Immunocytochemical detection of cytokines and chemokines in Langerhans cells and in vitro derived dendritic cells

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Abstract

We have developed a direct immunocytochemical technique to identify cytokine and chemokine production in epidermal Langerhans cells (LC) and in vitro derived CD14−, CD1a+, CD83+, CD40+ dendritic cells (DC) at the single cell level. Formaldehyde fixation combined with saponin permeabilization preserved cellular morphology and generated a characteristic juxtanuclear staining signal due to the accumulation of cytokine to the Golgi organelle. This approach was used for the assessment of TNF-α, IL-6, IL-8, IL-10, IL-12, GM-CSF, MIP-1α, MIP-1β and RANTES producing cells. In contrast, a diffuse cytoplasmic staining was evident for IL-1ra, IL-1α and IL-1β production. IL-1ra and IL-1α were expressed in 10–25% of unstimulated cultured cells, while all the other cytokines were undetectable. IL-1ra, IL-1α and IL-1β were also the dominating cytokines, expressed in up to 85% of the DC, after 3 h of LPS stimulation. A significantly lower number of cells (0–5%) synthesized TNF-α, IL-6, IL-10, IL-12 and GM-CSF. The incidence of chemokine producing cells (IL-8, RANTES, MIP-1α, MIP-1β) peaked 10 h after LPS stimulation in up to 60% of the DC. Both immature CD83− and mature CD83+ DC as well as LC had a similar cytokine production pattern. Thus, in comparison to monocytes, LPS stimulation of DC generated a lower incidence of TNF-α, IL-6, IL-10 and IL-12 producing cells while IL-1 was expressed in a comparable number of cells.

Introduction

Dendritic cells (DC) represent a highly specialized bone marrow derived subpopulation of leukocytes. They are considered to be the most potent antigen presenting cells and to play a critical role in initiation of primary immune responses (Steinman, 1991). Immature DC present in blood and peripheral nonlymphoid tissues engulf microbes by endocytosis or phagocytosis. Provided with adequate chemokine and cytokine signals they become activated and initiate migration to the T cell rich areas of regional lymph nodes or the spleen (Sallusto et al., 1995; Steinman and Swanson, 1995). Following maturation, DC decrease in their antigen processing capacity (Romani et al., 1989) and change into more immunostimulatory cells, characterized by the expression of high levels of MHC class II molecules, adhesion molecules and costimulatory receptors. Thus, mature DC undergo phenotypic and functional changes in order to be able to initiate a primary immune response in naive CD4+ and CD8+ T lymphocytes.

In order to optimize activation of naive T cells, DC are likely to produce specific cytokines directing T helper cells into a Th1 or Th2 type of responses. Indeed, mature DC have been found to express mRNA for a wide range of cytokines. A constitutive synthesis of mRNA for IL-6, IL-8, IL-10, TNF-α and TGF-β1 has been reported in CD83+ blood DC. Activation by phorbol myristate acetate (PMA) further increased IL-1α, IL-1β, IL-9, TNF-β, IFN-γ, GM-CSF, M-CSF and G-CSF mRNA expression (Zhou and Tedder, 1995a). However, several studies have been unable to verify mRNA translation to protein production. In vitro differentiated DC have been shown to express mRNA for IL-1α and β, IL-6, IL-10, IL-12, GM-CSF, IFN-α and TNF-α when exposed to HIV-1 while actual protein synthesis was only detectable for IL-6 and IFN-α (Ghanekar et al., 1996). In addition, lipopolysaccharide (LPS) stimulated DC have been shown to express increased number of mRNA copies for TNF-α without evidence for translation (McKenzie et al., 1995).

The aim of the present study was to induce cytokine production following different modes of activation and develop a method where preserved cellular morphology allowed immunocytochemistry to be used for identification of cytokine protein production at the single cell level. Moreover, we wished to study in vitro derived DC expanded from peripheral blood monocytes in different states of maturation for their ability to express costimulatory molecules in relation to cytokine producing capacity. A comparison to Langerhans cells (LC), isolated from human skin, was also performed.

Section snippets

In vitro differentiation of dendritic cells

DC were generated from peripheral blood mononuclear cells (PBMC) as earlier described (Romani et al., 1994; Sallusto and Lanziavecchia, 1994). In brief, PBMC from blood donors were isolated from EDTA-blood by density centrifugation on Ficoll Hypaque density gradient (Pharmacia, Uppsala, Sweden), resuspended in culture medium (RPMI 1640 with 2 mM l-glutamine (Gibco, Paisley, UK), supplemented with 10% fetal bovine serum (FBS) (Life Technologies) plus 1% streptomycin and penicillin (Gibco) and

Cell surface expression of in vitro derived DC and isolated LC

In order to generate DC from adherent blood monocytes, we supplemented the media with different cytokines and growth factors at defined time points. Initially, the cells were cultured in IL-4 and GM-CSF. TNF-α or Macrophage conditioned medium (MCM) were added after 5–6 days of culture in favor of inducing mature DC (Fig. 1). When cells were analyzed at day 9, the in vitro derived DC had developed a distinct morphology characterized by a large sized cytoplasm and dendritic portions. Staining for

Discussion

DC develop potent immunostimulatory properties during a process of maturation that requires a number of cytokine interactions. We used IL-4 and GM-CSF in order to generate an immature CD14− CD1a+ CD83± population of DC from peripheral blood. However, TNF-α or Macrophage conditioned medium (MCM) supplementation differentiated the cells into more mature CD83+ cells with an enhanced expression of the T cell costimulatory molecules CD80 and CD86. Epidermal Langerhans cells were also found to

Acknowledgements

We thank Dr. Tomas F. Tedder, Duke University Medical Center, Durham, NC for kindly providing the CD83 mAb, Dr. Staffan Pauli, Mabtech, Nacka, Sweden for the CD40 mAb, Dr. Maurice Gately, Hoffman La Rouche, Nutley, NJ for the IL-12 mAb and Dr. Monica Tsang, R&D Systems, Minneapolis, MN for all the biotinylated polyclonal affinity purified cytokine-specific antibodies. We also thank Dr. Leif Perbeck, Dept. of Surgery, Huddinge University Hospital for providing the skin biopsies. This study was

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