A novel, complement-mediated way to enhance the interplay between macrophages, dendritic cells and T lymphocytes

Molecular Immunology 47 (2009) 438–448

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A novel, complement-mediated way to enhance the interplay between macrophages, dendritic cells and T lymphocytes Noémi Sándor a , Domonkos Pap a , József Prechl b , Anna Erdei a,b , Zsuzsa Bajtay a,b,∗ a b

Department of Immunology, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1/C, Budapest H-1117, Hungary Research Group of Immunology of the Hungaria Academy of Sciences, Eötvös Loránd University, Pázmány Péter sétány 1/C, Budapest H-1117, Hungary

a r t i c l e

i n f o

Article history: Received 3 August 2009 Received in revised form 13 August 2009 Accepted 28 August 2009 Available online 30 September 2009 Keywords: Dendritic cell Complement C3 T cell activation Macrophages

a b s t r a c t Recently it has been reported that human C3-deficiency is associated with impairments in dendritic cell differentiation. Here we investigated how complement C3 influences the phenotype and functional activity of human dendritic cells. We show that human monocyte-derived dendritic cells (MDCs) when incubated with native, hemolytically active C3, bind the activation fragments of C3 covalently. This reaction directs MDCs to increase expression of MHCII, CD83 and CD86, moreover it results in a significantly enhanced secretion of TNF-␣, IL-6 and IL-8. A further functional consequence of C3b-fixation is the elevated capacity of the dendritic cells to stimulate allogeneic T cells. The distinct role of covalently fixed C3-fragments is strongly supported by our results obtained with MDCs where CD11b expression was downregulated by siRNA. To reveal the possible in vivo significance of the present findings we modelled a phenomenon occurring during inflammation, where C3 is produced locally by activated macrophages. In these cocultures MDCs were found to fix substantial amounts of macrophage derived C3-fragments on their cell membrane. Our data provide compelling evidence that antigen presenting cells arising in complement-sufficient environment mature to competent stimulators of T cells. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction The complement system is one of the principal components of both the innate and adaptive immunity. The deposition of activation fragments derived from its major component C3, allows the binding of antigens to cells expressing different complement receptors (CR). The ligand for CR1 (CD35) is C3b, while CR2 (CD21) interacts mainly with the final degradation product; C3d(g). While CR1 is expressed on a wide variety of cell types, CR2 is restricted to B lymphocytes and follicular dendritic cells. CR3 (CD11b/CD18) and CR4 (CD11c/CD18) are members of the ␤2 -integrin family, and are both able to bind iC3b. These receptors are expressed on several cell types, including murine and human DCs and their most important role is the binding and phagocytosis of pathogens opsonized by C3-fragments (Le Cabec et al., 2002). DCs are known as sentinels of the immune system that can be found all over the body. Depending on the activation stimuli in the microenvironment they can mediate several functions. The basic importance of DCs in generating immunity is underlined by their unique ability to activate naive T lymphocytes, linking innate and

∗ Corresponding author at: Eötvös Loránd University, Institute of Biology, Department of Immunology H-1117, Pázmány Péter sétány 1/C, Budapest, Hungary. Tel.: +36 13812175; fax: +36 13812176. E-mail address: [email protected] (Z. Bajtay). 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.08.025

adaptive immunity this way. For the efficient activation of T cells DCs have to provide three separate signals, such as presentation of antigens, expression of costimulatory molecules and production of cytokines (Banchereau et al., 2000; Reis e Sousa, 2006; Shortman and Liu, 2002). In the absence of inflammation, DCs in the peripheral tissues take up apoptotic cells, process self-antigens and migrate constitutively to the draining lymph node. Without danger signals however, they do not express costimulatory molecules, therefore fail to stimulate T cells. Maturation of DCs can be induced by various stimuli including microbial products, immune complexes and inflammatory cytokines (Banchereau et al., 2000; Shortman and Liu, 2002). The influence of different elements of the complement system on various functions of DCs has been studied by several authors, mainly by analyzing the role of C3 (Reis e Sousa et al., 2007; van Kooten et al., 2008; Zhou et al., 2007). It had been shown that binding of iC3b-opsonized apoptotic cells to CR3 bearing DCs induce the generation of tolerogenic type DC (Ehirchiou et al., 2007; Sohn et al., 2003; Verbovetski et al., 2002). Others demonstrated that C3 plays a role in antigen presentation and showed that in C3 KO animals this function of DCs and macrophages is strongly impaired (Zhou et al., 2006). Also in a murine model system, it had been shown that DC-derived C3 is required for full T cell activation and development of a Th1 phenotype (Peng et al., 2006). The important role of C3 in the differentiation of DCs has been clearly demonstrated recently by Ghannam et al. (2008), studying a human C3-deficient case.

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Component C3 is the most abundant complement protein in serum (1.2–1.5 mg/mL). Its primary source is the liver, however several cell types – including macrophages – are able to synthesize this acute phase protein (Colten et al., 1986). It contains an intrachain thioester bond, which is exposed by the conformational alteration caused by the limited proteolysis of the protein. Nascent C3b generated this way possesses an active thioester group with the transient capability of its revealed carbonyl group to esterify hydroxyl groups of the activating surface – i.e. various cell membrane structures and antigens. By its covalent attachment, C3b is central to the function of complement system; it is a subunit of both C3 and C5 convertases, it amplifies complement cascade, labels cells for phagocytosis and stimulates the adaptive immune response (Janssen and Gros, 2007; Law et al., 1984; Law and Dodds, 1997). Production and covalent binding of C3 to the human monocytic cell line U937 had been proven by Maison et al. (1989), moreover, C3 from normal murine serum was found to bind to Lewis lung carcinoma cells that do not express CRs mainly through covalent binding (di Renzo et al., 1999). Although the acceptor molecule could not be identified, it had been shown that after internalization of bound C3 the growth of these cells was induced and mediated by C kinase (Longo et al., 2005). We have demonstrated earlier that macrophages and B cells are able to fix C3-fragments covalently on their surface, resulting in their enhanced capacity to present antigens (Papp et al., 2008; Erdei et al., 1992; Kerekes et al., 1998). Macrophages are known to secrete a wide variety of proteins, including all the components of the complement system (Andrews et al., 1995; Morgan and Gasque, 1997), moreover macrophage derived C3 has been shown to locally opsonize various particles and pathogens leading to their more efficient phagocytosis (Ezekowitz et al., 1984). Although many data support the in vivo relevance of covalently fixed C3b (Biro et al., 1992; Erdei et al., 1991; Ezekowitz et al., 1983; Fabry et al., 1985; Gergely et al., 1985; Janssen and Gros, 2007; Kerekes et al., 1998; Law et al., 1984; Law and Dodds, 1997; Maison et al., 1989; Papp et al., 2008; Longo et al., 2005) the acceptor-site could be identified so far only on B lymphocytes, but not on the other cell types. On B cells complement receptor type 2 (CR2, CD21) had been demonstrated to be the major covalent binding-site (Marquart et al., 1994; Mold et al., 1988), but since this receptor is not expressed by macrophages, DCs and erythrocytes, additional cell membrane molecules have to be identified which are able to fix C3-fragments covalently. To gain a better understanding of the immunoregulatory role of complement we focused on the phenotypical and functional changes caused by the covalent interaction of C3b with DC. We examined how covalently fixed C3b influences maturation of the cells and their capacity to stimulate alloreactive T lymphocytes. We also investigated the possible significance of our findings by modeling the in vivo situation, where DCs and macrophages are in close vicinity in the peripheral lymphoid tissues. Our results demonstrate that DCs are able to fix locally produced, macrophage derived C3fragments and reveal for the first time that acceptor-bound C3b increases the capacity of these antigen presenting cells to stimulate T lymphocytes. Our results provide convincing explanation of the recently published data of Ghannam et al. (2008), who have shown that primary human C3-deficiency escorts a total lack of DC maturation. 2. Materials and methods 2.1. Reagents and antibodies For the separation and culture of cells the following materials were used: Ficoll-Paque (Amersham, Uppsala, Sweden), gelatine (Merck, Germany), CellGro serum-free DC medium (CellGenix, Germany), recombinant human (rHu) IL-4 (PromoCell, Germany), rHu GM-CSF (Leucomax, Novartis, Switzerland). LPS,

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PMA, methylamine hydrochloride and FITC-dextran were all from Sigma (Hungary). Carboxy-fluorescein-succinimide-ester (CFSE) was obtained from Molecular Probes (Invitrogen, USA), saponin from Fluka (Switzerland). The following first and secondary antibodies were used for FACS analysis: FITC-labelled anti-biotin mAb (BN-34) from Sigma (Hungary), Alexa488-labelled goat-anti-mouseIg from Molecular Probes. Mouse anti-hu CD83 (mIgG1, HB15A17.11) from Serotec (UK), biotinylated mouse anti-hu CD80 (mIgG1, L307.4), biotinylated mouse anti-hu CD86 (mIgG2b, IT2.2), FITC-labelled anti-hu-MHCII (mIgG2a, Tü39) and APC-labelled anti-hu CD14 mAb from BD PharMingen (USA), PE-conjugated mouse-anti-hu mannose receptor (mIgG1, 3.29B1.10) from Immunotech (France), PE-conjugated anti-LAMP-1 (mIgG1, H4A3) from Santa Cruz (USA) and polyclonal rabbit-anti-hu-C3c-FITC and monoclonal anti-hu CD11b-RPE from Dako (Denmark). CD11b ligand binding site specific monoclonal antibody TMG6-5 (mIgG1) was obtained from István Andó, BRC, Szeged, Hungary. For inhibiting the attachment of activated C3-fragments to monocyte-derived dendritic cells (MDCs), goat-anti-human C3 (Fab )2 antibody was used from Cappel (ICN, USA). HRPO-conjugated anti-hu C3 was also obtained from Cappel. 2.2. Isolation of C3 Human C3 was isolated from freshly drawn serum of healthy donors by fast protein liquid chromatography (FPLC) on a MonoQ HR5/50 column as described by Basta and Hammer (1991). Fractions containing intact C3 were pooled, dialyzed against PBS, concentrated and kept at −70 ◦ C until use. The presence of endotoxin contamination in the purified, concentrated C3 preparation was less than 0.1 EU/mL as measured by the limulus amoebocyte lysate (LAL) assay (Cambrex, MD, USA). To prevent the covalent binding of C3 in certain experiments C3 and serum pretreated by methylamine was used (Kerekes et al., 1998). 2.3. Generation and treatment of human MDCs by C3 MDCs were isolated from buffy coat obtained from healthy donors and provided by the Hungarian National Blood Transfusion Service as described previously (Csomor et al., 2007). Informed consent was provided for the use of blood samples according to the Declaration of Helsinki. Cells were cultivated in CellGro serum-free medium supplemented with 100 ng/mL rHu GM-CSF and 1500 U/mL rHu IL-4. On day 5 CD14− immature MDCs (imMDCs) were washed and cultured in wells pretreated as follows. In one set of the experiments 24-well tissue culture plates were coated with 150 ␮g/mL purified, native human C3 in serum-free medium (1 h at 37 ◦ C) then washed. Cells cultured for 2 days in these wells are referred to as “MDCs cultured on immobilized C3”. In parallel experiments the same number of cells were incubated with 150 ␮g/mL purified human C3 in serum-free medium in Eppendorf tubes at 37 ◦ C in a 5% CO2 thermostat (30 min), then washed and resuspended in fresh medium. These samples are referred to “MDCs treated with native C3”. For control samples imMDCs cells were left untreated. To generate maMDCs, cells were resuspended in serum-free medium containing 1 ␮g/mL LPS. Phenotypical (flow cytometry) and functional (MLR, cytokine measurement) studies were carried out at day 7. 2.4. Follow-up of DC maturation The maturation of MDCs was assessed by monitoring the expression of the following cell membrane molecules: CD80, CD83, CD86, MHCII and MR. To detect these markers cells were stained with

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Fig. 1. Covalent binding of C3b to MDCs. (A) imMDCs were treated with the indicated concentrations of native C3 or MA-C3, as described in Section 2. The amount of cell-bound C3 was assessed by cytofluorimetry using anti-C3c-FITC. MFI-values are shown (based on these data C3 was used at 150 ␮g/mL concentration in further experiments). (B) At day 5 imMDCs were treated at 37 ◦ C for 30 min with tenfold diluted normal human serum (black line), or methylamine-treated serum (grey line), then washed extensively and stained with anti-C3c-FITC. (C) At day 5 imMDCs were treated with intact, native C3 (solid line), with methylamine-inactivated C3 (dotted line) or with PMSF plus native C3 (dashed line) at 37 ◦ C for 30 min, then washed extensively and stained with anti-C3c-FITC. (D) At day 5 imMDCs were pretreated with TMG6-5 anti-CD11b antibody at 100 ␮g/mL for 30 min prior to treatment with native C3 (grey line) and as control with native C3 (black line) only. Cells were stained with anti-C3c-FITC. Histograms filled in grey represent control. Measurements were carried out by FACSCalibur cytofluorimeter using CellQuest software and analyzed by FCSExpress software. Results presented are representative of five independent experiments.

FITC- or PE-conjugated antibodies and analyzed by cytofluorimetry. To assess the functional activity of MR, imMDCs on day 7 were incubated with 1 mg/mL FITC-dextran for 1 h at 37 ◦ C. After washing three times in PBS, cells were labelled with anti-MR-PE and analyzed by FACS.

ter employing CellQuestPro (Becton Dickinson) software for data acquisition and FCSExpress software for data analysis. Results are expressed as MFI calculated by subtracting mean fluorescence intensity (MFI) of the appropriate isotype-control from each sample.

2.5. Generation and activation of human monocyte-derived macrophages (MM)

2.8. Confocal laser scanning microscopy

Monocytes were purified from buffy coat as described above, and cultured in serum-free medium supplemented with rHu GMCSF. Half of the medium was replaced every other day. At day 5, CD14+ macrophages were activated with 20 ng/mL PMA for 1.5 h at 37 ◦ C. 2.6. Measurement of the amount of cell-derived C3 To determine whether macrophages and MDCs produce C3, the amount of the complement protein in the supernatant of cultured cells was measured by ELISA. We used the human C3specific (Fab )2 fragment of goat IgG as capture antibody and HRPO-conjugated goat-anti-human C3 as secondary antibody. 2.7. Flow cytometry For phenotypic analyses, DCs were incubated with the indicated antibodies for 20 min at 4 ◦ C, then washed twice in FACS buffer (PBS, 1% FCS, 0.1% Na-azide), as suggested by the manufacturer. Isotype matched antibodies were used as control in each case. For intracellular staining cells were fixed in PBS containing 2% formaldehyde and permeabilized in PBS containing 0.2% saponin. Samples were analyzed using a FACSCalibur flow cytome-

Cells were treated with 150 ␮g/mL C3 for 30 min at 37 ◦ C, then fixed, permeabilized and stained with FITC-labelled anti-C3c and PE-labelled anti-LAMP-1. Samples were analyzed by an Olympus IX81 confocal microscope applying Fluowiev500 software. 2.9. MLR studies MDCs treated as described above were transferred to 96-well plates at day 7 and further cultured with allogeneic T cells at the indicated DC:T cell ratios. In certain experiments MDCs used in the MLR were fixed with 1% formaldehyde at 4 ◦ C for 20 min immediately after treatment with C3. As control, the MLR assay was also performed employing the supernatant of C3-treated MDCs, taken after 24 h, in the absence of stimulator cells. T lymphocytes were labelled with 0.5 ␮M CFSE prior to use. After 4 days of coculturing, the proliferation of T cells was assessed by analyzing CFSE labelled cells by cytofluorimetry. Data of three independent experiments are given as mean fluorescence intensity. 2.10. Cytokine measurements The culture supernatants of MDCs were analyzed 24 h after the various treatments. Measurements for IL-6 and TNF-␣ were carried out in duplicates, using the R&D duoset sandwich ELISA system,

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while to measure IL-8 the BenderMedsystem Instant ELISA was employed. Data are presented as mean ± SD, p < 0.05 of five experiments. 2.11. Cocultures of MDCs and MMs For these experiments MDCs and MMs had been differentiated from the monocytes of same donor. On day 5, 5 × 105 macrophages were activated with 20 ng/mL PMA for 90 min at 37 ◦ C or in the control sample left untreated. Then the cells were further incubated in fresh serum-free medium in the presence of 106 DCs. After coculturing for 24 h, DCs were removed and analyzed for C3-binding by flow cytometry. 2.12. Phagocytosis of sheep red blood cells (SRBCs) SRBCs were loaded with 5 ␮M CFSE and cocultured with activated MMs overnight for opsonization by macrophage derived C3-fragments. Opsonized SRBCs, and for control, non-opsonized erythrocytes were added to imMDCs at a 1:1 ratio and incubated for 1 h at 37 ◦ C. Non-phagocytosed SRBCs were lysed, and MDCs ingesting SRBCs were determined by FACS analysis. 2.13. Internalization of cell-bound C3 To follow the fate of cell-bound C3-fragments MDCs were incubated with 150 ␮g/mL C3 for 5 min at 37 ◦ C. After removing unbound C3 by washing, and the cells were kept in medium at 37 ◦ C in a CO2 incubator for the indicated time periods. After fixation cells were stained for either surface-bound or internalized C3 and analyzed by FACS. 2.14. CD11b siRNA CD11b silenced MDCs were differentiated according to the method of Prechtel et al. (2007), with a minor modification suggested by the manufacturer, i.e. Gene Pulser Electroporation Buffer was used for the transfection instead of OptiMEM. We used commercially available predesigned Qiagen AllStar Negative Control siRNA and Qiagen Genome Wide predesigned siRNA for CD11b (Hs ITGAM 5). Cells were transfected twice during the protocol to generate MDCs; first freshly isolated monocytes and then the differentiating DCs at day 3. Transfection was carried out each time with 20 ␮g siRNA. CD11b expression was analyzed by cytofluorimetry on day 5 and the MLR assay was also carried out on the same day. 2.15. Statistics Student’s t-test was performed with Sigmaplot 9.0 (Systat software). Data are presented as mean ± SD, p < 0.05 were considered significant. 3. Results

Fig. 2. Time-dependent internalization of C3-fragments bound covalently to MDCs. (A) MFI-values of extracellular C3-fragments detected by staining with anti-C3cFITC after incubation of MDCs with native C3. The control line represents MFI-values of non-treated cells kept in culture medium and stained with the FITC-labelled antibody. (B) MFI-values of extra- and intracellular C3 detected by staining with anti-C3c-FITC, after incubation of MDCs with native C3. The control line represents MFI-values of non-treated cells kept in culture medium and stained with the FITC-labelled antibody. Measurements were carried out using FACSCalibur cytofluorimeter employing CellQuest software, and analyzed by FCSExpress software. Results presented are representative of five independent experiments. (C) Confocal microscopic picture of MDCs treated with native C3 at 37 ◦ C for 30 min. Cells were permeabilized and stained with anti-C3c-FITC (green) and anti-LAMP-1-PE (red). The left panel shows staining with anti-C3c-FITC, the middle panel shows C3 plus LAMP-1, the right panel shows DIC picture of MDCs. Measurements were made on Olympus IX81 confocal microscope using 60× objective plus digital magnification and Fluowiev500 software. Pictures are representative of five independent experiment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

3.1. MDCs fix C3b covalently Previously it has been found that B lymphoblastoid cells, macrophages and B cells are able to fix C3-fragments covalently on their surface and this binding enhances the antigen presenting capacity of these cells (Kerekes et al., 1998; Papp et al., 2008). Our aim was to reveal whether DCs also are able to fix C3-fragments in a similar way, and if they do so, how covalent binding affects the function of the cells. We found that after incubation with purified native C3 dose dependent deposition of C3-fragments onto imMDCs occurs, in contrast to MA-C3, when the covalent

binding-site is inactive (Fig. 1A). To assess the possible physiological relevance of this reaction, we analyzed whether the cells are also able to bind C3-fragments when incubated in NHS with intact complement system. As shown in Fig. 1B, a strong C3-binding was detected also under these conditions. In these experiments however, we could not differentiate between covalently fixed and receptor-bound C3-fragments. In order to set apart these two reaction types, we employed C3 and serum pretreated with MA. This nucleophilic agent inactivates the thioester group in C3, consequently the complement protein is unable to bind covalently to

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Table 1 Expression of surface molecules on C3-treated MDCs. imMDC CD83 CD80 CD86 MHCII MR

5.79 5.28 10.08 89.30 25.49

± ± ± ± ±

maMDC 0.35 1.18 1.46 25.42 4.98

13.34 8.07 30.88 142.34 15.81

± ± ± ± ±

MDCs treated with native C3 ***

2.21 0.79* 6.35*** 20.1* 1.87*

7.38 6.44 25.98 137.21 22.49

± ± ± ± ±

**

0.41 0.75 2.01*** 11.3* 4.4

MDCs cultured on immobilized C3 4.57 4.61 9.1 95.42 29.68

± ± ± ± ±

0.71 0.89 2.00 13.5 0.99

Mean fluorescence intensity calculated from three independent experiment and its standard deviation is shown. Significant differences from imMDCs are calculated with Student’s t-test. * p < 0.05. ** p < 0.01. *** p < 0.005.

the OH-groups on various cell membrane proteins (Kerekes et al., 1998; Nishida et al., 2006; Law et al., 1980). As shown in Fig. 1A and C, MA-treated C3 binds significantly less efficiently to MDCs than native C3, suggesting that most of the detected cell membrane C3fragments are fixed covalently. Similar data were obtained using MA-treated serum (Fig. 1B). Although under the conditions applied engagement of CR3 by the generated C3-fragments on MDCs was marginal only, being aware of the importance of this integrin in various functions of DCs we set out to analyze its possible involvement in more detail. To block CD11b we used the ligand binding-site specific TMG6-5 antibody prior to treatment with native C3. In this case no change in the amount of cell-bound C3 was detected (Fig. 1D), proving that the complement protein does not bind to CR3 on MDCs under the experimental conditions employed. It also has to be mentioned that saturation of CR3 and CR4 by their ligand, iC3b occurred at a much lower concentration of the C3-fragment (data not shown), demonstrating that these two types of interactions are indeed different. The involvement of CR1 as a possible further binding-site can also be excluded, since this receptor could not be detected on MDCs even when using several various CD35-specific antibodies (not shown). As in the native form of C3 the thioester group is inactive and becomes exposed only in nascent C3b which is generated upon cleavage of intact C3, we set out to investigate whether cell membrane proteases are involved in the activation. To this end MDCs were pretreated with PMSF, a serine esterase inhibitor before incubation with intact C3. As seen in Fig. 1C the inhibitor almost completely blocked the binding of C3-fragments to the cell surface, in good agreement with the results of Biro et al. (1992), demonstrating the role of cell membrane proteases in the cleavage of the complement protein.

3.3. Covalently fixed C3b enhances the expression of CD83, CD86 and MHCII on MDCs In the next set of experiments we analyzed how the interaction of MDCs with active and inactive C3 affects the phenotype and function of these cells. Immature MDCs were cultured on immobilized C3 or treated with native C3 in suspension, followed by culturing. In the former case the interaction with CR3 might take

3.2. Internalization of cell-bound C3-fragments As we found that MDCs fix C3-fragments covalently (Fig. 1), our next aim was to reveal the fate of the surface-bound complement protein. First we analyzed whether it is internalized by the cells. To this end we treated MDCs with purified native C3 and assessed the amount of C3-fragments present on the cell surface after different time intervals. Our results show that DCs internalize most of the surface-bound complement protein in 30 min, but interestingly, even 48 h after the initial reaction, some C3-fragments could still be detected on the cell surface (Fig. 2A). When both intra- and extracellular C3 were measured, after a time-dependent decrease the amount of C3 remained constant up to 48 h (Fig. 2B). The intracellular localization of C3 was also demonstrated by confocal microscopy. MDCs were incubated with native C3, then the saponin permeabilized cells were labelled with anti-C3-FITC and anti-LAMP-1-PE. As seen in Fig. 2C, C3-fragments could be detected on the cell surface and partially also in the intracellular compartment colocalizing with LAMP-1.

Fig. 3. Expression and function of MR on MDCs are not influenced by C3-treatment. (A) Expression of MR on non-treated imMDCs, LPS-matured MDCs, native C3-treated and immobilized C3-treated MDCs. Cells were stained by anti-MR-PE. The filled grey histogram represents the isotype-control. (B) Functional analysis of MR expressed by non-treated imMDCs, LPS-matured MDCs, native C3-treated and immobilized C3-treated MDCs. Cells were allowed to phagocytose FITC-dextran for 1 h at 37 ◦ C, than washed and analyzed by flow cytometry. Measurements were carried out on FACSCalibur cytofluorimeter using CellQuest software, and analyzed by FCSExpress software. Results presented are representative of three independent experiments.

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Fig. 4. MDCs bearing covalently bound C3-fragments have elevated potency to stimulate alloreactive T cells. (A) MDCs cultured on immobilized C3, treated with native C3, stimulated with LPS and left untreated were cocultured in 96-well plates with 0.5 ␮M CFSE loaded allogeneic T lymphocytes at a DC:T ratio of 1:5. Proliferation of T cells was analyzed at day 5 by flow cytometry. Data shown are the results of three independent experiments (mean ± SD). Student’s t-test was used to compare the effects of various treatments with that of imMDCs. *p < 0.05; **p < 0.005; ***p < 0.001. (B) Number of proliferating T cells kept in the culture supernatant of C3-treated MDCs, cocultured with C3-treated MDCs fixed in 1% formaldehyde immediately after C3-treatment, with C3-treated MDCs in the presence of anti-human C3 F(ab )2 and with MDCs cultured on immobilized C3. The lowest bars represent the positive and negative controls – i.e. the alloreactivity of LPS-matured MDCs and non-treated, imMDCs. Data shown are the results of three independent experiments (mean ± SD). Student’s t-test was used to compare the effects of various treatments with that of imMDCs. *p < 0.05; **p < 0.005; ***p < 0.001. (C) Expression of CD11b on the surface of non-transfected imMDCs, on control siRNA transfected cells and on CD11b specific siRNA transfected MDCs was measured by flow cytometry using anti-hu CD11b-RPE antibody. Data shown are the results of three independent experiments (mean ± SD). (D) Stimulation of alloreactive T cells by C3-treated MDCs transfected with control siRNA and CD11b specific siRNA was measured by employing CFSE loaded T lymphocytes at a DC:T ratio of 1:5. Proliferation of T cells was analyzed at day 5 by flow cytometry. Data shown are the results of three independent experiments (mean ± SD).

place, but no covalent attachment can occur, since the adherence of C3 to the plastic surface inactivates the complement protein. In the second case, treatment with native C3 allows imMDCs to fix C3b mostly covalently, as shown in Fig. 1. Monitoring the amount of various cell membrane molecules 2 days after C3-treatment we found that after incubation of imMDCs with native C3 strongly elevates the expression of the maturation marker CD83, the costimulator molecule CD86 and MHCII. In contrast, when the cells were cultured on immobilized C3, we could not detect any change in the expression of these molecules. Interestingly, the expression

of CD80, an additional costimulatory molecule, was influenced by neither treatment (Table 1). Immature DCs readily capture foreign antigens and present peptides on their MHC to T cells. The high endocytotic capacity mediated by MR is a characteristic feature of imMDCs, and during maturation usually its downregulation occurs (Fig. 3). In our experiments the amount of MR on the cell surface did not change after treatment with either native or immobilized C3 (Fig. 3A), while in the LPS-stimulated control sample it had been dowmodulated. To assess weather the function of this receptor is also unaffected after

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Fig. 5. MDCs bearing covalently bound C3b induce the production of TNF-␣, IL-6 and IL-8. Cytokines were measured from 24 h supernatants of differently treated MDCs by ELISA. (A) TNF-␣ and IL-6 production by MDCs treated with various amounts of native C3. As controls, LPS-matured MDCs and imMDCs were used. (B) TNF-␣, IL-6 and IL-8 production by native C3-treated MDCs and cells cultured on immobilized C3. The same controls were used as above. Data are represented as mean ± SD of five independent experiments. Statistically significant alterations compared to the cytokine production of imMDCs were calculated employing the Student’s t-test. *p < 0.05; **p < 0.005; ***p < 0.001.

C3-treatment, we measured the MR-mediated phagocytosis of dextran by the cells. Our data show that the functional activity of MR is also unchanged after treatment of imMDCs by native or immobilized C3 (Fig. 3B). Again, in the control sample of LPS-matured MDCs phagocytosis was strongly decreased, in good agreement with data found in literature. 3.4. MDCs matured after the covalent fixation of C3b are strong stimulators of alloreactive T cells As we found that after treatment of MDCs with native C3 the expression of CD83, CD86 and MHCII is enhanced, it was important to see whether the T cell stimulatory capacity of MDCs is also affected by the interaction with C3. Data shown in Fig. 4A demonstrate that MDCs matured after the covalent fixation of C3b are as potent stimulators of allogeneic T cells as LPS-matured MDCs, albeit they express somewhat less costimulators and MHCII (Table 1). In our attempt to find out the underlying mechanism we analyzed whether the cell-bound C3-fragments might facilitate the adhe-

sion between MDCs and T cells. This assumption is plausible since it is known that a certain population of CD4+ T cells express CR1 (Cohen et al., 1989), thus they are able to interact with C3b present on MDCs. To test whether this “complement-bridge” might be formed during the MLR, we performed the T cell proliferation assay employing MDCs that were fixed immediately after treatment with native C3, to prevent costimulator upregulation. We found that T cell proliferation was also significantly enhanced under these conditions (Fig. 4B). It also has to be pointed out that the allostimulatory activity of MDCs did not change when the covalent attachment of C3b had been blocked (Fig. 4B), or when MDCs were cultured on immobilized C3 (Fig. 4A). Moreover, to exclude the possible activatory role of MDC-derived factors (such as cytokines), we analyzed the T cells’ proliferating capacity cultured in the supernatant of C3-treated MDCs and we found no enhancement (Fig. 4B). Although CR3 expressed on MDCs does not seem to play a crucial role in our experiments, we addressed their possible involvement by performing the MLR experiments with native C3-treated MDCs expressing strongly reduced amounts of CD11b (Fig. 4C). As seen

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Fig. 6. MDCs do not produce C3 but become opsonized by macrophage derived C3. (A) C3 produced by PMA activated human MMs and MDCs derived from the same donor was measured by ELISA. Mean values ± SD of five independent experiments are shown. (B) C3-deposition on MDCs cocultured with non-activated MMs (left panel) and PMA activated MMs (right panel) was assessed by cytofluorimetry. Both cell types were derived from the same donor’s monocytes. Cell membrane bound C3-fragments were visualized by FITC-labelled anti-human C3c and MDCs were identified based on their negativity for CD14. Results presented are representative of five independent experiments. (C) MFI-values of anti-C3c-FITC staining on MDCs (i.e. CD14− cells) after coculturing with non-activated MMs and PMA activated MMs. Control indicates MDCs incubated in the absence of MMs. Mean values ± SD of five independent experiments are shown. (D) Flow cytometric analysis of SRBCs opsonized by activated MMs. Cell membrane bound C3-fragments were visualized by FITC-labelled anti-human C3c. (E) Phagocytosis of CFSE labelled SRBCs opsonized by PMA activated MMs, non-opsonized erythrocytes and as control, after incubation at 4 ◦ C. MFI-values were obtained after FACS analysis. Mean values ± SD of five independent experiments are shown.

in Fig. 4D, there is no difference between the activity of the RNAsilenced and non-treated cells – i.e. both were potent stimulators of allogeneic T cells. This demonstrates that the possible ligation of CD11b by C3-fragments on DCs is not involved in the enhanced T cell stimulation. 3.5. C3-treatment of MDCs increases the production of pro-inflammatory cytokines It is well-known that DCs produce a great variety of molecules including pro-inflammatory cytokines. Since we found that covalently fixed C3-fragments affect both the phenotype and T cell stimulatory capacity of MDCs, we set out to analyze whether their cytokine production is also influenced. We measured the amount of IL-6, TNF-␣ and IL-8 in the supernatants of MDCs collected 24 h after complement-treatment. Our results show that MDCs bearing covalently fixed C3b secrete significantly increased amounts of the pro-inflammatory cytokines IL-6 and TNF-␣ (Fig. 5A), and the effect depends on the amount of C3 employed, in contrast to MDCs cultured on immobilized C3 (Fig. 5B). As positive control, LPS-matured cells were used, which are a good source of these pro-inflammatory cytokines. The chemokine IL-8 is known to induce T cell migration into the lymph nodes. Since native C3-treated MDCs proved to be very potent stimulators of T cells, we also analyzed their IL-8 production. As shown in Fig. 5B treatment of MDCs with native C3 results in elevated production of IL-8, while culturing on immobilized C3 does not affect the cells’ chemokine release at all. Parallel to the enhanced synthesis of cytokines, NF-␬B translocation in MDCs treated with native C3 was also detected (data not shown). 3.6. Human MDCs do not secrete complement C3 Activated macrophages are known for long to produce high amounts of complement proteins, particularly C3, and local complement production by these cells is suggested to play an important

role in vivo (Sheerin et al., 2008). Although the presence of C3 mRNA had been shown in human MDCs, the release of the protein by these cells has never been demonstrated (Reis e Sousa et al., 2006, 2007). As we assumed that C3 affecting various functions of MDCs may also derive from the same cells, we assessed the amount of the complement protein released by the cultured cells. However, as shown in Fig. 6A, we could not detect any secreted C3, while in the culture supernatant of PMA activated MMs differentiated from the same donor’s monocytes and used as control in this experiment, high amounts of C3 could be detected. These data clearly show that human DCs do not secrete complement C3. 3.7. MDCs are able to fix macrophage derived C3 In the secondary lymphoid organs, where most of the cellular interactions needed to initiate adaptive responses take place, activated macrophages, DCs and T cells get in touch with each other. To find out whether our results might be relevant under such in vivo conditions, we investigated how activated macrophages might affect the function of DCs which are in their close vicinity. To this end PMA activated MMs and MDCs differentiated from the same donor’s monocytes were cocultivated for 1 day. Cytofluorimetric analysis clearly show that approximately 30% of MDCs fix C3fragments on their cell membrane (Fig. 6B right panel and C), which however, themselves do not produce the complement protein C3 (Fig. 6A). In the control sample, where MDCs were cocultured with non-stimulated MMs, no complement-fixation occurred (Fig. 6B left panel and C). As described earlier by Ezekowitz et al. (1984) macrophage derived C3 plays a role in the process of local opsonization, facilitating the uptake of antigens by CR bearing phagocytes. To assess the biological function and the physiological relevance of the elevated C3 production by MMs in our system, we incubated SRBCs with the activated phagocytes overnight, then measured the uptake of the erythrocytes by MDCs. We found that phagocytosis by the antigen presenting cells was strongly elevated when the particles were

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opsonized by C3 derived from activated MMs, but not in the control samples (Fig. 6D and E). All together these data suggest that a similar scenario might take place in vivo, where in the peripheral lymphoid tissues activated macrophages and DCs reside in close vicinity. 4. Discussion The complement system, present in all the body fluids, provides a key element in immunological events. Increasing amount of evidence prove the importance of its certain activation products in the initiation and regulation of adaptive immunity (Carroll, 2004; Kemper and Atkinson, 2007). The fundamental step of the complement cascade is the covalent binding of C3b, the activation product of the major protein C3, to the antigen (Law et al., 1984; Law and Dodds, 1997; Nishida et al., 2006). Component C3 has a unique internal thioester group in its ␣-chain that becomes transiently active after proteolytic cleavage of the intact molecule. Covalently bound C3b is known to mediate several important biological functions including formation of C3 and C5 convertases of both the alternative and classical pathways, opsonization, lymphocyte activation and chemotaxis. It has also been proven that local secretion of C3 by tissue macrophages plays a role in the opsonization and subsequent elimination of microorganisms and apoptotic cells (Ezekowitz et al., 1984; Morelli et al., 2003; Skoberne et al., 2006). Apoptotic cells are also known to activate complement system and bind its components, including C1q and C3-fragments. The uptake of the opsonized particles has been shown to be mediated by complement receptors C1qR, CR3, CR4, which play an important role in the maintenance of peripheral tolerance this way (Morelli et al., 2003; Nauta et al., 2002, 2004; Schmidt et al., 2006; Skoberne et al., 2006; Sohn et al., 2003; Verbovetski et al., 2002). CR1 (CD35) and CR2 (CD21), interacting with C3b and C3d fragments cannot be detected on DCs, however these cells do express complement receptors CR3 and CR4, which bind iC3b (Li and Zhang, 2003). While CR3 (CD11b/CD18) is a potent phagocytic receptor, the proper function of CR4 (CD11c/CD18) is not well characterized yet. In our present study we demonstrate for the first time that human DCs are able to bind C3b covalently when activation of C3 takes place on the cell surface. This reaction of DCs is independent of CR3 and can be almost totally abolished if C3 is inactivated previously. The covalent fixation of C3b had been first described in the case of erythrocytes and zymosan (Law and Levine, 1977), then B cells and monocytes/macrophages (Fabry et al., 1985; Gergely et al., 1985; Maison et al., 1989; Marquart et al., 1994; Mold et al., 1988). We have demonstrated earlier that covalently fixed C3 enhances the antigen presenting capacity of macrophages and B cells (Erdei et al., 1992; Kerekes et al., 1998; Papp et al., 2008). Using Lewis lung carcinoma cells which do not express complement receptors, di Renzo et al. found that C3 binds covalently to the cell membrane, and after internalization of the acceptor-bound C3b the growth of these cells is induced (di Renzo et al., 1999; Longo et al., 2005). Although many data support the functional role and in vivo relevance of covalently fixed C3b (Biro et al., 1992; di Renzo et al., 1999; Erdei et al., 1991; Ezekowitz et al., 1983; Fabry et al., 1985; Gergely et al., 1985; Janssen and Gros, 2007; Kerekes et al., 1998; Law et al., 1984; Law and Dodds, 1997; Longo et al., 2005; Maison et al., 1989; Papp et al., 2008) interestingly, the acceptorsite could be identified so far only on B lymphocytes, but not on any other cell types. In the case of B cells complement receptor type 2 (CR2, CD21) had been demonstrated as the major covalent binding-site (Marquart et al., 1994; Mold et al., 1988), which also acts as an initiator of the alternative pathway. This receptor however, is not expressed by macrophages, DCs, lung carcinoma cells and erythrocytes; consequently there must be additional cell mem-

brane molecules which may serve as C3b-acceptor-sites. There were already some attempts to identify these structures and the appearance of high molecular weight complexes containing C3fragments had been detected in the extract of C3-treated cells (Barro et al., 1991; Erdei et al., 1992; Law and Levine, 1977). The cell membrane molecules which bind C3 covalently however, still need to be identified. It is well accepted that the covalent interaction of C3b requires the presence of suitable placed acceptor-sites moreover, C3bfixation to surfaces is not discriminative of host and foreign surfaces (Budzko et al., 1976; Kerekes et al., 1998). Although nascent C3b may react with any accessible nucleophile it comes across, there seems to exist severe restrictions. This is underlined by the fact that T lymphocytes are unable to bind C3b covalently under similar conditions when APCs like B cells, macrophages and DCs do so (Papp et al., 2008). To identify the possible acceptor molecules on APCs further experiments are needed. Regarding the functional consequences of C3b-fixation, its immunomodulatory effect had been shown in the case of B cells and macrophages (Kerekes et al., 1998; Papp et al., 2008). Here we show that this reaction strongly affects DC maturation, as it significantly enhances the expression of CD83, CD86 and MHCII. It is intriguing and worth to point out however, that the level of MR, the important phagocytic receptor does not change at all in the meantime. We have also found that the covalent binding of C3b causes a significant enhancement of IL-8, TNF-␣ and IL-6 production by DC (Fig. 5). This is in contrast to the effect of ligation CR3 and CR4, which had been shown to inhibit DC maturation (Ehirchiou et al., 2007; Schmidt et al., 2006; Skoberne et al., 2006; Sohn et al., 2003). Our findings using CD11b silenced MDCs also exclude the role of CR3 in the above described effects of the covalent bound C3b (Fig. 4). Regarding the mechanism of the elevated T cell proliferation we propose that DC-bound C3b interacts with CR1 expressed on T lymphocytes and functions as an adhesion molecule between DCs and T cells. This suggestion is supported by the fact that employing anti-C3 F(ab )2 this enhancement was abrogated. The phenotype of MDCs stimulated by C3 was markedly different from MDCs matured by LPS stimulus (Table 1), showing that a “danger signal” and a physiological stimulus direct DC maturation into different ways. It has to be emphasized that we used a natural ligand – i.e. isolated, native C3, or NHS as source of the complement protein. Thus, our experimental system mimics the in vivo occurring situation and it is optimal for the analysis of the function of C3-fragments generated during local inflammatory processes. Earlier we and others described that macrophages and B cells are able to fix C3b covalently, moreover, C3-fragment deposition on the surface of these cells significantly enhances the proliferation of antigen-specific T cells (Kerekes et al., 1998; Papp et al., 2008; Maison et al., 1989; Marquart et al., 1994). It has also been shown that macrophages can interact with the complement-fragments produced by themselves (Ezekowitz et al., 1983, 1984; Maison et al., 1989). Based on these data it was important to see whether DCs are also able to secrete complement C3. Earlier Reis et al. showed that human DCs express mRNA for C3, however protein production has not been demonstrated (Reis e Sousa et al., 2006, 2007; Spirig et al., 2008). Here we clearly show that no C3 is secreted by human MDCs (Fig. 6). Based on these data we set up the hypothesis that macrophage derived C3 may affect the functional activity of DCs and demonstrate that DCs cocultured with activated macrophages indeed do acquire C3-fragments on their cell membrane, which derive from macrophages. Our present finding broaden the concept of local opsonization described earlier by Ezekowitz et al. (1984). Based on our present results we suggest that macrophage derived C3 is cleaved by enzymes expressed by DCs and/or macrophages, and the generated C3-fragments interact covalently with DCs present in their close

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vicinity – a situation easily occurring during immune responses in the lymph node. The in vivo relevance of our finding is strongly supported by the very recent data of Ghannam et al. (2008), who have shown that primary human C3-deficiency results in an important functional defect of imMDCs. They had demonstrated that macrophages of a C3-deficient patient were able to synthesize C3, however it could not be secreted by the cells. The total number of MDCs was found to be significantly lower in this patient, compared with healthy individuals. As a result of C3-deficiency an impairment of the antigen presenting capacity of PBMC was also observed with no B cell activation and the total lack of memory B cells. Our findings are in good accordance with these data and prove that C3 is indeed required for normal DC development. The unique capacity of DCs as sentinels of the immune system is to detect and integrate various signals and thus sense the momentary immune-state of the organism. Complement component C3 is an acute phase protein, which is also an indicator of the actual physiological state of the body. It is well-known that antigens or inflammatory stimuli activate macrophages to produce substantial amounts of C3. Thus, locally produced and activated C3 can induce maturation of DCs, resulting in the elevation of T cell responses. Our data provide compelling evidence that DCs arising in complement C3 sufficient environment mature to competent stimulators of T cells, while in the absence of C3 their development is totally abrogated (Ghannam et al., 2008). Acknowledgements This work was supported by the Hungarian Academy of Sciences and the Hungarian National Science Fund (OTKA) grants K63038 and K72026. References Andrews, P.A., Zhou, W., Sacks, S.H., 1995. Tissue synthesis of complement as an immune regulator. Mol. Med. Today 1, 202–207. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B., Palucka, K., 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18, 767–811. Barro, C.D., Villiers, C.L., Colomb, M.G., 1991. Covalent binding of non-proteolysed C3 to Jurkat T cells. Mol. Immunol. 28, 711–717. Basta, M., Hammer, C.H., 1991. A rapid FPLC method for purification of the third component of human and guinea pig complement. J. Immunol. Methods 142, 39–44. Biro, A., Sarmay, G., Rozsnyay, Z., Klein, E., Gergely, J., 1992. A trypsin-like serine protease activity on activated human B cells and various B cell lines. Eur. J. Immunol. 22, 2547–2553. Budzko, D.B., Lachmann, P.J., McConnell, I., 1976. Activation of the alternative complement pathway by lymphoblastoid cell lines derived from patients with Burkitt’s lymphoma and infectious mononucleosis. Cell Immunol. 22, 98–109. Carroll, M.C., 2004. The complement system in B cell regulation. Mol. Immunol. 41, 141–146. Cohen, J.H., Aubry, J.P., Revillard, J.P., Banchereau, J., Kazatchkine, M.D., 1989. Human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) belong to Fc gamma receptor-positive CD4-positive T cells. Cell Immunol. 121, 383–390. Colten, H.R., Strunk, R.C., Perlmutter, D.H., Cole, F.S., 1986. Regulation of complement protein biosynthesis in mononuclear phagocytes. Ciba Found. Symp. 118, 141–154. Csomor, E., Bajtay, Z., Sandor, N., Kristof, K., Arlaud, G.J., Thiel, S., Erdei, A., 2007. Complement protein C1q induces maturation of human dendritic cells. Mol. Immunol. 44, 3389–3397. di Renzo, L., Longo, A., Morgante, E., Mardente, S., Prodinger, W.M., Russo, M., Pontieri, G.M., Lipari, M., 1999. C3 molecules internalize and enhance the growth of Lewis lung carcinoma cells. Immunobiology 200, 92–105. Ehirchiou, D., Xiong, Y., Xu, G., Chen, W., Shi, Y., Zhang, L., 2007. CD11b facilitates the development of peripheral tolerance by suppressing Th17 differentiation. J. Exp. Med. 204, 1519–1524. Erdei, A., Fust, G., Gergely, J., 1991. The role of C3 in the immune response. Immunol. Today 12, 332–337. Erdei, A., Kohler, V., Schafer, H., Burger, R., 1992. Macrophage-bound C3 fragments as adhesion molecules modulate presentation of exogenous antigens. Immunobiology 185, 314–326.

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