Skin Cell-lymphocyte Reaction Research Articles

Accessory function of human Langerhans cells in the primary allogeneic T-cell response.

Beyond antigen processing and presentation, antigen-presenting cells must convey additional signals that are necessary for T-cell activation and proliferation. In this study we analyzed the accessory function of human Langerhans cells (LC) in the primary T-cell response to alloantigens. We used paraformaldehyde-fixed LC suspensions to dissociate membrane-associated accessory structures from soluble activating factors. When freshly prepared LC-enriched (eLC, 8-20% LC) epidermal cell suspensions were fixed, normal allogeneic T cells failed to proliferate. This defect cannot be overcome by the addition of either interleukin (IL)-1 and/or IL-6 during the mixed skin cell lymphocyte reaction (MSLR). By contrast, when eLC were first incubated for 3 d, and especially in the presence of interferon gamma (IFN-gamma) before fixation, they retained significant allostimulatory property that did not correlate with increased MHC class II antigen expression. Furthermore, we showed that once LC have been activated, IL-1, but not IL-6, could serve as a co-stimulatory factor in the primary allogeneic T-cell response. In conclusion, the data suggest that human LC accessory function is not constitutive but requires an activation step, which can be provided by IFN-gamma during LC-T-cell interaction.

A Potential Role for CD1a Molecules on Human Epidermal Langerhans Cells in Allogeneic T-Cell Activation

The structural similarities of CD1a molecules to major histocompatibility complex (MHC) class I antigens, as well as their expression on epidermal antigen-presenting cells suggest that CD1a molecules might be involved in the cutaneous immune response. In the present study, we investigated the effect of different anti-CD1a monoclonal antibodies (BL6, DMC1, and Na1/34) on T cell proliferation induced by allogeneic epidermal cells in vitro. A significant inhibition of the mixed skin cell-lymphocyte reaction was obtained with BL6 and DMC1 monoclonal antibodies (MoAb), which recognize the same epitope on CD1a molecule. The observed inhibition could not be related to a steric hindrance of MHC class II molecules, because Na1/34 MoAb, which reacts with another epitope on CD1a molecule, had no significant effect. BL6 and DMC1 MoAb interfered with an early event of T-cell activation, as shown by a time-course study. In the presence of these MoAb, the addition of exogenous interleukin 2 did not restore T-cell proliferation. Furthermore, the inhibitory effect of anti-CD1a MoAb was not mediated by a suppressor factor released by Langerhans cells (LC). These present data suggest that CD1a molecule may have an important function in self peptide presentation by human Langerhans cells.

A Method for the Rapid Isolation of Human Epidermal Langerhans Cells Using Immunomagnetic Microspheres

Because Langerhans and indeterminate cells are the only epidermal cells that express the specific CD1a surface antigen T6, we have used immunomagnetic monodisperse polymer microspheres for positive selection of human epidermal Langerhans and indeterminate cells. Epidermal cells in suspension are successively incubated with a murine monoclonal anti-T6 antibody of the IgG1 subclass and then with magnetic beads coated with a sheep anti-mouse IgG1. Rosetted cells are obtained and then easily separated from the non-rosetted cells using a magnet. The two cell fractions are characterized by phase contrast microscopy, immunofluorescence, electron microscopy, and the skin cell-lymphocyte reaction. All the rosetted cells (1.5 to 5% of the total epidermal cells) express T6 antigen by indirect immunofluorescence and under the electron microscope possess all the ultrastructural characteristics of Langerhans cells. Moreover, the rosetted Langerhans cells remain functional: Under the electron microscope they internalize by receptor-mediated endocytosis gold labeled anti-T6 antibody, and in the skin cell-lymphocyte reaction they stimulate allogeneic lymphocytes. In contrast, the rosette depleted cell fraction is deprived of T6 positive cells and unable to stimulate allogeneic lymphocytes. The immunomagnetic depletion of epidermal cells is a simple and rapid method to isolate functional human Langerhans cells with good yield and high purity (97%). This technique should be of value in the study of the pharmacology of Langerhans cells and in the investigation of the interactions of Langerhans cells with keratinocytes or lymphocytes.

Loss of allogeneic T-cell activating ability and Langerhans cell markers in human epidermal cell cultures

Human Langerhans cells are the only epidermal cells that express the T6 and HLA-DR antigens and are responsible for the in vitro allogeneic T-cell proliferative responses in the mixed skin cell lymphocyte reaction (MSLR). To investigate the presence of Langerhans cells in normal human epidermal cell cultures, epidermal cell suspensions obtained from normal human skin specimens and from the subsequent epidermal cell cultures were analyzed by indirect immunofluorescence for the presence of T6 and HLA-DR determinants. In parallel, MSLRs were conducted with suspensions of cultured epidermal cells as stimulatory cells. These studies present evidence that when human epidermal cells are grown in culture, they loose both the ability to stimulate the proliferation of allogeneic T lymphocytes in vitro and their expression of HLA-DR and T6 antigens. The T6 antigens were lost during the first 2 weeks of culture, while HLA-DR determinants were still expressed by a small number of cells and were progressively lost through duration of cultures. The loss of HLA-DR antigens closely paralleled the progressive inability of human epidermal cells in culture to stimulate allogeneic T cells in MSLR.

Enrichment of Murine and Human Langerhans Cells with Solid Phase Immunoabsorption Using Pan-Leukocyte Monoclonal Antibodies

Using a solid phase immunoabsorption (panning) technique, we have employed pan-leukocyte monoclonal antibodies to enrich and deplete murine and human Langerhans cells from cell suspensions of normal skin. Langerhans cell-enriched fractions contained 80-99% mononuclear cells, almost all of which had the ultrastructural features of Langerhans cells. These results are comparable to those achieved by panning for human Langerhans cells with anti-Leu-6(T6) antibody. Similarly, less than 1% of these cells were detectable in Langerhans cell-depleted fractions and such fractions were incapable of stimulating allogeneic lymphocytes in the skin cell-lymphocyte reaction. We conclude that panning with pan-leukocyte antibodies is an effective means of enriching or depleting Langerhans cells from heterogeneous skin cell suspensions and can yield results similar to those achieved with more Langerhans cell-specific reagents such as anti-Leu-6(T6). These findings are of particular significance to the enrichment and depletion of murine Langerhans cells since they express no known correlate of the human Leu-6(T6) antigen.

Loss of HLA-DR Expression by Human Epidermal Cells After Growth in Culture

These studies present evidence that when human epidermal cells are grown in culture they lose both the ability to stimulate the proliferation of allogeneic T lymphocytes in vitro and their expression of HLA-DR antigens. Our results also show that epidermal cells incubated with anti-HLA-DR serum lose their ability to stimulate the proliferation to allogeneic T lymphocytes in a mixed skin cell-lymphocyte reaction.

Inhibition of a Langerhans Cell-Mediated Immune Response by Treatment Modalities Useful in Psoriasis

Neither the pathogenesis of psoriasis nor the mechanism whereby seemingly diverse therapies alter the disease is understood. In this study, several antipsoriatic agents were tested for their effects on the skin cell lymphocyte reaction (SLR), an immunologic assay in which HLA-DR antigens on Langerhans cells (LC) stimulate proliferation of allogeneic lymphocytes. Every agent tested (cortisol, methotrexate, hyperthermia, anthralin) inhibited the SLR at therapeutic dose levels. By contrast, a variety of antibiotics, an anti-inflammatory agent, and lithium carbonate and propranolol, two drugs known to be ineffective in psoriasis, failed to inhibit the SLR. Finally, we have shown that hyperthermia and anthralin treatments are toxic for LC whereas they have little or no effect on keratinocyte viability. These results suggest that antipsoriatic agents may act in psoriasis by alteration or killing of LC.

Separation of Human Skin Cells by Velocity Sedimentation into Functionally Distinct Fractions

The epidermis consists of a heterogeneous population of cells including Langerhans cells, Merkel cells, melanocytes, and keratinocytes in various stages of differentiation. The current study was undertaken to determine if skin cell suspensions can be separated into morphologically and/or functionally distinct fractions. Skin cells were suspended by trypsinization and separated into multiple fractions by velocity sedimentation. Certain fractions reproducibly stimulated proliferation of allogeneic lymphocytes in the skin cell lymphocyte reaction, whereas other fractions, containing larger cells, supported growth of keratinocyte colonies in cell cultures. These results indicate that stimulation in the skin cell lymphocyte reaction and growth of keratinocyte colonies are mediated by distinct cells, separable by velocity sedimentation.