritic processes of LC decreased within 2 h and cell density decreased dramatically within 6 h after DNCB application. LCs also repopulated more rapidly in the epidermis. Electron microscopically, in ACD, we observed that lymphocyte-like cells apposed to LCs; LCs were activated and damaged; however, in PICD, we found neither the apposition of lymphocyte-like cells to LCs, nor the activation of LCs. LCs play an important role in the convalescence phase as well as in the early and later phases of contact allergic reaction.     

Skin irritant reactivity following experimental cumulative irritant contact dermatitis

Despite the frequency of irritant contact dermatitis, very little is known about the duration of barrier function impairment following cumulative irritant contact dermatitis. We studied post-irritation irritant reactivity by assessing the response to SLS irritation in previously irritated sites. Cumulative irritant contact dermatitis was induced on the forearms of 15 volunteers aged 18 to 50 years by repeated occluded application of 0.5% SLS I h per day over 3 weeks. 3, 6 and 9 weeks later, previously irritated and unirritated control sites were challenged with 2% SLS under occlusion for 23 h. Irritation was assessed by visual scoring, transepidermal water loss (TEWL) as an indicator of epidermal barrier function, and capacitance as a parameter of epidermal water content. While no difference in irritant reactivity between pre-irritated and unirritated sites was observed 3 weeks following irritant contact dermatitis, there was a significant hyporeactivity of previously irritated skin as expressed by clinical scores, TEWL and capacitance at 6 and 9 weeks. Our results indicate that epidermal barrier function remains altered even 9 weeks after cumulative irritant contact dermatitis. With regard to patch testing, post-irritation hyporeactivity might be a cause of false-negative tests on previously irritated sites.     

A novel monoclonal antibody to a distinct subset of cutaneous dendritic cells.

A monoclonal antibody was generated by immunizing rats with Langerhans cell (LC)-enriched epidermal cells obtained from BALB/c mouse earskin after epicutaneous application of the contact sensitizer 2,4-dinitrofluorobenzene (DNFB). The antibody 4F7 detects in normal mouse skin, few dermal cells showing the morphologic, phenotypic, and functional properties of accessory dendritic cells, but lacking Birbeck granules. The capacity to stimulate allogenic T cells in the mixed leucocyte reaction resembles that of freshly isolated LCs. After DNFB application, an increased number of 4F7+ dendritic cells are found in the dermis and, in addition, some labeled dendritic cells occur in the epidermis. Some of the latter cells exhibit cytoplasmic Birbeck granules. Remarkably, there is no increase of the 4F7+ cells in the regional lymph nodes after DNFB treatment. These data suggest that the 4F7 antibody labels distinct dendritic cells of the mouse skin that are involved in the mediation of contact sensitization and probably represent immature LCs.