Cytokines and chemokines in the initiation and regulation of epidermal Langerhans cell mobilization

Langerhans cells (LC) are members of the wider family of dendritic cells. LC reside in the epidermis where they serve as sentinels of the immune system, their responsibilities being to sample the external environment for changes and challenges and to deliver information (antigen) to responsive T lymphocytes within skin draining lymph nodes. The ability of LC to migrate from the epidermis to regional lymph nodes is therefore of pivotal importance to the induction of cutaneous immune responses. The journey that LC have to make from the skin has a number of requirements. Initially it is necessary that LC disassociate themselves from surrounding keratinocytes and are liberated from other influences that encourage their retention in the epidermis. Subsequently, migrating LC must successfully traverse the basement membrane of the dermal-epidermal junction and make their way, via afferent lymphatics, to draining lymph nodes. Effective entry into lymph nodes is necessary, as is correct positioning of cells within the paracortex. There is increasing evidence that both cytokines and chemokines, and their interaction with appropriate receptors expressed by LC, orchestrate the mobilization and movement of these cells. We here consider the parts played by these molecules, and how collectively they induce and direct LC migration.     

Modulation of MHC class II+ Langerhans cell numbers in corticosteroid treated epidermis by GM-CSF in combination with TNF-α

Abstract It is important to understand how dendritic cells (DC) are recruited, maintained and stimulated to migrate from tissues to lymph nodes. This is because DC are potent initiators of primary immune responses and candidates for vaccine development. Identification of factors which could lead to increased numbers of DC in tissues could affect immune responses by modulating their interaction with antigen which penetrates the tissue. To identify cytokines which could increase DC in tissues we tested the ability of GM-CSF, TNF-α and IL-6 to partially prevent steroid depletion of Langerhans cells (LC) from the epidermis. Cytokines diluted in serum-containing medium were compared with cytokines diluted in albumin-containing, serum-free medium in order to determine a minimum combination of cytokines required to increase LC and the effect of serum on the LC-increasing activity of cytokines. In the presence of serum, GM-CSF or TNF-α could increase LC frequency compared to the control; but in the absence of serum neither of these cytokines were effective unless they were combined with each other. In the presence of serum the combination of GM-CSF with TNF-α was ineffective. The data support the hypotheses that GM-CSF and TNF-α are both important in regulating LC numbers in the epidermis in vivo. Serum may modulate how each of these cytokines, separately or in combination, affect LC frequency in the epidermis–GM-CSF and TNF-α separately probably interact with other factors present in serum to increase LC frequency, whereas in combination it is possible that these separate effects are cancelled in the presence of serum. TNF-α and GM-CSF together, in the absence of serum, form one combination of a minimum number of cytokines which can regulate LC frequency in the epidermis; and IL-6 alone, or in combination with GM-CSF, does not increase LC frequency.     

Interleukin 1β and the stimulation of Langerhans cell migration: comparisons with tumour necrosis factor α

Epidermal Langerhans cells (LC) and the cells into which they mature are believed to play a pivotal role in cutaneous immune function. The induction phase of contact sensitization is associated with the migration of LC from the skin and their accumulation as dendritic cells (DC) in lymph nodes draining the site of exposure. We have demonstrated previously that tumour necrosis factor α (TNF-α), an epidermal cytokine produced by keratinocytes, provides one signal for LC migration. We describe here experiments designed to evaluate the influence of interleukin 1β (IL-1β), a product exclusively of LC in murine epidermis, on LC migration, LC morphology and DC accumulation, and to compare the effects of this cytokine with those of TNF-α. Both cytokines induced a significant reduction in the frequency of epidermal LC and the arrival of DC in draining lymph nodes. Changes in both parameters were induced more rapidly following intradermal administration of TNF-α than were observed after treatment with IL-1β. However, the reduction in LC frequency was more persistent with IL-1β. Both cytokines caused the activation of LC, characterized by the acquisition of a more dendritic morphology and the increased expression of Ia molecules. These results demonstrate that IL-1β and TNF-α can each stimulate the migration of epidermal LC, but that the changes induced by these cytokines are not identical. Received: 9 September 1996     

Adhesion molecules in lesions of American cutaneous leishmaniasis

Abstract Accessory signals, which include adhesion molecules, MHC-II molecules and cytokines. are necessary to foster the interaction between memory T cells and epidermal cells, that is required to promote cutaneous inflammatory responses. American cutaneous leishmaniasis (ACL) is characterized by a spectrum of immunological manifestations, and is a prototype disease for the study of regulatory mechanisms involved in immune protection against protozoal infection. In the present study, we show that diffuse cutaneous leishmaniasis (DCL) epidermis contains keratinocytes that do not express ICAM-I and HLA-DR molecules. Langerhans cells (LC) are within normal values or somewhat lower, and a very few cells expressing the HB15 molecule a new described member of the Ig superfamily are found in such lesions. Mucocutaneous leishmaniasis (MCL) epithelium shows an increased expression of ICAM-1 and HLA-DR molecules, few HBI5⁺ cells, and an absence of epithelial LC. Localized cutaneous leishmaniasis (LCL) epidermis displays ICAM-⁺ keratinocytes organized in patches, a uniform expression of HLA-DR, hyper-plasia of LC, and numerous HBI5⁺ cells. In all forms of the disease, infiltrating T cells express more LFA-1β than LFA-1α, but LFA-1β⁺ cells are more abundant in LCL granulomas. In contrast, there are more LFA-lα⁺ cells in DCL and MCL than in LCL granulomas. LCL lesions also show the highest numbers of HB15⁺ cells within the granu-loma. These results indicate the importance of adhesion molecules in ACL lesions, and open new possibilities for therapeutic schemes oriented towards the control of cell migration.     

UVB-Dependent Modulation of Epidermal Cytokine Network: Roles in UVB-Induced Depletion of Langerhans Cells and Dendritic Epidermal T Cells

The epidermis of mice consists of three cellular components, i.e., keratinocytes, Langerhans cells (LC), and dendritic epidermal T cells (DETC). Each epidermal subpopulation produces a different set of cytokines, thereby forming a unique cytokine milieu. These cytokines, in turn, support the survival and growth of LC and DETC and regulate their immunological functions. LC and DETC play important, but distinct, effector roles in protective immunity against antigens that are generated in or penetrate into the epidermis. Acute or chronic exposure of mice to ultraviolet B (UVB) radiation is known to impair this cutaneous immunity, as evidenced by the failure to induce T cell-mediated immune reactions, by the generation of antigen-specific immunological unresponsiveness, and by the development of skin cancers. Importantly, these changes are associated with reduced densities of LC and DETC in UVB-exposed skin, suggesting that the deficiency in these epidermal leukocytes may account for some of the deleterious influences of UVB radiation on skin. Here I will review the recent advance in our understanding of the mechanisms by which UVB radiation may deplete LC and DETC from epidermis. More specifically, I will discuss the following possibilities: a) UVB-mediated suppression of the production of relevant growth factors for LC and DETC, b) UVB-induced abrogation of surface expression of growth factor receptors, and c) UVB-triggered apoptotic cell death in epidermal leukocytes.     

Stimulation of Langerhans cell migration by tumor necrosis factor alpha (TNF-alpha).

Following topical exposure of mice to skin-sensitizing chemicals, Langerhans cells (LC), many of which bear antigen, are stimulated to migrate via the afferent lymphatics to draining lymph nodes. Consistent with the acquisition of potent immunostimulatory activity, LC while in transit to lymph nodes, are subject to a functional and phenotypic maturation thought to be mediated by granulocyte/macrophage colony-stimulating factor (GM-CSF) and possibly other epidermal cytokines. An interesting question is the nature of the stimulus that initiates the migration of LC from the epidermis. We have examined the influence of intradermal tumor necrosis factor alpha (TNF-alpha), another epidermal cytokine, on the accumulation of dendritic cells (DC) in draining lymph nodes. Murine, but not human, recombinant TNF-alpha caused a rapid and concentration-dependent increase in the frequency of DC in draining nodes. The conclusion drawn is that local production of TNF-alpha provides one signal for LC migration during cutaneous immune and inflammatory responses.     

Wie entsteht ein Ekzem?

New experimental results on the role of T cells and keratinocytes have led to a better understanding of eczematous inflammation and can help explain both the clinical and histological pictures of eczema. Besides activated endothelial cells and adhesion molecules, a complex interaction of numerous chemokines controls the recruitment of T cells from the blood vessels and their migration into the dermis and epidermis. Activated T cells damage the epidermis by pro‐inflammatory cytokines and can induce apoptosis of individual keratinocytes through “killer molecules”. Cleavage of adhesion molecules on keratinocytes leads to spongiotic changes. Keratinocytes then activate repair mechanisms, which cause acanthosis and parakeratosis in chronic eczema.     

Spleen dendritic cells exhibit altered morphology and increased allostimulatory capacity after short-term culture.

Ia-bearing dendritic cells (DC) are a class of bone marrow-derived antigen-presenting cells that appear to possess an increased capacity to stimulate resting T lymphocytes. DC from different tissues share several morphologic, phenotypic and functional attributes. For example, freshly isolated DC from spleen resemble phenotypically and functionally freshly isolated Langerhans cells (LC) from epidermis; in addition, during short-term culture both DC and LC undergo several parallel changes including modifications affecting phenotype, capacity to present protein antigens, and ability to route surface Ia molecules into intracellular acidic compartments (J Immunol 1990: 145: 2820-2826). In the present study we show, using immunoelectron microscopy with anti-Ia and anti-33D1 monoclonal antibodies, freshly isolated DC in suspension to have a smooth cell surface with few and short cytoplasmic projections. By contrast, cultured DC display conspicuous bulbous cytoplasmic protrusions. In addition, spleen DC following culture for 24-48 hours exhibit an increased ability to stimulated allogeneic T lymphocytes in the primary mixed leukocyte reaction. These changes, similar to those described for freshly isolated and cultured LC respectively, further substantiate the close relationship between DC and LC.     

朗格汉斯细胞在银屑病中的作用研究进展

【摘要】 朗格汉斯细胞（LC）是表皮内重要的免疫细胞，具有抗原提呈功能，在炎症状态下经过成熟、活化后，迁移至局部淋巴结并活化初始T细胞，启动免疫应答。近年对LC在银屑病发病中的研究显示，LC具有促进炎症和抑制炎症的双重作用。对LC起源的研究显示，LC有两个亚群，分别具有促炎和抑炎作用，参与不同的免疫应答过程，可能与银屑病免疫异常有关。本文综述LC在银屑病中的研究进展。     

Role of Langerhans cells in epidermotropism of T cells

Summary Certain T lymphocytes display a specific affinity for the epidermis (epidermotropism). Recent studies have suggested that Ia⁺ Langerhans cells (LCs) are possible targets for the epidermotropism. A variety of self-Ia-reactive cloned T cells were tested for their ability to migrate into the epidermis following intradermal inoculation into the footpads of syngeneic mice. Clone BB5 was chosen as representative of the epidermotropic T cells. We investigated whether the depletion of Ia⁺ LCs from the epidermis by tape-stripping could alter the migration of BB5 cells into the epidermis. The epidermal invasion of BB5 cells was markedly impaired in those mice whose LCs were depleted by 95% after repetitive tape-stripping. Because production of epidermal-derived thymocyte activating factor (ETAF) by the epidermal cells was augmented after repetitive tape-stripping, the diminished migration of BB5 cells into tape-stripped epidermis did not result from a decrease in ETAF production which is thought to attract T cells chemotactically. These results suggest that Ia⁺ LCs may play an inductive role in the preferential migration of T cells into the epidermis.     

Lymphocyte subsets and Langerhans cells/indeterminate cells in erythema multiforme

A peroxidase-antiperoxidase study using monoclonal antibodies directed against T and B lymphocytes and Langerhans cells/indeterminate cells (LC/IC) was undertaken in order to understand more clearly the changes observed in erythema multiforme. At the various stages of development, from normal skin to target lesions, the quantity of inflammatory cells differed, but in each case the number of T8+ (cytotoxic/suppressor) cells was greater than the number of T4+ (helper/inducer) cells in the epidermis, whereas the latter exceeded the former in the dermis. Concomitant with the initial epidermis changes, there was an increase in the number of T6+ (LC/IC) cells in the upper and lower epidermis. With slight to moderate basal unit destruction, the number of LC/IC in the upper epidermis exceeded those in the lower epidermis. With severe basal unit destruction, there was a loss of LC/IC in the lower epidermis as detected by T6 reactivity. In fully formed blisters, the LC/IC in the upper half of the epidermis were decreased in parallel with the degree of epidermal necrosis. The character of the lymphocytic inflammatory infiltrate and redistribution in LC/IC are similar to those findings described in allergic contact dermatitis. The clinical, histologic, and immunopathologic changes in erythema multiforme appear to be due in part to cellular immune mechanisms with the lymphocyte as the predominant effector cell, and our data suggest a possible role for LC/IC in this disorder.     

Human epidermal langerhans cells express the immunoregulatory enzyme indoleamine 2,3-dioxygenase

Langerhans cells (LC) are a special subset of dendritic cells integrating cutaneous immunity. The study of LC function is of major interest not only for efforts of vaccine design and immunotherapy but also for gaining an insight into the pathogenesis of immune-mediated cutaneous diseases and neoplasias. Recently, defined antigen-presenting cells were described that express indoleamine 2,3-dioxygenase (IDO) and inhibit T cell proliferation in vitro and in vivo. Here, we show that stimulation with interferon-gamma (IFN-gamma) induces the expression of functionally active IDO in highly purified human epidermal LC. The induction of IDO after stimulation of LC with IFN-gamma seems to follow a defined kinetic with rapid upregulation followed by a downregulation after about 24 h of culture. Accordingly, proliferation of T cells induced by anti-CD3 antibodies was modulated by supernatants of IFN-gamma-activated human epidermal LC. Importantly, downregulation of T cell proliferation by supernatants of 24 h IFN-gamma-activated LC was prevented by inhibition of IDO. These results indicate that LC not only have the capacity to stimulate but also to inhibit T cells, and suggest that LC possess an immunoregulatory function in promoting T cell tolerance by production of IDO.     

Narrowband–UVB irradiation decreases the production of pro-inflammatory cytokines by stimulated T cells

Narrow-band ultraviolet B (UVB) phototherapy is an effective treatment for psoriasis. Owing to its limited penetration, the direct effects of UVB are mostly restricted to cells residing in the epidermis and papillary dermis, and are associated with epidermal depletion of Langerhans’ cells (LC) and T cells. It has been argued that the depletion of the skin-resident T-cell population may be due to a combination of UVB-induced apoptosis and decreased recruitment from the blood due to lower expression of the required adhesion and homing molecules. We have previously demonstrated that UVB treatment can alter the expression of adhesion molecules by blood lymphocytes, and as these can be influenced by cytokines, the aim of this study was to investigate whether UVB irradiation can also influence the cytokine production of circulating T cells. Four patients with active chronic plaque psoriasis were treated daily with narrow-band 312 nm UVB irradiation and blood samples obtained before treatment and weekly thereafter for 2 weeks. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured with a streptococcal superantigen or a conventional streptococcal antigen preparation, and cell culture supernatants were assayed for various cytokines. When stimulated with the superantigen, PBMCs from UVB-treated psoriasis patients secreted greater amounts of the anti-inflammatory cytokine IL-10, and showed markedly decreased production of IL-1β, IL-2, IL-5 and IL-6 compared to the pre-treatment values; the production of IFN-γ, IL-8 and IL-12p70 were also decreased but did not reach statistical significance. Thus, the combination of UVB-induced apoptosis, increased secretion of anti-inflammatory cytokines and decreased trafficking to the skin may help to explain the beneficial effects of UVB treatment on psoriasis and why disease remission can sometimes be sustained for a prolonged period.     

Further evidence for the self-reproducing capacity of Langerhans cells in human skin

The limited number of Langerhans cells (LC) in the epidermis is one of the main reasons for the technical difficulties in resolving the question of LC kinetics. In the present paper, we describe a method to evaluate the LC replication potential in epidermis. The procedure is based on the specific incorporation of bromodeoxyuridine (BrdU), a thymidine analogue, into the DNA during the S-phase of the cell cycle. Mice, bearing human skin grafts, were injected s.c. every 6 h for up to 17 days with BrdU. At different times, the incorporated BrdU as well as the human epidermal LC were revealed on skin sections using anti-BrdU and OKT-6 monoclonal antibodies, respectively. After 6 h, 4.9% of the LC were labeled with BrdU. Then, the number of OKT-6(+) BrdU(+) cells increased in a linear manner and achieved 34% at 120 h, 67% at 240 h, and 94% at 400 h during the course of continuous labeling procedures. Based on this result we calculated a total cell cycle time of 392 h (16.3 days) and 12 h for the S-phase for human epidermal LC. Applying this technique, we were able to show also that 48 h after local treatment with 12-O-tetradecanoylphorbol-13-acetate or after stripping, the number of BrdU-labeled LC was considerably increased. Furthermore, after i.p. injection of colchicine in the nude mouse, human epidermal LC undergoing mitosis were evidenced by electron microscopy in the graft. From these results we conclude that the LC are actively cycling--therewith a self-reproducing cell population in human epidermis.     

Langerhans cell migration is modulated by N-sulfated glucosamine moieties in heparin

Dendritic cell (DC) migration into and out of tissues is important for the generation of primary immune responses to antigens encountered in tissues. In order to study the mechanisms involved in DC migration we used a skin explant system and quantitated the number of Langerhans cells (LC), which are immature precursors of DC in skin-draining lymph nodes, remaining in the epidermis in response to incubation with various biomolecules. This paper shows that LC trafficking in epidermis is a metabolically active process that is modulated by heparin, specifically by N-sulfated glucosamine moieties in heparin. This is the first demonstration of structural specificity in the biochemical requirements for DC migration in a tissue and therefore is important to understanding DC migration in general.     

Migration of Langerhans cells in an in vitro organ culture system: IL-6 and TNF-alpha are partially responsible for migration into the epidermis

Although it is well established that epidermal Langerhans cells (LC) originate from bone marrow, little is known about the mechanism of this migration into the epidermis from bone marrow. In order to clarify the mechanism of this migration, we constructed an in vitro model. LC were depleted by daily topical application of clobetazole propionate (CP) solution onto the ear of Balb/c mice. Seven days later, ear skin was cut off, separated and co-cultured dermal-side-up with syngeneic (Balb/c), semisyngeneic ((C3H x Balb/c)F1), or allogeneic (C3H) epidermal cells (EC) for 3 days. We found (1) that a marked migration of donor LC into the recipient epidermis was observed in the LC-depleted skin, (2) that only syngeneic LC actively migrated into the recipient epidermis; however, the migration of semisyngeneic and allogeneic LC was detected at very low levels, (3) that the migratory capacity of donor LC was directly proved by a biolabeling technique using donor EC labeled with PKH-26, and (4) that anti-IL-6 and anti-TNF-alpha antibodies inhibited the migration of donor LC into the recipient epidermis. These data demonstrate that the resident LC have the potential to traffic through the dermis into the epidermis in a highly syngeneic-specific fashion, and that IL-6 and TNF-alpha are partially responsible for promoting this migration.     

Modulation of Ia+ Langerhans cell numbers in vivo by cultured epidermis derived supernatants and by GM-CSF

Abstract This paper demonstrates that epidermal cells in culture produce an activity which can increase the frequency of Ia⁺ epidermal Langerhans cells (LC). This was achieved by treating mice topically with a mixture containing supernatant derived from primary culture of murine epidermis (ES) and a synthetic corticosteroid, triamcinolone acetonide (TAG). The presence of the supernatant in the mixture partially protected the Ia⁺ LC from depletion by the steroid. The Ia⁺ LC frequency increasing activity was measured as the difference between the Ia⁺ LC frequency due to treatment with steroid mixed with supernatant and the Ia⁺ LC frequency due to treatment with steroid mixed with negative control medium. The mean frequency of Ia⁺ LC in epidermis treated with TAC mixed with ES was 606(SD 43) cells/mm², as compared with 486 (SD 68) cells/mm² in the epidermis treated with TAC mixed with control medium. The activity appeared to be caused by (a) proteinaceous factor(s). A fraction of ES which was retained above a ≥10 KDa molecular weight cut-off membrane was capable of partially protecting Ia⁺ LC frequency from TAC depletion. Supernatants from cultured lymph nodes, dermis as well as the squamous cell carcinoma lines T7 and T79, but not the human osteosarcoma cell-line 143B, also contained similar activities. We demonstrate that GM-CSF also increased the number of Ia⁺ epidermal LC when applied topically to mouse skin in this system. Therefore, using this Ia⁺ LC frequency modulation system, we propose that GM-CSF is one example of a cytokine which may be involved in the regulation of Ia⁺ LC numbers in epidermis and that epidermal cells produce factors which can increase the number of Ia⁺ LC.     

Defined in situ enumeration of T6 and HLA-DR expressing epidermal Langerhans cells: morphologic and methodologic aspects

An essential prerequisite for the in situ enumeration of epidermal Langerhans cells (LCs) is the unequivocal identification of the desired cell type. We have examined over 250 cryostat sections of normal human skin to analyze morphologic and methodologic problems underlying the quantification of epidermal LCs, defined by anti-T6 (OKT6) and anti-HLA-DR (OKIal) immunoperoxidase staining. Our findings show that OKT6 reactivity of dendritic processes in cross-sectioned epidermis yields microscopic images which are not easy to analyze objectively. The morphology that we find leads us to categorize dendritic cells into 3 arbitrary types of T6+ LC profiles. In addition we describe criteria for the assessment of OKT6 staining patterns relating to the dendritic state of epidermal LCs. Preliminary quantitative data on this issue are discussed in relation to: epidermal thickness; the thickness of skin tissue sections; and the discrepancy between the number of T6+ and HLA-DR+ LCs. We hope that the principles outlined in this report may serve to overcome potential methodologic problems with quantitation of T6+ epidermal LCs in skin sections.     

Differential localization of ICAM-1 and HLA-DR expression on epidermal basal surface in mycosis fungoides and lichenoid reaction

Scanning electron microscopy with immunogold labeling revealed that epidermal keratinocytes expressed ICAM-1 (intercellular adhesion molecule-1) and HLA-DR molecules on their surfaces in patterns that differed in mycosis fungoides (MF) and lichenoid reaction (LR). ICAM-1 molecules, visualized as deposits of gold particle, were visualized as clusters adjacent to the junctions interconnecting the keratinocytes of MF lesions. LFA-1 (leukocyte function-associated antigen-1) molecules were seen as granules on the surfaces of all infiltrates, most of which also expressed ICAM-1. HLA-DR molecules were seen continuously along the borders of the individual keratinocytes, thus producing a cobblestone appearance on the epidermal undersurface. In contrast, ICAM-1 and HLA-DR were found only sparsely on the undersurface of the epidermis from LR. These findings may help to explain the differing histological features of MF and LR: ICAM-1 molecules present on the intercellular junctions of MF epidermis lead the LFA-1-bearing cells to migrate into the interspaces, thus producing epidermotropism. These cells aggregate by means of co-expressed ICAM-1 to thus produce Pautrier's microabscess. In LR, the minimal expression of ICAm-1 on the epidermal undersurface leaves most infiltrates within the dermis, thus producing a band-like infiltrate.     

Epidermal Langerhans cells efficiently mediate CD1a-dependent presentation of microbial lipid antigens to T cells

Langerhans cells are a critical component of skin immunity, capable of capturing protein antigens in the epidermis and presenting them to specific T cells in the context of major histocompatibility complex class II molecules. Recently, a major histocompatibility complex independent pathway of lipid antigen presentation has been identified and is mediated by molecules of the CD1 family (CD1a, CD1b, CD1c, and CD1d). Because Langerhans cells are professional antigen-presenting cells and express CD1a molecules prominently, we hypothesized that Langerhans cells might play a role in T cell responses directed against not only peptide antigens but also lipid antigens. Here, we show that freshly isolated immature Langerhans cells as well as mature Langerhans cells that have migrated from the epidermis are efficient in presenting foreign microbial lipid antigens to specific T cells whereas dermal dendritic cells express much less CD1a molecules and function inefficiently. Further, we found that Langerhans cells migrating from epidermal sheets that were exposed to microbial lipid antigens expressed lipid-antigen-loaded CD1a molecules on the cell surface, resulting in activation of specific T cells. These results underscore an outstanding ability of Langerhans cells to mediate CD1a-dependent lipid antigen presentation. Thus, Langerhans-cell-mediated skin immunity may involve T cell recognition of both peptide and lipid antigens.