Impact of α-defensins1–3 on the maturation and differentiation of human monocyte-derived DCs. Concentration-dependent opposite dual effects
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,
References (43)
- et al.
Direct bacterial protein PAMP recognition by human NK cells involves TLRs and triggers alpha-defensin production
Blood
(2004) - et al.
Monocyte-derived dendritic cells formed at the infection site control the induction of protective T helper 1 responses against Leishmania
Immunity
(2007) - et al.
Antigen presentation by monocytes and monocyte-derived cells
Curr. Opin. Immunol.
(2008) - et al.
A clinical grade cocktail of cytokines and PGE2 results in uniform maturation of human monocyte-derived dendritic cells: implications for immunotherapy
Vaccine
(2002) - et al.
Chemokines: a new classification system and their role in immunity
Immunity
(2000) - et al.
How dendritic cells and microbes interact to elicit or subvert protective immune responses
Curr. Opin. Immunol.
(2002) - et al.
Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils
J. Biol. Chem.
(1996) - et al.
CD83 regulates lymphocyte maturation, activation and homeostasis
Trends Immunol.
(2008) - et al.
Dendritic cell CD83: a therapeutic target or innocent bystander?
Immunol. Lett.
(2008) - et al.
Human neutrophil peptides induce interleukin-8 production through the P2Y6 signaling pathway
Blood
(2006)