Human monocytes represent a competitive source of interferon-α in peripheral blood

Clinical Immunology (2008) 127, 252–264

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y c l i m

Human monocytes represent a competitive source of interferon-α in peripheral blood Leo Hansmann a , Sabine Groeger a , Werner von Wulffen b , Gregor Bein a , Holger Hackstein a,⁎ a b

Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig-University Giessen, 35385 Giessen, Germany Department for Internal Medicine, Justus-Liebig-University Giessen, 35385 Giessen, Germany

Received 10 May 2007; accepted with revision 17 January 2008 Available online 14 March 2008

KEYWORDS Monocyte; IFN-α; Human

Abstract Interferon-alpha (IFN-α) has a critical role in antiviral immunity and plasmacytoid dendritic cells (pDCs) have been demonstrated as the principal IFN-α source after Toll-like receptor (TLR) 7 and 9 stimulation. Little is known about the contribution of pDC-independent IFN-α sources to total IFN-α production capacity of human peripheral blood. Using an array of pathogen associated molecular patterns (PAMPs), Poly(I:C)/Dotap represented the second strongest IFN-α stimulus in total PBMC. Poly(I:C)/Dotap induced three times more IFN-α, when compared to TLR7-stimulation (R848) and four times less, when compared to TLR9-stimulation. Dotap (mediator of cellular uptake) dramatically increased Poly(I:C)-induced IFN-α production. Sorting experiments and ELISpot assays revealed that monocytes and not myeloid DCs are the main IFN-α source after Poly(I:C)/Dotap stimulation. ELISpot analyses demonstrated the highest IFN-α spot numbers after Poly(I:C)/Dotap stimulation. Although pDCs produced highest IFN-α levels per cell, monocytes represent a competing IFN-α source in total PBMC due to their high frequency. © 2008 Elsevier Inc. All rights reserved.

Introduction Human interferons are of high importance for innate and adaptive immunity. They are classified into type I interferons which is IFN-α (leukocyte interferon) and IFN-β (fibroblast interferon) as well as type II interferon that is IFN-γ. Type I Interferons represent a group of cytokines with antiviral, ⁎ Corresponding author. Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig University Giessen, Langhansstr. 7, Giessen, D-35390 Giessen, Germany. Fax: +49641-9941509. E-mail address: [email protected] (H. Hackstein).

antiproliferative and immunomodulatory effects [1]. IFN-α in particular has successfully been used in clinical therapy of viral infections, such as hepatitis C as well as in treatment of solid and haematopoietic malignancies [1–3]. Especially in the early immune response, IFN-α is an important player priming activation of dendritic cells (DCs) [4,5], macrophages and NK cells [6–8]. In human CD4+ T cells IFN-α supports Th1 development and suppressing IL-4 and IL-5 production, IFN-α blocks Th2 differentiation [9]. In lymphoid cells IFN-α has been shown to enhance the expression of histocompatibility antigens [10]. Successful host defence against viral infections needs early production of type I interferon and subsequent activation of a cellular cytotoxic response. The acute IFN and

1521-6616/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2008.01.014

Interferon-α production by human monocytes inflammatory response against virus infections is mediated by cellular pattern recognition receptors (PRRs) that recognise specific molecular structures or products of viral replication [11]. Toll-like receptors are a class of membranebound PRRs that are not only capable of detecting microbial infections but are also involved in cellular maturation [11– 13]. Different cell types of the immune system have their own TLR expression patterns. Thus, human monocytes show high expression of TLR2 while not expressing TLR3 and only weak levels of TLR7 and TLR9 [14]. pDCs are characterised by high expression of TLR7 and TLR9 that is significantly higher than in B-cells, whereas TLR3 is highly expressed in CD11c+ imDCs [14–16]. pDCs respond to TLR9 ligand CpG as well as to TLR7 ligands (guanosine-/ uridine-rich ssRNA and Resiquimod) producing high levels of IFN-α [15–17]. Thus, CpG specifically induces IFN-α in pDCs [18–21]. The synthetic TLR7 agonists R848 and ssRNA have been shown to induce IFN-α production in a TLR7 specific manner [17,22–25]. Poly(I:C) (dsRNA) is a synthetic ligand for TLR3 and has been demonstrated to induce in a TLR3independent manner high amounts of IFN-α via retinoic acidinducible gene (RIG)-like helicases (RLHs) RIG-I and melanomadifferentiation associated gene 5 (MDA5) [26]. Many viruses produce double-stranded RNA during their replication cycle. It is either their genetic material (for some RNA viruses) or an essential intermediate or coproduct for viral RNA synthesis. Besides high IL-12 production, Poly(I:C) provides phenotypical DC maturation [27,28]. Further, Poly(I:C) can induce high levels of Type I interferons in murine myeloid DCs [29]. Before 1999 there was a longstanding discussion about the precise phenotype and origin of the principal type I interferon producing cell type. A lineage negative, CD4+ HLA-DR+ cell type named “natural type I interferon producing cell” (IPC) [30,31] was identified to be responsible for high IFN-α production. Identity of IPCs and certain DC subtypes was suspected because of many similarities between these cell types [32,33]. At that time even monocytes have been described to be the main producers of IFN-α in response to Sendai virus infection [34]. The confusion was thought to be clarified in 1999, when pDCs were described to be the principal source for IFN-α [35]. Given the fact, that pDCs represent the major IFN-α producers on a per cell basis the question arises whether human monocytes can be stimulated to produce similar amounts of IFN-α. Moreover, with respect to peripheral blood as a compartment, we have analysed how much IFN-α can be induced in this compartment. We have found that besides pDCs, human monocytes have the capacity to produce high amounts of IFN-α after stimulation with Poly(I:C)/Dotap. Since monocytes are more frequent in peripheral blood than pDCs we found that stimulation of monocytes through Poly(I:C)/Dotap induces higher total IFN-α levels in PBMC than TLR7 stimulation of pDCs. Our data indicate that human monocytes may represent an underestimated source of IFN-α in peripheral blood.

Materials and methods Subjects Cells were isolated from buffy coats or Heparin-anticoagulated samples of human blood donors after they gave their

253 informed consent. All included blood donors are male Caucasians aged between twenty and forty years and were found healthy in an orienting physical examination.

Preparation of PBMC PBMC were isolated from Heparin-anticoagulated blood samples (20 ml) or buffy coat preparations using Ficoll-Paque® (SERVA Electrophoresis GmbH, Heidelberg, Germany) density gradient centrifugation according to the manufacturer's instructions.

Positive selection of monocytes using magnetic or fluorescent labelled antibodies Monocytes were positively selected with magnetic beads or fluorescent antibodies and subsequently sorted with autoMACS™ (Miltenyi Biotec, Bergisch Gladbach, Germany) or FACSVantage SE cell sorter with DiVA option (BD Biosciences, San Jose, CA, USA). In the autoMACS procedure PBMC were incubated with anti CD14 MicroBeads (Miltenyi Biotec) and passed through a magnetic separation column employing the magnetic cell sorter autoMACS. Reagents were used according to the manufacturer's instructions. Purity of positively selected monocytes was N 98% according to CD14 expression determined by flow cytometry. In the following, these monocytes are referred to as “positively selected MACS monocytes”. For purification of monocytes with FACS, PBMC were incubated with anti CD14-FITC- and appropriate isotype control antibodies (BD Biosciences). After labelling, cells were sorted using FACSVantage SE cell sorter. In the following, the positive fraction is referred to as “FACS monocytes”, the negative fraction is referred to as “FACS non-monocytes”. The purity of FACS monocytes was N 98%, purity of FACS non-monocytes was N 99%.

Purification of negatively selected monocytes, DC-depleted monocytes and pDCs Reagents were used according to the manufacturer's standard protocol. Negatively selected monocytes were purified from PBMC using the Monocyte Isolation Kit II (Miltenyi Biotec) for indirect labelling of non-monocytes (anti-CD3, -CD7, -CD16, -CD19, -CD56, -CD123 and -CD235a). After magnetic labelling, cells were passed through a magnetic separation column (AutoMACS™; sensitive depletion program “depletes”) resulting in two fractions: “negatively selected monocytes” and “non-monocytes”. Unless otherwise indicated, monocytes were purified through negative selection and referred to as “monocytes”. The corresponding positive fractions were referred to as “non-monocytes”. Purity of negatively selected monocytes was N 90% according to CD14-FITC expression determined by flow cytometry (FACSCalibur™ flow cytometer; BD Biosciences, San Jose, CA, USA). DC-depleted monocytes were obtained after depletion of CD1c+ CD11cbright CD123low myeloid type 1 DCs (mDC1s), CD123bright CD11c− pDCs as well as CD1c− CD11c+ CD123− myeloid type 2 DCs (mDC2s) from the negatively selected monocyte fraction using biotinylated antibodies against blood dendritic cell Ag- (BDCA-) 1 (CD1c), BDCA-2 and BDCA-3 [36]

254 as well as anti biotin MicroBeads (Miltenyi Biotec). For this depletion up to 2 × 107 monocytes were resuspended in 130 μl PBS without magnesium and calcium (PAN Biotech GmbH, Aidenbach, Germany) containing 2 mM EDTA (Merck KGaA, Darmstadt, Germany) and 0.5% BSA (SERVA Electrophoresis GmbH, Heidelberg, Germany). First 30 μl FcR Blocking Reagent (Miltenyi Biotec) and subsequently biotin labelled anti BDCA-1, anti BDCA-2- and anti BDCA-3 antibodies were added (14 μl each). After 10 min incubation at 4 °C in the dark and one washing step, 30 μl anti biotin MicroBeads were added and incubated for 15 min at 4 °C in the dark. Labelled cells were depleted using magnetic cell sorter autoMACS™ (sensitive depletion program “depletes”). DC depletion was controlled by using the Blood Dendritic Cell Enumberation Kit (Miltenyi Biotec) and FACSCalibur™ flow cytometer (BD Biosciences). The DC depleted negatively selected monocytes will be referred to as “DC-depleted monocytes”. pDCs were purified from PBMC using the BDCA-4 dendritic cell isolation kit (Miltenyi Biotec). Cells were labelled with anti BDCA-4 antibodies coupled to colloidal paramagnetic MicroBeads and passed twice through a magnetic separation column employing the magnetic cell sorter autoMACS™. Purity of the cells was at average 94%. For quantification of pDCs and purity determination, cells were stained with anti BDCA-2-PE (Miltenyi Biotec) and analysed by flow cytometry using BD Biosciences FACSCalibur™ flow cytometer. All FACS data were analysed using CELLQuest, WinMDI 2.8, or Weasel software.

Cultivation and stimulation All cells were cultured in 96-well round-bottom plates (Greiner, Frickenhausen, Germany) in 200 μl medium, comprising RPMI 1640 with L-glutamine, penicillin/streptomycin, 10% heat-inactivated FCS Gold (PAA Laboratories, Linz, Austria), nonessential amino acids (Sigma-Aldrich, Steinheim, Germany), sodium pyruvate and HEPES (GIBCO, Invitrogen Corporation, Auckland, New Zealand). Stimulation was performed with the following stimulants: Poly(I:C) (Sigma-Aldrich, Munich, Germany) in a concentration of 50 μg/ml was observed to be optimum for stimulation (supplementary Fig. 1B), 1 μg/ml R848, 3 μg/ml CpGODN2216 (referred to as “CpG”), 5 μM CpG-ODN2006, 100 ng/ml PAM3CSK4, 2 × 107 cells HKLM, 10 μg/ml LPS, 10 ng/ml Flagellin, 0.5 μg/ml Imiquimod, 10 ng/ml FSL-1 (all InvivoGen, San Diego, California, USA), 10 μg/ml ssRNA40 (sequence: 5′-gcc cgu cug uug ugu gac uc-3′, MWG Biotech AG, Ebersberg, Germany). Immediately before application Dotap Liposomal Transfection Reagent (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate, Roche Applied Science, Penzberg, Germany) was mixed with the stimulants and used in a final concentration of 50 μg/ml that was determined as optimum for stimulation (supplementary Fig. 1B). In ELISpot experiments CpG was also combined with 5 μg/ml Dotap since CpG alone did not induce spots in this assay. The stimulants were supplemented to the cell culture at t = 0 min and cells were stimulated for 19 h at 37 °C and 5% CO2 unless otherwise described. Controls consisted of medium alone and medium with Dotap. Costimulation experiments were performed with: rh IL-1a, rh IL-1b, rh IL-3, rhIL-5, rh IL-6, rh IL-7, rh IL-8, rh IL-9, rh IL-11,

L. Hansmann et al. rh IL-12, rh IL-19, rh IL-21, rh IL-22, rh MIP-1α, rh MIP-1β, rh MIP-3β, rh G-CSF, rh M-CSF, rh IGF-1, rh IGF-2, rh TGF-β3, rh IFN-β1a, rh IFN-β1b, rh TNF-α, rh TNF-β, rh CCL-1, rh CCL-2, rh CXCL-10, rh Rantes, rh Eotaxin (all from Immuno Tools, Friesoythe, Germany), rh sCD40L (Immuno Tools; ProSpecTany TechnoGene Ltd., Rehovot, Israel), IL-4 (Strathmann Biotec GmbH & Co. KG, Hamburg, Germany), and IL10 (ReproTech, INC, Rocky Hill). If not differently described, all costimulants were used in a concentration of 20 ng/ml and were added to the cells 20 min before stimulation. Culture supernatants were collected after 19 h of stimulation and frozen at −20 °C.

CD14 and Fcγ-receptor blocking 3 × 106 cells were incubated with 15 μl anti CD14 MicroBeads (Miltenyi Biotec) in a volume of 300 μl medium. After 15 min incubation time at 4 °C, cells were washed and 1 × 106 cells were taken out for stimulation. In additional experiments, unlabelled, functional grade purified anti-human CD14 antibody (eBioscience, San Diego, CA, USA) was added in different concentrations of 10 μg/ml and 25 μg/ml. Controls consisted of equally concentrated, unlabelled, functional grade purified isotype controls (eBioscience). The effect of anti biotin MicroBeads was investigated adding 10 μl of antibodies to a total volume of 200 μl of medium at t = 0 min of stimulation. Fc-receptor blocking experiments were performed with sodium azid free antibodies against CD16, CD32 (BD Biosciences) and CD64 (eBioscience) in concentrations of 5 μg/ml, 10 μg/ml and 15 μg/ml. Controls consisted of azid free isotype controls (eBioscience). In all experiments, the respective antibodies were added 15 min prior to stimulation.

IFN-α-inhibition with BDCA-4 MicroBeads 5 × 106 PBMC were resuspended in 150 μl PBS containing 2 mM EDTA and 0.5% BSA and subsequently 50 μl FcR Blocking Reagent (Miltenyi Biotec) were added. After 5 min incubation at 4 °C in the dark 50 μl BDCA-4 MicroBeads were added followed by another 12 min of incubation. Consecutively, cells were washed one time and resuspended in medium for stimulation.

Phenotyping of monocytes At t = 0 h and t = 24 h, monocytes were analysed using flow cytometry. Surface staining was performed with CD14-FITC, CD1a-APC, HLA-DR-PE and appropriate isotype controls (all BD Biosciences) according to the manufacturer's instructions. For intracellular staining with CD68-PE (BD Biosciences), TLR3-PE (eBioscience) and appropriate isotype controls (BD Biosciences), we used the BD Cytofix/Cytoperm™ Fixation/ Permeabilization Solution Kit (BD Biosciences). After fixation in 250 μl Cytofix/Cytoperm for 15 min, cells were washed twice in 1 ml Permeabilization solution and subsequently incubated with the respective antibodies for 30 min at 4 °C in the dark. Finally cells were washed twice with Permeabilization solution and resuspended in buffer for analysis.

Interferon-α production by human monocytes

255

Detection of cytokines ELISA After thawing the cell culture supernatants, human IFN-α was measured by ELISA, using Human IFN-α ELISA Kit (PBL Biomedical Laboratories, Piscataway, New Jersey, USA). The ELISA is specific for human IFN-αA, α2, αA/D, αD, αK and α4b. No cross-reactivity with human IFN-γ or human IFN-β. The specific activity of the interferon used as standard is based on international standard for human IFN-α [37]. Because the interferon samples are titrated against the international standard, the values can be determined in U/ ml as well as pg/ml. The conversion factor of about 3–5 pg/U is applicable for human IFN-α [38]. IFN-β was detected with Human IFN-Beta ELISA Kit (PBL Biomedical Laboratories). Extinctions were measured using SLT SPECTRA ELISA-reader (SLT Labinstruments, Crailsheim, Germany). ELISpot For ELISpot analysis, cells were stimulated in 96-well flatbottom plates with PVDF membranes (MultiScreen-IP, Milli-

Figure 2 Poly(I:C)/Dotap induces second highest total IFN-α levels in PBMC. 1 × 106 PBMC were stimulated with Poly(I:C)/ Dotap or pDC-specific CpG, R848 and ssRNA/Dotap. Highest IFNα amounts were detected after stimulation with CpG followed by Poly(I:C)/Dotap whereas ssRNA/Dotap or R848 induced only moderate cytokine production. Poly(I:C)/Dotap induced significantly higher IFN-α production than TLR7 stimulation (p b 0.01 for ssRNA and p b 0.02 when compared to R848 stimulation, Mann–Whitney U-Test). Results are representative for n = 16 independent experiments.

pore Corporation, Billerica, MA). The plates were coated with anti IFN-α antibody (Mabtech AB, Hamburg, Germany). After 19 h stimulation, cytokines were visualised using ELISpot-Assay for Human IFN-α (Mabtech AB). Antibodies were specific for human IFN-α subtypes 2a, 2b and 2c. Spots were counted and measured using ELISpot Bioreader®-4000 and Bioreader© Generation 8.5 software, Patch 0.5 (BIO-SYS GmbH, Karben, Germany).

Cell counting All cells with the exception of purified pDCs were counted employing Sysmex cell counter KX-21N (Sysmex Deutschland GmbH, Düsseldorf, Germany). pDCs were counted in a dilution of 30 μl cell-suspension + 10 μl Trypan-Blue in a Neubauer counting chamber.

Statistical analysis Statistical analysis was performed using the Mann–Whitney U-test or Wilcoxon test, when appropriate. All tests were performed two-tailed. For all tests SPSS® software, version 12 (SPSS Inc., Chicago, IL) was used and a probability b 0.05 was considered significant.

Results Poly(I:C) causes high IFN-α production in PBMC Figure 1 Poly(I:C)/Dotap induces marked IFN-α production in PBMC. 1 × 106 PBMC were stimulated with an array of PAMPs in combination with or without Dotap (50 μg/ml). High IFN-α production was detected after stimulation with Poly(I:C)/Dotap besides the pDC-specific stimulants CpG, ssRNA/Dotap or Imiquimod/Dotap. The difference between CpG/Dotap and Poly(I:C)/Dotap induced IFN-α is statistically not significant. Results are representative for n = 3 independent experiments.

IFN-α is usually induced after stimulation of TLR7 or TLR9 and pDCs expressing these TLRs are supposed to represent the principal source of this cytokine [35]. We analysed the IFN-α inducing capacity of an array of PAMPs, representing ligands for TLR1-9, in human PBMC in order to determine the capacity of these agents to contribute to IFN-α in peripheral blood. As expected we

256 measured high amounts (mean of n = 3 independent experiments) after TLR9 stimulation with its synthetic ligand CpG (2863.9 pg/ml) and after TLR7 stimulation with the synthetic ligands Imiquimod (257.5 pg/ml) and ssRNA40 (560.8 pg/ml) in combination with Dotap (mediator of cellular uptake) (Fig. 1). Unexpectedly we detected marked IFN-α production after stimulation with Poly(I:C) and Dotap (1614.7 pg/ml). With respect to PBMC as compartment, Poly(I:C)/Dotap induced higher IFN-α production than pDC-specific TLR7 stimulation via ssRNA40 or Imiquimod (Fig. 1). In these experiments Dotap was used to facilitate the cellular uptake and to stabilize Poly(I:C) as well as ssRNA40 and Imiquimod. PAM3CSK4, HKLM, LPS, Flagellin and FSL-1 caused no significant IFN-α production in PBMC.

Dotap increases Poly(I:C) mediated IFN-α production The IFN-α inducing effect of Poly(I:C) in PBMC was first described by Field et al. in 1967 [39]. Further groups described Poly(I:C) as an IFN-α inducer in different subtypes of PBMC [40] but with respect to Poly(I:C) stimulation alone, only low IFN-α amounts were detected. Dotap is a cationic liposome reagent for transfection of DNA, RNA, oligonucleotides, ribonucleoprotein particles and proteins, in general negatively charged molecules. Since Poly(I:C) is detected by intracel-

L. Hansmann et al. lular pattern recognition receptors TLR3, MDA5 and RIG-I, addition of Dotap is reasonable. Addition of Dotap to Poly(I:C) enhanced the IFN-α inducing capacity of Poly(I:C) in PBMC twelve-fold (140.2 pg/ml without and 1614.7 pg/ml with Dotap, mean of n = 3 independent experiments). No major enhancing effects of Dotap (50 μg/ ml) on IFN-α production were detected after stimulation with R848, CpG, PAM3CSK4, HKLM, LPS, Flagellin or FSL-1 (Fig. 1). We did not observe cytotoxic side-effects of Dotap ± Poly (I:C) using Trypan-Blue staining.

Poly(I:C)/Dotap is the second strongest stimulant for IFN-α in PBMC To compare the overall IFN-α inducing capacity of pDC-specific R848, ssRNA40 and CpG with Poly(I:C)/Dotap in peripheral blood we stimulated equal numbers of 1×106 PBMC from healthy blood donors. Poly(I:C)/Dotap induced the second highest amounts of IFN-α (1053.6 pg/ml) when compared to pDC-specific CpG (4227.6 pg/ml) and ssRNA40/Dotap (301.3 pg/ml) or R848 (344.5 pg/ml) (mean values of n =16 independent experiments) (Fig. 2). Thus, in total PBMC, Poly(I:C)/Dotap induced about three times more IFN-α than pDC-specific TLR7 ligands ssRNA40 (pb 0.05) or R848 (pb 0.05) (Mann–Whitney U-test) (Fig. 2). Dose dependent effects for different Poly(I:C), CpG and R848 concentrations (±Dotap) were determined in titration experiments (supplementary Fig. 1).

Figure 3 Poly(I:C)/Dotap stimulates highest cell numbers in PBMC when compared to CpG or R848. 2.5 × 105 PBMC were stimulated with Poly(I:C)/Dotap, R848 or CpG/Dotap. In this experiment we combined CpG with Dotap (5 μg/ml) to avoid an interaction of CpG with the PVDF microtiter-plate membrane. (A) Most spots were detected after Poly(I:C)/Dotap stimulation followed by CpG and R848. Poly(I:C) induced significantly higher spot numbers than CpG (p b 0.05) and R848 (p b 0.05) (Wilcoxon test) (B) Spots after Poly(I:C)/ Dotap are smaller than spots after R848 or CpG/Dotap stimulation, indicating that CpG and R848 induce higher IFN-α production per cell. Results are representative for n = 7 independent experiments.

Interferon-α production by human monocytes

IFN-α producers after Poly(I:C)/Dotap are more frequent than after CpG or R848 stimulation To get an impression about the frequencies of cells producing IFN-α among PBMC, we used ELISpot technique. Identical amounts of 250,000 PBMC were stimulated with CpG/Dotap, R848 or Poly(I:C)/Dotap. The results showed most spots (mean of n = 7 independent experiments) after Poly(I:C)/ Dotap (371 spots), only a few spots after R848 (81 spots) and even less spots after CpG/Dotap (244 spots) (Fig. 3A). Thus we revealed that Poly(I:C) stimulation effected most cells from PBMC. Further we could show that CpG and R848 induced larger sized spots than Poly(I:C) (Fig. 3B). This shows that pDCs produce high amounts whereas Poly(I:C)/Dotapstimulated cells produce only weak amounts of IFN-α per cell. Nevertheless, because of their high number in peripheral blood, cells stimulated by Poly(I:C)/Dotap produce more total IFN-α than R848 stimulated pDCs.

257 To monitor the phenotype of monocytes, we performed FACS stainings for CD14, CD68, CD1a, HLA-DR and TLR3 at t = 0 h and t = 24 h of Poly(I:C)/Dotap stimulation (supplementary Fig. 2). CD14 expression was down-regulated after 24 h in unstimulated cells whereas Poly (I:C)/Dotap stimulation inhibited this down-regulation. The expression of CD68, CD1a, HLA-DR and TLR3 was comparable at t = 0 h and t = 24 h in the two groups (±Poly(I:C)/Dotap) (supplementary Fig. 2).

Direct comparison of monocytes' and pDCs' capacity to produce IFN-α

Monocytes are the main source of IFN-α after Poly (I:C)/Dotap stimulation

Although numerous studies have demonstrated the unsurpassed capacity of pDCs to produce high amounts of IFN-α, little is known about the IFN-α producing capacity of monocytes directly compared to pDCs. We stimulated pDCs and monocytes with the most effective TLR7 and TLR9 ligands as well as Poly(I:C)/Dotap (Fig. 6A). The results showed that pDCs stimulated with CpG produced unsurpassed IFN-α amounts (32,217.8 pg/ml) whereas IFN-α after R848 stimulation was markedly reduced (957.1 pg/ml)

In 1989 Lepe-Zuniga described induction of about 20 U/ml IFN-α in human monocytes after stimulation with Poly(I:C) [40]. To characterise the cell type that is responsible for the strongly increased IFN-α production after Poly(I:C)/Dotap stimulation, we performed cell sorting and depletion experiments. The following PBMC populations were sorted: (1) Ficoll-density gradient isolated PBMC, (2) monocytes (CD3−, CD7−, CD16−, CD19−, CD56−, CD123−, and CD235a−), (3) DC-depleted monocytes (depletion of CD1c+ CD11cbright CD123low mDC1s, CD123bright CD11c– pDCs and CD1c– CD11c+ CD123– mDC2s from monocytes), (4) non-monocytes (CD3+, CD7+, CD16+, CD19+, CD56+, CD123+, and CD235a+). Remaining DC subsets in the sorted PBMC fractions are displayed in Fig. 4B–H. Each fraction was stimulated with Poly(I:C)/Dotap and IFN-α amounts were measured in the supernatants using ELISA (Fig. 4A). Monocytes produced the highest amounts of IFN-α (3980.7 pg/ml) whereas non-monocytes produced only very weak IFN-α levels (207.1 pg/ml) (mean of n = 4 independent experiments). Importantly, IFN-α did not change upon depletion of all major DC subsets from monocytes (3597.3 pg/ml in the DC-depleted fraction), indicating that human monocytes but not DCs represent the major source of IFN-α after Poly(I:C)/Dotap stimulation (Fig. 4A). Moreover, the IFN-α levels positively correlated with the percentage of CD14+ cells in the different fractions. The ELISA results were confirmed by an IFN-α specific ELISpot assay (Fig. 5). PBMC, monocytes and non-monocytes were stimulated with Poly(I:C)/Dotap. Highest spot numbers were detected after Poly(I:C)/Dotap stimulation of monocytes (315 spots). Accordingly, the ELISpot analysis indicated a marked increase of spot numbers upon depletion of nonmonocytes from the PBMC fraction (166 spots in the PBMC) and only a few spots remaining in the non-monocytes fraction (54 spots) (Spot numbers are mean values of n = 4 independent experiments) (Fig. 5). These data suggest that monocytes and not myeloid or plasmacytoid DCs represent the main producers of IFN-α after Poly(I:C)/Dotap stimulation in human PBMC.

Figure 4 Monocytes are the principal IFN-α producers after Poly(I:C)/Dotap stimulation. (A) 1 × 106 PBMC, non-monocytes, monocytes and DC-depleted monocytes were stimulated with Poly(I:C)/Dotap. We observed an enhancement of IFN-α production after depletion of non-monocytes from PBMC. In contrast, IFN-α production in the monocytes fraction was not affected by subsequent depletion of all major DC subsets. IFN-α production by monocytes was significantly higher than by PBMC (p b 0.05 for monocytes and for DC-depleted monocytes, Mann–Whitney Utest). (B–G) DC subsets were identified after staining monocytes and DC-depleted monocytes with CD14, CD19, BDCA-1, -2 and -3 monoclonal antibodies. Displayed are 50,000 events. (B) The “leukocytes” region excludes debris and platelets. (C) Definition of “R2” through exclusion of B-cells, monocytes, granulocytes and dead cells. (D) To identify mDC1s and pDCs, this dot plot is gated on “leukocytes” and “R2”. (E) This dot plot is also gated on “leukocytes” and “R2” to identify mDC2s. (F, G) Final cluster resolutions of pDCs, mDC1s and mDC2s in monocytes before (F) and after (G) DC depletion. They are gated on “leukocytes” and “R2” and (mDC1s or pDCs or mDC2s). (H) shows the relative frequencies/leukocytes of mDC1s, pDCs and mDC2s in PBMC, monocytes and DC-depleted monocytes. Data represent mean values of n = 4 independent experiments.

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Figure 4 (continued ).

L. Hansmann et al.

Interferon-α production by human monocytes

259 Since inhibition of IFN-α production in BDCA-4 selected pDCs after viral or CpG stimulation has been reported [41, 42], we investigated the BDCA-4 effect in our experimental setup and stimulated BDCA-4 MicroBeads-labelled or unlabelled PBMC with CpG or R848. IFN-α production was significantly reduced in BDCA-4 MicroBeads-labelled PBMC after stimulation with CpG but not after R848 stimulation (Fig. 6B). Therefore IFN-α levels in BDCA-4 isolated, CpGstimulated pDCs may be underestimated.

Modulation of Poly(I:C) induced IFN-α production in monocytes through cytokines

Figure 4 (continued ).

(Fig. 6A). On a per cell basis, R848 stimulation of pDCs still induced higher IFN-α levels when compared to equal numbers of Poly(I:C)/Dotap-stimulated monocytes (197.8 pg/ml) (mean of n = 6 independent experiments) (Fig. 6A). No significant IFN-α levels were detected after stimulation of pDCs with Poly(I:C)/Dotap or Monocytes with CpG or R848 (Fig. 6A).

We questioned whether Poly(I:C) induced IFN-α production in monocytes could be significantly modulated through costimulation with cytokines and analysed a total of 33 cytokines (IL-1a, IL-1b, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-19, IL-21, IL-22, MIP-1α, MIP-1β, MIP3β, G-CSF, M-CSF, IGF-1, IGF-2, TGF-β3, IFN-β1a, IFN-β1b, TNF-α, TNF-β, CCL1, CCL2, CXCL10, Rantes, Eotaxin, and sCD40L). These experiments indicated only moderate stimulatory effects of IFN-β-1a (60 ng/ml) and moderate inhibitory effects of TNF-α on Poly(I:C)/Dotap mediated IFN-α production in monocytes. IFN-α levels were enhanced from 4378.1 pg/ml to 5714.7 pg/ml after costimulation with IFN-β-1a whereas TNF-α

Figure 5 IFN-α specific ELISpot confirms monocytes as the principal IFN-α source after Poly(I:C)/Dotap stimulation. 2.5 × 105 PBMC, monocytes or non-monocytes were stimulated with Poly(I:C)/Dotap. Most spots were detected in the monocytes fraction in contrast to less spots in PBMC and only a few spots remaining in the non-monocyte fraction. Spot numbers are representative for n = 4 independent experiments.

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L. Hansmann et al. First IFN-α production was detected after 8 h, reaching its maximum at 24 h of stimulation (Fig. 7). IFN-β levels were first detected after 8 h of stimulation and longer stimulation periods (up to 24 h) did not result in increase of IFN-β production (Fig. 7). Although IFN-β was not produced prior to IFN-α, it still might be involved in the increase of IFN-α levels during longer stimulation periods (8 h–24 h, Fig. 7).

MicroBeads suppress Poly(I:C)/Dotap induced IFN-α production in monocytes

Figure 6 (A) pDCs produce highest IFN-α amounts per cell. 4 × 104 pDCs or 5 × 105 monocytes were stimulated with CpG, R848 or Poly(I:C)/Dotap. After ELISA measurement, IFN-α amounts were scaled to an equivalent of 4 × 104 cells. pDCs produced unsurpassed amounts of IFN-α in response to CpG followed by R848 stimulation. Monocytes produced only weak amounts, when stimulated with Poly(I:C)/Dotap. No significant IFN-α production was detected after stimulation of pDCs with Poly(I:C)/Dotap or monocytes with CpG or R848. Results are representative for n = 6 independent experiments. (B) BDCA-4 MicroBeads suppress IFNα-production after CpG- but not after R848 stimulation. 5 × 105 PBMC were labelled with BDCA-4 MicroBeads and subsequently stimulated with CpG or R848. IFN-α-production after CpG stimulation was suppressed in BDCA-4 MicroBeads-labelled cells (p b 0.05, Wilcoxon test) whereas no significant effect on R848 stimulation was detected. Results are representative for n = 7 independent experiments.

Before isolating negatively selected monocytes we purified monocytes through positive selection with anti CD14 MicroBeads. Using this technique, we observed an inhibitory effect of CD14 MicroBeads on Poly(I:C)/Dotap mediated IFN-α production. Positively selected MACS monocytes produced less IFN-α than the corresponding PBMC fractions (Fig. 8A) questioning the conclusion that monocytes represent the critical IFN-α producing population after Poly(I:C)/Dotap stimulation (2818.1 pg/ml in PBMC vs. 1072.5 pg/ml in positively selected MACS monocytes after separation, mean of n = 6 independent experiments) (Fig. 8A). In contrast, when monocytes were isolated by depletion of non-monocytes from PBMC we observed substantially increased IFN-α production in the negatively selected monocyte fraction, in comparison with the corresponding PBMC fraction (Fig. 8B). Accordingly, the corresponding non-monocytes exhibited only residual IFN-α levels (Fig. 8B). In order to analyse the mechanism of IFN-α suppression, we stimulated negatively selected monocytes after pre-incubation with CD14 MicroBeads, MicroBeads coupled to an irrelevant antibody (anti biotin), purified CD14 antibodies and isotype control antibodies. These experiments showed that CD14 MicroBeads profoundly inhibited IFN-α production (Fig. 8C). This inhibitory effect of antibody-coupled MicroBeads was CD14-independent, since MicroBeads coupled to an irrelevant antibody inhibited IFN-α production in a similar manner (Fig. 8C). Accordingly, unlabelled purified CD14 antibody (10 μg/ml and 15 μg/ml) exhibited no inhibitory effects on IFN-α production (Fig. 8D) indicating that the MicroBead particles were responsible for

suppressed IFN-α production to 3200.2 pg/ml. (mean of n=3 independent experiments). The differences in IFN-α production after costimulation with IFN-β-1a and TNF-α were statistically not significant when compared to controls.

Monocytes simultaneously produce IFN-α and -β after Poly(I:C)/Dotap stimulation Since IFN-β can enhance Poly(I:C)/Dotap mediated IFN-α production, we questioned if IFN-β is produced by monocytes prior to IFN-α.

Figure 7 IFN-α and IFN-β are produced simultaneously in monocytes after Poly(I:C)/Dotap. 5 × 105 monocytes were stimulated with Poly(I:C)/Dotap and IFN-α and -β were measured after 0 h, 4 h, 8 h, 16 h and 24 h. Both cytokines were detectable as early as 8 h of stimulation. IFN-α levels continuously increased from 656.1 pg/ml at 8 h to 2660.1 pg/ml at 24 h whereas IFN-β levels remained at the 8 h level of 225.6 pg/ml (266.0 pg/ml at 24 h). Results are representative for n = 4 independent experiments.

Interferon-α production by human monocytes

261

Figure 8 Suppression of IFN-α production with MicroBeads. (A) 1 × 106 Positively selected MACS monocytes, non-monocytes and PBMC were stimulated with Poly(I:C)/Dotap (n = 6). Positively selected MACS monocytes produced less IFN-α than the corresponding PBMC fractions (p b 0.05, Mann–Whitney U-test). (B) Monocytes were isolated by labelling and depletion of non-monocytes (n = 8) and 1 × 106 cells of the different fractions were stimulated with Poly(I:C)/Dotap. Monocytes produced higher levels of IFN-α than PBMC (p b 0.05, Mann–Whitney U-test). (C) Response of 1 × 106 monocytes on Poly(I:C)/Dotap stimulation in presence of CD14 MicroBeads or anti biotin MicroBeads (n = 2). IFN-α levels strongly decreased after adding of CD14 MicroBeads or anti biotin MicroBeads during stimulation. (D) 5 × 105 monocytes were stimulated with Poly(I:C)/Dotap in presence of anti-CD14, -CD16, -CD32 and -CD64 antibodies as well as isotype controls in concentrations of 10 μg/ml (n = 4). Antibodies against CD14 and Fcγ-receptors did not significantly influence Poly(I:C) mediated IFN-α production in human monocytes.

the IFN-α suppression. To control that Fc-receptor binding was not responsible for these inhibitory effects, we performed additional experiments with anti-CD16- (FcγRIII), anti-CD32(FcγRII) and anti-CD64- (FcγRI) antibodies (5 μg/ml, 10 μg/ml and 15 μg/ml) indicating that Fc-receptor binding did not suppress IFN-α production in monocytes (Fig. 8D).

Discussion IFN-α is essential for antiviral immunity and several reports have demonstrated that TLR-expressing DCs, primarily pDCs, represent the principle IFN-α producing cells in the immune system [29,35]. Based on the discovery of novel cytoplasmatic dsRNA receptors MDA-5 and RIG-I, the question was arising how much IFN-α can be produced by non-DCs in peripheral blood. This question is of clinical relevance, since many viruses that are potent IFN-α inducers replicate in the cytoplasm and are unlikely to expose their nucleic acid structures at early stages (before cell lysis) to TLRs. Moreover, although many studies have focused on pDCs or other leukocyte subsets, little is known about the overall IFN-α

producing capacity in peripheral blood as compartment. In this study we have analysed systematically the IFN-α inducing capacity of different PAMPs in human PBMC. Our data reveal that Poly(I:C), mimicking dsRNA, represents the second strongest total IFN-α inducer in PBMC, when complexed to Dotap, facilitating its cellular uptake. Based on these results we have analysed the cellular source of IFN-α in Poly(I:C)/Dotap-stimulated PBMC and identified monocytes as major producers. dsRNA is detected by different PRRs: TLR3, protein kinase R (PKR), the DExD/H-box helicase RIG-I and MDA 5 [11,26,43]. TLR3 is predominantly expressed on CD11c+ imDCs, whereas monocytes do not express TLR3 [14]. The stress kinase PKR can bind dsRNA that results in phosphorylation of IĸB and thus promotes IFN-α/β gene expression. Further PKR is involved in dsRNA mediated apoptosis. Other studies report a virus dependent enhancement of TLR3- and PKR expression in certain cell types [6,44–49]. Although PKR may influence IFN-α production after Poly(I:C) stimulation, recent studies identified MDA-5 and RIG-I to have a predominant role in recognition of single-stranded as well as double-stranded RNA [50–52]. MDA 5 and RIG-I, members of the IFN inducible DExD/Hbox helicase family, exhibit N-terminal caspase recruitment

262 domains (CARD) and a C-terminal helicase domain [53]. The helicase domain with intact ATPase activity is responsible for dsRNA recognition whereas the CARD domains transmit downstream signals resulting in activation of NF-ĸB and IRF-3. Subsequent gene activation by these factors induces antiviral functions such as IFN-α production [50,53–55]. PKR and retinoic acid inducible gene protein as well as MDA 5 product represent intracytoplasmatic receptors that can recognise dsRNA in a TLR3 independent manner and are key-players in detection as well as eradication of replicating viral genomes [11]. Poly (I:C) in particular was recently shown to be recognised by MDA-5 and not RIG-I [56]. With respect to a recent paper by Diebold et al. one might have been expected that non-plasmacytoid (i.e. myeloid and lymphoid-related) DCs represent the major producers of IFNα after stimulation with PAMPs mimicking dsRNA [29]. Diebold and colleagues have demonstrated in a murine system that non-plasmacytoid DCs, transfected with dsRNA, are able to produce comparable high amounts of IFN-α like pDCs. Surprisingly, we have found in the human system that depletion of myeloid and plasmacytoid DC subsets via biotinylated BDCA-1, -2, and -3 antibodies did not reduce the IFN-α producing capacity of human PBMC after Poly(I:C) stimulation. In contrast, we have observed a sharp reduction in IFN-α, both by ELISA and ELISpot analysis, after depletion of monocytes from human PBMC. These results were confirmed by experiments with purified monocytes and suggest that human monocytes might represent an underestimated source of IFN-α in human peripheral blood. It is important to note, that antibody-coated microbead-particles strongly suppressed IFN-α production by monocytes. Thus, analyses that are using CD14-microbead selected monocytes are prone to underestimate the capacity of monocytes to produce IFN-α. >We have found that this suppression of IFN-α in human monocytes is not related to the CD14 epitope or unspecific binding to Fcγ-receptors, but associated with MicroBeads. MicroBeads are superparamagnetic Fe-oxid particles that are coupled to antibodies. They are approximately 50 nm in size and biodegradable. Further studies are necessary to analyse the mechanism of MicroBead-associated IFN-α production. Possible explanations are either interference of the MicroBeads with functional maturation of monocytes and subsequent modulated cytokine responses or interference of the endocytosis pathway with IFN-α production capacity of monocytes. To our knowledge we have presented in this paper for the first time quantitative ELISpot data on IFN-α production of human leukocytes. It should be noted, that in ELISpot analyses we have combined CpG with Dotap because CpG alone induced no spots. We have suspected an interaction of the PVDF ELISpot membrane with CpG because control stimulations of the same cells in conventional microtiter plates without PVDF ELISpot membranes induced very high IFN-α levels as indicated by ELISA. The suspected interaction of CpG with the PVDF ELISpot membrane was avoided by adding Dotap in a concentration of 5 μg/ml to stabilise CpG. One might argue that the spot numbers after Poly(I:C)/Dotap stimulation of PBMC are relatively low when compared to CpG stimulation of PBMC (Fig. 3A). In our opinion this is primarily a matter of sensitivity of the ELISpot assay, since monocytes produce far lower IFN-α levels than pDCs (Fig. 6A).

L. Hansmann et al. In summary, in the presented study we have compared by ELISA and ELISpot assays the capacity of human PBMC and different leukocytes subsets to produce IFN-α after stimulation with PAMPs. Our results indicate that monocytes can produce large amounts of IFN-α after Poly(I:C)/Dotap stimulation. With respect to their higher absolute numbers in peripheral blood and in direct comparison to TLR7- or TLR9-stimulated pDCs, adequately stimulated monocytes may represent a hitherto underestimated source of IFN-α in human blood.

Acknowledgments We gratefully acknowledge Angelika Nockher, Christina Lang and Gabriela Haley for their excellent technical assistance. This work was supported by grants from the Bundesministerium für Bildung und Forschung (Germany), Nationales Genomforschungsnetz (NGFN1 IE-S08T03, NGFN2 NIE-S14T30) and by Grant GE-S13T01 (to G.B. and H.H.) and the excellence cluster cardiopulmonary system (DFG).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.clim.2008.01.014.

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