Published online before print August 7, 2007
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-defensins 1–3
,1,
,,2
* Service of Immunology,
Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS)-AIDS Research Group, and
Service Infectious Diseases and AIDS Unit, Hospital Clínic Universitari de Barcelona, Catalonian Center for HIV Vaccines (HIVACAT), School of Medicine, University of Barcelona, Barcelona, Spain
2 Correspondence: Service of Immunology, Hospital Clínic Universitari de Barcelona, Villarroel 170, 08036 Barcelona, Spain. E-mail: tgallart{at}clinic.ub.es
ABSTRACT
Defensins are effector molecules of the innate immunity with a broad antimicrobial spectrum, including HIV. They also link innate and adaptive immunity, displaying chemotactic activity for monocytes, T cells, and dendritic cells (DCs). 
-Defensins 1–3 are mainly produced by neutrophils, but their production by other leukocyte subsets has also been reported. Herein, we studied whether monocyte-derived DCs (MDDCs), which are regarded as a model for myeloid DCs, produce -defensins 1–3. We found that immature MDDCs (imMDDCs) produce -defensins 1–3 mRNA, but this production is undetectable or barely detectable following 48 h of maturation with the proinflammatory cytokine cocktail (IL-1β+IL-6+TNF-) or LPS. It is surprising that -defensins 1–3 production was up-regulated when exposed to each one of the proinflammatory cytokines alone, especially IL-1β. -Defensins 1–3 produced by imMDDCs were mainly secreted peptides. Production and secretion of -defensins 1–3 by imMDDCs can have biological relevance for the antigen processing of pathogens and can contribute to understanding differences in susceptibility to infections, an issue of special interest in the field of HIV infection.
Key Words: human neutrophil peptides 1–3 • real-time PCR • innate immunity
Human defensins are small (3–6 kDa), cationic, and cysteine-rich peptides [1
] with a wide, antimicrobial spectrum against bacteria, fungi, parasites, and viruses, including HIV [2
3
4
]. Besides their activity as effector molecules of the innate immunity, human defensins link adaptive immunity, acting as chemotactic factors for T cells, monocytes, and immature dendritic cells (imDCs) [5
, 6
]. Human defensins are classified into two different subfamilies: - and β-defensins [1
]. Neutrophils are the main source of human -defensins 1–3 {also named human neutrophil peptides 1–3 (HNP 1–3) [1
, 7
]}, although they have also been found in other leukocyte subsets such as NK cells, B cells, 
T cells, monocytes, and macrophages [3
, 4
]. We hypothesized that myeloid DCs, the most potent, professional APCs [8
, 9
], would also produce -defensins 1–3. We have investigated this aspect by using monocyte-derived DCs (MDDCs), which are a valid model of in vivo myeloid DCs [10
].
MDDCs were generated from human monocytes as described previously [11
, 12
] with minor modifications. Monocytes (>95% CD14+, without polymorphonuclear neutrophil contamination) were differentiated for 5 days to imMDDCs with 1000 units/mL IL-4 (Strathmann Biotec AG, Hamburg, Germany) and 1000 units/mL GM-CSF (Peprotech, London, UK). Mature MDDCs (mMDDCs) were obtained by an additional 48 h culture with the proinflammatory cytokine cocktail [13
] (10 ng/mL IL-1β, 1000 units/mL IL-6, 1000 units/mL TNF-, Strathmann Biotec AG) or 500 ng/mL LPS (Sigma-Aldrich Co., St. Louis, MO, USA) added on Day 5. For some experiments, the different cytokines were added separately. The purity and immunophenotype of the imMDDCs and mMDDCs were assessed by flow cytometry analysis using commercially labeled mAb against surface markers [12
]. A real-time RT-PCR was developed to evaluate the expression of -defensins 1–3 mRNA. Total RNA was extracted with the PureLink Micro-to-Midi Total RNA purification system (Invitrogen Corp., Paisley, Scotland), and cDNA was generated as reported [12
]. The cDNAs were amplified using LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche, Penzberg, Germany). Real-time PCR was carried out for 45 cycles using the LightCycler instrument (Roche). Specific primers (Sigma-Aldrich) were selected according to the GeneBank database resource as follows: -defensins 1–3, amplimer 105 bp, forward 5'-CTTGGCTCCAAAGCATCC-3', reverse 5'-GAACCTGCATCTACCAGGGA-3'; β-2-microglobulin, amplimer 107 bp, forward 5'-ACA CAA CTG TGT TCA CTA GC-3', reverse 5'-CAA CTT CAT CCA CGT TCA CC-3'. PBMCs were used as a positive control [14
]. Amplified products were identical to the published sequences of -defensins 1–3 cDNA by sequence analysis (data not shown). To calculate relative levels of -defensins 1–3 mRNA, β-2-microglobulin mRNA levels were used as an endogenous control [12
, 15
] to normalize mRNA quantities. Relative mRNA levels were calculated using the following equation [16
]: Relative mRNA expression = 2 – (Ct of -defensins 1–3–Ct of endogenous control) x 103, where Ct is on threshold cycle value. All cDNA samples were amplified in duplicates. For intracellular staining of -defensins 1–3, MDDCs were first surface-stained with FITC-conjugated HLA-DR (BD Biosciences, Erembodegem-Aalst, Belgium), then fixed and permeabilized with the Cytofix-Cytoperm Plus kit (BD Biosciences), and incubated with biotinylated mAb mouse anti-human defensins 1–3 (Clone D21, Hycult Biotechnology, Uden, The Netherlands) for 30 min and then with PE-conjugated streptavidin (1/100, BD Biosciences) during 30 min. For some of these experiments, cells were pretreated with monensin [17
] (Golgi Stop, BD Biosciences) for 8 h. The negative control was FITC-conjugated isotype control (BD Biosciences) and mouse IgG1 isotype control biotin conjugate (Southern Biotechnology, Birmingham, AL, USA). The levels of -defensins 1–3 in cell-free supernatant were quantified using the commercial HNP 1–3 ELISA test kit (Hycult Biotechnology).
Transcripts of -defensins 1–3 were detected in imMDDCs and monocytes, but the relative expression of -defensins 1–3 mRNA in imMDDCs was higher than in monocytes. In contrast, -defensins 1–3 were not detected or barely detectable in mMDDCs (Fig. 1A
). It should be noted that the viability of imMDDCs and mMDDCs was similar (>90%).
 View larger version (34K): [in a new window] |
Figure 1. Production of -defensins 1–3 by MDDCs. (A) Real-time RT-PCR analysis revealed -defensins 1–3 mRNA expression in monocytes and imMDDCs but in mMDDCs, was barely or nondetectable. The relative expression of -defensins 1–3 mRNA was higher in imMDDCs than in monocytes from the same donor. Fresh peripheral blood samples were used. Total RNA was extracted from MDDCs, and monocytes were donor-matched. Total RNA (1 µg) was reverse-transcribed into cDNA and tested by real-time RT-PCR for -defensins 1–3. The relative expression levels of -defensins 1–3 were normalized versus β-2-microglobulin. Box-and-whisker plots of data found in three independent experiments from different donors are shown. Boxes represent interquartile ranges; the horizontal bar within each box indicates the median; whiskers indicate the 10th and 90th percentiles. (B) Intracellular staining and flow cytometry analysis did not detect intracellular -defensins 1–3 in MDDCs, but (C) after treatment with monensin for 8 h, intracellular -defensins 1–3 was detected in imMDDCs and also in mMDDCs in a lower proportion. Double-staining for HLA-DR and -defensins 1–3 was performed. HLA-DR-positive cells were selected, and the histograms correspond to cells positive for intracellular -defensins 1–3. More than 100,000 events were analyzed in each histogram. By applying the Kolmogorov-Smirnof test to the analysis of histograms, the differences between imMDDCs and mMDDCs were statistically significant (P<.001). Data shown are representative of three independent experiments. (D) ELISA quantification revealed that -defensins 1–3 were secreted into the culture supernatants of imMDDCs. After maturation, these levels tended to decrease. Box-and-whisker plots of data found in four independent experiments from different donors are shown. Boxes represent interquartile ranges; the horizontal bar within each box indicates the median; whiskers indicate the 10th and 90th percentiles.
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-defensins 1–3 was investigated by flow cytometric analysis, with negative results in imMDDCs and mMDDCs (Fig. 1B)
. As defensins have been described as secreted peptides [1
, 7
, 18
], we repeated the assay after treatment of MDDCs with monesin [17
] for 8 h to accumulate the -defensins 1–3 peptides inside the cell. Flow cytometry analysis after monensin treatment revealed that 9.18% of imMDDCs contained intracellular -defensins 1–3 peptides, whereas this occurred in only 1.03% of mMDDCs. These differences between imMDDCs and mMDDCs were statistically significant (P<.001; Fig. 1C
). These data suggested that -defensins 1–3 would have been secreted into the culture supernatants. Thus, we next investigated the presence of -defensins 1–3 in the culture supernatants of the imMDDCs (Day 5) and mMDDCs (Day 7) by ELISA. For control purposes, monocytes from which MDDCs were obtained were also cultured in parallel, and -defensins 1–3 were measured at Days 5 and 7. In the supernatants of the cultured imMDDCs and monocytes, -defensins 1–3 were detected, and the levels were clearly higher in imMDDC supernatants (range 165–2345 pg/mL) than in monocyte supernatants (range 0–913 pg/mL; data not shown), whereas in the mMDDC supernatants, the levels of -defensins 1–3 tended to decrease, reaching a similar range to that found in the monocyte culture supernatants (Fig. 1D)
. These results are in agreement with real-time RT-PCR and intracellular protein detection results. The decrease of -defensins 1–3 in the mMDDC supernatants compared with the imMDDC supernatants was not a result of their absorption to culture wells, as centrifuged supernatants of imMDDCs cultured in parallel without cells for 48 h showed no significant differences. This decrease is probably a result of the lack of synthesis in mMDDCs, although there is the possibility that it might also reflect some degree of consumption by imMDDCs during their maturation process. Further studies are required to assess this possibility, which would be consistent with the fact that -defensins 1–3 chemoattract imDCs [5
, 6
] and with the role of -defensins 1–3 in initiating the interaction between lung epithelia and CD4+ T lymphocytes by up-regulating cell adhesion molecules [19
]. In addition, β-defensins induce maturation of mouse DCs [20
]. The inter-individual variability in the amount of -defensins 1–3 produced by imMDDCs is consistent with the differential mRNA expression in PBMCs, depending on the copy number polymorphism of -defensin genes DEFA1 and DEFA3 [21
].
We also analyzed the effect of each one of the proinflammatory cytokines of the maturation cocktail. For this purpose, imMDDCs were cultured in parallel for 48 h with each cytokine of the maturation cocktail, IL-1-β, IL-6, and TNF-, and with all the cytokines together. Relative expression of -defensins 1–3 mRNA increased in all samples after stimulation with IL-1β for 48 h. IL-6 and TNF- increased slightly or maintained -defensins 1–3 mRNA levels compared with imMDDCs. In all cases, after maturation with the whole maturation cocktail, -defensins 1–3 mRNA levels were undetectable or barely detectable (Fig. 2A
). These data were consistent with ELISA results, which showed the highest levels of secreted -defensins 1–3 in the IL-1β-stimulated supernatants and the lowest peak after stimulation with the maturation cocktail (Fig. 2B)
. Two dose-activity ranges have been found for -defensins: micromolar for microbicidal activity and nanomolar for chemotactic activities [1
]. Thus, the IL-1β-mediated, strong up-regulation in the production and secretion of -defensins 1–3 by imMDDCs could be of physiological relevance in a proinflammatory microenvironment. An increased production by IL-1β stimulation has also been found for β-defensins in PBMCs and airway epithelia [14
, 22
].
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Figure 2. Regulation of -defensins 1–3 production by MDDCs after stimulation with cytokines. (A) Real-time RT-PCR analysis revealed an increase of -defensins 1–3 mRNA expression after stimulation with IL-1β for 48 h. IL-6 and TNF- stimulation for 48 h maintained or lightly increased -defensins 1–3 mRNA relative expression with respect to imMDDCs. It is interesting that -defensins 1–3 mRNA expression was almost undetectable after stimulation with the maturation cocktail (IL-1β, IL-6, and TNF-). (B) ELISA experiments revealed induction of -defensins 1–3 secretion into the MDDC medium in response to stimulation with IL-1β for 48 h. IL-6 and TNF- lightly induced or maintained -defensins 1–3 secretion with respect to imMDDCs. After stimulation with the maturation cocktail, -defensins 1–3 secretion into the MDDC medium was decreased. Samples were obtained from buffy coats. Box-and-whisker plots of data found in four independent experiments from different donors are shown. Boxes represent interquartile ranges; the horizontal bar within each box indicates the median; whiskers indicate the 10th and 90th percentiles.
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-defensins 1–3 became undetectable (Fig. 3A
), and the levels of -defensins 1–3 in the supernatants of LPS-maturated MDDCs tended to diminish compared with those found in imMDDC supernatants (Fig. 3B)
. mDCs and imDCs have different programs [8
, 9
, 13
]. imDCs are specialized in capturing antigen and in processing it, whereas they are poorly able to present the antigen and can even induce tolerance. The opposite occurs with mDCs, which are committed to present the antigen peptides to T cells and have lost the capacity to capture antigen and to process it. Thus, it is conceivable that full maturation may also involve changes in the production of -defensins. As LPS, which acts by a different pathway than the cytokine cocktail, induced the same effects as this cocktail, we believe that other systems of promoting full maturation of DCs will also result in the down-regulation of -defensins 1–3 production. Experiments are in progress to test this hypothesis.
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Figure 3. Production of -defensins 1–3 after maturation with LPS. (A) Real-time RT-PCR analysis revealed -defensins 1–3 mRNA expression nondetectable after maturation of MDDCs with LPS. (B) ELISA quantification revealed decreased levels of -defensins 1–3 after maturation with LPS, similar to the levels found after maturation with the cytokine cocktail. Samples were obtained from buffy coats. Values are mean ± SD of duplicates.
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-defensins 1–3 by imMDDCs would contribute to the recruitment of monocytes, naïve T cells, and other imMDDCs to the inflammatory microenvironments at mucosal sites of infection. Moreover, the antimicrobial effect of -defensins 1–3 would favor an efficient processing of inactivated pathogens without DCs becoming infected. The variability in the production of -defensins 1–3 by imMDDCs could be another host determinant of the different inter-individual susceptibility to infections, an issue of particular interest in the field of HIV infection.
ACKNOWLEDGEMENTS
This study was supported mainly by research grants FIS03-1200 and FIS2006-1259 (T. G.) from the Spanish Ministry of Health, SAF2005-05566 (J. M. G.) from the Spanish Ministry of Education and Science, FIPSE36536-2005 (T. G.) from the Foundation for the Investigation and Prevention of AIDS in Spain, and ISCIII-RETIC RD06/006 from the Spanish Cooperative Network of AIDS Research Groups from the Ministry of Health; the "Fundación Máximo Soriano Jiménez" also supported this study.
FOOTNOTES
1 These authors contributed equally to this work. 
Received May 10, 2007; revised July 4, 2007; accepted July 4, 2007.
REFERENCES
-defensin-1 in anti-HIV-1 innate immunity J. Clin. Invest. 115,765-773[CrossRef][Medline]-Defensins can have anti-HIV activity but are not CD8 cell anti-HIV factors AIDS 17,F23-F32[CrossRef][Medline]- and β-defensins in human peripheral blood Eur. J. Clin. Invest. 33,82-87[CrossRef][Medline] expression in human plasmacytoid and monocyte-derived dendritic cells J. Leukoc. Biol. 74,1125-1138-defensin genes DEFA1 and DEFA3 Hum. Mol. Genet. 14,2045-2052This article has been cited by other articles:
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