© 2012 Nature America, Inc. All rights reserved. Received Received 11 October 2011; accepted 12 June 2012; published online 26 August 2012; Ohio, USA. of Immunology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. Dermatology, University of Lübeck, Lübeck, Germany. of Medicine, Cincinnati, Ohio, USA. Biology, Center for Structural and Cell Biology in Medicine, University of Lübeck, Lübeck, Germany. Autoimmunity, German Rheumatism Research Center, Berlin, Germany. and Central Glycoanalytics, Institute of Laboratory Medicine and Pathobiochemistry, Charité Medical University, Berlin, Germany. College of Medicine, Cincinnati, Ohio, USA. 1 of independently cells mast and , as such effector functions ratio) A/I the upregulating ratio of Fcto activating inhibitory peritonitis and nephritis autoimmune experimental Fc Activating impact on activating Fc and Fc between Fc inhibitory property of IgG1 ICs, as it promotes the association Notably, high of galactosylation IgG N-glycans is crucial for this skin blisters in experimental bullosa epidermolysis acquisita. inflammatory responses C5a effector functions This pathway blocks ERK1/2 C5aR-mediated phosphorylation, Fc inositol domain–containing phosphatase (SHIP) downstream of dectin-1, resulting in of phosphorylation Src homology 2 functions. IgG1 ICs promote the association of Fc lectin–like dectin-1 suppress (C5aR) (ICs), the inhibitory IgG receptor Fc is limited. Here we demonstrate that IgG1 immune complexes arthritis Despite the key role of C5a in allergic asthma many of the properties proinflammatory of complement homeostasis contributes to host defense, immune surveillance and Complement is an ancient danger-sensing system that Fred D Finkelman Nathaniel L Harris Veroniqué Blanchard Marcela Rosas M Christian Karsten andgalactosylation association of Fc Anti-inflammatory activity of IgG1 mediated by Fc nature medicine nature Institute Institute for Systemic Research, University of Lübeck, Lübeck, Germany. g RIIB RIIB and spleen tyrosine kinase downstream of dectin-1. g 4 RIIB RIIB exert properties beyond anti-inflammatory their , , sepsis 14 10 These These authors contributed equally to this work. should Correspondence be addressed to J.K. ([email protected]). , 1 g 1 1 RIIB RIIB and dectin-1. Thus, IgG1 galactosylated . Activating Fc Activating . γ . . C5a and its G receptor –coupled mediate 1 Rs and C5aR promote inflammatory responses in in responses inflammatory promote C5aR and Rs 1 . C5a decreases the cellular activation threshold by threshold activation cellular the decreases C5a . 1 5 3 2

and cancer , , Jacqueline U McDonald . . C5a also recruits and activates inflammatory cells advance online publication online advance 2 2 , , , Gabriele Köhl 12 in in vitro g 1 in in vivo 4 , , Rs. 14 13 , , André Winkler γ , , Manoj K Pandey , , Gordon D Brown Rs eventually drive proinflammatory proinflammatory drive eventually Rs 9 6 Department Department of Respiratory Diseases Research, Boehringer Ingelheim Pharma GmbH, Biberach, Germany. and C5a-dependent , , knowledge about its regulation , , including peritonitis and 3 Institute Institute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff, UK. g 7 , RIIB RIIB and the C-type 8 arthritis , 1 , , Eva Wex 11 Department Department of Biology, Chair of Genetics, University of Erlangen, Germany.Erlangen-Nuremberg, 5 , Constanze , HessConstanze 3 3 , , autoimmune γ 9 , Selinda , J Selinda Orr 2 Rs Rs (known as the I alveolitis IC , 6 ,

14 g , , Marc Ehlers RIIB RIIB with , , Julia Figge 9 , Ralf , Ludwig Ralf 2 6 . Section Section of Infection and Immunity, University of Aberdeen, Aberdeen, UK. 1 0

3 1 , , Markus Berger 5 1 , , Regina Kilchenstein 13 , , Delyth M Reid , are protected from IC-mediated inflammation despite the presence of Fc activating Fc lation was significantly reduced upon reduced pretreatment and 107.3-IC lation was was significantly ( manner and dose-dependent shown) not (data WT from neutrophils (CR3) CD18 the and CD11b involve neutrophils of recruitment ( inflammation of C5a-dependent tion demonstrating a crucial role for Fc not but WT in OVA-IC–inducedblocked peritonitis (OVA)-specific ICs is mediated by C5a by induced ovalbumin ously inflammation shown that the peritoneal not have an anti-inflammatory effect (data not shown). We have previ ( WT and in peritoneum the into migration C5a-mediated reduced TNP-OVAand 107.3 (107.3-IC) clone IgG1 comprising IC (TNP)-specific trinitrophenyl the of tion myeloid cells by binding of ICs to activating Fc circulating of activation incidental prevent to mechanism protective Fc inhibitory to IgG1 of binding preferential The mice. in serum of abundant most the is IgG1 ( strains mouse both in peritoneum the into recruited were or (WT) wild-type BALB/c functions. effector C5a-mediated suppressing ICs IgG of property anti-inflammatory an be could tion protec such underlying mechanism one that We C5a. hypothesized 5 Fig. 1 Fig. Department Department of Medicine, Cincinnati Veterans Affairs Medical Center Cincinnati, & Jörg Köhl 10 The mechanisms underlying C5aR-mediated extravasation and tissue To test our we hypothesis, C5a injected into cavity the ofperitoneal γ 2 Rs Division Division of Cellular and Molecular Immunology, University of Cincinnati d , Detlef , Zillikens Detlef o i 2 : a 1 . . FcR and and 0 Fcer1g . 1 0 3 γ 8 Supplementary Fig. 1 Fig. Supplementary γ 8 -chain–deficient ( -chain–deficient Division Division of Emergency Medicine, University of Cincinnati College Rs but do express the inhibitory Fc inhibitory the express do but Rs / n −/− m γ 1 . , mice but not in Fc 4 2 2 RIIB RIIB and dectin-1 6 , , Dominique Petzold 8 , , Irina V Majoul 6 2 10 1 1 4 Supplementary Fig. 2 Fig. Supplementary , , Philip R Taylor , , Falk Nimmerjahn . C5a upregulated CD11b expression on on expression CD11b upregulated C5a . Fcer1g Fcer1g 1 3 γ 30 min before C5a administration administration C5a before min 30 −/− RIIB in 107.3-IC–mediated inhibi ). Monomeric 107.3 antibody did did antibody 107.3 Monomeric ). γ mice. As expected, neutrophils neutrophils As expected, mice. RIIB-deficient ( RIIB-deficient 5 −/− Laboratory Laboratory of Tolerance and 1 Fig. 1 Fig. 1 7 . . Pretreatment with 107.3-IC ) mice express no functional ) mice express no functional , , Richard T Strait β 2 b γ 4 heterodimer heterodimer integrin Rs ). , γ 3 RIIB is considered a considered is RIIB

Fcer1g 1 , 10 2 ), and this upregu this and ),

. Intravenous injec γ Department Department of 4 RIIB. These mice mice These RIIB. 11 Glycodesign Glycodesign 7 s r e t t e l Fcgr2b Fcgr2b , Institute Institute of −/−

ie n a in mice 12 Division Division −/− −/− Fig. 1 Fig. ) ) mice mice, mice, 8 , a ). ).  - - - - -

© 2012 Nature America, Inc. All rights reserved. of [Ca ([Ca release calcium phospholipase the intracellular in pathway, phosphate increase an to C–inositol leading and (ERK1/2) 2 and 1 kinases extracellular signal-regulated kinases (MAP) protein mitogen-activated the of tion inhibition. of mechanism general more a suggesting Fcer1g and WT- of migration neutrophil CXCL2-dependent also but C5aR only not blocked 107.3-ICs that found we Moreover, inflammation. Fc activating of only ( migration neutrophil on of C5a-mediated 107.3-ICs effect inhibitory Fc the with from not but ( macrophages peritoneal and neutrophils marrow–derived bone C5a-mediated of inhibited dose-dependently 107.3-ICs from cells among adhesion neutrophil C5a-dependent reduced also tion from with cells absent in Values experiments. × 10 (5 C5a × 10 (5 C5a with treatment histogram, gray (background); C5a of absence the in phosphorylation ERK histogram, A8 antagonist receptor C5a Gr-1 in phosphorylation ERK1/2 C5a-mediated on 107.3-ICs of ( right). the on (histogram Gr-1 marker the of expression the and plot) dot (left pattern (SSC) granularity and (FSC) size cell ( cells derived ( intensity. fluorescence mean MFI, mice. ( neutrophils in response to C5a ( Figure 1 s r e t t e l  c a Fig. Fig. 1 Supplementary Fig. 4 Fig. Supplementary

d , 5R xrs ay f t booia fntos hog activa through functions biological its of many exerts C5aR d Neutrophil Neutrophil Number of neutrophils

) Impact of 107.3-ICs on CD11b expression ( expression CD11b on 107.3-ICs of ) Impact 2 adhesion (%) adhesion (%) per mm 100 150 200

Fcer1 Untreated 2+ 50 10 15 20 10 15 20 25 30 35 −/− e 0 0 5 0 5

and C5a + 107.3-IC ] IgG1-ICs IgG1-ICs inhibit C5a-mediated inflammatory responses i - - but not in bone cells marrow–derived from WT and 0.1 0.1 107.3-IC 0.01 107.3-IC 1 Medium −/−

−8 C5a γ Supplementary Fig. 3a Supplementary WT f *** Fcgr2b RIIB-specific blocking antibody Ly17.2 abrogated the the abrogated Ly17.2 antibody blocking RIIB-specific Fcer1g C5a (nM) C5a (nM) M; 1 min) + 107.3-ICs or C5aRA at the indicated concentrations. Results in in Results concentrations. indicated the at C5aRA or + 107.3-ICs 1 min) M; ) from WT, ) from Fcgr2b but not not but 107.3-IC 1 1 1 1 * µ Fcgr2b –/ –/ g ml – – µ g ml −/− 2+ –1 γ a Number of neutrophils Rs Fcgr2b , Fcgr2b ] –1 ** b 2 mice. Pharmacological targeting of Fc of targeting Pharmacological mice. 0 i Fcer1g 0 ). Thus, 107.3-ICs inhibit the functions not not functions the inhibit 107.3-ICs Thus, ).

) per mm , −/− 1 d 2 200 100 150 ∆ Untreated 2 . 107.3-ICs blocked the C5a-mediated rise rise C5a-mediated the blocked 107.3-ICs . 50 – 71−73 but also of complement-mediated acute acute complement-mediated of also but 0 f -derived -derived cells (

e C5a + 107.3-IC are means means are a g −/− −/− Number of neutrophils ) or OVA-ICs comprising OVA and OVA-specific rabbit IgG (

−/− SSC-H Fcer1g 1,02 2 (C5aRA) on C5a-mediated ERK1/2 phosphorylation in bone marrow–derived neutrophils (graph on the right). Empty Empty right). the on (graph neutrophils marrow–derived bone in phosphorylation ERK1/2 C5a-mediated on (C5aRA) per mm C5a 25 51 76 100 150

mice ( mice Untreated mice ( mice or or *** 50 0 4 6 2 8 107.3-IC 0 107.3-IC 2 0 Fcgr2b –/ – ** – 56 d 0.01 ** FSC-H * e ± ) ) from WT and Fig. Fig. 1 ** WT Fig. 1 Fig. 51 * , s.e.m. ( s.e.m. f ( 0.1 * 7 2 ) C5a-mediated migration of bone marrow–derived neutrophils ( neutrophils marrow–derived bone of migration ) C5a-mediated µ g ml −/−

Supplementary Fig. Supplementary 3b Number of neutrophils 68 1 per mm2 mice with or without 107.3-ICs. ( 107.3-ICs. without or with mice c 100 150 200 –1 1,024 d c

). 107.3-IC administra ). 107.3-IC Untreated 50 ) ). Next, we found that that found we Next, ). ) and iC3b-dependent adhesion ( adhesion iC3b-dependent ) and 0

n C5a + 107.3-IC

= 5–10 per group). * group). per = 5–10 Number of neutrophils 2 per mm C5a Fcgr2b

Counts 100 150

10 Untreated 50 25 50 75 107.3-IC 0 0 0 107.3-IC 10 Fcer1g Fcer1g Gr-1 –/ 0 – Fcer1g 10 0.01 ** 1 + ** in in vivo * 10 WT bone marrow–derived neutrophils (three graphs on the left) and the effect of the the of effect the and left) the on graphs (three neutrophils marrow–derived bone WT ( Gr-1APC

0.1 * µ −/− −/− 2 b g ml –/ 10

– Number of neutrophils γ 3 mice mice 1 2 RIIB RIIB 10 –1 and per mm 100 200 300 , 4 ) e ), ), 0 - - Ag Ctrl P h Number of neutrophils

in in vitro 2 < 0.05, ** < 0.05, Counts

10 per mm Peritonitis WT 100 150

25 50 75 Untreated Ab Ctrl 50 tyrosine-based activation motif (ITAM) in the the in (ITAM) motif activation tyrosine-based gation enables the Src kinase that phosphorylates the immunoreceptor atively regulates immune cell activation. Mechanistically, such coaggre from from cells myeloid of chemotaxis C5a-mediated block 107.3-ICs SHIP. of Because activation and binding allows then ITIM phosphorylated inhibitory motif (ITIM) within the Fc release. calcium and intracellular phosphorylation ERK1/2 both mation by an Fc inflam C5a-mediated block IgG1-ICs that demonstrate results these the C5aR antagonist A8 in magnitude to what we found when C5aR signaling was blocked with ( in bone marrow–derived neutrophils from WT but not Fig. 5a of ERK1/2 in Gr1 marrow–derived neutrophils ( Gr1 in phosphorylation ERK1/2 C5a-mediated on 107.3-ICs from not but 0 0 Peritonitis WT 10 107.3-IC 0 Fig. 1 P-ERK1/2 0 Ctrl IgG1 IC-mediated coaggregation of Fc γ Rs to also phosphorylate the immunoreceptor tyrosine-based tyrosine-based immunoreceptor the phosphorylate also to Rs by an Fc 10 Fcgr2b g 0.01 d ) Gating of bone marrow–derived neutrophils according to their their to according neutrophils marrow–derived bone of ) Gating

1 107.3-IC Fcer1g WT + 107.3-IC *** ) in bone marrow–derived neutrophils from from neutrophils marrow–derived bone ) in

h Peritonitis 10 ), ), which is inhibited by 107.3-ICs in a manner dose-dependent ( and 0.1 + µ P 2 g ml *** –/ < 0.01, *** < 0.01,

10 Peritonitis b 1 – 3 ) with or without 107.3-ICs in WT,

γ Fcer1g −/− Peritonitis –1 Supplementary Fig. 5b RIIB-dependent mechanism. RIIB-dependent ( 10 c ) Fcgr2b and and 4

mice, the effect does not result from classical pairing pairing classical from result not does effect the mice, –/– f Fcgr2b advance online publication online advance γ 2+ 10 + RIIB-dependent mechanism leading to leading mechanism inhibition of RIIB-dependent Ca peak height 25 50 75 Fcgr2b 1,000 1,500 2,000 bone marrow–derived neutrophils ( 0 0 –/– g 10 500 are representative of at least three independent independent three least at of representative are

0 107.3-IC −/− C5a + 107.3-IC –/– P 0 10 < 0.001. + ∆ mice ( mice 1 C5a 71−73 10 * 2 107.3-IC 0.1 WT

107.3-IC c e 10

µ MFI CD11b ) and increase in [Ca in increase ) and 1,000 1,050 1,100 g ml (5 × 10 3 Fig. 1 800 850 900 950 Fig. 1 Fig. 10 –1 Untreated 4 −8 107.3-IC α

10 2+ M; 1 min); magenta histogram, histogram, magenta 1 min); M; 25 50 75 g ). This inhibitory effect was similar Ca peak height -chain of Fc 0 0 1,000 1,500 2,000 −5 f ). C5a promotes phosphorylation 10 Fcer1g ). Next, we tested the impact of impact the tested we Next, ).

500 0.01 0 M) γ C5a + 107.3-IC RIIB with activating Fc 0 10 a ( 0.1 µ , 1 C5a h 1 b g ml –/ Fcer1g 5 10 Fcer1g ) Dose-dependent impact impact ) Dose-dependent ) ) Peritoneal migration of – ( * 2 Fig. 1 107.3-IC 1 107.3-IC 1 –1 µ 10 Fcer1g g ml ) γ 3 –/ 2+

γ RIIB –1 – 10 −/− -chain of activating activating of -chain MFI CD11b ] 1,000 1,050 1,100 nature medicine nature i 4 h in bone marrow– bone in 800 850 900 950 or

). Taken together, 2+ 80 20 40 60 −/− Untreated

1 Ca peak height 0 Supplementary 2 1,000 2,000 3,000 Fcgr2b Fcgr2b 107.3-IC . The tyrosine- and and C5a + 107.3-IC 0

0.01 Fcgr2b C5a Fcgr2b Fcgr2b −/− −/− γ ( 5 C5aRA Rs neg µ + 0.1

× 107.3-IC mice. g ml 10 bone bone –/– mice –5 –/

−/− M –1

– 1 ) - - -

© 2012 Nature America, Inc. All rights reserved. kinases mediate tyrosine phosphorylation of the Fc of the phosphorylation tyrosine mediate kinases of IgG1-IC ( effect inhibitory the abrogated completely genistein inhibitor kinase 1 min and by that of the inhibition tyrosine phosphorylation tyrosine after occurred effect inhibitory the that noted we IgG1-ICs, of effect neutrophils. in functions Fc require ICs (ref. dectin-1 lacking vector control the with not but isoform dectin-1B the with blocked by 107.3-ICs in ( dectin-1-deficient from neutrophils marrow–derived bone of tion toward C5a ( Ly6G only findings, these Ly-6G all almost on expressed is are C5aR neutrophils of bone marrow–derived Ly-6G of 25–30% about is, that pattern; same the follows expression C5aR 30–40% that found We Ly-6G IgG1-IC. of of effect inhibitory the for cial 6 Fig. neutrophils in dectin-1 of internalization promotes rapid laminarin polymer soluble the not but curdlan polymer ( required is indi dectin-1 that cating migration, neutrophil C5a-mediated on inhibitory of 107.3-IC effects inhibitory the blocked largely curdlan or laminarin with 107.3-ICs. of effect inhibitory the on curdlan agonist dectin-1 the and laminarin inhibitor dectin-1 the of impact neutrophils on expressed is and motif like symbol we ( dectin-1 on focused phosphorylation, SHIP and ITIM ing Fc of partners new potential for Fc activating of * ( ( (pMXs-IZ) alone vector with or (D1B) isoform dectin-1B the with transfected line cell Clec7a from and mice WT 129S6/SvEv from neutrophils marrow–derived bone of chemotaxis mediated ( 10 bars, Scale C5aR. and dectin-1 Ly-6G are cells migrated All microscopy. confocal by analyzed and C5aR and dectin-1 Ly-6G, of expression (10 ( histogram. right top the in shown is neutrophils Fc cytometry. flow by determined as histogram), left (top neutrophils Ly-6G on plot) right bottom the in shown control isotype plot; contour left (bottom expression dectin-1 and ( (bottom) curdlan or (top) laminarin with pretreatment vitro in neutrophils marrow–derived bone of migration ( dectin-1. on depends migration neutrophil 2 Figure nature medicine nature two the that Wefound dectin-1. in motif ITAM-like the and ITIM d c a n Clec7a P ) Bone marrow cells that migrated toward C5a C5a toward migrated that cells marrow ) Bone C5a-mediated on 107.3-ICs of ) Impact = 3 per group). Values are means means are Values group). = 3 per , To search for the signaling pathways underlying this inhibitory inhibitory this underlying pathways signaling the for search To < 0.05, ** < 0.05, e −8 ) Impact of 107.3-IC treatment on C5a- on treatment 107.3-IC of ) Impact M for 30 min) were stained for membrane membrane for stained were min) 30 M for ), suggesting that the surface availability of dectin-1 is cru is dectin-1 of availability surface the that suggesting ), hi −/− when dectin-1 is blocked by blocked is dectin-1 when −/−

neutrophils express dectin-1and C5aR ( C5aR dectin-1and express neutrophils mice ( mice The inhibitory effect of IgG1-ICs on IgG1-ICs of effect inhibitory The n hi = 3 per group). ( group). = 3 per ) ) mice bone marrow–derived neutrophils express dectin-1. dectin-1. express neutrophils marrow–derived bone Fig. Fig. P Fig. hi < 0.01, *** < 0.01, 19) ( 19) γ neutrophils that express express that neutrophils d RIIB and dectin-1 to inhibit C5a-mediated effector effector C5a-mediated inhibit to dectin-1 and RIIB γ ) or a ) or 3

1 Rs with Fc with Rs 2 8 a hi c (

Fig. 2 Fig. and bone marrow–derived marrow–derived bone γ ). ). 107.3-ICs failed to inhibit C5a-mediated migra Fig. RIIB expression of Ly-6G of expression RIIB advance online publication online advance Clec7a Supplementary Fig. 8a Supplementary Clec7a

2 e P d and and b < 0.001. ). C5a-mediated migration was, however, was, migration ). C5a-mediated ) C5aR (CD88) (CD88) ) C5aR hi γ −/− RIIB. To search Tosearch RIIB. dectin-1 neutrophil neutrophil −/− Supplementary Fig. 7 Fig. Supplementary hi µ γ neutrophils retrovirally transduced ± Fig. 2 Fig. m. m. RIIB driv RIIB neutrophils ( neutrophils s.e.m. s.e.m. e Clec7a )

+ C5aR

a ). The particulate particulate The ).

), as it harbors an ITAM- an harbors it as ), 1

hi 6 - +

. First, we evaluated the the evaluated we First, . neutrophils migrated migrated neutrophils Invitro dim ). Phosphorylated Src ). Phosphorylated Fig. 2 Fig. Fig. 2 Fig. 1 a d dectin-1 7

( Number of Number of neutrophils Number of neutrophils

γ 2 2 2 Supplementary Supplementary RIIB-associated RIIB-associated

neutrophils per mm per mm per mm 100 150 100 b preincubation preincubation ). Thus, IgG1- Thus, ). b 20 40 60 80 50 20 40 60 80 Untreated Untreated ). In line with with In line ). 0 0 0 ). ). About 10% + 107.3-l + 107.3-l + laminarin − + curdIan *** *** . . Fc β -glucan -glucan WT

* 107.3-l C 107.3-l C *** *** γ + laminarin RIIB RIIB *** + curdIan+ *** + C C - - -

** Clec7a and dectin-1 in response to 107.3-IC administration ( administration 107.3-IC to response in dectin-1 and Syk with Fc SHIP or Phosphorylated colocalize phosphorylated not but WT from neutrophils marrow–derived bone in phosphorylated ( Syk ( microscopy, Finally, by confocal treatment 107.3-IC following isoform phosphopeptide Syk-binding the in a with Syk pulled phosphorylated we down manner, dectin-1–dependent a in phosphorylation Syk 8b Figs. ( chemotaxis neutrophil C5a- on mediated IgG1-ICs of effect inhibitory the inhibi reversed Syk completely or tor, 61-3606 BAY piceatannol, R406, inhibitors, kinase mice deficient (Syk) kinase tyrosine ( neutrophil on 107.3-ICs of effect Src-family kinase inhibitors I and II completely blocked the inhibitory IgG to Fc of binding for essential is moiety Fc the of Asn297 at N-glycan The and Fcwith dectin-1 proinflammatory C5a-mediated for functions. effector crucial influx calcium and phosphorylation ERK1/2 C5a-mediated blocks then pathway This Fc within ITIM the of phosphorylation tyrosine to linked also are dectin-1 of downstream phosphorylation Syk and Tyrosine phosphorylation. Syk to leading ( min 5 after declined that in rapid and transient Syk and 1–3 within min SHIP phosphorylation 1 Movies Supplementary b

–10 CD88 Ligation of dectin-1 results in the phosphorylation of spleen spleen of phosphorylation the in results dectin-1 of Ligation To identify the mechanism underlying the interaction of IgG1-ICs interaction the Tounderlying mechanism the identify dectin-1, engage IgG1-ICs that suggest data these Taken together, 10 10 10 Fcgr2b 2 3 4 5 –/ 101 134 –10 34 67 – 0 47.90% 11.09% 10 Clec7a 1 10 0 – γ 2 10 d Dectin 1 107.3-IC Untreated Rs −/− 10

1 Ly-6G and 3 2 10 72,31 −/− 3 or or 2 2 , the serum collectin mannose-binding , lectin mannose-binding the collectin serum 10 14.23% 26.78% 1

4 10 9 and pharmacological targeting by four different Syk by different four targeting and pharmacological neutrophil cell line transduced with the dectin-1B dectin-1B the with transduced line cell neutrophil Clec7a ). To further demonstrate that IgG1-IC promotes promotes IgG1-IC that demonstrate further To ). 3 10 5 10 γ 4 RIIB, we focused RIIB, on we focused the Fc glycan composition. 2 γ 0 e –10 RIIB and subsequent SHIP phosphorylation. phosphorylation. SHIP subsequent and RIIB Rat IgG2b . Genetic deletion of Syk in inducible Syk- inducible in Syk of deletion Genetic . 10 10 10 − Number of / 149 198 2 3 4 5 −

2 –10 50 99 – Supplementary Fig. 10a Fig. Supplementary

mice in response to 107.3-IC treatment. treatment. 107.3-IC to response in mice neutrophils per mm 0 99.35% 0.33% 5 10 100 1 40 60 80 20 ). Notably, 107.3-IC treatment resulted resulted treatment 107.3-IC Notably, ). 10 0 0 Rat IgG2b 2 10 Fc 10 1 Clec7a 3 10 γ 91,40 Fig. 3b Fig. RIIB D1B 10 2 * i. 3d Fig. 0.12% 0.20% 4 10 Fig. 3 Fig. –/ 10 3 –

+ 5 10 , 4 upeetr Fg 8e Fig. Supplementary c ). c f , ) and SHIP ) ( and SHIP e ** Clec7a and and pMXs-IZ –/ , – b Supplementary

+ s r e t t e l ). Fig. 3f Fig. 107.3-IC Untreated Fig. 3 Fig. 2 4 and the Dectin-1 CD88 Ly-6G , γ g g RIIB RIIB and and ) ) are 2 ). ).  2 -

© 2012 Nature America, Inc. All rights reserved. hmtxs C1b peuain n ices i [Ca and WT from in neutrophils marrow–derived bone increase and upregulation CD11b chemotaxis, C5a-mediated the inhibited LoGalH5-ICs not but (HiGalH5-ICs) ( 1 Table glycan Fc contentgalactose 8.8%) ( (LoGalH5-Ab; form degalactosylated a and 96.9%) tent con galactose glycan Fc (HiGalH5-Ab; form galactosylated highly antibody H5 degalactosylated we or galactosylated galactosylation, N-glycan of importance the assess directly To ( neutrophils marrow of bone chemotaxis C5a-mediated inhibit not did antibody H5 or 7B4 of composed ICs ( tively (ref. H5 and 7B4 , ylation. The of N-glycan galactosylation two other TNP-specific IgG1 galactos by glycan 81.8% MALDI-TOF,antibody 107.3 we observed ( galactosylation N-glycan the mined thought previously than more accessible is and dynamic glycan Fc that demonstrated analysis resonance nuclear recent magnetic A glycan. Fc any with interact to shown be not has by a expressed pathogens broad range of fungal C1q molecule collectin-related 20 bars, Scale experiments. independent ( two of representative are Results feature. IsoSurface the and software analysis image IMARIS and microscopy confocal by acquired images of Z-stacks using spots) (white colocalization show insets The rows. second the in neutrophils fluorescent white by demonstrated is treatment Fc with p-SHIP and p-Syk of Co-localization rows). fourth and third (second, 107.3-ICs with incubation after WT, from neutrophils ( (p-SHIP) SHIP ( (p-Syk) Syk of phosphorylation ( * a in Values 107.3-ICs. of presence the in chemotaxis neutrophil marrow–derived bone C5a-mediated on R406 by blockade kinase ( treatment. ( mice knockout Syk conditional from isolated neutrophils marrow–derived ( I ( inhibitors kinase ( genistein by phosphorylation tyrosine of blockade specific of absence or presence the in 107.3-ICs without or with neutrophils marrow–derived bone of Fc of downstream SHIP of and dectin-1 of downstream Syk and tyrosine of phosphorylation promote and kinase 3 Figure s r e t t e l  ( (SialH5) H5 antibody galactosylated of highly sialylation Additional from not f Syk d a Supplementary Fig. Supplementary 14 Supplementary Fig. 12 Fig. Supplementary P –

, ) C5a-mediated chemotaxis of bone bone of chemotaxis ) C5a-mediated g e γ < 0.05, ** < 0.05, Number of RIIB. ( RIIB. ) Impact of 107.3-IC treatment on treatment 107.3-IC of ) Impact are means means are 2 flox/flox neutrophils per mm 100

Untreated 20 40 60 80 0 Supplementary Fig. 12 Fig. Supplementary

). N-glycan composition had no effect on binding binding antigen on effect no had composition N-glycan ). IgG1-ICs activate Src Src activate IgG1-ICs a ) with or without tamoxifen tamoxifen without or ) with + 107.3-IC Fcgr2b – e c ***

) Impact of Syk tyrosine tyrosine Syk of ) Impact + genistein ) C5a-mediated chemotaxis chemotaxis ) C5a-mediated P ± < 0.01, *** < 0.01, g s.e.m. ( s.e.m. 107.3-IC *** ) in bone marrow–derived marrow–derived bone ) in −/− *** b + genistein+ ) or II ( II ) or or or Fcgr2b n Clec7a = 3–5 per group). group). per = 3–5 ). ). Immune complexes comprised of HiGalH5 Supplementary Fig. Supplementary 12 and and P c −/− ). ). < 0.001. < 0.001. a b

or or ) or Src Src ) or

28), was only 24.6% and 29.7%, respec 29.7%, and 24.6% only was 28), −/− f Number of Supplementary Table 1 Supplementary ) ) or 2 2 and and

Clec7a neutrophils per mm 5

100 150 mice ( mice . Dectin-1 recognizes recognizes . Dectin-1 50

Untreated 0

Supplementary Table 1 Supplementary

+ Src kinase inh. l

Supplementary Fig. 11 Fig. Supplementary −/−

+ 107.3-IC µ Supplementary Fig. Supplementary

m. **

mice. Depicted are immunofluorescence signals in the absence of 107.3-ICs (first rows) or obtained 3 min 3 min obtained or rows) (first 107.3-ICs of absence the in signals immunofluorescence are Depicted mice.

Supplementary Fig. Supplementary + Src kinase107.3-IC inh. **

f + ** 2 and 6 P-Syk Fc . . However, dectin-1 2 in vitro in Fcer1g 7 γ . When we deter we When . RIIB l Supplementary Supplementary ) did not alter alter not did ) β -1,3-glucans -1,3-glucans −/− 2+ , yielding a yielding , Number of c ] mice but mice 2 i

). IgG1- ). among among neutrophils per mm 13a 100 150 ) ) of the Dectin-1 Fc 50

Untreated 0 13a γ RIIB – + Src kinase Inh. II

c + 107.3-IC ). ). ). ). - - - - ***

+ Src kinase Inh. II

autoimmune diseases mediated by C5a. In line with this view, data data view, this with line In C5a. by mediated diseases autoimmune in response effector inflammatory the aggravate may dectin-1 that suggest data our autoimmunity, spontaneous develop not do mice ( of HiGal-ICs effect protective the in of dectin-1 role key the firming ( of Fig. model this development in the lesions reduced cutaneous treatment LoGalH5-IC not but IC ( EBA of development the from that found We collagen. VII type mouse against IgG directed of rabbit transfer by the induced disease (EBA) acquisita bullosa of epidermolysis model an experimental in C5 for role a key suggest ( peritoneum the into recruitment neutrophil on inhibitory C5a-mediated of HiGalH5-ICs effect the abrogated and neutrophils recruited on internalization dectin-1 in of resulted curdlan of administration intravenous not but WT perito of neum the into recruitment neutrophil C5a-mediated suppressed ( effect inhibitory the to contributes IgG1, is, that isotype, IgG the also but chemotaxis C5a-mediated block not did IgG2a-ICs TNP-specific galactosylated ( effect inhibitory the 107.3-IC Supplementary Fig. 19 Fig. Supplementary **

In vivo In +

Dectin-1 P-Syk 18 Supplementary Fig. 15a Fig. Supplementary ). HiGal-ICs were not protective in in protective not were HiGal-ICs ). , HiGalH5-ICs but not LoGalH5-ICs almost completely completely almost LoGalH5-ICs not but HiGalH5-ICs , in in vitro Tamoxifen 107.3-IC d

g Number of advance online publication online advance neutrophils per mm2 Fig. 4 Fig. P-SHIP Fc 100 150 50 , suggesting that not only the glycan composition composition glycan not the that only , suggesting γ 0 RIIB Fcgr2b + – – – b upeetr Fg 15a Fig. Supplementary and ). Although dectin-1–deficient humans and humans dectin-1–deficient ). Although 2 * WT 9 ** , a subepidermal autoimmune blistering blistering autoimmune subepidermal a , + – * γ −/− RIIB and dectin-1 in response to 107.3-IC 107.3-IC to response in dectin-1 and RIIB Supplementary Fig. 16 Fig. Supplementary + + Dectin-1 Fc or or Supplementary Fig. 17 Fig. Supplementary – γ + – – – RIIB c Syk Clec7a ). ** i. 4c Fig. *** C5ar1 flox/flox + – ** + + −/− −/− , mice ( mice Dectin-1 P-SHIP d e mice were protected protected were mice and and Clec7a Number of

neutrophils per mm2 nature medicine nature ). Notably, highly highly Notably, ). Clec7a 100 150 200 Fig. 4 Fig. 50

Supplementary Supplementary Untreated 0 ). Previous data ). Previous

−/− + R406 + 107.3-IC Fcgr2b WT 107.3-IC WT unstimulated Clec7a + 107.3-IC ). HiGalH5- ). mice, con mice, −/− a * ). Further, Further, ). ) or three three ) or –/ –/ – – * 107.3-IC 107.3-IC + R406 - - © 2012 Nature America, Inc. All rights reserved. ICs as donor and an Alexa647-labeled Toll-like receptor 4–specific 4–specific receptor Toll-like Alexa647-labeled an and donor as ICs Cy3-HiGalH5- or donor as Cy3-LoGalH5-ICs used we when FRET of 20% ( efficiency FRET a found we acceptor, as antibody dectin-1–specific Alexa647-labeled an and donor as HiGalH5-ICs Cy3-labeled Using of promotes Fc association the not LoGalH5-ICs but HiGalH5-ICs that (FRET) transfer energy resonance Förster by found we approach, second a In status. galactosylation their of ent Fc of absence the in that demonstrate data These shown). not (data cells) (BWZ line cell lymphoma or a dectin-1–transfected recombinant dectin-1–Fc Fig. Fig. Fc bind First, and/or we H5-ICs LoGalH5-ICs found that HiGalH5-ICs, Fc of ciation inflammation. autoimmune-driven models litis from in Values acceptor. as Alexa647 to conjugated antibody TLR4-specific ( control a negative As acceptor. as Alexa647 to conjugated antibody dectin-1–specific and donor ( line). black the by (marked value starting the with compared as intensity fluorescence of change the indicate Arrows cells. marrow bone on staining acceptor of images) (bottom absence or images) (top presence the in photobleach acceptor Alexa647 to conjugated antibody dectin-1–specific during means in values (AUC); curve the under area the of assessment by quantified as 4–12), (days EBA experimental of course the during mice IC–treated 100 bars, Scale separation. dermal-epidermal toward point arrows Black groups. treatment LoGal-H5-IC and HiGalH5-IC PBS, in 12) (day EBA experimental in membrane basement the at row) (fourth C3 and row) (third collagen VII ( group). per ( HiGalH5-ICs without or with C5a of injection (i.p.) intraperitoneal to response in migration neutrophil peritoneal on pretreatment curdlan of ( 4 Figure nature medicine nature as ( acceptor antibody g a d a ) FRET efficiency following acceptor bleaching in bone marrow cells treated with either Cy3-HiGalH5-ICs ( Cy3-HiGalH5-ICs either with treated cells marrow bone in bleaching acceptor following efficiency ) FRET WT,in 107.3-ICs without or with C5a to response in neutrophils of migration ) Peritoneal To directly assess whether N-glycan galactosylation drives the asso γ AUC Number of IBtasetd hns hmtr vr cls ( cells ovary hamster Chinese RIIB-transfected

(% affected surface area) 5 21 20

40 60 80 20 Untreated neutrophils (× 10 ) experimental models of autoimmune arthritis autoimmune of models experimental 0 0 2 4 6 8 ± ) with similar affinities. In contrast, such ICs do not bind bind not do ICs such contrast, In affinities. similar with ) ad o eobnn mue Fc mouse recombinant to and ) HiGalH5-IC C5a + HiGaIH5-IC

s.e.m. * s.e.m.

Fig. Fig. 4e PBS High Fc glycan galactosylation is crucial for the inhibitory effect of IgG1-ICs IgG1-ICs of effect inhibitory the for crucial is galactosylation glycan Fc High LoGalH5-IC C5a + LoGaIH5-IC * * *

c C5a 3 ) Development of cutaneous lesions (first row), subepidermal split formation (second row), linear deposition of rabbit antibody to type type to antibody rabbit of deposition linear row), (second formation split subepidermal row), (first lesions cutaneous of ) Development γ WT 1 RIIB and dectin-1, we performed a two-step approach. two-step a performed we dectin-1, and RIIB ** point toward a role for dectin-1 as a modulator of of modulator a as dectin-1 for role a toward point – P g < 0.05, ** < 0.05, ** and

e γ advance online publication online advance RIIB, IgG1-ICs do not bind dectin-1 independ dectin-1 bind not do IgG1-ICs RIIB, Intensity Intensity Fig. 4e Fig. Supplementary Fig. 22 Supplementary 85 35 40 45 50 55 60 65 70 75 80 85 50 55 60 65 70 75 80 Number of 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 5 0 5 0 Untreated neutrophils (× 10 ) 0 2 4 6 8 P

< 0.01. ( < 0.01. C5a + HiGaIH5-IC – 10 10 g

). C5a + LoGaIH5-IC Fcgr2b 15 15 C5a 20 20 HiGalH5-IC 25 25 Time (s) –/ Time (s) e – , f 30 30 ) Time-dependent change in fluorescence intensity of Cy3-HiGalH5-ICs ( Cy3-HiGalH5-ICs of intensity fluorescence in change ) Time-dependent γ 35 35 IBF ( RIIB-Fc 40 40

). ). Notably, we found no Number of 5 45 45 Untreated neutrophils (× 10 ) γ 5 0 1 2 3 4 50 RIIB with dectin-1. dectin-1. with RIIB 50 55 55 Supplementary Supplementary Supplementary Supplementary

3 60 60

C5a Clec7a 0 HiGaIH5-IC or encepha or C5a

f LoGaIH5-IC + Intensity Intensity –/ – 400 450 500 550 600 650 700 750 800 350 400 450 500 550 600 650 700 750 800 850 300 350 C5a

+ 5 0 5 0 g are means means are b - - - 10 10 Number of neutrophils ( 105) 15 15 × Methods and any associated references are available in the the pape the of version in available are references associated any and Methods M hypogammaglobulinemia or common variable immunodeficiency with patients in phenomena autoimmune of incidence increased the contributeto mayalso effect This glycoform. agalactosyl flammatory mation in autoimmunity and infection before IgGs switch to the proin feedback loop to control complement and -mediated a as inflamIgG1-ICsmayserve galactosylated highly of effect inhibitory the infections and disease bowel inflammatory erythematosus, autoimmune conditions, including rheumatoid arthritis, systemic tions glycans from IgGserum are under agalactosylated steady-state condi mediated by sialylation properties of Fc-linked N-glycans anti-inflammatory the from the distinct is of mechanism This functions effector proinflammatory chemoattractant GPCRs C5aR and CXCR2 ( the blocks that way Fc the of signaling cooperative promotes molecules IgG1 of tosylation Untreated 0 2 4 6 8 20 20 eth Collectively, our findings demonstrate that high N-glycan galac N-glycan high that demonstrate findings our Collectively, γ µ LoGalH5-IC IB ih etn1 rslig n n niioy inln path signaling inhibitory an in resulting dectin-1, with RIIB m. ( m. 25 25 Time (s) Time (s) 3 ± 3 s.e.m. ** . This frequency markedly increases in experimental and human 30 30 o in vivo in

d C5a

ds HiGaIH5-IC ) Clinical disease severity in PBS-, LoGalH5IC– and HiGalH5- and LoGalH5IC– PBS-, in severity disease ) Clinical 35 35 C5a + curdlanC5a ** * 40 40

Fcgr2b +

and promotes the association of Fc of association the promotes and HiGaIH5-IC 45 45 * 50 50 * n + 55 −/− = 10), Cy3-HiGalH5-ICs were used as donor and and donor as used were Cy3-HiGalH5-ICs = 10), r 55 . curdlanC5a or or 60 60 + Clec7a c n = 30) or Cy3-LoGalH5-ICs ( Cy3-LoGalH5-ICs or = 30) g −/− PBS HiGaIH5-IC/anti-dectin1 mice ( mice Percentage e

) and Cy3-LoGalH5-ICs ( Cy3-LoGalH5-ICs ) and FRET efficiency

LoGaIH5-IC/anti-dectin1 0.1 0.2 0.3 0.4 n = 5 per group). ( group). = 5 per HiGalH5-IC Supplementary Supplementary Fig. 23 3

γ HiGaIH5-IC/anti-TLR4 2 RIIB and dectin-1. dectin-1. and RIIB . . About 25–35% of Fc s r e t t e l n = 20) as = 20) b LoGalH5-IC ) Impact ) Impact n f = 5 a ) donors ) donors 3 , 3 b . Thus, . online online , d are are

3 4 ). ). .  - - - - -

© 2012 Nature America, Inc. All rights reserved. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at version of the pape The authors declare competing interests:financial details are available in the anddesigned coordinated the study, analyzed the data and wrote the manuscript. cells. M.E. provided inputscientific and coordinated the glycan analysis. J.K. HiGalH5-IC and H5-IC binding to Fc recombinant IgG1 7B4 antibody and recombinant mouse Fc Syk-knockout mice and the protocol for ERK phosphorylation studies with neutrophils. E.W. provided the conditional TNP-OVA and helped with initial IC studies. N.L.H. and G.K. performed the provided material and advice for the FRET experiments. R.T.S. and F.D.F. provided assays using such mice and with G.D.B. and D.M.R. provided the analysis. A.W. and C.H. performed the the peptide pull-down experiments. M.B., D.P. and V.B. did the MALDI-TOF cell lines, and helped with assays using such lines. S.J.O. performed P.R.T., andM.R. J.U.M. provided neutrophil the dectin-1–transduced and IgG VII–specific for the EBA studies and helped with EBA-related data analysis. performed the EBA studies. andR.L. D.Z. provided the rabbit collagen type characterization of bone marrow cells, and some antigen binding of C.M.K. and M.K.P. conducted key studies and analyzed the data. J.F. assessed the 7B4 hybridoma clones. discussions and B. Heyman (Uppsala University, Sweden) for providing the H5 and the fluorescence spectroscopy measurements, T. Peters and L. Wollin for helpful assistance with the surface plasmon resonance analysis, T. Gutsmann for help with G.D.B. was supported by the Wellcome Trust. We thank T. Köhli for technical 5 to M.E. P.R.T. is a Medical Research Council (UK) Senior Fellow (G0601617). EXC306/1 to D.Z., andR.L. J.K., and by Deutsche Forschungsgemeinschaft EH221- Forschungsgemeinschaft, GRK1727; project 8 and SFB/TR22; project A21) to J.K., This work is supported by the German Research Foundation (Deutsche Note: Supplementary information is available in the s r e t t e l  COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING AUTHOR CONTRIBUTIONS A

cknowledgments

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© 2012 Nature America, Inc. All rights reserved. of the membrane were stained with Diff-Quick. The numbers of migrated migrated of and were of mm per the number counted, cells fields in high-power five cells numbers The Diff-Quick. with stained were membrane the of side on bottom the cells and the removed were membranes the Subsequently, nol (100 inhibitors I kinase Src-family and II (at 10 (100 genistein inhibitor phosphorylation tyrosine the IgG2a-ICs, IgG1-ICs, or IgG1 monomeric of concentrations indicated the with treated were cells experiments, In some °C. at 37 min 30 for incubated and wells top a with 3- overlaid and (Neuroprobe) chamber chemotaxis Boyden micro a of (10 a at BSA) 10 × 5 of 2% density containing (GBSS medium chemotaxis in resuspended were assay. Chemotaxis personnel. by certified by approved für UmweltLandwirtschaft, and (Ministerium und ländliche Räume, Kiel, Committee Germany) and Use performed and reviewed Care Animal were the of authorities studies local and mouse All institutional appropriate guidelines. the with national accordance in handled and age of been described recently have mice Syk-knockout Inducible LoGalH5-ICs. and HiGalH5-ICs H5, naive with experiments chemotaxis in and peritonitis in used were mice background for the production of cell lines (see below). C57BL/6. below). (see lines cell of production the for background deficiency was also backcrossed for eight generations onto the C57BL/6 genetic C57BL/6 control mice were obtained from our (C57BL/6. own breeding colonies. Dectin-1 background C57BL/6 (129S6/SvEv. background 129S6/SvEv isogenic the on mice ground) were from purchased Taconic Dectin-1–deficient (Laven, Denmark). (Bar Laboratory Harbor, ME). Mice. from was Millipore. from was membrane fluoride from RPMI polyvinylidene Fluoromount-G The was Healthcare. GE Kit Labeling Antibody Supplemented laboratories. CyDye PAAAmersham Cy3 Technology. from SouthernBiotech. was Biosearch medium from 1640 was BSA coupled Fc Mouse and (SCF) were (IL-3) -3 and from IL-6 Sigma-Aldrich. were factor from Peprotech. cell stem curdlan, Laminarin, Corning. from Poly- Calbiochem. from were 61-3606 from were I and II BAYinhibitors and Syk inhibitors The Src kinase R406 Sigma-Aldrich. and piceatannol genistein, 4-hydroxytamoxifen, Calcein, (rhC5a), C5a human Recombinant cells. hybridoma from produced was 7B4 described as cells HEK293 in transfection transient by produced was and IgG1 to IgG2a TNP-specific original the ing BD IgG1 The mouse Pharmingen. TNP-specific 7B4 was by generated switch elsewhere described has been A8 antagonist receptor C5a The (1:100). Biomedicals MP from was C3 to antibody goat The Merz Baxter, & from Dade. was Diff- solution staining Quick (1:100). Dako from was IgG rabbit to antibody pig FITC-labeled The Invitrogen. from were zeocin and Fluo-4-AM 546, Fluor Alexa detection TSA 1:20), (SAv-Alexa405; 405 Fluor Streptavidin–Alexa Biologicals. Novus from was Syk to antibody the Signaling; Cell from were 1:20) (C87C1; p-Syk to antibody and peroxidase) horseradish to (conjugated IgG 1:20), (Tyr1020; p-SHIP1 1:20), (Tyr352; p-Syk 1:20), (Thr202/Tyr204; kinase MAP p-p44/42 to antibodies rabbit the instructions; manufacturer’s the per as Bioproducts) Phycolink Peridinin-chlorophyll-protein complex (PerCP)-labeling kit (Europa with and to Ly-6B.2 F4/80 were labeled The antibodies was from eBioscience. 1:100) to (MTS510; TLR4 antibody The Alexa647-labeled from AbD Serotec. were 1:20) (10/92); to PE and or to (conjugated 1:20) CD88 Alexa647 (2A11); FITC (7/4; 1:100), dectin-1 (conjugated to allophycocyanin (APC) or Alexa647 as used described Fc Alexa488-labeled Pharmingen; Ly-6G 1:100), and (M1/70; 1:100) (1A8; were Gr-1 1:100) from (RB6-8C5; BD Reagents. O doi:10.1038/nm.2862 NLINE METH NLINE µ −7 m polycarbonate membrane. Then, 50 50 Then, membrane. polycarbonate m M) were diluted in chemotaxis medium, placed in the bottom wells wells bottom the in placed medium, chemotaxis in diluted were M) Female BALB/c or C57BL/6 mice were purchased from the Jackson Jackson the from purchased were mice C57BL/6 or BALB/c Female µ M), R406 M), (10 R406 γ Monoclonal phycoerythrin (PE)-labeled antibodies to CD11b CD11b to antibodies (PE)-labeled phycoerythrin Monoclonal IBF ad CHO-mFc and RIIB-Fc 2 1 and were bred at Charles River. All mice were used at 8–12 weeks 6 1 cells ml cells 1 O ; to F4/80 (Cl:A3-1; 1:100), Ly-6B.2- ; 1:100), to Monoclonal antibody (Cl:A3-1; F4/80 Bone marrow–derived cells or peritoneal macrophages macrophages peritoneal or cells marrow–derived Bone DS µ M), M), Syk (10 inhibitor −1 . The chemoattractants C5a (10 C5a chemoattractants The . 1 Fcer1g 5 . The mouse TNP-specific IgG was from . 107.3 The TNP-specific mouse γ Clec7a RIIB-specific antibody (Ly17.2; was antibody 1:500) RIIB-specific γ −/− IB ee sd s described as used were RIIB l and -lysine–coated microtiter plates were were plates microtiter -lysine–coated −/− µ 2 8 M) M) or the Syk inhibitors piceatan µ ), and the 129S6/SvEv and the the and 129S6/SvEv the and ), . The TNP-specific mouse IgG2a IgG2a mouse TNP-specific The . Fcgr2b l of the cells were placed in the the in placed were cells the of l µ M) M) or BAY (10 61-3606 −/− mice (on BALB/c back −8 Clec7a M) or CXCL2 CXCL2 or M) β -estradiol -estradiol Clec7a µ 3 −/− 5 M), the the M), TNP- . ∆ ), the the ), µ 71−73 M). M). −/− 2 - - -

slides. At least >20 different microscopic fields were evaluated as described were evaluated fields At microscopic >20 slides. different least Cytospin of staining Diff-Quick by determined were fluid lavage peritoneal 100 nM, (200 inflammation. peritoneal C5a-mediated diffusion. gel Ouchterlony by determined was formation IC and ratios, different at bodies IgG1 anti TNP-specific TNP-OVAwith 10:4. was incubated were complexes added to the OVA solution and incubated overnight. The final TNP/OVA ratio 500 in dissolved TNP- of mg 50 Then, 9.6. pH 500 with mixed and saline of ml 5 in OVA of dissolved was complexes. of TNP-OVAPreparation and of TNP-OVA/anti–TNP-OVA IgG1 immune samples. triplicate of value mean the as expressed are Results microscopy. assisted computer by calculated was followed by an i.p. injection of OVA-specific rabbit IgG (800 (800 IgG rabbit OVA-specific of injection i.p. an by followed (OVA, Ovalbumin model. i.v., was 20 injected mg Sigma) weight; kg per body peritonitis. complex Immune (50 LoGalH5-ICs or H5-ICs HiGal (0.5–50 107.3-ICs with (i.v.) intravenously treated pre were mice experiments, some In beads. True BD count using cytometry Ly-6G experiments, some In anesthesia. Femurs, tibias and humeri were removed, placed in PBS on ice ice on PBS in placed removed, were humeri and tibias Femurs, anesthesia. preparation. Neutrophil antibody. C3 anti-mouse goat and IgG antibody microscopy using direct immunofluorescence an pig FITC-labeled anti-rabbit by detected were deposits C3 IgG and H&E. with stained were sections mice, previously as described microscopy immunofluorescence and assessment histopathological for used microscopy. and immunofluorescence Histopathology account. into activity disease maximal and onset disease both taking points, time different the at progression disease the for obtained (AUC) curve the under of area the calculation by compared was groups Disease treatment analyses. different the of histological outcome for obtained were samples tissue additional 12, day On taken. were biopsies tail and lesions, skin by affected area surface of body percentage as expressed and determined 7 was 4, severity days disease 12, and On d. 12 of period time a over evaluated was progression Disease (50 107.3-ICs or LoGalH5-ICs HiGalH5-ICs, PBS, either of i.v.injections ment, day. treat For second every IgG affinity-purified collagen–specific, VII type of 100 injections subcutaneous of three by a total induced EBA was mental EBA followed published protocols model. acquisita bullosa epidermolysis Experimental before skin mouse on microscopy Reactivity of the isolated IgG fractions was confirmed by immunofluorescence Lab). (Bio-Rad chromatography affinity Affi-Gel and 757-967) acids (amino collagen VII type of mouse fragments His-tagged recombinant using fication reported previously as G affinity protein using chromatography by affinity purified were sera rabbit described as collagen VII type mouse of collagen–specific VII type antibodies. pathogenic of purification and Generation 50 of concentration a at 107.3-ICs with i.v. pretreated were mice experiments, cytospin slides. Neutrophil numbers were evaluated as outlined above. In some 10 and PBS, with once were washed cells 10 ml of Peritoneal with PBS. lavaged was cavity peritoneal described as Biomedicals) ICN µ µ g per kg body weight. body kg per g g per kg body weight) were started on day –1 and repeated on days 3 and 6. Rabbits were immunized with recombinant forms of the NC1 domain TNP-OVA complexes were generated as described as generated TNP-OVAwere complexes µ l, i.p.). After 6 h, mice were killed and neutrophil numbers in in numbers neutrophil and killed were mice h, 6 After i.p.). l, µ l DMSO resulting in 100 mg/ml 100 in resulting DMSO l 2 5 2 9 cells in 200 200 in cells 9 . Isolated IgG was further subjected to affinity puri affinity to subjected further was IgG Isolated . . To assess morphological skin alteration in diseased in . diseased alteration Toskin morphological assess Mice were killed by cervical dislocation under under dislocation cervical by killed were Mice hi neutrophil numbers were enumerated by flow flow by enumerated were numbers neutrophil ε We used the reverse passive Arthus reaction reaction Arthus passive reverse the Weused -aminocaproyl-succinimide ester (OSU) was was (OSU) ester -aminocaproyl-succinimide 1 1 . Mice were killed 6 h after injury, and the the and injury, after h 6 killed were Mice . in vivo in µ g per kg body weight). body kg per g µ l of PBS were used for preparation of of preparation for used were PBS of l 2 use. 2 9 9 with minor Briefly,modifications. . IgGs from immune and normal normal and immune from IgGs . Mice were injected with C5a C5a with injected were Mice −1 µ g per kg body weight), weight), body kg per g TNP-OSU, which was was which TNP-OSU, Induction of experi of Induction nature medicine nature Tissue samples were µ l 1 M NaHCO M 1 l µ 1 g per mouse; mouse; per g 3 . Briefly, 1 g 1 Briefly, . µ g rabbit g rabbit 1 1 3 - - - - - .

© 2012 Nature America, Inc. All rights reserved. duction duction of stable termed precursors, in 1.5 selected bias toward neutrophil progenitor outgrowth. Two days after infection cells retrovirus Hoxb8 were and SCF for 3 d as described directed by the manufacturer. The lin (lin) age from the femurs of C57BL/6. female In vitro Fcgr2b antibody.MAP kinase cells from In bone marrow–derived some experiments, ERK1/2 for anti-phospho-p44/42 rabbit a analyzed with staining intracellular by subsequently phosphorylation and min 1 for nM) (50 C5a with lated A8 cultured were mice phosphorylation. of ERK1/2 Analysis (Ver. software Tree FlowJo 7.5, Star). the of Ca the of assessment by calculated was (10 (10 nM), and recording was continued for another 90 s. The increase in [Ca for 30 was added C5a s. was recorded Then signal The background (BD). eter on Ly-6G gated then were Cells PBS. using away washed was dye Non-incorporated min. 30 for (Invitrogen) 5 with and loaded collected [Ca calcium intracellular in Increase observed that by washing. before washing after remaining by signal calculated fluorescence was the adhesion dividing percentage The PBS. with twice washing after (BioTekand before Reader Instruments) Fluorescence Microplate an FLX800 1 or (0.1 107.3-ICs with preincubated were cells calcein-labeled experiments, In some surface complement-coated marrow cells were labeled with calcein (2 (10 IgM human with were prepared by of surfaces coated poly- incubation 3–mediated assay. (Version 3.0). software Express FCS using Dickinson) (Becton FACSCalibur a on analyzed and collected were Data ice. on min 45 for control isotype ing correspond the or PE to conjugated antibody anti-CD11b with stained then 100 1 Pharmingen; (BD antibody 2.4G2 with tors were blocked with ice-cold PBS. For flow cytometric analysis of CD11b expression, Fc recep once washed and ice on placed were cells Then, min. 10 for °C 37 at nM) (10 100 in suspended C5a-induced upregulation of upregulation CD11b expression. C5a-induced F4/80 were cells adherent that of the majority revealed analysis Flow cytometric for used migration. cell PBS were with steps and cells were washing adherent cells removed by several Nonadherent °C. 37 at h 4 for plates microtiter plastic on adhere to allowed were cells Peritoneal PBS. 5 of ml with sterile flushed gently was cavity toneal Macrophage preparation. neutrophils. marrow–derived bone of viability the affect not flow cytometer. hours of Forty-eight did treatment with 4-hydroxy-tamoxifen II LSR BD a using staining 7-AAD and exclusion blue trypan by determined nol for 48 h. Controls were treated accordingly with ethanol only. Viability was (pen/strep) and 10% FCS in the presence 0.6 l mM 2 with supplemented medium 1640 RPMI in cultured were cells mice, from neutrophils in depletion Syk For marker. Ly-6G the for tive were isolated cells of 80% and as cells, staining, by posi stained polymorphonuclear defined Diff-Quick than More PBS. with flushed subsequently and nature medicine nature -glutamine, 100 U/ml benzylpenicillin and 0.1 mg/ml dihydrostreptomycin dihydrostreptomycin mg/ml 0.1 and benzylpenicillin U/ml 100 -glutamine, ∆ µ 71−73 l l of FACS buffer for 30 min on ice). Cells were washed once with PBS and −/− generation of neutrophils. of generation + for 30 min at 37 °C. Bone marrow–derived cells were then stimu then were cells marrow–derived Bone °C. 37 at min 30 for µ cells using the mouse lineage cell depletion kit (Miltenyi Biotec) as as Biotec) (Miltenyi kit depletion cell lineage mouse the using cells mice were used. were mice g ml g µ −1 g/ml g/ml of puromycin for 10 d. Typically, this in resulted the pro ) for 30 min at 37 °C. The fluorescence was measured using using measured was fluorescence The °C. 37 at min 30 for ) 1 µ 9 and then immediately cultured in SCF and and SCF in cultured immediately then and l of GBSS buffer containing 2% BSA and treated with C5a C5a with treated and BSA 2% containing buffer l of GBSS ± µ 107.3-ICs (0.1 or 1 1 or (0.1 107.3-ICs g ml g high To obtain mouse peritoneal macrophages, the peri µ 3 −1 ± 7 CD11b M M of the Ca before being with infected the pMXs-IP:FL-ER- C5a (0.1, 1 or (0.1, C5a 10 × 10 ) and 10% human serum as described as serum human 10% and ) hi cells and analyzed on an LSRII flow cytom flow on an LSRII analyzed and cells Clec7a Bone marrow–derived cells were pooled pooled were cells marrow–derived Bone high My − cells were prestimulated with IL-3, IL-6 2+ Bone marrow–derived cells from WT cells marrow–derived Bone 2+ eloid eloid . µ 2+ ] peak using the kinetic plug-in tool tool plug-in kinetic the using peak i g g ml . −/− -sensitive fluorophore Fluo-4-AM Fluo-4-AM fluorophore -sensitive Bone marrow–derived cells were were cells marrow–derived Bone µ µ g ml g P M M 4-hydroxy-tamoxifen in etha mice and were depleted of and mice were line depleted recursor recursor −1 ) ) for 30 min and added to the −1 Neutrophils (1 × 10 ) or the C5aR antagonist antagonist C5aR the or ) −9 l M) for 10 at min 37 °C. -lysine precoated plates precoated -lysine H Complement (iC3b)- oxb µ g per g 10 per 8 from C57 β -estradiol to -estradiol Syk 6 3 cells in cells 6 6 flox/flox . Bone Bone . ) ) were B 2+ L/ ] 6 ------i

(MyPH8-B6). The cells were maintained in SCF and were separated by SDS-PAGE, transferred to PVDF membrane and analyzed analyzed and membrane PVDF to SDS-PAGE, by transferred separated were Laemmli buffer was added and samples were heated for 5 min at 95 °C. Samples reducing washed, were pull-downs for 4 Peptide h. Technologies) (Life beads cell 20 with incubated Research), were lysates Protein Peptide MQDEDGpYITLNIKNK-biotin, (pClec2: phosphopeptide biotinylated described using previously a experiments pull-down peptide For fluoride). phenylmethylsulfonyl 1 mM EDTA,Na-deoxycholate, 0.25% 630, Na IGEPAL-CA- 1% NaCl, mM 150 7.4, pH Tris-HCl mM (50 buffer RIPAlysis 10 with °C 37 at 30 min at 37 °C. Ten in were 100 million resuspended blotting. western and pull-down Peptide morphology. nuclear typical by their as Ly-6B.2 by flow cytometry and of present are cells the 90–98% identified represent routinely neutrophils ml ng 20 (both factor colony–stimulating and SCF to remove the medium with times B6. was verified by flow cytometry. For 50 described viously as pre retrovirus (pMXs-IZ) control or empty (pMXs-IZ:dectin-1B) specific MyPH8-B6. the binding curves for the different dilutions were overlaid, and the equilibrium 15 10 of rate flow constant a at chip sensor the over passed were samples the binding, of Fordetection nM). 10 at (starting added were PBS in and as served a reference cell. dilutionsSerial of HiGalH5-ICs or LoGalH5-ICs protein of any addition without but protocol immobilization the to subjected 1 M ethanolamine hydrochloride-NaOH for 7 min. A second flow chamber was increase of 6,000 resonance units. Unbound reactive groups were saturated with Fc chip. For immobilization, sensor the to added were volumes equal in diimide M 0.05 N chip, CM5 the of matrix dextran carboxylated coupling. the of amine activation via For Healthcare) (GE chip sensor CM5 the to Fc coupled °C. was 25 at system Healthcare) (GE 3000 Biacore a using performed Fc analysis. resonance plasmon Surface software. IMARIS the using analyzed were example, (for interest of Fc points locate and visualize to analysis) isosurface rendering and (volume Surpass IMARIS with operating Switzerland), AG, Bitplane 7.0.1.1, (version software IMARIS by analysis image for used then were Stacks extension. full their in cells show to stack per slices 20–30 with 0.1 analysis. image Three-dimensional software. 2.1c Fluoview the using performed was capturing and analysis Image objective. oil NA:1.35 60×- microscope a with confocal Germany) (Olympus, 100 FV Olympus the using obtained were Images G. Fluoromount- with mounted then were Cells °C. at 4 min 15 Abs for specific further incubated with Alexa488-labled Fc Fc For (Invitrogen). manufacturer the case of SHIP, using the tyramide signal amplification kit as described by the for phospho-Syk detection with an Alexa568-labeled secondary antibody, or, in stained then and washed were Cells h. 24 for °C 4 at incubated were cells the and were added, antibodies or phospho-SHIP Phospho-Syk (BD Bioscience). or neutrophils. without IgG1-ICs at 37 °C. Cells were then fixed with BD Cytofix/Cytoperm marrow–derived 3 (1, bone or 5 with min) periods time for different were preincubated Neutrophils of staining Immunofluorescence antibodies. anti-Syk and anti-pSyk with blotting by western -hydroxysuccinimide and 0.2 M -hydroxysuccinimide γ γ γ

µ min. min. BIAevaluation software 3.2 RC1 was used for analysis of the data. First, Clec7a RIIB RIIB was diluted to 34 RIIB RIIB with HiGalH5-ICs and surface LoGalH5-ICs, plasmon resonance was RIIB, dectin-1 and p-Syk expression). For co-localization studies, data studies, sets and ForRIIB, p-Syk expression). dectin-1 co-localization µ g/ml g/ml of zeocin to maintain the reconstituted cells. Expression of dectin-1B m m using an Olympus FV 1000 (Olympus, confocal microscope Germany) −/− Clec7a :pMXs-IZ or MyPH8-B6. :pMXs-IZ µ 3 −/− g IgG1-ICs for the indicated times. Cells were lysed with with lysed were Cells times. indicated the for IgG1-ICs g 8 , but using MyPH8 culture medium supplemented with with supplemented medium culture MyPH8 using but , cells were reconstituted with the isoform dectin-1B with µ g g ml high −1 µ CD117 . . Injection of 5 g of peptide bound to streptavidin agarose agarose streptavidin to bound peptide of g N β γ in vitro RIIB and dectin-1 staining, samples were were samples staining, dectin-1 and RIIB -estradiol before differentiation for 4 d in differentiation before -estradiol -ethyl- Z-stacks were taken with an interval of interval an with taken were Z-stacks To determine the interactions between between To interactions the determine Clec7a low γ RIIB-specific and/or APC-dectin-1– generation of neutrophils, MyPH8- cells and cells on preparations Cytospin N The cells were serum starved for for starved serum were cells The -(dimethylamino-propyl) carbo -(dimethylamino-propyl) −/− 3 VO :dectin-1B were washed three three were washed :dectin-1B µ l l receptor resulted in a signal 4, β leupeptin, aprotinin and aprotinin leupeptin, -estradiol -estradiol as described µ 2 2 l l DPBS and stimulated derived from Clec-2 Clec-2 from derived doi:10.1038/nm.2862 −1 µ ). By day 4, 4, day By ). l min l γ −1 RIIB RIIB for for 3 7 ­ - .

© 2012 Nature America, Inc. All rights reserved. Subsequently, cells were fixed using diluted Cytofix solution from BD. FRET was incubated for 3 min with 1 above and as antibody described anti–dectin-1 with Alexa647-labeled stained transfer. energy resonance Förster tool. Glyco-peakfinder the and GlycoWorkbench software the using evaluated were data The accumulated. were shots laser 3,000 from and mode, ionization mass spectra positive were in recorded reflector Spectra performed on a glucose ladder, and dihydroxybenzoic acid was used as matrix. and laser a with a equipped LIFT-MS/MS was facility. Smartbeam Calibration Spectra were recorded on an Ultraflex III mass spectrometerspectrometry. mass (Bruker Daltonics)MALDI-TOF by analyzed and protocols standard to ing were N-glycans by purified C18 accord and carbon columns, graphitized permethylated resulting the and (Sigma-Aldrich), PNGaseF with digested were described viously etry. Determination of IgG1 glycan composition by MALDI-TOF mass spectrom spectrometry. mass TOF ELISA, and N-glycosylation of IgG Fc fragments was characterized by MALDI- bywere measured by reactivities and IgG1 (Pierce) Anti-TNP verified ELISA. assays BCA by determined were antibodies IgG1 glycosylated differentially the of Concentrations PBS. against dialyzing and chromatography G protein by enzymes from were separated for 48 h at IgG 37 °C. antibodies (GK80120) was with IgG1 incubation by H5 °C. degalactosylated 37 at h 48 for (Calbiochem) acid CMP-sialic of mg 0.625 by with 25 mU incubation of human 48 h at to exchange 37 buffer 50 °C. After mM MES pH IgG 6.0, was sialylated in 1 ml 50 mM MOPS pH 20 for 7.2 with mM (Calbiochem) MnCl2 galactose 75 mU of human ously tion and/or sialylation was performed in a two-step procedure media as with protein-G-Sepharosedescribedculture andcell previ dialyzed from against PBS. purified were Antibodies penicillin/streptomycin. 1% and BioScience) (Sheffield RL/UF Primatone 0.03% with supplemented (GIBCO) antibody (clone H5) was grown for antibody production in RPMI high glucose In vitro model. affinity state steady a 1:1 assuming constant dissociation the calculate to and binding analyte of curves saturation create to used was of response Then, the equilibrium each was response experiment determined. doi:10.1038/nm.2862 Analysis of N-glycan composition of IgG samples were performed as pre 3 2 . Briefly, 5 mg of antibody were galactosylated by rotating incubation with remodeling of IgG antibodies. 3 β 9 1,4-galactosyltransferase (Calbiochem) and 2 mg of UDP- (Calbiochem) 1,4-galactosyltransferase . Briefly, tryptic glycopeptides obtained from IgG from samples obtained glycopeptides . Briefly, tryptic µ g ml −1 Cy3-labeled HiGalH5-ICs or LoGalH5-ICs. Bone marrow–derived neutrophils were were neutrophils marrow–derived Bone α β 2,6-sialyltransferase (Calbiochem) and (Calbiochem) 2,6-sialyltransferase -galactosidase (10 mU) from Prozyme Prozyme from mU) (10 -galactosidase The mouse anti-TNP IgG1 hybridoma In vitro galactosyla - - - - -

was done by one-way analysis of variance (ANOVA) or ANOVA on ranks. ranks. ANOVAon or (ANOVA) variance of analysis one-way by done was groups distributed or non-normally normally two of than more means of the two-tailed a groups, distributed normally two between differences Toanalyze assumed. was distribution normal sizes, sam ple small to regard With Software). (Systat package statistical 3.5 version analyses. Statistical acceptor. the of photodestruction complete acceptor. nonbleached of presence the in Cy3 of intensity fluorescence normalized the ( efficiency FRET of interest on the cell surface using Olympus’s FV1000 software (Ver.03.0). The regions different in recorded was Alexa647 of and Cy3 intensity fluorescence mean Then, achieved. was of photodestruction 100% when and stopped time real in monitored was process bleaching The power. laser maximum the of 50% at laser 635-nm the used we photobleaching, For label. Alexa647 the of image was acquired once before acceptor bleaching and after photodestruction 5 of time dwell at a pixel on pixels × 320 320 to set was set resolution the and was automatic, diameter Pinhole objective. oil NA 1.40 60× a with microscope, Olympus FV1000 (Olympus Europa Holding GmbH, Germany) laser scanning an with imaged were donor. Cells the of emission fluorescent the in increase acceptor.antibody anti–dectin-1 In FRET, the bleaching acceptor results in an Alexa647 the of photobleaching after and before donor Cy3-LoGalH5-IC or Cy3-HiGalH5-IC the of intensity fluorescence the determining by measured 38. 37. 36. 35. values mean as expressed and experiments from 3–5 individual data were taken If not otherwise, stated (ANOVA method on ranks). Dunn’s or (ANOVA) test Tukey the using performed was pair comparison wise difference, significant a showed groups the of values mean the When 39.

Rosas, M. Rosas, G.G. Wang, 3 receptor Complement E.J. interactions. Brown, & V.M. Holers, D.C., Anderson, antibody-Fc-receptor I.L., Graham, Analyzing J.V. Ravetch, & F. Nimmerjahn, eeol S. Wedepohl, myeloid cell programming and the progression of . of progression the and programming cell myeloid Hoxb8. conditional using tyrosine for required(1994). 1139–1147 is CD11b/CD18) 2, beta phosphorylation M of paxillin alphain adherent and integrinnonadherent neutrophils. Mac-1, (CR3, Biol. Mol. Methods GalNAc and GalNAc-GalNAc motifs. GalNAc-GalNAc and GalNAc (2008). 3549–3557 µ s. Cy3 was excited at 539 nm, and Alexa647 was excited at 635 nm. An An nm. 635 at excited was Alexa647 and nm, 539 at excited was Cy3 s. I D represents the normalized fluorescence intensity of Alexa647 after et al. et t al. et t al. et The induction of inflammation by dectin-1 by inflammation of induction The E ) was calculated as calculated ) was uniaie rdcin f arpae o nurpis x vivo ex neutrophils or macrophages of production Quantitative Statistical analysis was performed using the SigmaStat SigmaStat the using performed was analysis Statistical

-lcn nlss f eobnn Lslci rvas sulfated reveals L- recombinant of analysis N-glycan 415 , 151–162 (2008). 151–162 , ± Nat. Methods Nat. s.e.m. P < 0.05 was considered a significant difference. difference. a significant considered < was 0.05 J. Proteome Res. Proteome J. E

= 1 – ( 3 , 287–293 (2006). 287–293 , I DA t -test was used. Comparison Comparison used. was -test

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