Changes in expression of Ia, Thy-1, and Ly-5 antigens in epidermal cells during delayed contact sensitivity reactions in mice: flow cytometric analysis

Ia antigen-bearing (Ia+) Langerhans cells have attained an important position as immunocompetent cells in the epidermis. Recently there have been successive reports on other new possible candidates for immunocompetent cells in the epidermis, i.e., Ia+ keratinocytes and dendritic Thy-1 antigen-bearing (Thy-1+) epidermal cells which also express Ly-5 antigen and asialo-GM1. Based on our previous findings that in allergic contact sensitivity reactions, keratinocytes express Ia antigen 3-9 days postchallenge, in this report, we have attempted to define more clearly the dynamic changes of Ia+ keratinocytes and dendritic Thy-1+ epidermal cells by enumeration of the precise percentages of Ia+, Thy-1+, and Ly-5 antigen-bearing (Ly-5+) cells in epidermal cells at various times of the challenge phase in allergic contact sensitivity reactions by use of a fluorescence-activated cell sorter. By 24 h postchallenge, the percentages of Ia+, Thy-1+, and Ly-5+ cells showed hardly any change. There were approximately 2% Ia+ cells, 50% Thy-1+ cells which consist of 2 populations (i.e., 45% weakly Thy-1 antigen-positive cells and 4% strongly Thy-1 antigen-positive cells), and 3.5% Ly-5+ cells. From 48 h postchallenge, however, the percentage of Ia+ cells and that of Thy-1+ cells began to increase and reached a plateau, with approximately 20% Ia+ cells and 70% Thy-1+ cells, respectively, at 120 h postchallenge. The change of the percentages of Ly-5+ cells appears to correspond to that of strongly Thy-1 antigen-positive cells. Only at 48 h postchallenge, Ly-5+ cells and strongly Thy-1 antigen-positive cells showed a small increase in number, comprising approximately 10% of the epidermal cells. These data suggest that among Thy-1+ epidermal cells, strongly Thy-1 antigen-positive cells correspond to dendritic Thy-1+ epidermal cells, and in contact sensitivity reactions in mice, dendritic Thy-1+ epidermal cells show only a minor dynamic change in contrast to Ia+ cells, in which more than 15% of keratinocytes express Ia antigen from 48 h postchallenge.     

Dynamic changes in epidermal Ia-positive cells in allergic contact sensitivity reactions in mice

In mice sensitized with trinitrochlorobenzene, serial changes in epidermal Ia-positive cells were studied at various times after challenge. Until 3 days post-challenge, the Ia-positive cells consisted only of dendritic Langerhans cells; their number was decreased but they were significantly enlarged, with extending dendrites. Some Langerhans cells were also found surrounding a hair follicle, extending their dendrites toward the follicle like the spokes of a wheel. From 3 to 9 days after challenge, keratinocytes also began to express Ia antigens in the epidermis in addition to Langerhans cells, whose size diminished. This suggests that there are two phases in the response of the epidermal Ia antigens in contact sensitivity reactions, i.e. an early phase in which enlarged Langerhans cells are the only Ia-positive cells in the epidermis, and a late phase in which keratinocytes take over as the major Ia-positive cells, while Langerhans cells resume their original size. Ia antigen expression on keratinocytes in this late phase probably plays a crucial role in completely eliminating allergens deposited on the keratinocytes.     

Quantitation of cutaneous Langerhans cells of sarcoidosis patients

Langerhans cells play a role in cell-mediated immune reactions which are often depressed in sarcoidosis. We examined the epidermis of 17 anergic patients with sarcoidosis (Kveim-reactive and/or biopsy-proved) for the number of Langerhans cells in noninvolved skin and in any cutaneous sarcoidal lesions. Skin biopsies of 10 healthy volunteers served as controls. In comparison to controls, the epidermis overlying noninvolved (p less than 0.05), sarcoidal (p less than 0.0005), and Kveim-reactive (p less than 0.005) skin contained significantly fewer detectable Ia and T6 antigen-bearing Langerhans cells. The reductions within noninvolved skin were most pronounced in patients with multisystem disease. Lower epidermal Langerhans cell densities, in comparison to controls, were detected in both prednisone-treated and untreated patients. Epidermis overlying sarcoidal skin of untreated patients contained significantly fewer Ia and T6 antigen-bearing Langerhans cells (p less than 0.05, p less than 0.0025, respectively) than epidermis from noninvolved skin. Whether reduced numbers of cutaneous Langerhans cells are due to either a local and/or systemic effect of sarcoidosis, or reflect the anergic state of these patients is unknown.     

Expression of OKM5 antigen on epidermal cells in mycosis fungoides plaque stage

To characterize and quantitate potential antigen-presenting cell subsets in the epidermis of patients with cutaneous T-cell lymphoma, epidermal cells in suspension were obtained from involved and uninvolved skin. Involved epidermis contained increased numbers of OKT6+HLA-DR+ Langerhans cells and a variable number of OKM5+ epidermal cells (ECs) in all mycosis fungoides (MF) patients tested (N = 14). The OKM5+ EC population from involved epidermis of MF patients were heterogeneous and comprised both OKM5+HLe1- keratinocytes and OKM5+HLe1+ leukocytes. Uninvolved epidermis, in 6 of 14 patients with MF, contained a small number of OKM5+ leukocytes; however, no OKM5+ keratinocytes were detected. Neither OKM5+ leukocytes nor OKM5+ keratinocytes were detected in the epidermis obtained from healthy controls. The increased number of potential antigen-presenting cells, that is, OKT6+HLA-DR+ Langerhans cells and OKM5+HLA-DR+ monocytic leukocytes, in the epidermis of patients with MF may be important for the activation of abnormal T cells contained within the epidermis of these patients. Such activated T cells may release gamma-interferon and induce expression of both HLA-DR and OKM5 antigens on keratinocytes. OKM5+ keratinocytes are present in the epidermis of patients with MF, but not in normal skin, and may thus play a role in the pathogenetic mechanisms of mycosis fungoides by recruitment of immunocompetent cells to the epidermis.     

Thy-1 antigen-bearing dendritic cells populate murine epidermis

Two distinct cell populations, melanocytes and Langerhans cells (LC), have been recognized previously to possess dendritic configuration in normal mammalian epidermis. Employing immunofluorescence microscopy with monoclonal antibodies against Thy-1.2 antigen to identify cells in whole mounts of murine epidermis, we have identified a third dendritic cell population which differs from both LC and melanocytes. Thy-1 antigen-bearing (Thy-1+) epidermal cells are primarily dendritic, although round and angular forms may be found. They are distributed relatively evenly across skin surfaces, although densities vary greatly from site to site and from strain to strain. Densities were highest in ear epidermis from the pigmented strain B10.A (580 cells/mm2), a value approaching that of epidermal LC, and were lowest in ear epidermis from the albino strain BALB/c (5 cells/mm2). Thy-1+ epidermal cells possess neither Ia antigens nor substantial amounts of melanin, and their surface distributions are disparate from those of both LC and mature melanocytes. We propose that at least some of these cells are T lymphocytes whose malignant counterparts account for cutaneous T-cell lymphomas.     

Epidermal Langerhans cells,dermal dendritic cells,and keratinocytes in viral lesions of skin and mucous membranes: an immunohistochemical study

Summary We wanted to evaluate the eventual expression of viral antigens and MHC class II products by keratinocytes as well as the alterations of epidermal Langerhans cells and dermal dendritic cells in viral lesions of skin and mucous membranes. Therefore we investigated 68 biopsy specimens of protracted viral lesions, such as warts, condylomas, and mollusca contagiosa, and of rapidly resolving viral lesions such as herpes simplex virus infection. For this we used immunohistochemical staining techniques and several monoclonal and polyclonal antisera. In most cases investigated viral antigens (human papilloma virus antigens or herpes simplex virus type 1 antigens) could be demonstrated in keratinocytic nuclei. Except for a few viral lesions in which epidermal Langerhans cells were rather numerous, epidermal Langerhans cells were reduced in number or absent in almost all viral lesions. Moreover, epidermal Langerhans cells and dermal dendritic cells showed changes in morphology, distribution, and immunophenotype. These alterations may be caused by a toxic effect of the virus on dendritic cells. HLA-DR⁺ keratinocytes could be identified in few viral lesions onlyl HLA-DQ⁺ keratinocytes were not seen. Possible explanations for this lack of MHC class II expression by keratinocytes are discussed.Aspirant of the NFWO (National Fund for Scientific Research)     

Acrosyringium and follicular infundibulum: immunomorphologic aspects]

Acrosyringium and infundibulum are epidermal structures with different morphology, but functional similarities regarding re-epithelialization, transport of antigens, and attraction of infiltrates. Immunomorphologically, the infundibula revealed an immunoreactivity of Langerhans cells and keratinocytes comparable with that of the interfollicular epidermis, whereas the acrosyringia reacted differently because of the permanent expression of MHC class II antigens and reduced Langerhans cells in the perisyringial region. Our findings suggest that these epidermal compartments differ with regard to their individual immunologic situations. While the mechanisms of epidermal immunity known from the interfollicular epidermis are possibly the same in the infundibula, we have to expect a basically different immunologic situation in the acrosyringia.     

Modulation of expression of epidermal Langerhans cell properties following in situ exposure to glucocorticosteroids

Langerhans cells (LC) have been implicated as antigen-presenting and target cells in contact allergic, cell-mediated reactions. We have examined the effects in guinea pigs, rats, and humans of in situ (epicutaneous) exposure to glucocorticosteroids (GCS) on the expression of epidermal LC markers. Reductions in the number of Fc-rosetting, C3b-rosetting, and immune-associated (Ia) antigen-bearing LC occurred in a dose-related fashion, with the degree of such reductions dependent upon the specific GCS employed. These reductions were determined to be reversible following cessation of exposure to GCS. T6 antigenicity, another cell surface marker of human LC, was little affected by GCS exposure. Simultaneous immunofluorescent staining for T6 and Ia antigenicity within human epidermis of amcinonide treated skin detected reduced numbers of T6+/Ia+ cells with a concomitant increase in T6+/Ia- cells. The data presented suggest a selective reduction in the expression of immunologically important receptors and antigens by LC which may be involved in steroid-responsive contact allergic reactions.     

Accessory and adhesion molecules expressed on murine epidermal Langerhans cells and the modulation by cytokines.

Langerhans cells (LC) are MHC class II (Ia)-positive dendritic cells that act as an antigen-presenting cells for T cell-dependent immune responses. LC originate from cells in bone marrow and migrate into the epidermis through blood vessels. LC also migrate from the epidermis to regional lymph nodes after antigen stimulation where they present antigens to T cells. These are the essential features of LC. The morphological and functional properties of LC are modulated by external stimuli or various cytokines. In this review we focus on the accessory and adhesion molecules on LC and describe how these molecules are modulated by cytokines. We also describe the molecules participating in the migration of LC into and from the epidermis. Moreover, we introduce our data obtained from purified murine epidermal LC and from the transgenic mice overexpressing several cytokines in the epidermis.     

Behavior of cutaneous Langerhans' cells and skin reactivity after gold sodium thiomalate treatment of pemphigus vulgaris

Skin biopsies from pemphigus vulgaris patients in the acute phase of disease and after successful GST therapy were studied in respect to their capacity to express OKT6, OKIa1 and HLA-DR antigens. In comparison to controls, the epidermis overlaying noninvolved skin contained significantly (p = 0.0001) fewer detectable Ia and T6 antigen-bearing Langerhans' cells. Lower epidermal Langerhans' cell density in comparison to controls was detected in both prednisolone-treated and untreated patients. A normalization of prior defective antigen expression was observed in patients in remittence after GST therapy. This correlated with a normal delayed type hypersensitivity reaction (Multitest Mérieux).     

Growth and differentiation of human epidermal cultures used as auto- and allografts in humans

Human keratinocytes from small skin specimens were grown on mouse 3T3 cell feeder layers into epidermal sheets free from Langerhans cells and MHC class II antigen. These were found to be suitable for the permanent coverage of wounds when used as autografts or allografts. We report here the ultrastructural differentiation of this cultured epidermis after grafting onto autologous or allogeneic recipients. The cultured epidermis was a thin but multilayered Malpighian epithelium composed of keratinocytes at different stages of differentiation. The dermo-epidermal basement membrane was newly synthesized during the first few days following transplantation onto de-epidermized wounds. The analysis of keratins and examination of various keratinocyte membrane antigens by immunofluorescence indicated that full terminal epithelial differentiation was only achieved after in vivo transplantation of the cultured epidermis. Langerhans cells, absent in cultures, progressively colonized the grafts, while melanocytes, not detectable in sections of the cultures, were identified among the keratinocytes 2 weeks after grafting.     

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.     

In situ characterization of cellular infiltrates in lupus vulgaris indicates lesional T-cell activation.

Skin biopsy specimens from nine patients with lupus vulgaris were examined in situ by means of monoclonal antibodies directed against phenotypes of lymphocyte subsets, Langerhans cells, HLA-DR antigens, and interleukin 2 receptor. The epidermis showed prominent changes, including intense expression of HLA-DR on keratinocytes, increase in epidermal cell layers, moderate to high Langerhans cell hyperplasia, and infiltration by CD3+ pan-T cells as well as CD8+ (cytotoxic/suppressor) and CD4+ (helper/inducer) T cells. The predominant lymphocyte in the dermal granulomas was the activated CD3+ T cell, expressing major histocompatibility complex class II antigens and interleukin 2 receptor. CD4+ and CD8+ cells were randomly distributed among the epithelioid cells, which showed intense staining for major histocompatibility complex class II antigens. In all except two patients, the CD4+ population was greater than that of the CD8+ cells. CD1+ Langerhans cells were scattered in moderate numbers in the dermal granulomas. Acid-fast bacilli were conspicuously absent in the biopsy specimens. These features suggest that T-cell activation and Langerhans cell hyperplasia are prominent features of dermal tuberculosis.     

The functional maturation of Langerhans cells in CTL induction during tissue culture

In the previous report, we investigated the effects of tissue culture on the APC function of murine epidermal Langerhans cells (LC) in the induction of allo-CTL in vitro, and found that (1) cultured ear LC expressed increased amounts of Ia antigens on their cell surface, and (2) they induced extremely enhanced levels of CTL over those produced by freshly prepared ear LC and also (3) cultured tail LC were proved to be able to induce CTL for the first time. It seemed that tissue culture decreased the functional heterogeneity among the murine ear LC and tail LC in addition to that among Ia+ APC in spleen and in epidermis. In this study, we investigated the culture conditions that increase the APC function of LC. Our data indicate that LC cultured with dermal components exhibited more enhanced APC function than LC cultured in single cell suspension with only epidermal cells. Recent studies indicate that IL-1 and GM-CSF, which keratinocytes release, are essential for freshly prepared LC to mature into highly efficient APC that resemble splenic dendritic cells. We found dermal factors are more important than epidermal ones for LC to mature in tissue culture.     

A surface glycoprotein complex related to the adhesive receptors of the VLA family, shared by epidermal Langerhans cells and basal keratinocytes

A murine monoclonal antibody, designated K20, was raised by immunization with a human malignant T-cell line. It reacted specifically with membrane glycoprotein complexes on early haematopoietic cells, T cells, and monocytes. In epidermis, K20 specifically reacted with Langerhans cells and basal keratinocytes, as demonstrated by double labeling experiments. Membrane immunoprecipitation analysis demonstrated that the antigen identified by K20 on lymphoid cells and epidermal cells was different. While on lymphoid cells, K20 recognized glycoprotein complexes made of a constant 130-kD subunit associated with subunits of higher molecular weight ranging from 150 to 200 kD, a complex of 105-145 kD was precipitated from Langerhans and basal cells. Metabolic labeling studies demonstrated that these proteins were synthesized by the basal cells. The antigen identified by K20 was thought to belong to the integrins, a family of cell surface receptors that play a role in cell adhesion, cell interactions, wound healing, and immune defense mechanisms. K20 is the first monoclonal antibody that specifically recognizes a membrane antigen common to Langerhans and basal cells. Additionally, K20 is the first of five reported monoclonal antibodies to have been characterized on the epidermal cells that detect antigens shared by lymphoid subpopulations and normal basal keratinocytes.     

HLA class I antigen (heavy and light chain) expression by langerhans cells and keratinocytes of the normal human epidermis: ultrastructural quantitation using immunogold labelling procedure

Summary Using an immunogold labelling procedure, we quantified the density of major histocompatibility (MHC) class I antigens on the surface of Langerhans cells (LCs) and keratinocytes of the normal human epidermis. According to ultrastructural features, keratinocytes were divided into three subpopulations: stratum basalis (SBK), stratum spinosum (SSK), and stratum granulosum keratinocytes (SGK), and analyzed separately. For this purpose, three monoclonal antibodies (MCAs) were employed: an anti-HLA A,B,C, an anti-B₂-microglobulin (B₂-m), and a polymorphic anti-HLA A2 Aw69 MCA. Under electron microscopy, quantitative analysis demonstrated: (a) the presence of a high amount of HLA monomorphic determinants on SBK and SSK and moderate but significant labelling of SGK; (b) the very weak density of MHC class I antigens on the surface of epidermal LCs; (c) the expression, at an identical level, of the HLA heavy chain common determinant (HLA A,B,C), B₂-m, and the alloantigen HLA A2 by all epidermal cells (ECs) apart from SGKs and LCs that presented far fewer HLA A2 sites than monomorphic determinants (B₂-m and HLA A,B,C); (d) the absence of HLA class I on corneocytes and a moderate labelling of melanocytes. A knowledge of the precise quantitative distribution of HLA class I antigens among various cell subpopulations of the normal human epidermis would be very useful for the study and followup of cutaneous malignancies that are known to lose these molecules as well as for the understanding of immune responses, especially allospecific, that involve the skin.     

In vitro model of essential fatty acid deficiency.

The polyunsaturated fatty acids linoleic acid (18:2, n-6) and arachidonic acid (20:4, n-6) are essential for normal skin function and structure, both as eicosanoid precursors and as components of lipids forming cell membranes. Adult human keratinocytes grow optimally in serum-free medium (MCDB 153) that contains no fatty acids. These keratinocytes expand rapidly and produce normal epidermis upon in vivo grafting. Analysis of lipid extracts of epidermis and of cultured keratinocytes was done to determine the fatty acid composition of cells grown in essential fatty acid (EFA)-deficient medium. Gas chromatography and high-performance liquid chromatography analyses were done of the fatty acids in the entire cell and in a thin-layer chromatography separated fraction containing those lipids that form cellular membranes. Comparison of snap-frozen epidermis and epidermal basal cell suspensions to passage 1 to 4 cultures shows that the cells are in an extreme essential fatty acid-deficient state by the first passage. The amount of the saturated fatty acids 16:0, 18:0, and 14:0 is unchanged by culture. The polyunsaturated fatty acids are found to be significantly decreased, the cells balancing their lack with a significant increase in the relative abundance of the monounsaturated fatty acids, 18:1 and 16:1. Greater than 85-90% of the fatty acids was found in lipids associated with membranes and no unusual fatty acids were detected. Because the serum-free medium is fatty acid free and the cells cannot synthesize essential fatty acids, the rapid division of the cells results in the predominance of an extreme EFA-deficient cell type. The essential fatty acid-deficient keratinocyte is an excellent adult, normal epidermal cell model that can be used to study EFA deficiency and the effect of the eicosanoid and fatty acids on cell function and structure.     

Thy-1阳性树枝状表皮细胞及郎格罕细胞的比较观察

观察了青、老年小鼠及大鼠不同部位皮肤内的Thy-1阳性树枝状表皮细胞(Thy-1⁺dEC)及郎格罕细胞(LC).于C57BL/6Ss小鼠,Thy-1⁺dEC通常比LC大;Thy-1⁺dEC的Thy-1抗原主要存在于细胞膜,而LC的Ia抗原较均匀分布,Thy-1⁺dEC突起常比LC短,有的甚至并不明显,正常角肮细胞表面也有少量Thy一抗原,但无Ia抗原.二种细胞于老年动物都少于青年动物.于Lewis/Ss及BN/Ss大鼠,未见到Thy-1⁺dEC.我们认为Thy-1⁺dEC可能具有免疫学功能.对人的Thy-1⁺dEC相应物的探索,也许能在皮肤病发病机理的研究中展开一条新的途径.     

The expression of MHC class II (Ia) antigens on mouse keratinocytes following epicutaneous application of contact sensitizers and irritants

The expression of MHC class II (Ia) antigens on mouse keratinocytes was studied following both the induction and elicitation of contact sensitivity, and after primary irritant reactions. IA+ and IE+ keratinocytes were detected, using an indirect immunofluorescence assay on epidermal sheets, only after the induction and elicitation of contact sensitivity with the sensitizers oxazalone, picryl chloride and 2,4-dinitrochlorobenzene but not with formaldehyde. Ia+ keratinocytes were not detected after epicutaneous application of the non-sensitizing irritants croton oil, SDS and anthralin, or following attempted sensitization of nude mice, suggesting that the expression of Ia antigen on keratinocytes during contact sensitivity reactions is T-cell mediated. Because Ia antigen expression on keratinocytes could be detected only several days after induction or elicitation of contact sensitivity, and contact sensitization could also be demonstrated to occur independently of aberrant Ia expression, Ia+ keratinocytes cannot be involved in the initiation of these reactions. However, they might be important in exerting an immunomodulatory influence during the later stages of the responses to certain sensitizers.     

Antigenic profile of human acrosyringium

The demonstration of HLA-DR on human acrosyringium has led to the suggestion that eccrine epithelium, through its interaction with certain molecules, might play an active role in epidermal immune responses. An immunohistochemical study was undertaken to identify the antigenic profile of acrosyringium in normal skin and following the intradermal administration of a T-lymphocyte-derived cytokine, interferon gamma (IFN-gamma). Acrosyringium in normal skin, in contrast to interappendageal epidermis, was found to lack CD1a+ Langerhans cells. However, antigens CD36 (OKM5) and L1 (MAC387) were uniquely expressed by keratinocytes immediately adjacent to the distal portion of acrosyringium. Constitutive expression of each class II MHC antigen, namely HLA-DR, DP and DQ was observed on luminal acrosyringial cells. EMB11 antigen (CD68), a mononuclear cell determinant, was similarly expressed on acrosyringial epithelium in normal skin. Following intradermal administration of IFN-gamma, intercellular adhesion molecule-1 (ICAM-1) (CD54) was induced on acrosyringial epithelium and the expression of HLA-DR was intensified. A range of other markers including CD3, CD4, CD8, CD11a, CD11b and CD15 were not expressed by acrosyringium either in normal skin or after administration of IFN-gamma. Expression of antigens associated with cell-mediated immune mechanisms on acrosyringium is consistent with the hypothesis that it may have an immunological role in epidermal immune responses.