Elsevier

Clinical Immunology

Volume 131, Issue 3, June 2009, Pages 374-384
Clinical Immunology

Impact of α-defensins1–3 on the maturation and differentiation of human monocyte-derived DCs. Concentration-dependent opposite dual effects

https://doi.org/10.1016/j.clim.2009.01.012Get rights and content

Abstract

α-defensins1–3 are potent antimicrobial molecules that also link innate and adaptive immunity, depending on the concentration range. However, their effects on the biology of human DCs remain largely unknown. We analyzed the impact of different concentrations of α-defensins1–3 on the maturation and differentiation of monocyte-derived DCs (MDDCs). Low doses of α-defensins1–3 up-regulated CD83, CD86 and HLA-DR expression, increased TNF-α, IL-1β, IL-12p40, IL-10 and IL-8 secretion, and slightly augmented allostimulatory capacity. By contrast, high doses down-regulated CD86 and HLA-DR expression, TNF-α, IL-1β, IL-12p40 and IL-10 secretion and allostimulatory capacity, whereas strongly up-regulated IL-8. Furthermore, during the MDDC differentiation process, high doses of α-defensins1–3 affected CD14, CD11c and CD86 expression and strongly up-regulated IL-8. Results suggest that α-defensins1–3 might modulate the maturation and differentiation of MDDCs in vivo and therefore could be of special interest in the field of vaccine development.

Introduction

Defensins are important effector molecules of the innate immunity. They are very conserved molecules present in all vertebrates consisting of small (3–6 kDa) highly cationic peptides with a broad antimicrobial spectrum [1], [2]. In humans, two different subfamilies are found based on structural characteristics, α-defensins and β-defensins [1]. Six types of α-defensins have been described in humans, α-defensins 1 to 4 are produced by leukocytes and α-defensins 5 and 6 by Paneth cells. α-defensins1–3, also known as human neutrophil peptides 1–3 (HNP1–3), were firstly described in neutrophil granules [1], [3], constituting approximately 9% of the total neutrophil protein. Although neutrophils are the main source for α-defensins1–3, other leukocyte subsets also produce them [4], [5], [6], including immature dendritic cells (DCs) as we recently reported [7].

Apart from their direct antimicrobial activity, α-defensins are also able to modulate the adaptive immune response acting as chemotactic factors for T cells, monocytes, immature DCs, macrophages and mast cells [8], [9], [10], suggesting that α-defensins in vivo might participate in the recruitment of these cells to the sites of infection. Moreover, α-defensins have been reported to modify surface molecules and certain cytokine secretion in monocytes and lung epithelial cells in humans [11], [12]. In addition, in the murine system, β-defensin-2 has been shown to induce a TLR-4-dependent maturation of immature DCs [13] and more recently, human β-defensin-3 has been found to activate human antigen-presenting cells through TLR-1 and TLR-2 [14]. However, the effects of α-defensins1–3 on human DCs remain largely unknown.

DCs are the most potent antigen presenting cells, which play a key role in the initiation and regulation of the adaptive immune response [15], [16], [17]. DCs are present in an immature state in peripheral tissues where they internalize and process antigens. Antigen capture in a pro-inflammatory environment drives DCs throughout a maturational process characterized by the up-regulation of MHC class II and co-stimulatory molecules and the secretion of cytokines that increases their capacity to stimulate T cells [15], [16], [17]. Monocyte-derived DCs (MDDCs) are a valid ex vivo model of myeloid DCs [18], [19]. The differentiation of peripheral blood CD14+ monocytes into immature MDDCs is characterized by the loss of CD14 expression [20] and the up-regulation of CD11c and other surface markers. The maturation of these MDDCs can be induced by a pro-inflammatory cytokine cocktail [7], [21] that produces the up-regulation of HLA-DR, co-stimulatory molecules and the expression of CD83, a marker for mature DCs [22].

DCs may interact with the α-defensins1–3 released by neutrophils or other cells in the primary inflammatory focus [1]. Considering the important role of DCs in the initiation of the adaptive immune response and given the lack of information on this issue, we wanted to investigate the impact of different concentrations of α-defensins1–3 over the maturation and differentiation of DCs.

Section snippets

Generation of MDDCs and isolation of monocytes

MDDCs were generated from human monocytes as previously described [1], [7], [23], [24]. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats from volunteer healthy donors by standard Ficoll density gradient centrifugation and incubated on cell culture dishes for 2 h at 37 °C. To obtain DCs, adherent cells (> 95% CD14+) were washed and differentiated for 5 days to immature monocyte-derived DCs (imMDDCs) in complete XVIVO-15 medium with 50 μg/ml gentamycin (Braun B.) and

Alteration of the morphology of MDDCs after α-defensins1–3 stimulation

Adherent PBMCs were cultured for 5 days in the presence of IL-4+GM-CSF, which resulted in their differentiation into imMDDCs. They were then washed and cultured for two additional days with IL-4+GM-CSF in the absence or presence of increasing doses of α-defensins1–3, ranging from 0.25 to 20 μg/ml. To assess the effect of α-defensins1–3 on the cocktail-induced maturation of imMDDC, they were also cultured in parallel for two days with the same doses of α-defensins1–3 plus the Mat-cocktail

Discussion

In the present study we demonstrate that α-defensins1–3 are able to modulate the ex vivo maturation and differentiation of human normal MDDCs. This immunomodulatory effect showed a dose-dependent biphasic behavior that was reflected in terms of morphologic and immunophenotypical changes, cytokine production and allostimulatory activity. To our knowledge, no other studies have approached these activities of α-defensins1–3 over the human normal MDDCs.

DCs are key cells in the initiation and

Acknowledgments

M R-G is the recipient of a research award “Emili Letang” (Hospital Clínic, Barcelona). This study was supported mainly by Research Grants FIS03-1200 and FIS2006-1259 (T. G.) from the Spanish Ministry of Health, and by the Research Grant NIH NO1-AI-50028 (T.M.M). Additional support was also received from the research grants SAF2005-05566 (J. M. G.) from the Spanish Ministry of Education and Science, FIPSE36536-2005 (T. G.), the Foundation for the Investigation and Prevention of AIDS in Spain,

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