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.     

Large increase of Langerhans cells in human skin lymph derived from irritant contact dermatitis

In order to monitor the kinetics of Langerhans cells in the afferent lymph during contact dermatitis, a superficial peripheral lymph vessel draining the skin of the upper and medial part of the foot was cannulated by means of microsurgery on the lower leg of four healthy volunteers. After 2 days an irritant contact dermatitis was induced by application of 10% sodium lauryl sulphate to the area of skin drained by the cannulated lymph vessel. Three days later the spontaneously regressing skin reaction was treated with clobetasol propionate in two of the subjects. Lymph was collected twice daily for 8 days. Langerhans cells were identified by immunofluorescence microscopy of cytocentrifuge slide preparations from the lymph, using a monoclonal anti-CDla antibody.
In the late phase of the contact dermatitis the output, i.e. both the absolute number and the percentage of Langerhans cells in the lymph dramatically increased. At the end of the experiment, when there were no remaining clinical signs of contact dermatitis, the Langerhans cell output still markedly exceeded the initial values. These results are the first direct evidence in humans that migration of Langerhans cells from the skin to the regional lymph nodes is a major feature of irritant contact dermatitis.     

Allergic contact dermatitis

Contact dermatitis is an inflammatory skin condition induced by exposure to an environmental agent. Eczema and dermatitis are used synonymously to denote a polymorphous pattern of skin inflammation characterized at least in its acute phase by erythema, vesiculation and pruritus. Substances responsible for contact dermatitis after single or multiple exposures are non protein chemicals, i.e. haptens, that induce skin inflammation through activation of innate skin immunity (irritant contact dermatitis) or both innate and acquired specific immunity (allergic contact dermatitis). The present review will focus on allergic contact dermatitis, a delayed-type hypersensitivity reaction, which is mediated by hapten-specific T cells. Recent advances in the pathophysiology of ACD have shown that the occurrence of ACD, as well as its magnitude and duration, is controlled by the opposite functions of CD8 effector T cells and CD4 regulatory T cells. From these studies ACD can be considered as a breakdown of cutaneous immune tolerance to haptens.     

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.     

Freshly isolated Langerhans cells negatively regulate naïve T cell activation in response to peptide antigen through cell-to-cell contact

BACKGROUND: Epidermal Langerhans cells (LCs) have been believed to function as professional antigen-presenting cells (APCs). However, LC-ablated mice reportedly suffer from severer contact hypersensitivity (CHS) upon cutaneous challenge with hapten than wild-type mice, suggesting LCs as regulators of adaptive immune responses in the skin. OBJECTIVE: This study was designed to address the possible regulatory roles of LCs in the balanced primary adaptive immune responses to protein antigens. METHODS: LCs were freshly isolated from skin of BALB/c mice (>95% positive for MHC class II). Na?ve CD4+ T cells reactive to ovalbumin (OVA) were purified by FACS-sorting from lymph node cells of DO11.10 BALB/c mice, labeled with CSFE, and incubated with OVA peptide in the presence of splenic dendritic cells (DCs) and/or LCs. Cell division frequencies were determined by the degree of serially diluted expressions of CSFE in the individual CD4+ T cells. RESULTS: Approximately 70% of them underwent cell division when na?ve CD4+ T cells were activated by OVA presented by splenic DCs. In contrast, LCs only very modestly induced their cell division. Furthermore, LCs inhibited the cell division induced by splenic DCs, and this regulatory action was abolished by prevention of their contact to other cells, but not by the treatment with neutralizing antibodies against IL-10 or TGF-beta, well-established regulatory cytokines. CONCLUSION: LCs negatively regulate the primary adaptive T cell response, presumably allowing well-controlled immune response in the skin.     

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.     

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.     

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     

IL-10-producing Langerhans cells and regulatory T cells are responsible for depressed contact hypersensitivity in grafted skin

Although skin grafting is a common surgical technique, the immunological state of grafted skin remains unelucidated. An experimental model has shown that the development of murine contact hypersensitivity (CHS) is depressed when mice are sensitized with a hapten through full-thickness grafted skin. We explored the immunological mechanisms underlying this hyposensitization, focusing on the fate of Langerhans cells (LCs). When FITC was applied to grafted skin, FITC-bearing LCs were capable of migrating to the draining lymph nodes. Epidermal cell suspensions isolated from the grafted skin produced a high amount of IL-10 as assessed by real-time PCR. Adoptive transfer of immune lymph node cells from the sensitized mice suppressed the CHS response of recipients in an antigen-specific manner. CD4(+)CD25(+) but not CD4(+)CD25(-) T cells purified from lymph node cells were responsible for this suppression. Finally, we detected high expression of receptor activators of nuclear factor kappa-B ligand (RANKL) in the grafted skin, and found that recombinant RANKL stimulated LCs to produce IL-10. These findings suggest that the hyposensitization of CHS through the grafted skin is not attributable merely to the reduction of LC number but that IL-10-producing LCs exert a downmodulatory effect by inducing regulatory T cells.     

Heterogeneity of dermal OKT6+ cells in inflammatory and neoplastic skin diseases

This immunopathologic study of both normal and pathologic skin specimens (contact dermatitis [CD], lichen planus [LP], cutaneous T cells lymphoma [CTCL], and histiocytosis X [HX]) allowed as to differentiate four types of dermal OKT6+ cells: (1) cells with the same morphologic features as epidermal Langerhans cells (LCs), rarely found in either normal or pathologic dermis; (2) cells structurally similar to LCs but lacking Birbeck granules (BGs), found mainly in CD and LP; (3) larger cells rich in cytoplasmic organelles, only 5% of which contained BGs. They were especially common CTCL; and (4) cells typical of HX.     

The HECA-452 epitope is highly expressed on lymph cells derived from human skin

The cutaneous lymphocyte-associated antigen (CLA), recognized by the monoclonal antibody HECA-452, is a cell surface glycoprotein that binds specifically to E-selectin. CLA is present on most T cells at sites of cutaneous immune response and has been shown to be important in lymphocyte homing to the skin. It is expressed only by a minor subset of peripheral T cells and is absent on thymocytes. We have analysed (using a FACScan flow cytometer) the expression of CLA on human lymph cells derived from normal skin, from ultraviolet (UV)-irradiated skin and from allergic contact dermatitis. Whereas in the peripheral blood CLA was expressed on < 20% of CD4 +, CD8 + and CD56 + cells (natural killer cells), > 60% of CD4 +, CD8 + and CD56 + cells isolated from skin-derived lymph expressed CLA. Furthermore, > 90% of CD1a + dendritic lymph cells were positive for CLA. UV irradiation of the skin and induction of an allergic contact dermatitis did not change CLA expression on lymph cells, although lymph flow and cell output increased. These results provide further evidence for an important role of CLA in cell homing to the skin.     

Isolation of human skin-derived lymph: flow and output of cells following sodium lauryl sulphate-induced contact dermatitis

Summary By means of microsugery a peripheral subcutaneous lymph vessel draining a defined skin area was isolated and cannulated on the lower leg of six healthy volunteers. Lymph was collected over a period of 8 days. During the first 2 days baseline values for lymph flow and output of cells were established. A contact dermatitis was then induced in the drained skin area by the application of 10% sodium lauryl sulphate. All six probands developed a mild to moderate irritant contact dermatitis. Lymph flow as well as output of cells increased with the intensity of the skin reaction. Subsequent local treatment with clobetasol propionate decreased the cell output, but the lymph flow increased further. Neither lymph flow nor output of cells returned to the initial baseline values at the end of the study, when the clinical signs of contact dermatitis had completely disappeared. During the experiment significant individual variations were found, with means ranging from 0.10 to 0.48 ml/h for lymph flow and from 8700 to 174000/h for cells, which probably depended mainly on the different topographies and calibres of the cannulated lymph vessels.     

CD8+ T cells are effector cells of contact dermatitis to common skin allergens in mice

Allergic contact dermatitis (ACD) to strong experimental haptens is mediated by specific CD8+ T cells. Here, we show that similar mechanisms occur for weak haptens, which comprise the vast majority of chemicals responsible for human ACD. We used a model of ACD, that is, the contact hypersensitivity reaction, to test for the allergenicity of three weak haptens involved in fragrance allergy. ACD to weak haptens could not be induced in normal mice. In contrast, mice acutely depleted in CD4+ T cells developed a typical ACD reaction to the three weak fragrance allergens that peaked 24 hours after challenge. Priming of CD8+ T cells was observed in draining lymph nodes 5 days after sensitization and development of ACD was associated with the infiltration of activated CD8+ T cells in the challenged skin. CD8+ T cells were effectors of the ACD reaction as in vivo treatment with depleting anti-CD8 mAbs abrogated the ACD responses and as purified CD8+ T cells could adoptively transfer ACD to naive recipients. In conclusion, our data demonstrate a dominant role of CD8+ T cells as initiators of ACD to weak haptens, and suggest that CD8+ T cells may represent potential targets for preventing or treating ACD.     

Contact dermatitis II. Clinical aspects and diagnosis

Contact dermatitis (CD) is an altered state of skin reactivity induced by exposure to an external agent. "Eczema" and "dermatitis" are often used synonymously to denote a polymorphic pattern of inflammation of the skin characterized, at least in its acute phase, by erythema, vesiculation and pruritus. Substances that induce CD after single or multiple exposures may be irritant or allergic in nature. The clinical presentation may vary depending on the identity of the triggering agent and the reactivity of the subject, but in all cases the lesions are primarily confined to the site of contact. According to the mechanism of elicitation, the following types of contact reactions may be distinguished: (1) allergic contact dermatitis (ACD); (2) irritant contact dermatitis (ICD); (3) phototoxic and photoallergic contact dermatitis, and (4) immediate type contact reactions. The present review will focus on allergic contact dermatitis. ACD is the clinical presentation of contact sensitivity in humans. The pathophysiology of the contact sensitivity reaction has been reviewed in a preceding issue of this journal [1].     

The role of CD4+ and CD8+ T cells in contact hypersensitivity and allergic contact dermatitis

Allergic contact dermatitis (ACD) and contact hypersensitivity (CHS) are delayed-type hypersensitivity reactions which are mediated by hapten specific T cells. During the sensitisation phases, both CD4+ and CD8+ T cell precursors are activated in the draining lymph nodes by presentation of haptenated peptides by skin dendritic cells. Subsequent hapten skin painting induces the recruitment of T cells at the site of challenge which induces inflammatory signals and apoptosis of epidermal cells, leading to the development of a skin inflammatory infiltrate and of clinical symptoms. There have been major controversies on the respective roles of CD4+ and CD8+ T cells in the development of the CHS inflammatory reaction. Experimental studies from the last 10 years have demonstrated that, in normal CHS responses to strong haptens, CD8+ type 1 T cells are effector cells of CHS while CD4+ T cells are endowed with down-regulatory functions. The latter may correspond to the recently described CD4+ CD25+ regulatory T cell population. However, in some instances, especially those where there is a deficient CD8 T cell pool, CD4+ T cells can be effector cells of CHS. Ongoing studies will have to confirm that the pathophysiology of human ACD is similar to the mouse CHS and that the CHS response to weak haptens, the most frequently involved in human ACD, is similar to that reported for strong haptens.     

Phenotype of Langerhans cells in human afferent skin lymph derived from allergic contact dermatitis

Abstract By means of microsurgical lymph cannulation human skin lymph derived from the late phase of an elicitation reaction to diphenylcyclopropenone was sampled. Cells were isolated by centrifugation and then treated with mouse anti-CDl a mnonoclonal antibodies and sheep antimouse antibody-coated Dynabeads. Ultrastructural and immunocytochemical analyses revlaled anti-CDl a/Dynabead-rosetted CDl a- and protein S-100-positive cells which did not express monocyte surface markers, but surface antigens such as HLA-DR, ICAM-I and, in part, LFA-3. In comparison to freshly prepared human epidermal Langerhns cells (LC), a large fraction of these cells contained no or markedly fewer Birbeck granules and exhibited extensive ruffling of the surface. These data suggest that the phenotype of LC in skin lymph derived from the elicitation phase of allergic contact dermatitis is similar to LC cultured in vitro. In the functional concept or LC or our time, these cells correspond to the dendritic cells designated as “veiled”.     

sICAM-1, sE-selectin and sVCAM-1 are constitutively present in human skin lymph and increased in allergic contact dermatitis

The expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin is important for the regulation of the leucocyte traffic into and in inflammatory dermatoses. ICAM-1, VCAM-1 and E-selectin were initially identified as cell-surface proteins, but recent evidence suggests that they also exist in a soluble form. The collection of human afferent lymph exclusively deriving from a selected skin area allows insight into local pathomechanisms as well as signal transmission in skin disorders. In the present study we measured the concentrations of the soluble adhesion molecules (sAM) sICAM-1, sVCAM-1 and sE-selectin in human skin lymph derived from normal untreated skin, irritant contact dermatitis (CD) and the induction and elicitation phases of allergic CD. The strong elicitation reactions of allergic CD produced an increase in sAM output to about three times the baseline values but in the weaker irritant CD we observed no increase at all. In the induction phase of allergic CD the concentrations during the first 9 days of the experiment remained unchanged, as in the lymph derived from normal untreated skin, but were slightly increased thereafter. To our knowledge, no in vivo data exist on the local involvement of sAM in irritant and allergic CD in humans. Our results provide evidence of increased concentrations of sAM mainly in strong allergic CD. Received: 12 June 1996     

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.