0000000000150219
AUTHOR
Mansour Mohamadzadeh
Freshly isolated mouse 4F7+ splenic dendritic cells process and present exogenous antigens to T cells.
The antibody 4F7 was reported to recognize an epitope expressed on dendritic cells (DC) from various tissues. To study the ability of splenic 4F7+ dendritic cells to process antigen for presentation to CD4+ T cells, DC were enriched using a separation procedure avoiding overnight culture which could lead to an altered phenotype. These DC were used as antigen-presenting cells (APC) in stimulation cultures of major histocompatibility complex class II-restricted T cells. It was found that they induce antigen-dependent lymphokine production by T cells and therefore could present exogenous antigens. These processing takes place intracellularly, because fixation abrogates presentation to T cells.…
Increased Level of Intracellular MHC Class II Molecules in Murine Langerhans Cells Following In Vivo and In Vitro Administration of Contact Allergens
Treatment of murine Langerhans cells (LC) with contact allergens results in increased internalization of cell membrane constituents and therefore in depressed cell-surface expression of major histocompatibility complex (MHC) class II molecules during the first hours after haptenization. In this presentation we show that this downregulation of cell-surface-expressed Ia-antigens is accompanied by an augmentation of the intracellular pool of MHC class II molecules. Rat MoAb 2G9 was developed, which recognizes IA and IE molecules of the d-haplotype. This MoAb competes with the murine MoAb MK-D6 for binding sites to IAd-molecules. After blocking the cell-surface-expressed molecules with 2G9 and …
Induction of inflammatory cytokines in murine keratinocytes upon in vivo stimulation with contact sensitizers and tolerizing analogues
In order to elucidate the role of keratinocytes (KCs) in the induction of contact sensitivity, we applied various contact sensitizers [2,4-dinitrofluorobenzene (DNFB), urushiol, 3-n-pentadecylcatechol (PDC), 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone)] and tolerizing compounds [2,4-dinitrothiocyanobenzene (DNTB), 5-methyl-3-n-pentadecyl-catechol (5-Me-PDC)] onto the earskin of non-sensitized Balb/c mice. In addition, we applied croton oil as a non-sensitizing, but stimulatory agent. Cytokine production was demonstrated by Northern blot hybridization of the total cellular RNA extracted from epidermal cells depleted by Langerhans cells and Thy 1+ dendritic cells using radiolabeled DNA …
Human Keratinocyte-Derived IL-12 Affects LC-Induced Allogeneic T-Cell Responses
Our laboratory has recently defined the cytokines involved in the early induction phase of contact sensitivity that help LC to become more potent APC and that help to direct this classical Thl immune response1,2.
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. So…
In vitro analysis of the phenotypical and functional properties of the 4F7+ cutaneous accessory dendritic cell
The monoclonal antibody 4F7 detects a molecule on dermal and epidermal Ia+ dendritic cells (DCs), and some of these cells are Birbeck granule-containing cells. Here we report on the phenotypical and functional characteristics of these cells which were highly enriched by 4F7-labelled immunomagnetic beads. The ultrastructural, immunocytochemical and cytochemical analyses of these preparations showed cells with the typical characteristics of DCs. The cells were found to express the DC marker NLDC145, but not 33D1. The C3bi receptor and marker F4/80 were only expressed by epidermal 4F7+ cells. The capacity of freshly isolated 4F7+ epidermal and dermal DCs to activate allogeneic T cells in a mix…
Induction of hapten-specific tolerance by interleukin 10 in vivo.
Interleukin 10 (IL-10) is released during the induction phase of contact sensitivity and was shown in prior functional studies to convert epidermal Langerhans cells (LC) from potent inducers of primary immune responses to specifically tolerizing cells in vitro. To investigate whether IL-10 also subserves the function of a tolerizing agent in vivo ears of BALB/c or C3H mice were injected intradermally with 1-2 micrograms of recombinant mouse (rm)IL-10 8 h before epicutaneous application of 3% trinitrochlorobenzene (TNCB; a contact allergen). As a control, mice were injected with phosphate-buffered saline or IL-10 plus neutralizing amounts of anti-IL-10 mAb. 5 d later, mice were challenged wi…
Cytokine expression and antigen-presenting capacity of 4F7 + dendritic cells derived from dermis, spleen, and lymph nodes
We took advantage of the recently generated 4F7 mAb, which recognizes an epitope expressed on dendritic cells (DC) from different tissues, to freshly isolate and positively sort for these cells and to characterize their cytokine pattern and antigen-presenting capacity in comparison with epidermal Langerhans cells (LC). RT-PCR and Northern blot analyses demonstrated constitutive mRNA expression of MIP-1 gamma, MIP-1 alpha, C10, and IL-1 beta in both 4F7+ DC and LC. Lipopolysaccharide (LPS) treatment resulted in the upregulation of mRNA expression of all four cytokines and in a newly detected signal for TNF alpha. Immunoblot analysis showed constitutive secretion of MIP-1 gamma, with LPS trea…
Regulation of cytokine expression by interferon-alpha in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of interleukin-1 receptor antagonist
We investigated the effects of interferon-alpha (IFN-alpha) on the expression of cytokines by human bone marrow stromal cells. Production of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), and interleukin-1 beta (IL-1 beta) in stromal cell layers was induced by incubation with IL-1 alpha, tumor necrosis factor (TNF), or lipopolysaccharide (LPS). Addition of IFN-alpha to such stimulated cultures resulted in a strong downregulation of mRNA expression of GM-CSF and IL-1 beta. Similarly, the protein levels of GM- CSF and IL-1 beta were significantly reduced by IFN-alpha, whereas G- CSF production was only moderately inhibited. In contrast, IFN-alpha markedly …
Heat-stable antigen is expressed by murine keratinocytes and delivers costimulatory signals in T-cell activation.
Heat-stable antigen (HSA), expressed by various antigen-presenting cells (APC), has been described as a costimulatory molecule for CD4+ T cells. Recently, we observed that HSA also serves as an important costimulatory molecule on epidermal Langerhans cells (LC). During these studies, low levels of HSA staining were also detected on normal murine keratinocytes (KC). To investigate whether HSA also is involved in T-cell activation by KC, normal murine KC or the spontaneously transformed KC cell-line PAM 212 were treated with PDB or PMA to induce HSA-expression. FACS analyses showed induction of HSA expression on normal murine KC, as well as PAM 212 cells. In functional assays PDB or PMA-treat…
Modulation of Contact Sensitivity Responses by Bacterial Superantigen
Superantigens are potent modulators of the immune system, especially T cells. Therefore, we determined the influence of superantigens on the T-cell-mediated immune response, contact sensitivity. We chose the combination of staphylococcal enterotoxin B (SEB) as superantigen and 2,4-dinitrofluorbenzene (DNFB) as the contact sensitizer, because in BALB/c mice SEB reacts almost exclusively with V beta 8+ T cells, and these cells are capable of transferring contact sensitivity to DNFB from sensitized donors to naive syngeneic recipients. Pretreatment with a single intradermal injection of 50 ng SEB 24 h before DNFB exposure at the same site on the lower abdomen enhanced the induction of contact …
Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions
Incubation of human keratinocytes with nanomolar concentrations of Staphylococcus aureus alpha-toxin leads to irreversible depletion of cellular ATP. The toxin forms hexamers in the target cell membranes, and rapid transmembrane flux of K+, Na+, and 86Rb+ is observed. Unexpectedly, pores formed in keratinocytes through application of low but lethal doses of alpha-toxin appeared to be considerably smaller than those formed in erythrocyte membranes. They permitted neither rapid influx of Ca2+ or propidium iodide, nor efflux of carboxyfluorescein. Larger pores allowing flux of all three markers did form when the toxin was applied at high concentrations. Flux of monovalent ions and reduction in…
Enhanced expression of IL-8 in normal human keratinocytes and human keratinocyte cell line HaCaT in vitro after stimulation with contact sensitizers, tolerogens and irritants.
Abstract To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensi-tizer (2,4-dinitrofiuorobenzene, 3-n-penladecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Intcrleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irri…
Epidermal Cells Enhance Interleukin 4 and Immunoglobulin E Production After Stimulation with Protein Allergen
Exposure to certain allergens via epithelial tissues is the primary route for tile induction of immunoglobulin E–dependent allergies of the immediate type associated with atopic diseases. In order to address the question whether and how epithelial cells might contribute to the induction or increase of T H2 -dependent IgE production, we performed co-culture experiments of syngeneic epidermal cells and cells from the associated lymphoid tissue or spleen (responder cells) of BALB/c mice primed with ovalbumin in vivo . In the presence of ovalbumin in vitro , immunoglobulin E but not immunoglobulin G 2a production was significantly enhanced by the addition of epidermal cells, and separation of e…
Processing and Presentation of Protein and Parasite-Derived Antigens by 4F7+ Dendritic Cells
The dendritic cell (DC), a trace component of splenocytes is the principal cell type required for a primary mixed lymphocyte reaction.1 Splenic DC are described as antigen presenting cells (APC) capable to generate immunogenic fragments of intact protein antigens for presentation to MHC class II restricted T cells,2 in contrast to former findings.3 Langerhans cells (LC) as potent APC are members of the dendritic cell lineage forming a system of potent APC, first identified by Steinman and Conn.4 Importanly Schuler and Steinman have subsequently presented experimental evidence suggesting that LC represent immature DC.3 During infectious diseases caused by parasites of the genus Leishmania LC…