Activation Of Langerhans Cells Research Articles

Gene expression profiling in the human keratinocyte cell line, hacat exposed to urushiol isolated from sap of korean lacquer tree (Rhus verniciflua Stokes)

Rhus verniciflua Stokes (RVS) is a common poison ivy that causes allergy dermatitis. However, RVS has been widely used in Korea as a traditional food preservative and herb. RVS sensitization on skin involves the activation of Langerhans cells and keratinocytes, as well as T cell-mediated reaction. While keratinocytes are the first line of immune defense against injury and infection on the skin, there is barely documented about immune-related gene profiles of keratinocytes following RVS treatment To clarify this issue, we investigated the changes in gene expression in HaCaT cells upon stimulation of RVS extracts, either urushiol or non-urushiol (allergen-removed- extract: non-urushiol) by employing the Human Whole Genome Oligo 12-plex chip (44,049 elements). We identified 154 upregulated (P=0.0708) and 196 downregulated (P=0.021) genes that underwent a greater than 2-fold change in expression level from urushiol-treated HaCaT cells. In non-urushiol treated- HaCaT cells, we found 207 upregulated (P=0.00688) and 251 downregulated (P=0.00157) genes with a greater than 2-fold change in expression levels. Collectively, these data provides for the first time RVS-specific genes expression pattern corresponding to gene expression profiles. Our findings could help in understanding the mechanism by which RVS affects the skin.

Short Papers in Pharmaceutical Technology

Introduction and Objectives Epidermal Langerhans cells (LCs) present an attractive and accessible target for deoxyribonucleic acid (DNA) vaccination. Excised human skin is perhaps the most representative preclinical model attainable for immunological studies involving human LCs. In terms of DNA vaccination, explant viability and artefactual LC activation in response to culture conditions are important issues to be addressed in any potential ex-vivo human skin model. This study aims to optimise such a model for ex-vivo DNA vaccination studies by ascertaining the structural and functional viabilities of human skin explants, and minimising artefactual LC activation.

A Major Role for the Minor Capsid Protein of Human Papillomavirus Type 16 in Immune Escape

High-risk human papillomavirus (HPV) infection of the cervical epithelium is causally linked with the generation of cervical cancer. HPV does not activate Langerhans cells (LC), the APC at the site of infection, leading to immune evasion. The HPV protein responsible for inducing this immune escape has not been determined. We demonstrate that LC exposed to the minor capsid protein L2 in HPV16L1L2 virus-like particles do not phenotypically or functionally mature. However, HPV16L1 virus-like particles significantly induce activation of LC. Our data suggest that the L2 protein plays a specific role in the induction of this immune escape of HPV16 through the manipulation of LC. This novel function is the first immune modulating action attributed to the L2 protein and adds significantly to our understanding of the mechanism of HPV immune escape.

Langerhans Cells Suppress Contact Hypersensitivity Responses Via Cognate CD4 Interaction and Langerhans Cell-Derived IL-10

Mice lacking epidermal Langerhans cells (LC) develop exaggerated contact-hypersensitivity (CHS) responses due to the absence of LC during sensitization/initiation. Examination of T cell responses reveals that the absence of LC leads to increased numbers of hapten-specific CD4 and CD8 T cells but does not alter cytokine expression or development of T regulatory cells. CHS responses and Ag-specific T cells are increased in mice in which MHC class II is ablated specifically in LC suggesting that direct cognate interaction between LC and CD4 cells is required for suppression. LC-derived IL-10 is also required for optimal inhibition of CHS. Both LC-derived IL-10-mediated suppression and full LC activation require LC expression of MHC class II. These data support a model in which cognate interaction of LC with CD4 T cells enables LC to inhibit expansion of Ag-specific responses via elaboration of IL-10.

Gram-positive bacteria enhance HIV-1 susceptibility in Langerhans cells, but not in dendritic cells, via Toll-like receptor activation

AbstractAlthough numerous studies have shown a higher risk of acquiring HIV infection in the presence of other sexually transmitted diseases, the biologic mechanisms responsible for enhanced HIV acquisition are unclear. Because Langerhans cells (LCs) are suspected to be the initial HIV targets after sexual exposure, we studied whether microbial components augment HIV infection in LCs by activating Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) pattern recognition receptors. We found that TLR1/2 and TLR2/6 agonists dramatically enhanced both HIV susceptibility and replication in immature monocyte-derived LCs, whereas TLR3-5, TLR7-9, and NOD1,2 agonists did not significantly affect HIV infection. The same infection-enhancing effects were observed when LCs were incubated with other related bacterial components as well as with whole Gram+ bacteria. In resident LCs in human skin, TLR2 agonists also significantly increased HIV susceptibility. By contrast, TLR2 agonists and related bacterial components decreased HIV susceptibility in monocyte-derived dendritic cells (DCs). We found that TLR2 activation of LCs, but not DCs, resulted in a significant down-regulation of APOBEC3G, which is a cellular restriction factor for HIV. Given these data, we hypothesize that ligation of TLR2 by Gram+ bacterial products may underlie enhanced sexual transmission of HIV that occurs with concomitant bacterial sexually transmitted disease infections.

Skin application of the nonsteroidal anti-inflammatory drug ketoprofen downmodulates the antigen-presenting ability of Langerhans cells in mice

Ketoprofen (KP) is widely used as a topical nonsteroidal anti-inflammatory drug that inhibits prostaglandin (PG) biosynthesis. As PGE(2) upregulates the antigen-presenting activity of Langerhans cells (LCs), i.e. migration to lymph nodes and expression of immunocompetent molecules, modulation of LC functions resulting from topical application of KP is an issue to be clarified. To investigate the in vivo effect of KP application to the skin and the in vitro effect of KP addition to the culture on the antigen-presenting ability of murine LCs. Methods Ears of BALB/c mice were painted with picryl chloride (PCl) hapten, KP or both. An immunofluorescence study of epidermal sheets and a flow cytometric analysis of epidermal cell suspensions from the treated ears were performed. PCl altered the morphology of LCs and reduced their number, and simultaneous application of 10% KP maintained LC morphology and number. KP at 5% or 10% clearly decreased the PCl-augmented expression of major histocompatibility complex class II and CD86 on LCs. In cultivation of freshly isolated epidermal cells, 5 mmol L(-1) KP inhibited the culture-promoted expression of these molecules on LCs, whereas 100 micromol L(-1) indomethacin was not inhibitory. The further addition of PGE(2) to the KP-containing epidermal cell culture did not restore the expression of these molecules. Moreover, topical application of 10% KP to the sensitizing sites suppressed the development of contact hypersensitivity to PCl. KP may have the potential to inhibit the antigen-presenting ability of LCs, in a PGE(2)-independent manner.

Commentary 5

Studies on the pathogenesis focussing on melanocyte biology, melanin biochemistry or skin immunity have yielded a variety of hypotheses to explain the disappearance of melanocytes in vitiligo, including a genetic predisposition, increased oxidative stress and toxic metabolites, neurochemical factors and autoimmunity (1). Whereas these factors probably all contribute to the development of depigmentation (convergence theory) (2), their interaction is not clearly defined. This commentary presents a multidisciplinary view of the pathogenesis of vitiligo (Fig. 1). Schematic overview of the pathogenesis of vitiligo. Melanin synthesis in melanocytes is a tightly regulated process. Defects in the protective mechanism in the skin that scavenge radicals and toxic intermediates of melanin production may lead to vitiligo. The increased levels of oxidative stress in the vitiligo skin leads to the deactivation of catalase (3). H202 can also oxidize (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin to 6-biopterin, which is cytotoxic to melanocytes. Although this melanocyte death may account for part of the depigmentation, dying melanocyte fragments will be taken up by epidermal Langerhans cells. The uptake of self-antigens from dying melanocytes in the absence of activation signals does not activate the Langerhans cells and will therefore not lead to immunity against melanocytes. Concurrent external stress factors, such as wounding, high dose of UV radiation or hormonal changes, however, activate Langerhans cells and dendritic cells in the skin, leading to breakage of tolerance (4). Clinically, these stress factors induce progression of depigmentation. The Koebner phenomenon results from stress factors at remote skin sites that induce local activation of Langerhans cells and reactivation of anti-melanocyte immunity, leading to the formation of new lesions. The lower threshold for breakage of tolerance in patients with vitiligo is illustrated by the increased incidence of autoimmune diseases in patients with vitiligo (5-12). Normally, regulatory T cells maintain peripheral tolerance by suppressing autoreactive T-cell activity. In patients with vitiligo, however, decreased levels of regulatory T cells in the skin lower the threshold for autoimmunity. Activated Langerhans cells containing melanocyte antigens migrate to the lymph nodes and present melanocyte antigenic peptides bound to HLA molecules to T cells. The association of vitiligo vulgaris with certain HLA class II alleles (13-15) indicates the dominance of peptides binding to these HLA types in inducing immunity. In the lymph nodes, melanocyte-reactive CD8+ and CD4+ T cells are activated to proliferate and migrate to the skin, resulting in increased levels of T cells reactive with tyrosinase, gp100 or MART-1 in peripheral blood (16-19), as well as autoantibodies against these antigens (20-22). Moreover, the number of circulating MART-1-reactive T cells expressing CLA in patients with vitiligo correlated with the extent of depigmentation (18). In perilesional skin, CD8+ T cells, macrophages and to a lesser extent CD4+ T cells were found (23, 24). Infiltrating CD8+ T cells expressed skin homing, cutaneous leukocyte-associated antigen (CLA) and T-cell activation markers CD25, perforin and granzyme B (25), and colocalized with disappearing melanocytes. Finally, perilesional melanocyte-reactive CD8+ cytotoxic T cells were shown to be capable of actively killing melanocytes in autologous skin tissue (J.G. van den Boorn, D. Konijnenberg, T.A.M. Dellemijn, J.P.W. van der Veen, J.D. Bos, C.J.M. Melief, F.A. Vyth-Dreese and R.M. Luiten, unpublished data). Taken together, the pathogenesis of vitiligo results from changes in biochemical processes in the skin that trigger autoimmunity, which is enhanced by genetic predisposition to autoimmunity.

The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test – human cell line activation test (h-CLAT)

Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20-95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65-90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of "CD86 > or = 150 or CD54 > or = 200" resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.

Production of IL-8 in THP-1 cells following contact allergen stimulation via mitogen-activated protein kinase activation or tumor necrosis factor-.ALPHA. production

Contact allergens induce in vitro and in vivo the activation of dendritic cells (DC) and Langerhans cells (LC), which includes the up-regulation of surface marker expression (e.g. CD86, CD54) and cytokine production (e.g. TNF-alpha, IL-1beta, IL-8). The mitogen-activated protein kinase (MAPK) pathway also has a crucial role in this activation. However, the extent of MAPK involvement in the IL-8 production during DC/LC activation is not well understood. Earlier, we reported that contact allergens activated THP-1 cells, human monocytic cell line, like LC/DC in vitro. In this study, we further characterize the mechanism of IL-8 production using THP-1 cells as surrogate DCs. First, we evaluated the potential of 23 chemicals with different skin sensitization potencies to predominantly induce IL-8 production in vitro. Next we investigated the role of MAPK signaling and TNF-alpha, which is known to have autocrine effects on DC activation (e.g., IL-8 production). Inhibition of extracellular signal-regulated kinase (ERK), one of the MAPK pathways, suppressed the IL-8 production induced by both 2,4-dinitrochlorobenzene (DNCB) and nickel sulfate (NiSO(4)), and inhibition of p38 MAPK, a second MAPK pathway, significantly suppressed IL-8 production induced by only DNCB. Additionally, neutralization of TNF-alpha activity suppressed IL-8 production in THP-1 cells exposed to DNCB and NiSO(4). In conclusion, IL-8 production was predominantly induced in THP-1 cells following allergen stimulation, and MAPK pathways and TNF-alpha were involved in the IL-8 production induced by DNCB and NiSO(4). A better understanding of the mechanism of DC activation in vitro might lead to the clarification of the in vivo skin sensitization mechanism.

Prevention of paclitaxel-evoked painful peripheral neuropathy by acetyl- l-carnitine: Effects on axonal mitochondria, sensory nerve fiber terminal arbors, and cutaneous Langerhans cells

Prophylactic treatment with acetyl- l-carnitine (ALCAR) prevents the neuropathic pain syndrome that is evoked by the chemotherapeutic agent, paclitaxel. The paclitaxel-evoked pain syndrome is associated with degeneration of the intraepidermal terminal arbors of primary afferent neurons, with the activation of cutaneous Langerhans cells, and with an increased incidence of swollen and vacuolated axonal mitochondria in A-fibers and C-fibers. Previous work suggests that ALCAR is neuroprotective in other nerve injury models and that it improves mitochondrial dysfunction. Thus, we examined whether the prophylactic efficacy of ALCAR was associated with the prevention of intraepidermal terminal arbor degeneration, the inhibition of Langerhans cell activation, or the inhibition of swelling and vacuolation of axonal mitochondria. In animals with a confirmed ALCAR effect, we found no evidence of a neuroprotective effect on the paclitaxel-evoked degeneration of sensory terminal arbors or an inhibition of the paclitaxel-evoked activation of Langerhans cells. However, ALCAR treatment completely prevented the paclitaxel-evoked increase in the incidence of swollen and vacuolated C-fiber mitochondria, while having no effect on the paclitaxel-evoked changes in A-fiber mitochondria. Our results suggest that the efficacy of prophylactic ALCAR treatment against the paclitaxel-evoked pain may be related to a protective effect on C-fiber mitochondria.

Selective 5-Lipoxygenase Expression in Langerhans Cells and Impaired Dendritic Cell Migration in 5-LO-Deficient Mice Reveal Leukotriene Action in Skin

5-Lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes (LTs), which are implicated in immune reactions. Recently, it was shown that FITC-triggered epidermal Langerhans cell (LC) emigration to draining lymph nodes (LNs) is impaired in LTC4 export pump (multidrug resistance-associated protein 1)-deficient mice. Here, we sought genetic evidence for a role of endogenous LTs in dendritic cell function through the study of 5-LO-deficient mice. Though DC numbers in skin, spleen, and peripheral LNs were similar in both 5-LO-deficient and wild-type (WT) mice, DC homing from skin to draining LNs induced by FITC was reduced by 75% in 5-LO-deficient mice. Moreover, in WT mice, all epidermal LCs, dermal langerin+ LCs, and subsets of dermal macrophages and langerin+ LCs in T-cell areas of skin-draining LNs markedly expressed 5-LO. However, the enzyme was noticeably absent in all DC subsets of the dermis, thymus, spleen, Peyer's patches, mesenteric LNs, and mucosal surfaces of lung and intestine. As all epidermal cells other than LCs lacked 5-LO and because differentiation and activation of DCs generated from 5-LO-deficient mice in vitro were normal, these data support a selective role of endogenous LTs in DC homing following skin sensitization.

Immunological activation following transcutaneous delivery of HR-gp100 protein

Transcutaneous immunization aims at taking advantage of the skin's immune system for the purpose of immunoprotection. In the present study, we evaluated the potential of topical delivery of a recombinant melanoma protein, HR-gp100, derived from a shortened sequence of the native gp100 gene. The protein was applied on the skin, with and without the addition of two forms of heat labile enterotoxin (nLT and LTB). HR-gp100 fused to Haptide, a cell penetrating 20mer peptide (HR-gp100H) was also tested. Topical HR-gp100 and HR-gp100H application on the ears of mice elicited the production of specific antibodies, and transcutaneous delivery to intact human skin induced dose-dependent LC activation. nLT and LTB also activated LC, but did not further increase the activation induced by HR-gp100. These results show that HR-gp100, an antigenic tumor-derived protein, activates the immune system following transcutaneous delivery, as shown by both Langerhans cell activation and induction of antibody production.

Human Langerhans-cell activation triggered in vitro by conditionally expressed MKK6 is counterregulated by the downstream effector RelB

AbstractEnvironmentally exposed epithelial Langerhans cells (LCs) encounter diverse innate stress signals, which lead to the activation of complex intracellular signaling cascades. Among these, p38 MAPK is consistently phosphorylated. For which aspects of LC activation triggering of p38 signaling is sufficient remains to be elucidated. We show that conditional induction of a dominant active form of MAPK kinase 6 (d.a.MKK6), a direct upstream kinase of p38, in LCs efficiently induces the up-regulation of costimulatory molecules and enhances their T-cell stimulatory capacity. These immediate effects showed no or only a minor requirement for classical NF-κB signaling. Concomitant with LC activation, d.a.MKK6 induced the alternative NF-κB member RelB, whose nuclear localization marks mature DCs. Specific inhibition of nuclear RelB during d.a.MKK6-induced LC activation further enhanced their maturation state. This observation was validated using the p38 activator anisomycin, thus suggesting a novel LC intrinsic control mechanism regulated by RelB.

Drug eruption caused by the nonionic contrast medium iohexol. “Recall‐like phenomenon” appearing on an area previously affected by herpes zoster

We report a case of "recall-like phenomenon" caused by nonionic contrast medium. A 62-year-old woman suffering from postherpetic neuralgia developed erythematous plaques 12 h after an intercostal nerve block under X-ray guidance using iohexol (Omnipaque) as contrast medium. The erythematous plaques were preferentially located in the sites where she had experienced herpes zoster 4 months previously. The lesions cleared spontaneously leaving no pigmentation. Both patch testing and intradermal testing with iohexol and ioversol were positive. We postulate that local immunological changes in the skin, such as an increased number and/or accelerated activity of Langerhans cells and mast cells in the herpes zoster lesions, were responsible for this phenomenon. This "recall-like phenomenon", occurring preferentially in skin previously affected by herpes zoster, could facilitate understanding of the pathology of drug eruptions.

Nicotinic Acid-Induced Flushing Is Mediated by Activation of Epidermal Langerhans Cells

The antidyslipidemic drug nicotinic acid (niacin) has been used for decades. One of the major problems of the therapeutical use of nicotinic acid is a strong cutaneous vasodilation called flushing, which develops in almost every patient taking nicotinic acid. Nicotinic acid-induced flushing has been shown to be mediated by the nicotinic acid receptor GPR109A and to involve the formation of vasodilatory prostanoids. However, the cellular mechanisms underlying this short-term effect are unknown. Here, we show that epidermal Langerhans cells are essential for the cutaneous flushing response induced by nicotinic acid. Langerhans cells respond with an increase in [Ca(2+)](i) to nicotinic acid and express prostanoid synthases required for the formation of the vasodilatory prostanoids prostaglandin E(2) and prostaglandin D(2). Depletion of epidermal Langerhans cells but not of macrophages or dendritic cells abrogates nicotinic acid-induced flushing. These data unexpectedly identify epidermal Langerhans cells as essential mediators of nicotinic acid-induced flushing and may help to generate new strategies to suppress the unwanted effects of nicotinic acid. In addition, our results suggest that Langerhans cells besides their immunological roles are also involved in the local regulation of dermal blood flow.

Immunopathogenesis of psoriasis

This paper reviews the pathogenesis of psoriasis, in particular, the immunological cascade in psoriasis. Psoriasis is an immune-mediated skin disease where the T cell plays a pivotal role in the pathogenesis of the disease. The critical steps involved in the pathogenesis include Langerhans cell activation and maturation by antigens in the skin, activation of the T cell by mature Langerhans cells, differentiation and expansion of T cells within the lymph nodes, trafficking of activated T cells from the lymph node to the skin and the subsequent release of cytokines. These cytokines are responsible for epidermal and vascular hyperproliferation and pro-inflammatory effects. Each of these steps provides an opportunity for biological agents designed to block the psoriatic immunological cascade. This paper reviews the immunopathogenesis of psoriasis. Biologic agents in psoriasis, to be published separately, reviews the new biologic therapies that aim to selectively block the immunological steps implicated in the pathogenesis of psoriasis outlined in this paper.

Paclitaxel- and vincristine-evoked painful peripheral neuropathies: Loss of epidermal innervation and activation of Langerhans cells

Experimental painful peripheral neuropathies produced by the chemotherapeutic drugs, paclitaxel and vincristine, are produced by relatively low doses that do not cause axonal degeneration in peripheral nerve. Using quantitative immunolabeling with the PGP9.5 antibody, we have investigated whether these painful neuropathies might be associated with degeneration that is confined to the region of the sensory fiber's receptor terminals in the skin. Because complete and partial nerve transections are known to cause an increase in PGP9.5 in epidermal Langerhans cells (LCs), we also examined whether this effect occurs in chemotherapy-treated animals. At the time of peak pain severity, rats with paclitaxel- and vincristine-evoked painful peripheral neuropathies had a significant decrease (24% and 44%, respectively) in the number of intraepidermal nerve fibers (IENF) in the hind paw glabrous skin and an increase (217% and 121%, respectively) in the number of PGP9.5-positive LCs, relative to control. However, neither loss of IENF nor an increase in PGP9.5-positive LCs was found in rats with a painful peripheral neuropathy evoked by the anti-HIV agent, 2′,3′-dideoxycytidine. We also confirmed that there is a decrease in IENF and an increase in PGP9.5-positive LCs in rats with neuropathic pain following a partial nerve injury (CCI model) and in rats with a complete sciatic nerve transection. Partial degeneration of the intraepidermal innervation suggests mechanisms that might produce chemotherapy-evoked neuropathic pain, and activation of cutaneous LCs suggests possible neuroimmune interactions that might also have a role.

Epicutaneous immunization converts subsequent and established antigen‐specific T helper type 1 (Th1) to Th2‐type responses

Epicutaneous immunization is a potential novel technique for topical vaccine delivery. It targets the immunologically rich milieu of the skin while having the advantage of being a non-invasive immunization procedure. By disrupting the stratum corneum of the epidermis a natural adjuvant effect can be achieved through activation of resident Langerhans cells. This negates the normal need for co-application of noxious adjuvants. Epicutaneous immunization on barrier-disrupted skin induces potent antigen-specific systemic immunity with a strong T helper type 2 (Th2) bias. We show here that epicutaneous immunization enhances the vigour of a subsequent T-cell response to the same antigen. The induced systemic Th2 response prevents the development of Th1 responses induced through injection of antigen in complete Freund's adjuvant (CFA). Prior epicutaneous immunization results in reduced production of antigen-specific interferon-gamma and immunoglobulin G2a (IgG2a) and enhanced interleukin-4, IgG1 and IgE responses to immunization with CFA. Moreover, epicutaneous immunization converts an established Th1 response to a Th2 response, as demonstrated by the specific reduction of interferon-gamma and IgG2a and the enhancement of interleukin-4 and IgE. This Th2 dominance of epicutaneous immunization may have direct therapeutic application as an immune-modulating procedure in Th1-dominant diseases such as autoimmune rheumatoid arthritis, type 1 diabetes, Hashimoto's thyroiditis and multiple sclerosis.

Assessment of usefulness of J774A.1 macrophages for the assay of IL-1β promoter activity

Existing data indicate that the increase of il-1β gene expression can be a promising marker of Langerhans cells activation after exposure to contact sensitizers. In this study, we were interested in development of an alternative in vitro screening test detecting such sensitizers. Two IL-1β reporter constructs containing the enhanced green fluorescent protein (GFP) gene and mouse IL-1β promoter fragments of varying lengths (−500bp and −4093bp) were used for transient transfections of J771A.1 murine monocyte-macrophage cells. As a result of the transfections performed using Lipofectamine reagent we did not observe any GFP fluorescence after stimulation of the cells with LPS as well as known sensitizers (potassium tetrachloroplatinate, dinitrochlorobenzene and nickel sulfate). Low transfection efficiency of J774A.1 cells (less than 0.1%) was confirmed using control plasmid containing GFP gene under the control of cytomegalovirus promoter. The fact that, using the same conditions, we were able to transfect murine fibroblasts 3T3-L1 with the control plasmid very efficiently, may support the theory of high metabolic activity of macrophages being responsible for the extremely low transfection efficiency. These data suggest limited suitability of J774A.1 cell line for transient transfections using cationic liposomes.

Type I Interferons Inhibit Maturation and Activation of Mouse Langerhans Cells

Type I interferons (IFN) have an essential role in antiviral defense, and they are produced upon viral infection in a variety of cells. IFN-alpha/beta treatment of immature dendritic cells (DC) is known to induce their phenotypic and functional maturation, but it remains unclear whether stimulation by this cytokine family influences the functions and maturation of Langerhans cells (LC). We used highly enriched (>95%) LC directly isolated from BALB/c mouse skin and addressed this issue, comparing LC with splenic CD11c(+) DC. Type I IFN-treated LC exhibited impaired ability to produce IL-12 and inflammatory cytokines, IL-6 and TNF-alpha, whereas IL-10 production was not augmented. In splenic DC, the production of inflammatory cytokines was rather enhanced by type I IFN treatment. With regard to chemokines, in both LC and splenic DC, type I IFN upregulated the production of inflammatory chemokines, such as CXCL10, CXCL11, CCL3, CCL4, and CCL5. Strikingly, IFN-beta treatment reduced the expression of CD40, CD54, CD80, and CD86 on LC, whereas IFN-beta-treated splenic DC showed enhanced expression of these molecules. Furthermore, IFN-beta-treated LC had impaired costimulatory activity for anti-CD3-induced proliferation of T cells. Finally, treatment with IFN-alpha/beta reduced the migratory capacity of LC to CCL21. These results indicate that type I IFN inhibit maturation and activation of LC in a direct manner. Our observations may provide a novel explanation for the reported inability of LC to act as potent antigen-presenting cells in cutaneous and mucosal viral infection.