ARTHRITIS & RHEUMATISMVol. 60, No. 10, October 2009, pp 3081–3090DOI 10.1002/art.24852© 2009, American College of Rheumatology
C1q Inhibits Immune Complex–Induced Interferon-�Production in Plasmacytoid Dendritic Cells
A Novel Link Between C1q Deficiency andSystemic Lupus Erythematosus Pathogenesis
Christian Lood,1 Birgitta Gullstrand,2 Lennart Truedsson,2 Anders I. Olin,2 Gunnar V. Alm,3
Lars Ronnblom,4 Gunnar Sturfelt,5 Maija-Leena Eloranta,4 and Anders A. Bengtsson5
Objective. C1q deficiency is the strongest riskfactor known for the development of systemic lupuserythematosus (SLE), since almost all humans with agenetic deficiency of C1q develop this disease. Low C1qserum concentration is also a typical finding in SLEduring flares, emphasizing the involvement of C1q inSLE pathogenesis. Recent studies have revealed thatC1q has a regulatory effect on Toll-like receptor–induced cytokine production. Therefore, we undertookthis study to investigate whether C1q could regulateproduction of interferon-� (IFN�).
Methods. Peripheral blood mononuclear cells(PBMCs) and plasmacytoid dendritic cells (PDCs) werestimulated with 3 known interferogenic stimuli andcultured with physiologic concentrations of C1q. IFN�production was determined by an immunoassay.
Results. C1q significantly inhibited PBMC IFN�production induced by RNA-containing immune com-plexes (ICs), herpes simplex virus (HSV), and CpGDNA. C1q also inhibited PDC IFN� production inducedby ICs and CpG DNA but increased PDC IFN� produc-tion induced by HSV. The regulatory role of C1q was notspecific for IFN� but was also seen for interleukin-6(IL-6), IL-8, and tumor necrosis factor �. We demon-strated binding of C1q to PDCs both by surface plasmonresonance interaction analysis and by flow cytometry,and we also demonstrated intracellular detection of 2C1q binding proteins.
Conclusion. Our findings contribute to the under-standing of why C1q deficiency is such a strong riskfactor for SLE and suggest an explanation for theup-regulation of the type I IFN system seen in SLEpatients.
Systemic lupus erythematosus (SLE) is a systemicautoimmune disease involving several organ systems,and it is also characterized by the presence of B cellhyperreactivity, autoantibodies, increased complementconsumption, and an ongoing production of type Iinterferon (IFN) (1). The elevated serum levels of IFN�in SLE patients have been proposed to have a significantrole in the pathogenesis of SLE. Administration ofrecombinant IFN� to patients with viral infections ormalignancies can lead to the development of antinuclearantibodies as well as to SLE or other autoimmune
Supported by grants from the Swedish Research Council(grants 15092 and 70297601), the Medical Faculty at Lund University,the Alfred Osterlund Foundation, the Crafoord Foundation, the Gretaand Johan Kock Foundation, King Gustaf V’s 80-Year Foundation,Lund University Hospital, the Dana Foundation, the Swedish Rheu-matism Association, the Ulla and Roland Gustafsson Foundation, theFoundation of the Swedish National Board of Health and Welfare, andCOMBINE (Controlling Chronic Inflammatory Diseases with Com-bined Efforts).
1Christian Lood, MSc: Lund University Hospital and LundUniversity, Lund, Sweden; 2Birgitta Gullstrand, BSc, Lennart Trueds-son, MD, PhD, Anders I. Olin, PhD: Lund University, Lund, Sweden;3Gunnar V. Alm, MD, PhD: Swedish University of AgriculturalSciences, Uppsala, Sweden; 4Lars Ronnblom, MD, PhD, Maija-LeenaEloranta, PhD: Uppsala University, Uppsala, Sweden; 5GunnarSturfelt, MD, PhD, Anders A. Bengtsson, MD, PhD: Lund UniversityHospital, Lund, Sweden.
Dr. Truedsson has received consulting fees and/or honorariafrom Shire Human Genetic Therapies (less than $10,000). Drs. Almand Ronnblom have received consulting fees, speaking fees, and/orhonoraria from Miltenyi Biotec (less than $10,000 each). Dr. Bengts-son has received consulting fees, speaking fees, and/or honoraria fromActive Biotech Research AB (less than $10,000).
Address correspondence and reprint requests to Anders A.Bengtsson, MD, PhD, Department of Clinical Sciences, Section ofRheumatology, Lund University Hospital, 223 62 Lund, Sweden.E-mail: [email protected].
Submitted for publication October 22, 2008; accepted inrevised form June 29, 2009.
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diseases (2–4). SLE patients have circulating IFN�-inducing factors, identified as immune complexes (ICs),which often contain RNA or DNA (5,6). These ICscould be endocytosed by the natural IFN�-producingcells, the plasmacytoid dendritic cells (PDCs), throughCD32, eventually inducing IFN� production throughToll-like receptor 7 (TLR-7) or TLR-9 stimulation (7,8).It has also been described that SLE serum, containingIFN�, induces maturation of monocytes to DCs withantigen-capturing ability (9). An important role of IFN�in SLE has been indicated by studies showing an over-expression of IFN�-induced genes in SLE patients com-pared with healthy controls, termed the type I IFNsignature (10–15).
The complement system has dual functions inSLE: on the one hand protective, but on the other handalso pathogenic (16). A major role has been addressed inthe clearance of apoptotic cells and ICs, since apoptoticcells are thought to be the main source of SLE autoan-tigens. A vicious circle with an impaired complementfunction, accelerating apoptosis, release of nuclear anti-gens, formation of autoantibodies, and eventually for-mation of ICs has been proposed (17). More than 90%of human individuals with a genetic C1q deficiencydevelop SLE. A deficiency in C1r/C1s or C4 is alsoassociated with SLE but at a lower frequency (18). Theelevated risk of developing SLE in C1q deficiencycompared with other hereditary complement deficien-cies might not solely be explained by differences in theclearance of apoptotic cells, but other functions of C1qmay also be important. It has recently been found thatC1q can regulate cytokine production from murine bonemarrow–derived DCs stimulated with TLR-4 or TLR-9ligands (19). It has also been demonstrated that bothC1q and mannose-binding lectin (MBL) can inhibitmonocyte cytokine expression (20). Taken together, thiswould indicate that C1q is not only important in theclearance of apoptotic material and ICs, but that it isalso a regulator of cytokine production.
Therefore, we investigated whether C1q couldregulate IFN� production in relevant cell populations.We found that C1q not only regulated the production ofIFN� but also that of other cytokines such asinterleukin-6 (IL-6), IL-8, and tumor necrosis factor �(TNF�).
MATERIALS AND METHODS
Preparation of IgG. Anti-RNP–positive sera from theDepartment of Clinical Immunology, Lund University Hospi-tal, Lund, Sweden were pooled, and IgG was purified on a
protein G column (Protein G Superose HR 10/2; PharmaciaLKB, Uppsala, Sweden). The column was equilibrated with 20mM sodium phosphate buffer (pH 7.0) and IgG was elutedwith 0.1M glycine-HCl (pH 2.8). The pH was adjusted to 7.0with 1M Tris (pH 9.0). The IgG fractions were concentratedand dialyzed against veronal buffered saline containing 0.15mM Ca2� and 0.5 mM Mg2� (pH 7.2).
IFN� inducers and inhibitors. To prepare necrotic cellmaterial, Jurkat cells were suspended at a concentration of 4 �107 cells/ml in Macrophage-SFM Medium (Invitrogen, GrandIsland, NY). The cells were frozen at –80°C for at least 30minutes, then thawed at 37°C followed by 3 more freeze-thawing periods of at least 10 minutes, after which the super-natant was collected by centrifugation at 400g for 5 minutesand stored at –80°C. Purified IgG at a concentration of 0.25mg/ml was mixed with necrotic material from Jurkat cellsupernatant at a concentration of 5% (volume/volume) tocreate ICs. Herpes simplex virus (HSV), treated according toLovgren et al (21), was used at the optimal concentration of10% (v/v). CpG DNA (Cybergene, Huddinge, Sweden) withthe sequence 5�-ggGGGACGATCGTCgggggG-3� was usedat a concentration of 0.15 �g/ml. The small letters indicatenucleotides with phosphorothioate backbone, the capital let-ters indicate nucleotides with phosphodiester backbone, andthe underlined letters indicate CpG dinucleotides.
C1q purified according to Tenner et al (22) was used atconcentrations ranging up to 70 mg/liter. The activity andpurity of the C1q preparation were demonstrated by a hemo-lytic assay and sodium dodecyl sulfate–polyacrylamide gelelectrophoresis. The C1q preparation did not contain detect-able levels of IgG measured by an enzyme-linked immunosor-bent assay with a detection limit of 0.1% of the normalconcentration (70 mg/liter). Transferrin (Sigma, St. Louis,MO) and human IgG (Immuno, Vienna, Austria) were alsoused. C1q collagen-like region (CLR) and C2 were purifiedaccording to previously described protocols (23–25) with mod-ifications. Separation of C2 from factor B was achieved byaffinity chromatography using N-hydroxysuccinimide–activated Sepharose (Amersham Pharmacia Biotech, Uppsala,Sweden) coupled with rabbit anti-C2 IgG. Recombinant MBLwas kindly provided by Dr. Jensenius, University of Aarhus,Aarhus, Denmark.
TLR inhibitors. All CpG DNA oligodeoxynucleotides(ODNs) with phosphorothioate backbone were generatedby Cybergene. The control ODN (5�-tcctgcaggttaagt-3�) wasused at a concentration of 1.4 �M, the TLR-7 inhibitor(5�-tgcttgcaagcttgcaagca-3�) was used at a concentration of 1.4�M, and the TLR-9 inhibitor (5�-tcctggaggggttgt-3�) was usedat a concentration of 5.6 �M according to Barrat et al (26).
Isolation of peripheral blood mononuclear cells(PBMCs) and PDCs. PBMCs from healthy donors were ob-tained by Lymphoprep (Axis-Shield PoC, Oslo, Norway)density-gradient centrifugation at 605g for 30 minutes, afterwhich the cells were washed 3 times at 360g for 5 minutes inMacrophage-SFM Medium supplemented with 20 mMHEPES (Invitrogen) and 50 �g/ml gentamicin (Invitrogen). Toavoid any prestimulation of the PDCs, we used a negativeselection method (27,28). The PBMCs were magneticallysorted using the Plasmacytoid Dendritic Cell Isolation Kit(Miltenyi Biotec, Auburn, CA) according to the manufactur-er’s instructions. The purity of the cells was analyzed by flow
3082 LOOD ET AL
cytometry (Epics XL-MCL; Beckman Coulter, Miami, FL)using antibodies against CD123 and CD303 (blood dendriticcell antigen 2 [BDCA-2]) (Miltenyi Biotec) and was �95%.
Culture of cells. The PBMCs and the PDCs wereadded to 96-well, flat-bottomed culture plates (TPP, Trasadin-gen, Switzerland) at final concentrations of 2 � 106 cells/mland 2 � 105 cells/ml, respectively. After adding the IFN�inducers, the culture volumes were adjusted to 100 �l withMacrophage-SFM Medium containing 20 mM HEPES, 50�g/ml gentamicin, 2 ng/ml granulocyte–macrophage colony-stimulating factor (Leukine; Berlex, Montville, NJ), and 500units/ml Intron A (Schering-Plough [Brinny] Company, Innis-hannon, Ireland) and incubated for 20 hours at 37°C with 5%CO2 and 97% humidity.
Immunoassay for IFN�. The amount of IFN� wasdetermined by dissociation-enhanced lanthanide fluoroimmu-noassay according to the protocol of Lovgren et al (21) usingthe LT27:293 and europium-labeled LT27:297 monoclonalantibodies, with modifications. Briefly, microtiter plates(AAAND-0001 Yellow Plate; PerkinElmer, Waltham, MA)were coated with antibody LT27:293 at a concentration of 10�g/ml. Europium-labeled antibody LT27:297 was added to-gether with the IFN� standard or the sample and incubated for1 hour at 37°C with continuous shaking. The wells were washed6 times with 0.05% Tween 20 in phosphate buffered saline(PBS) (pH 7.2), after which 100 �l of Enhancement Solution(PerkinElmer) was added at room temperature. The measure-ments were done immediately in a 1420VICTOR reader(PerkinElmer). This assay has a detection limit of 2 units/ml.
IL-6, IL-8, and TNF� measurements. For the detec-tion of IL-6, IL-8, and TNF�, the IMMULITE 1000 system(Siemens Healthcare Diagnostics, Deerfield, IL) was used forpooled culture supernatants according to the manufacturer’sinstructions.
C1q measurements. A flat-bottomed 96-well microtiterplate (Nunc-Immuno plates, Maxisorp; Nunc, Roskilde, Den-mark) was coated with 20 �g/ml rabbit anti-human C1q IgGF(ab�)2 in PBS and incubated at 4°C for 20 hours in a totalvolume of 100 �l/well. The culture supernatants (100 �l) wereadded and incubated for 2 hours at room temperature. Alka-line phosphatase–conjugated anti-C1q F(ab�)2 was added andincubated for 1 hour at room temperature, after which thephosphatase substrate (Sigma) was added and incubated for 90minutes. The absorbance was measured at 405 nm with aMultiskan Plus photometer (Labsystems, Helsinki, Finland).Between each step the plate was washed 3 times with PBScontaining 0.05% Tween 20.
Detection of cell death. The cells were analyzed for celldeath by flow cytometry after staining with fluorescein isothio-cyanate (FITC)–labeled annexin V and propidium iodide (PI)(BD Biosciences Pharmingen, San Diego, CA) according tothe manufacturer’s instructions.
Expression of C1q binding proteins. For detection ofsurface expression of C1q or C1q receptors (C1qR; specifi-cally, the receptor for the collagen part of C1q [cC1qR] andthe receptor for the globular parts of C1q [gC1qR]), isolatedPDCs (3 � 105 cells/ml) were incubated with conjugated ornonconjugated antibodies against gC1qR (74.5.2 or 60.11) andcC1qR (FMC75) (all from Santa Cruz Biotechnology, SantaCruz, CA), calreticulin (another name for cC1qR) (Mabtech,
Nacka Strand, Sweden), CR1 and C1q (both from Dako,Glostrup, Denmark), or �2�1 integrin (CD49b) (BD Bio-sciences Pharmingen) for 30 minutes at 4°C. As a secondaryantibody, FITC-labeled rabbit anti-mouse IgG (Dako) wasused. For the assessment of intracellular proteins, PDCs wereincubated with 4% paraformaldehyde for 20 minutes and thenwashed and incubated with 0.2% Triton X-100 (Sigma) in PBSfor another 20 minutes, after which the antibodies were added.For detection of C1q binding to PBMCs, 1 � 106 cells/ml wereincubated with or without C1q for 2 hours, washed, andincubated with conjugated antibodies against CD3, CD4,CD16, CD19, C1q (all from Dako), and CD8 (BeckmanCoulter, Fullerton, CA) before analysis by flow cytometry.
Detection of activation markers on PDCs. IC-stimulated PDCs were cultured overnight in the absence orpresence of C1q (70 mg/liter) and then analyzed by flowcytometry for expression of CD80, CD83, CD86 (Santa CruzBiotechnology), CD303, and HLA–DR (BD BiosciencesPharmingen). The PDCs were gated as either HLA–DR orCD303 positive in all analyses.
Detection of intracellular IgG and C1q in IC-stimulated PDCs. For detection of intracellular IgG and C1q,isolated PDCs were cultured for 6 hours in the presence ofdifferent stimuli. PDCs were fixated and permeabilized asdescribed before and then incubated for 30 minutes withantibodies directed against IgG (rabbit anti-human IgG;Dako) or C1q, together with the PDC-specific anti-CD303antibody, and finally analyzed by flow cytometry.
Surface plasmon resonance (SPR) interaction analy-sis. C1q, diluted to 10 mg/liter in 10 mM sodium acetate (pH4), was immobilized via amine coupling to CM5 sensorchipflow chambers (BIAcore, Uppsala, Sweden) at 2,300, 6,700,and 10,000 response units. Briefly, C1q was mixed with freshlyprepared 100 mM N-hydroxysuccinimide and 400 mM N-ethyl-N�-(dimethylaminopropyl)carbodiimide in equal volumes, andcapping of unreacted sites was achieved by a 1M ethanolamine(pH 8) injection. Antibodies to CD123 (IL-3 receptor) (Milte-nyi Biotec) and CD303 (BDCA-2) were immobilized similarlyto 620 and 840 response units, respectively. A flow chambersubjected to immobilization protocol but without any additionof protein was used as control (blank) for each experiment.PDCs and T cell suspensions (5 � 103 to 1 � 106 cells/ml inPBS) were injected at a flow rate of 2–5 �l/minute or 15�l/minute over the C1q or the antibody surfaces, respectively.Anti-C1q was injected at concentrations of 30–500 nM at aflow rate of 50 �l/minute. All experiments were performed at25°C in running buffer containing 10 mM HEPES (pH 7.5),150 mM NaCl, and 0.005% Surfactant P20 (BIAcore) and weremonitored in a BIAcore 2000 instrument. Between experi-ments, the surfaces were strictly regenerated with multiplepulses of 0.1M NaHCO3 (pH 12) and 2M NaCl for theanti-C1q antibody and 20 mM HCl and 50 mM NaOH/1MNaCl for the cells followed by an extensive wash procedureusing running buffer.
After x-axis and y-axis normalization of the obtaineddata, the blank curves from the control flow chamber(s) ofeach injected concentration or experiment were subtracted.Where applicable, the association (Ka) and dissociation (Kd)rate constants were determined simultaneously using the equa-tion for 1:1 Langmuir binding in the BIA Evaluation 4.1software (BIAcore).
C1q AND IFN� PRODUCTION IN SLE 3083
Statistical analysis. Data are presented as the mean �SD. P values were calculated by Wilcoxon’s signed rank test orthe Mann-Whitney U test.
Ethics. Permission was obtained from the ResearchEthics Committee of Lund University, Lund, Sweden.
RESULTS
C1q regulates IFN� production by PBMCs.PBMCs were cultured with 3 known interferogenicstimuli, ICs, HSV, and CpG DNA, in order to determinewhether C1q could regulate IFN� production. We con-firmed that IC-induced IFN� production was dependenton TLR-7 as described by others (data not shown) (29).
PBMCs were incubated with C1q to determine whetherthis molecule could regulate IFN� production. Additionof C1q to the cell cultures clearly inhibited IFN� pro-duction induced by all 3 stimuli, in a dose-dependentmanner (Figure 1A). As a negative control we usedtransferrin, and as a positive control we used human IgG(7). The production of IFN� was unaffected by trans-ferrin, but it was inhibited by human IgG for all 3 stimulitested (data not shown). Thus, IFN� production byPBMCs can be dose-dependently inhibited by C1q andalso, as described before, by human IgG.
C1q regulates IFN� production by PDCs. Todetermine whether the regulatory role of C1q was
Figure 1. Effect of different complement components on interferon-� (IFN�) production by peripheral blood mononuclear cells (PBMCs) orplasmacytoid dendritic cells (PDCs) cultured for 20 hours. A and B, PBMCs (A) or PDCs (B) were stimulated in culture with immune complexes(ICs), herpes simplex virus (HSV), or CpG DNA in the presence of different concentrations of C1q (up to 70 mg/liter). Results are the mean andSD of at least 6 experiments. C, When PDCs were cultured and stimulated with ICs in the presence of heat-inactivated serum (IA), C1q-deficientserum (C1qD), or medium (0), no inhibition of IFN� production was seen, while when C1q was added to C1q-deficient serum or when normalhuman serum (NHS) was used instead, IFN� production was markedly inhibited. Results were obtained with 20% serum and are the mean and SDof at least 4 experiments. D, PDCs were stimulated in culture with ICs together with mannose-binding lectin (MBL) up to 4 mg/liter and with C2,C4, or C1q collagen-like region (CLR) up to normal human serum concentration. Results are the mean and SD of at least 3 experiments. � � P �0.05; �� � P � 0.01; ��� � P � 0.005, versus medium control.
3084 LOOD ET AL
dependent on a direct interaction with the major IFN�-producing cells, the PDCs, we purified PDCs and stim-ulated them in culture in the presence of differentconcentrations of C1q. C1q dose-dependently inhibitedIFN� production induced by ICs (P � 0.002) and CpGDNA (P � 0.04) but increased HSV-induced IFN�production (P � 0.001) (Figure 1B). No production ofC1q could be detected (�35 ng/ml) in culture superna-tants from PDCs (data not shown). These results indi-cate that C1q acts directly on PDCs and regulates IFN�production by these cells. These findings were furtherconfirmed in experiments using serum from a persongenetically deficient in C1q. Neither C1q-deficient se-rum nor heat-inactivated serum could inhibit IC-inducedIFN� production. When C1q was added to the C1q-deficient serum or when control serum was used instead,IFN� production was inhibited (Figure 1C). BesidesC1q, we also investigated C2, C4, and MBL for regula-tory effects on IC-induced IFN� production. In addi-tion, the C1q CLR was tested. None of the testedmolecules affected IC-induced IFN� production by pu-rified PDCs when used in physiologic concentrations(Figure 1D). These results clearly demonstrate that theregulatory function is specific for C1q and that the intactC1q molecule or the globular heads are necessary.
C1q regulates the production of other proinflam-matory cytokines. We investigated whether the inhibi-tory effect of C1q was specific for IFN� production inPDCs by also measuring IL-6, IL-8, and TNF� levels inpooled culture supernatants from 6 experiments. Wefound that C1q inhibited IC- and CpG DNA–inducedIL-6, IL-8, and TNF� production and increased HSV-induced cytokine production (Figure 2). Thus, the re-sults demonstrate that C1q has a general regulatoryeffect on cytokine production, in particular by inhibitingthe action of ICs.
C1q does not induce increased cell death ormaturation. Since we observed a general inhibition ofthe production of several cytokines, it was important toexclude C1q-induced cell death. We therefore stainedcultured PDCs with annexin V and PI after 20 hours ofculture with C1q. The percentage of annexin V–positivecells in the presence of C1q (mean � SD 2.53 � 2.41)did not differ from that in control cultures (mean � SD1.39 � 1.16). This demonstrates that apoptosis was notincreased by C1q. We also demonstrated that the de-creased IFN�-producing ability did not depend on adecreased cell number by counting the number of livingcells per minute by flow cytometry (mean � SD 565.1 �231.7 and 504.5 � 105.3 in the presence of C1q and in
Figure 2. Effect of C1q on cytokine production. PDCs were cultured with different stimuli and different concentrations of C1q (up to 70 mg/liter)for 20 hours. Results with IFN� are the mean and SD of �6 experiments. Levels of the other cytokines were measured once in a culture supernatantpool consisting of 6 culture supernatants. IL-6 � interleukin-6; TNF� � tumor necrosis factor � (see Figure 1 for other definitions).
C1q AND IFN� PRODUCTION IN SLE 3085
control cultures, respectively). Accordingly, C1q did notalter the proportion of apoptotic or living cells, demon-strating that the decreased IFN� production did notdepend on an increase in cell death. C1q has previouslybeen shown to activate myeloid DCs (30), and wetherefore investigated the effect of C1q on maturationmarkers on IC-stimulated PDCs. Even though C1qsignificantly inhibited IFN� production, no difference inCD80, CD83, CD86, or HLA–DR expression was found(data not shown).
Binding of C1q to different cell populations.Binding of C1q to different PBMCs or purified PDCs
was tested by incubation of cells with or without C1q for2 hours. C1q binding ability was analyzed by flowcytometry. The leukocytes were dissected based ondifferences in CD markers, and we observed thatCD19� B cells, monocytes, and CD3–CD16� naturalkiller cells were able to bind C1q to their surface, whilenone of the T cell populations could bind significantamounts of C1q (Table 1). We also demonstrated thatpurified PDCs could bind C1q to their surface (Figure3A). The specificity of the binding was further demon-strated by using serum as the source of C1q. Heat-inactivated serum and C1q-deficient serum were incapa-ble of depositing C1q on PDCs, whereas C1q depositionwas seen when using control serum, even though this wasless pronounced compared with purified C1q prepara-tion (data not shown). This indicates that activated C1qand not C1 is necessary in the binding to the cell surface.Thus, we detected binding of C1q to the surface of allcell populations tested, with the exception of T cells,and, importantly, the PDCs were able to bind C1q.
Interactions between C1q and PDCs. To verifythe binding between C1q and PDCs, we used SPRinteraction analysis. The response of adsorbed cellsincreased as expected with increasing C1q density of thesurface, indicating that the response of PDCs that boundwas dependent on the density of C1q (Figures 4A andB). We also demonstrated that PDCs bound with asimilar response to the PDC-specific anti-CD303 anti-body (Figure 4D). Since we could not detect any bindingof C1q to T cells by flow cytometry, we used these cells
Figure 3. Detection of C1q and C1q binding proteins on the surface of different cell populations by flow cytometry. A, PBMCs or purified PDCswere incubated with normal human serum concentrations of C1q for 2 hours, and C1q binding was detected by flow cytometry. B, Shown isexpression of the different C1q binding proteins on permeabilized PDCs. Data are representative of 3 experiments. cC1qR � receptor for thecollagen part of C1q; gC1qR � receptor for the globular parts of C1q (see Figure 1 for other definitions).
Table 1. Detection of Clq bound to the surface of different cellpopulations among peripheral blood mononuclear cells defined byflow cytometry analysis*
Cell population†No Clqadded
Clqadded‡
Lymphocytes 0.62 � 0.15 1.40 � 0.57Monocytes 4.52 � 1.12 52.7 � 31.4CD3�CD16� NK cells 1.75 � 0.54 99.2 � 31.4CD19� B cells 1.19 � 0.68 4.84 � 2.53CD3� T cells 0.51 � 0.05 0.79 � 0.07CD3�CD4� T cells 0.52 � 0.05 0.71 � 0.09CD3�CD8� T cells 0.52 � 0.06 0.84 � 0.03CD123�CD303� PDCs 0.57 � 0.47 19.3 � 15.1
* Values are the mean � SD mean fluorescence intensity based on 3experiments. NK � natural killer; PDCs � plasmacytoid dendriticcells.† Lymphocytes and monocytes were defined by forward and sidescatter properties, and other cell populations were defined by expres-sion of CD markers.‡ Clq added to equal normal human serum concentration.
3086 LOOD ET AL
as negative controls. By comparing surface responsesbefore and after injection of the T cells, we found thatthey did not bind to any of the surfaces investigated (e.g.,CD123, CD303, or C1q). However, due to limitations inregeneration and accumulation of cell debris, it was notpossible to obtain dilution series suitable for kineticmeasurements of the PDC–C1q interaction using SPRinteraction analysis. We have thus demonstrated bindingof C1q to PDCs using 2 different methods, flow cytom-etry and SPR interaction analysis, and with the latterapproach we showed a strong binding.
Expression of cC1qR, gC1qR, CR1, and �2�1integrin. Since we observed binding of C1q to the PDCs,we examined the surface expression of the 4 known C1qbinding proteins, cC1qR, gC1qR, CR1, and �2�1 inte-grin (CD49b) (31), through which C1q could mediate itsinhibitory effect. None of the proteins were detected onthe cell surface of PDCs, even though several differentclones were used (data not shown). However, we iden-tified intracellular staining of both gC1qR and cC1qR(Figure 3B). Thus, we did not detect the potential C1qbinding surface protein, but instead we detected 2intracellular C1q binding proteins through which C1qcould possibly mediate its effect.
C1q increases the uptake of ICs in PDCs. Inorder to determine whether the ICs were differentiallybound and endocytosed by PDCs when bound to C1q,we measured intracellular IgG and C1q uptake in PDCsby flow cytometry. ICs were readily taken up after 6hours of incubation, and the addition of C1q did notdecrease the uptake, but rather increased it slightly. Wealso demonstrated that C1q itself was taken up by thePDCs in combination with ICs (Figure 5). These exper-iments were done with permeabilized cells, but we stillcould not determine conclusively whether IgG and C1qwere located intracellularly or at the cell surface. Wetherefore performed surface staining for the same sam-ples. We could not see any cell surface IgG, but, as inprevious experiments, binding of C1q to the cell mem-brane was observed when adding C1q only (Figure 3A).Experiments were also performed with HSV and CpGDNA. However, results of these experiments were in-conclusive since C1q could be detected both before andafter permeabilization, indicating that at least not allC1q was taken up with those stimuli. In summary, wefound that C1q was taken up together with ICs, but thatC1q alone only bound to the cell membrane. Thisindicates that C1q opsonized the ICs, thereby enhancing
Figure 4. Surface plasmon resonance interaction analysis sensor-grams demonstrating binding of plasmacytoid dendritic cells (PDCs)(A, B, and D) and different concentrations of anti-C1q (C) to C1qsurfaces (A–C) or PDC-specific antibodies (D). C1q was anchored tothe flow chambers of a CM5 Sensor Chip in different densities (6,700response units [RU] in A, 10,000 response units in B, and 2,300response units in C). Association of PDCs to anti-CD123 and anti-CD303 antibodies is shown in D. The chip was flowed with differentconcentrations of anti-C1q or with cell suspension (11 � 105 cells/ml).The stop of injection is denoted by an arrowhead. Note the differentresponse at the y-axis in A compared with that in B. The remainingbound cell response units and the affinity (Kd) of the antibody are alsoshown. The extensive regeneration of surfaces described in Materialsand Methods is not within the time frames shown. Data are represen-tative of 2 experiments.
Figure 5. Detection of IgG and C1q by flow cytometry. Purified PDCswere stimulated with medium, immune complexes (ICs), or ICstogether with C1q (IC-C1q) and cultured for 6 hours, after whichpermeabilized and nonpermeabilized cells were analyzed by flowcytometry for detection of IgG and C1q. PDCs were gated based ontheir expression of CD303 (blood dendritic cell antigen 2 [BDCA-2]).The percentage indicated in the upper right corner is the percentage ofdouble-positive cells compared with only BDCA-2–positive cells, thusexcluding all non-PDCs from the calculations. Data are representativeof 3 experiments. See Figure 4 for other definitions.
C1q AND IFN� PRODUCTION IN SLE 3087
their uptake and subsequently interfering with theirinterferogenic properties.
DISCUSSION
Deficiency states of the components of the clas-sical pathway of the complement system are associatedwith the development of SLE (16). C1q especially hasbeen suggested to play an important role since almost allindividuals with a genetic C1q deficiency develop SLE.A suggested mechanism has been impaired clearance ofapoptotic cells, illustrated by the C1q-knockout mousemodel (32), but this is highly dependent on backgroundgenetics. We have previously found a correlation be-tween serum levels of C1q and IFN� in SLE, supportingthe theory of a cytokine regulatory function of C1q (33).In SLE, the most prominent IFN� inducers are RNA- orDNA-containing circulating ICs (5), and we investigatedwhether C1q could regulate IC-induced IFN� produc-tion.
The major finding of the present investigation isthat C1q could inhibit IC-, HSV-, and CpG DNA–induced IFN� production by PBMCs, indicating a gen-eral inhibition since 3 different stimuli, acting on bothTLR-7 and TLR-9, were used. The inhibitory effect ofC1q was also clearly demonstrated on the major IFN�-producing cells, the PDCs, where IC- and CpG DNA–induced IFN� production was dose-dependently inhib-ited by C1q, but HSV-induced IFN� productionincreased. The increased HSV-induced cytokine produc-tion could possibly arise from increased uptake byunknown mechanisms. The cytokine regulatory functionof C1q was not specific for IFN� production and in-cluded a dose-dependent inhibition of IC- and CpGDNA–induced cytokines such as IL-6, IL-8, and TNF�.
We are convinced that C1q binds to PDCs, sincewe could see this interaction both with flow cytometryand with SPR interaction analysis. The use of SPRinteraction analysis in studies involved in cell–ligandinteractions is fairly new, and our results clearly demon-strate a binding comparable with that found previously(34,35). Flow cytometry of permeabilized PDCs alsoindicated the presence of a C1q binding protein, sincemore ICs were present intracellularly in the presencethan in the absence of C1q. A receptor for enhancedphagocytosis, C1qRp (CD93), has been described(36,37), but its ability to bind to C1q is controversial(38,39). C1q has been reported to be able to interactwith several other surface proteins, and we investigatedwhether any of the so-far-described C1q binding pro-teins could be detected in PDCs. Both cC1qR and
gC1qR are intracellular proteins in the endoplasmicreticulum and the mitochondria, respectively, but theyhave been reported to be expressed on the surface ofseveral types of cells (40,41). Interactions with gC1qRhave been reported to inhibit cytokine production by Tcells and DCs (41,42). We could not detect any surfaceexpression of those proteins, but both gC1qR andcC1qR were present intracellularly in PDCs. The other 2described C1q binding proteins, CR1 and �2�1 integrin,could not be detected on the surface or intracellularly inPDCs.
There may also be an as-yet-undefined C1q bind-ing protein on the surface of PDCs, through which C1qmediates its effects. Alternatively, C1q could interactwith either of the 2 identified intracellular C1q bindingproteins (cC1qR and gC1qR) in PDCs and therebyinhibit IFN� production. It is also possible that the C1qopsonization of ICs interferes with TLR-7 activation,either by physical hindrance, intracellular compartmen-talization, or activation of antiinflammatory pathwayssuch as the suppressor of cytokine signaling family asdescribed previously for gC1qR interactions (42).Clearly, more work is needed to elucidate the exactmechanism for this cytokine regulatory function of C1qand to identify the potential C1q binding protein.
C1q has also been described by others to havecytokine regulatory properties, and in line with ourfindings, soluble C1q has been demonstrated to inhibitlipopolysaccharide-induced cytokine production frommurine bone marrow–derived DCs (19). However, C1qhas also has been reported to increase cytokine produc-tion (20,30), but in those studies C1q was immobilizedand not able to participate in intracellular events. Thus,the cellular localization of C1q probably explains someof these differences in the cytokine regulatory propertiesof C1q.
In complement deficiencies of the classical path-way, there exists a hierarchy of disease susceptibilityaccording to the position of the missing component inthe complement activation pathway. This hierarchy hasalso been suggested to apply in the ability of thedifferent complement components to take part in theclearance of apoptotic cells (43), indicating that themain link between SLE and complement deficiencies isimpaired clearance of apoptotic cells. Recently reporteddata from our group, based on studies of sera fromindividuals with inherited complement deficiency states,could not confirm such a hierarchical association be-tween the different complement components in theclearance of apoptotic cells (44). This suggests that C1qdeficiency operates by mechanisms in SLE pathogenesis
3088 LOOD ET AL
other than improper clearance of apoptotic cells andICs. One such alternative mechanism could be based onthe ability of C1q to inhibit IFN� production.
In conclusion, we have described a novel functionof C1q in the regulation of IC-induced production ofIFN� and other cytokines by PDCs. These findings areimportant for the understanding of the role of C1q inprotection against the development of lupus, and theymay also be important when exploring new therapeuticstrategies aiming to down-regulate the activated PDCsin SLE patients.
AUTHOR CONTRIBUTIONS
Dr. Bengtsson had full access to all of the data in the studyand takes responsibility for the integrity of the data and the accuracyof the data analysis.Study design. Lood, Gullstrand, Truedsson, Olin, Alm, Ronnblom,Sturfelt, Eloranta, Bengtsson.Acquisition of data. Lood, Gullstrand, Olin, Bengtsson.Analysis and interpretation of data. Lood, Gullstrand, Truedsson,Olin, Alm, Ronnblom, Sturfelt, Eloranta, Bengtsson.Manuscript preparation. Lood, Gullstrand, Truedsson, Olin, Alm,Ronnblom, Sturfelt, Eloranta, Bengtsson.Statistical analysis. Lood, Gullstrand, Bengtsson.
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