Báo cáo khoa học: Direct interaction between CD91 and C1q docx

On the other hand, CD91 of phag-ocytic cells has also been suggested to interact with calreticulin translocated from the endoplasmic reticu-lum to the cell surface of apoptotic cells [24

Karen Duus1, Erik W Hansen2, Pascale Tacnet3, Philippe Frachet3, Gerard J Arlaud3,

Nicole M Thielens3and Gunnar Houen1

1 Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen, Denmark

2 Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, Denmark

3 Laboratoire d’Enzymologie Mole´culaire, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France

Introduction

The complement system is an important branch of

innate immune defence and there are three major

path-ways of complement activation that are currently

recognized: (a) the classical (antibody-dependent)

path-way; (b) the MBL (lectin) pathpath-way; and (c) the

alter-native pathway [1,2] The classical pathway is activated

upon antibody binding to target antigens and the

recognition of immune complexes C1q, the

recogni-tion unit of C1, has an important role in immune

com-plex clearance and complement activation Binding of

C1q to immune complexes is known to activate

the C1q-associated proteases, C1r and C1s Through cleavage of C4 and C2 by C1s, the C3- and C5-conver-tases are generated and the pore-like membrane attack complex (C5b-9) can be formed In addition to its role

in immune complex recognition, C1q has been shown

to bind both necrotic and apoptotic cells and to play

an important role in the scavenging of such cells [3–6] Timely removal of apoptotic and necrotic cells is imperative to avoid initiation of autoimmune reactions [7] and C1q deficiency results in systemic lupus erythe-matosus [8] C1q receptors have been suggested to play

Keywords

C1q; calreticulin; CD91; collectin; scavenger

receptor

Correspondence

G Houen, Department of Clinical

Biochemistry and Immunology, Statens

Serum Institut, Artillerivej 5, DK-2300

Copenhagen, Denmark

Fax: +45 32683149

Tel: +45 32683276

E-mail: gh@ssi.dk

(Received 18 November 2009, revised 27

May 2010, accepted 2 July 2010)

doi:10.1111/j.1742-4658.2010.07762.x

C1q-mediated removal of immune complexes and apoptotic cells plays an important role in tissue homeostasis and the prevention of autoimmune conditions It has been suggested that C1q mediates phagocytosis of apop-totic cells through a receptor complex assembled from CD91 (a-2- macro-globulin receptor, or low-density lipoprotein receptor-related protein) and calreticulin, with CD91 being the transmembrane part and calreticulin act-ing as the C1q-bindact-ing molecule In the present study, we observe that C1q binds cells from a CD91 expressing monocytic cell line as well as mono-cytes from human blood C1q binding to monomono-cytes was shown to be cor-related with CD91 expression and could be inhibited by the CD91 chaperone, receptor-associated protein We also report data showing a direct interaction between CD91 and C1q The interaction was investigated using various protein interaction assays A direct interaction between puri-fied C1q and CD91 was observed both by ELISA and a surface plasmon resonance assay, with either C1q or CD91 immobilized The interaction showed characteristics of specificity because it was time-dependent, satura-ble and could be inhibited by known ligands of both CD91 and C1q The results obtained show for the first time that CD91 recognizes C1q directly

On the basis of these findings, we propose that CD91 is a receptor for C1q and that this multifunctional scavenger receptor uses a subset of its ligand-binding sites for clearance of C1q and C1q bound material

Abbreviations

F-C1q, FITC-labelled C1q; FITC, fluorescein isothiocyanate; HSA, human serum albumin; LDL, low-density lipoprotein;

LPS, lipopolysaccharide; MM6, Mono Mac 6; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin; pNPP, para-nitrophenyl

phosphate; RAP, receptor-associated protein; SAP, serum amyloid P; SPR, surface plasmon resonance.

an important role in the function of C1q and the

prevention of autoimmune diseases [2,7,9] and several

candidates have been described, including CD93

(C1qRp), CD35 (CR1), C1qRO2 (CD59), a2b1

inte-grin, gC1qR (p33), megalin and calreticulin (cC1qR)

[9–16]

The most studied of the proposed receptors is

calret-iculin Calreticulin does not have a transmembrane

domain and therefore is incapable of signalling for

phagocytosis Accordingly, calreticulin has been

pro-posed to form a complex with CD91 on the surface of

phagocytes [17–23] On the other hand, CD91 of

phag-ocytic cells has also been suggested to interact with

calreticulin translocated from the endoplasmic

reticu-lum to the cell surface of apoptotic cells [24,25]

CD91 is a member of the low-density lipoprotein

(LDL) receptor gene family and comprises a receptor

with more than 30 ligands [26–31] Two chains of

CD91 form a heterodimer: the transmembrane b-chain

with a short cytoplasmic region and the extracellular

a-chain with four ligand-binding clusters formed from

31 ligand-binding type repeats [26,32] Of the

haemato-poietic cells, only monocytes, their precursors and

erythroblasts express CD91 [33]

In the present study, we investigated the direct

inter-action of CD91 with C1q and show that CD91 itself

recognizes C1q independently of calreticulin

Results

C1q interaction with peripheral blood monocytes

CD91 is a C1q receptor that is able to promote the

ingestion of apoptotic material coated with C1q We

used human peripheral blood to confirm that the CD91 positive cells were C1q binding (Fig 1A) and found that most of the C1q binding cells expressed CD91 CD91 has been reported to be present on mac-rophages and macrophage precursors [33] and this was confirmed by double staining of the cells with antibod-ies against CD91 and CD14 This revealed no popula-tion of single positive cells (Fig 1B), thus indicating that CD14 positive monocytes express CD91 Some 45–70% of the CD14 positive cells bound C1q with some person-to-person variability (Fig 1C) C1q bind-ing to other cell types was also observed (Fig 1A) and

Fig 1 C1q interaction with peripheral blood monocytes (A) Blood

cells were stained with PE anti-CD91 (x-axis) and F-C1q (y-axis).

The red blood cells were lyzed and remaining cells were analyzed

by flow cytometry Only monocytic cells gated by forward scatter

and side scatter is shown (B) Peripheral blood monocytes are

highly CD91 positive Blood cells were stained with

flourophore-conjugated antibodies recognizing CD14 and CD91 (left) or an

iso-type control (right) Red blood cells were lyzed and remaining cells

were analyzed by flow cytometry (C) C1q binding levels for three

individuals Blood cells were stained with F-C1q and PE anti-CD14.

The red blood cells were lyzed and remaining cells were analyzed

by flow cytometry C1q binding levels of white blood cells are

pre-sented on the x-axis with CD14 positivity on the y-axis (D) PBMCs

from human blood were stained with F-C1q with the addition of

the CD91 blocking protein RAP (100-fold molar excess), soluble

CD91 (10-fold molar excess) or the control protein HSA (100-fold

molar excess) After staining, C1q-positive cells were quantified by

flow cytometry F-C1q stained cells without inhibition were set as

100%.

therefore inhibition experiments were conducted with

isolated peripheral blood mononuclear cells (PBMCs)

These experiments showed that partial inhibition of

the C1q–monocyte interaction could be obtained with

soluble CD91 and the CD91 blocker,

receptor-associ-ated protein (RAP) (Fig 1D) Similar results were

obtained by Lillis et al [34], where only some

phago-cytotic inhibition was observed with monocytes

defi-cient for CD91 These results indicate that scavenging

of C1q complexes relies on a highly redundant

recep-tor system and that several receprecep-tors for C1q are likely

to exist

Interaction of C1q with CD91 expressing cells

The monocytic cell line Mono Mac 6 (MM6) was used

to examine C1q interaction with monocytic cells The

cell line has previously been described to express CD91

[35] However, in the present study, only a low level of

CD91 was identified on the surface of MM6 cells

(approximately 7% of cells were CD91 positive; data

not shown)

To increase surface expression of CD91, the cells were stimulated with lipopolysaccharide (LPS) As measured by flow cytometry, approximately 40% of the cells were found to be CD91 positive after LPS stimulation (Fig 2A), and the mean fluorescent inten-sity of the cells stained with fluorescently-labelled CD91 antibody was observed to increase Some 85%

of the CD91 positive cells were highly CD14 positive (data not shown)

When the stimulated MM6 cells were stained with fluorescein isothiocyanate (FITC)-labelled C1q (F-C1q), 1.2–9% of the cell population showed high fluorescence intensity, indicating C1q interaction The percentage of positive cells could be inhibited by RAP and unlabelled C1q (Fig 2B and results not shown) We used confocal scanning laser microscopy to visualize localization at the cell surface CD91 antibody bound MM6 at the cell surface and some (but not complete) co-localization was observed between the added F-C1q and CD91 expressed by the cells (Fig 2C), indicating that other receptor systems are likely to exist Staining

of the nuclei revealed no signs of apoptosis

Cell size Cell size PE anti-isotype control

0 FSC-H

1023

0 FSC-H

1023

2

1

0

300 30

0

RAP/C1q molar ratio

A

B

C

Fig 2 CD91 expression and C1q binding

on Mono Mac 6 cells (A) Flow cytometry of stimulated MM6 cells showing CD91 expression After LPS stimulation, CD91 expression was measured on MM6 cells by the addition of PE-labelled CD91 antibody (left) or an isotype control (right) and fluores-cence was measured by flow cytometry Approximately 40% of the LPS-stimulated cells were determined to be CD91 positive (45.52–6.13% = 39.39%) (B) C1q interac-tion with stimulated MM6 cells and RAP inhibition MM6 cells were stained with F-C1q with or without the addition of RAP RAP showed inhibition of the C1q interac-tion in 30- and 300-fold molar excess The results are presented with the relative bind-ing with F-C1q without the addition of RAP set as 1 (C) CD91 and C1q localization in MM6 cells analyzed by confocal laser microscopy Cells were stained with biotiny-lated antibodies against CD91 and streptavi-din coupled with Alexa Fluor 546 (red colour, i) and the addition of F-C1q (green, ii) Images i and ii are merged to produce an overlay plot (iii) Scale bar = 10 lm.

Evidence for a direct interaction between CD91

and C1q

Evidence for a direct interaction was obtained by surface

plasmon resonance (SPR) analysis Using C1q as the

soluble analyte and CD91 as the immobilized ligand,

several concentrations of C1q were used to reveal a

time-and concentration-dependent interaction (Fig 3A)

Furthermore, in the reversed configuration with C1q

immobilized and CD91 as the soluble analyte, an

interaction was observed (Fig 3B) Using an ELISA binding assay, CD91 showed a direct interaction with C1q coated on a polystyrene surface (Fig 4A) At a physiological salt concentration (0.15 m NaCl), the interaction was time-dependent, saturable and fast, being detectable after only a few minutes of incubation

By contrast, increasing the NaCl concentration to 0.65 m strongly inhibited the interaction (Fig 4A) In the opposite configuration, with CD91 immobilized, sol-uble C1q also bound and could be detected with C1q specific antibodies (Fig 4B) although some nonspecific interaction was observed between the polyclonal anti-body directed against C1q and CD91 The C1q–CD91 interaction was also observed when C1q was bound to immobilized IgM, indicating that CD91 binding is possi-ble with ligand-bound C1q (Fig 4C)

Kinetic analysis of the SPR binding data, showed that the best fit for the interaction between immobi-lized CD91 and soluble C1q was provided by a two-state reaction model (Table 1), suggesting that several interaction phases take place This was supported by the ELISA, which revealed that binding of soluble CD91 to C1q coated on the polystyrene surface was highly sensitive to 0.65 m NaCl in the early phase of the interaction (Fig 4A) By contrast, if CD91 and C1q were allowed to interact under physiological salt concentrations before the addition of 0.5 m NaCl, then the interaction gradually became insensitive to high salt (Fig 4D) This was suggestive of a two-state inter-action with an initial ionic binding step allowing subse-quent stable interaction

Interaction of CD91 with the globular domain of C1q and its collagen fragment was also analyzed by SPR analysis, using the latter proteins as soluble ligands In the case of the globular domain, the two-state binding model also provided the best fit More-over, the affinity of the interaction calculated by a Langmuir 1:1 model was approximately 20-fold lower compared to intact C1q (KD= 3.0· 10)7m) The C1q collagen fragment also bound immobilized CD91, and the reaction followed a classical 1 : 1 Langmuir model, with a KDin the nanomolar range (Table 1)

0

40

80

120

Time (s)

100

0

100

200

Time (s)

A

B

Fig 3 Interaction of CD91 with C1q, demonstrated by SPR

analy-sis (A) CD91 was immobilized by coupling of amino groups to a

carboxylated surface C1q was injected for 120 s at concentrations

in the range 2.5–50 n M (B) CD91–C1q interaction C1q was

immo-bilized by amine coupling to a carboxylated surface and soluble

CD91 was injected for 120 s at the indicated concentration in the

range 20–50 n M

Table 1 Comparison of the kinetic constants of the CD91 interaction between C1q, C1q globular heads and C1q collagen tails.

KD, Langmuir

1 : 1 model Chi squared

conformational change

4.04 · 10 5

M )1s)1

6.61 · 10)10S )1

5.09 · 10)3s)1 0.464 s)1

C1q globular region Two-state reaction with

conformational change

1.94 · 10 4

M )1s)1

3.28 · 10)9S )1

5.97 · 10)3s)1 1.87 · 10)7s)1

M )1s)1 2.04· 10)3s)1 3.95 · 10)9M 5.89

Several known interaction partners of CD91 and

C1q inhibit their interaction

To characterize the CD91–C1q interaction in more

detail, the ability of known ligands of either protein to

interfere with binding was investigated RAP is a

CD91 chaperone that has previously been shown to

block the interaction of CD91 with all its known

ligands [36,37] In agreement with this characteristic, RAP also strongly inhibited binding of CD91 to immobilized C1q (Fig 5A) a-2-macroglobulin and Pseudomonas exotoxin A are known ligands of CD91 and they also exerted some inhibition (15% and 40% inhibition, respectively) (data not shown) Calreticulin,

a protein known to act as a ligand for the collagen region of C1q and the proposed C1q-binding compo-nent of the CD91⁄ calreticulin receptor complex, signifi-cantly inhibited binding of CD91 to immobilized C1q

in the presence of 5 mm Ca2+ ions, with almost 45% inhibition at a 300 : 1 calreticulin⁄ CD91 molar ratio (Fig 5B) Inhibition was also observed in the absence

of Ca2+, although to a lesser extent

Serum amyloid P (SAP), another known ligand of C1q [38,39], abolished binding of CD91 to immobi-lized C1q in the presence of 5 mm Ca2+but had only

a slight inhibitory effect in the absence of Ca2+ (Fig 5C) A significant inhibition of the interaction by the sulphated polysaccharide fucoidan was also observed, with approximately 65% inhibition at a

600 : 1 fucoidan⁄ CD91 molar excess (Fig 5D) The physiological partner proteases of C1q, C1r and C1s were also tested as inhibitors, with inhibition observed only for C1r (Fig 5E) Inhibition was also observed with the globular region of C1q, confirming the results observed by the SPR data (results not shown)

Taken together, these inhibition experiments indicate that C1q occupy known ligand-binding sites on CD91 and that CD91 presumably interacts with C1q at

0

1

2

3

4

1500 1000

500 0

Time (min)

Physiological NaCl (0.15 M )

0.5 M additional NaCl (0.65 M )

A405

A

0

0.2

0.4

0.6

0.8

1

IgG (agg) IgM

IgG

b-CD91

C1q + b-CD91

A405

C

0

1

2

3

4

150 100

50 0

Time without NaCl (min)

A405

D

0

1

2

3

Anti-C1q

Anti-isotype control

A405

B

Fig 4 Interaction between CD91 and C1q, demonstrated by ELISA (A) Time dependence of the association between CD91 and C1q Biotinylated CD91 (b-CD91) was added to microtitre plates coated with C1q b-CD91 was allowed to incubate for the indicated time (from 10 min to 20 h) with a NaCl concentration of either 0.15

or 0.65 M Bound b-CD91 was quantified by incubation with alkaline phosphatase-conjugated streptavidin and pNPP (B) CD91 was immobilized on a microtitre plate and C1q or the control protein bovine serum albumin was allowed to interact Bound C1q was detected with rabbit antibodies recognizing C1q and alkaline phos-phatase-conjugated secondary antibodies Data are presented as the mean ± SD of two individual wells for one representative experiment (C) A microtitre plate was coated with the indicated protein; either IgG, IgM or IgG aggregated [IgG(agg)] by incubation

at 60 C for 20 min, b-CD91 was either added directly (grey bars)

or after a layer of C1q (white bars) and bound CD91 was detected

by incubation with alkaline phosphatase-conjugated streptavidin and pNPP (D) The time-dependent influence of 0.5 M NaCl on CD91 binding to C1q b-CD91 was added to a microtitre plate coated with C1q After the indicated amounts of time, 0.5 M NaCl was added and the incubation continued for a total incubation time of 2 h, before the bound b-CD91 was quantified by incubation with alkaline phosphatase-conjugated streptavidin and pNPP.

several sites, with one of them being at or near the C1r attachment site Calreticulin was only able to inhi-bit the interaction to 45%, despite the use of a very large excess Considering that calreticulin is known to interact strongly with C1q, this suggests that only a part of the binding sites on C1q are shared by calreti-culin and CD91 No interaction between CD91 and calreticulin could be observed, regardless of which of them was immobilized (results not shown)

Discussion

It is well established that C1q initiates the clearance of apoptotic material, although the identification of direct C1q-receptors has remained elusive CD91 has previ-ously been described to function as a receptor complex together with calreticulin In this receptor complex, calreticulin is suggested to act as the C1q-recognition molecule and CD91 as the phagocytic, transmembrane molecule

To determine whether C1q binding could occur on human blood cells, we first confirmed the high CD91 level on human blood monocytes We then analyzed C1q binding to human blood cells, confirming a mono-cyte interaction The interaction between C1q and PBMCs could be partially inhibited by RAP and solu-ble CD91 The interaction between C1q and CD91 was also investigated using the monocytic cell line MM6 known to present CD91 at the cell surface Fluorescent-labelled C1q was found to bind these cells

in a manner overlapping the areas of CD91 expression The partial co-localization suggests that other C1q receptors exist This was confirmed by the inhibition experiments where the interaction between C1q and MM6 cells could be partially inhibited by the addition

of RAP, suggesting that only part of the C1q interac-tion occurs specifically through CD91 or other mem-bers of the LDL receptor superfamily

CD91 and calreticulin have previously been described to function as a C1q receptor complex with calreticulin as the recognition unit of the complex Despite these reports, in the present study, we provide evidence that CD91 directly recognizes C1q

indepen-0

1

2

3

4

100 10

0

RAP/CD91 molar ratio

RAP

Ovalbumin

A405

0

1

2

3

4

Calreticulin/CD91 molar ratio

Without calcium

5 m M calcium

A405

A405

0

1

2

3

4

600 60

0

SAP/CD91 molar ratio

Without calcium

5 m M calcium

0

1

2

3

600 60

0

Fucoidan/CD91 molar ratio

A405

0

0.5

1

1.5

50 0

C1s

C1r

BSA

A405

CD91/serine protease molar ratio

A

B

C

D

E

Fig 5 Inhibition of the CD91–C1q interaction Biotinylated CD91 was added to microtitre plates coated with C1q, together with the indicated molar excess of inhibitor (A) RAP and the control protein ovalbumin; (B) calreticulin with and without the addition of 5 m M calcium; (C) SAP with and without 5 m M calcium; (D) fucoidan; and (E) C1r and C1s After 2 h of incubation, the amount of bound bioti-nylated CD91 was quantified by incubation with alkaline phospha-tase-conjugated streptavidin and pNPP Data are the mean ± SD of two individual wells for one representative experiment.

dently of calreticulin The CD91–C1q interaction

showed all of the signs for being specific because the

interaction was time- and concentration-dependent

Furthermore, the interaction was inhibited by several

known ligands of either protein Interaction was

observed both with commercially available C1q⁄ CD91

and purified C1q⁄ CD91 In several assays, the

interac-tion showed signs of two-state binding First, the

inter-action appeared to be salt sensitive at the early stages

but became salt insensitive at later stages Second,

when the interaction was investigated using SPR,

a two-state binding was indicated and, third, an

inter-action was observed both for the globular region and

with the collagen tail of C1q A likely hypothesis is

that CD91 binds C1q first through its collagen tail in a

salt-sensitive manner and then mediates a

salt-insensi-tive interaction with the globular head or vice versa

CD91 contains four ligand-binding clusters of which

RAP is known to block at least three [36,40] RAP is

specific for the receptors of the ‘LDL receptor

super-family’ and blocks all tested ligands of CD91 [36]

RAP completely inhibited binding between CD91 and

C1q, indicating that the interaction occurs through

one of more of these ligand-binding clusters Some

inhibition was also observed with the CD91 ligands

a-2-macroglobulin and exotoxin A These ligands

occupy the CD91 ligand-binding cluster II and IV,

thereby suggesting that C1q might interact with one or

both of these sites on CD91 [41,42]

Several interaction partners of C1q were also tested

for their ability to inhibit the interaction between

CD91 and C1q Fucoidan yielded inhibition of CD91

binding to C1q Fucoidan was reported to bind C1q at

a site that abolishes attachment of the serine proteases

[43] It is therefore possible that CD91 interacts with

C1q at a site near or at the serine protease binding

site This theory was supported by inhibition as a

result of the serine protease C1r, where a 50-fold

excess inhibited the interaction between CD91 and

C1q by approximately 40% C1r binds C1q at the

col-lagen tail at a site where the colcol-lagen tail is trimeric

without obtaining higher-order oligomeric forms

[44,45] Complete inhibition was also observed with

SAP SAP is known to bind C1q at sites on both the

collagen stalk and the globular region [38,39] and it is

therefore possible that CD91 interacts at one or both

of these sites The possibility that both the globular

region and the collagen region could be involved in

this interaction was strengthened by SPR data, where

either region showed an interaction with CD91 Taken

together, these inhibition experiments imply that the

CD91–C1q interaction occurs at one or more of the

ligand-binding clusters of CD91 On the C1q molecule,

the interaction site may be near or at the serine prote-ase attachment site, although several sites on the C1q molecule are assumed to be involved

Calreticulin is known to bind C1q and is a reported co-receptor of CD91 Only 45% inhibition was obtained in the case where calreticulin was used as an inhibitor of the C1q–CD91 interaction The calreticulin inhibition indicates that only a part of the binding sites

on C1q is shared between CD91 and calreticulin No binding was detected between CD91 and calreticulin in the ELISA, regardless of which of them was immobi-lized or in solution These results are somewhat intriguing because calreticulin has been reported to function as a co-receptor for CD91 in the binding of C1q and collectins [20,22,23] However, Donnelly et al [46] also were unable to detect distinct binding between calreticulin and CD91 and Walters and Berwin [47] reported that the uptake of calreticulin is different from another CD91 ligand, thereby suggesting that calreticulin is not a traditional ligand of CD91

In conclusion, the data obtained in the present study support the hypothesis that CD91 is a receptor for C1q and presumably detects and internalizes ligand bound C1q in the same way that it internalizes other ligands We therefore conclude that CD91 itself is a receptor for C1q On the basis of these findings, we propose a binding model for CD91 scavenging of C1q that is independent of calreticulin (Fig 6)

Materials and methods

Proteins and chemicals Ovalbumin, bovine serum albumin, p-nitrophenyl-phos-phate (pNPP) substrate tablets, FITC, N-hydroxy-succinim-idobiotin, a-2-macroglobulin, exotoxin A, fucoidan, LPS (Pseudomonas aeruginosa) and C1q were obtained from Sigma (St Louis, MO, USA) Alkaline phosphatase-conju-gated streptavidin and IgM were obtained from Dako (Glostrup, Denmark) MaxiSorp microtitre plates were obtained from Nunc (Roskilde, Denmark) CD91 (contain-ing RAP) and biotin-labelled CD91 antibody were obtained from BioMac (Chamalie`res, France) RAP was obtained from Innovative Research (Southfield, MI, USA) SAP was

a gift from N H H Heega˚rd (Statens Serum Institut, Copenhagen, Denmark) Adiponectin (trimeric form) was obtained from Abcam (Cambridge, UK) Human IgG and human serum albumin (HSA) were obtained from SSI (Copenhagen, Denmark) CM-5 sensorchips, surfactant P20, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydro-xysuccinimide and ethanolamine were obtained from GE healthcare, BIAcore (Uppsala, Sweden) Excell medium was obtained from Safc Biosciences (Hampshire, UK)

Penicillin, streptomycin and glutamine were obtained from

Gibco-BRL (NY, USA) Flowbuffer, phycoerytrin (PE)

mouse anti-human CD91, PE mouse anti-human CD14,

FITC mouse anti-human CD91, FITC mouse anti-human

CD14 and corresponding isotype control antibodies were

obtained from BD Biosciences (Franklin Lakes, NJ, USA)

C1r and C1s were obtained from R&D Systems (Abingdon,

UK) Rabbit anti human C1q polyclonal antibody was

obtained from Dako High-yield Lyse and streptavidin

labelled with Alexa Fluor 546 were obtained from

Invitro-gen (Carlsbad, CA, USA) Vectashield mounting medium

was from Vector Laboratories (Burlingame, CA, USA)

Purification of human placenta calreticulin

Human placenta calreticulin was purified using a previously

described procedure [48,49] The purified protein showed a

single band with an apparent molecular weight of 60 kDa

by SDS⁄ PAGE and a single peak by MALDI-TOF ⁄ TOF

mass spectrometry

Purification of C1q and C1q-derived fragments

C1q was purified from human plasma as described

previ-ously [50] The collagen-like fragments of C1q were

obtained by pepsin digestion and purified as described pre-viously [51] The fragments corresponding to the C1q glob-ular domains were generated by treatment of C1q with collagenase and purified by high-pressure gel filtration chro-matography as described previously [51]

Purification of CD91 Commercially obtained CD91 (BioMac) was used when not stated otherwise CD91 containing no RAP was a gift from

S K Moestrup (Institute of Medical Biochemistry, Univer-sity of A˚rhus, A˚rhus, Denmark), purified as described pre-viously [52], and was used to confirm the C1q interaction

Protein biotinylation The proteins subjected to biotinylation were dialysed against 0.1 m NaHCO3(pH 9.0) at 4C, followed by the addition

of N-hydroxysuccinimidobiotin in dimethyl sulphoxide (10 mgÆmL)1) to a final concentration of 4 mgÆmg)1CD91 The solution was incubated for 2 h at room temperature with end-over-end agitation, and then dialysed against NaCl⁄ Pi(10 mm NaH2PO4⁄ Na2HPO4, pH 7.3, 0.15 m NaCl)

at 4C Biotinylated CD91 was stored at)20 C until use

FITC labelling Commercially available C1q and HSA were dialysed against

50 mm sodium carbonate (pH 9.5) FITC (100 lgÆmg)1 pro-tein) was added and the reaction mixture was incubated with end-over-end agitation for 1 h in the dark Excess fluo-rescein was removed by dialysis against NaCl⁄ Piat 4C

ELISA Unless otherwise stated, incubations and washings were performed at room temperature on a shaking table using

100 lL per well for incubation and 200 lL per well for washing and blocking TTN buffer (0.025 m Tris–HCl, 0.5% Tween 20, 0.15 m NaCl, pH 7.5) was used for block-ing, incubation and washing

Unless otherwise stated, the C1q used was from Sigma (St Louis, MO, USA) Proteins were coated at 1 lgÆmL)1 onto the surface of the microtitre plates using 0.05 m sodium carbonate (pH 9.6) as coating buffer After coating overnight at 4C, plates were washed three times for 1 min, followed by blocking for 30 min Subsequently, incubation with or without biotinylated CD91 diluted to 1 lgÆmL)1 was carried out for 2 h, followed by another three washes Finally, the plates were incubated for 1 h with alkaline phosphatase-conjugated streptavidin diluted 1 : 1000 After another three washes, bound CD91 was quantified using pNPP (1 mgÆmL)1) in 1 m diethanolamine, 0.5 mm MgCl2

(pH 9.8) A405 was read with background subtraction at

Epidermal growth factor-type repeat

Ligand-binding repeat

Beta-propeller

C1q

Ligand bound

C1q

CD91

Apoptotic material

Phagocyte

Fig 6 Proposed model for CD91 interaction with C1q CD91 is

present on phagocytes and consists of two noncovalently bound

polypeptide chains; with the 85 kDa b-chain as the transmembrane

and the a-chain of 515 kDa with four ligand-binding clusters (blue

areas) Ligand interaction occurs through 31 similar ligand-binding

repeats (blue squares) distributed unequally between the four

ligand-binding clusters The CD91 a-chain also consists of

epider-mal growth factor-type repeats (grey circles) and b-propellers (black

stars) Interaction occurs at several sites on C1q and possibly

through one or more ligand-binding domains at CD91 CD91 is a

phagocytic receptor and is likely to internalize C1q bound material.

650 nm on a VERSAmax microplate reader (Molecular

Devices, Sunnyvale, CA, USA) All experiments were

per-formed at least twice and the results are presented as the

mean ± SD of two wells for one representative experiment

Inhibition ELISA

Inhibition of the CD91–C1q interaction was tested by

mix-ing biotinylated CD91, diluted to a concentration of

1 lgÆmL)1, with varying amounts of the indicated inhibitor

for 1 h The mixture was then added for 2 h to plates

coated overnight with C1q Subsequently, the plates were

washed, incubated with alkaline phosphatase-conjugated

streptavidin and developed as described above

SPR experiments

CD91 and C1q were immobilized on a BIAcore CM5

sensor-chip in NaCl⁄ Hepes-P20 (10 mm Hepes, 0.15 m NaCl,

3.4 mm EDTA, 0.005% surfactant P20) in accordance with

the manufacturer’s instructions, resulting in an

immobiliza-tion level of 13 000 and 17 000 relative units, respectively

Interaction between immobilized CD91 and C1q, the C1q

globular domain and collagen fragment was investigated

on a BIAcore 3000 instrument using NaCl⁄ Tris (10 mm

Tris–HCl, 0.15 m NaCl, pH 7.4) as running buffer

Inter-action between immobilized C1q and soluble CD91 was

investigated on a BIAcore 1000 instrument with the same

running buffer The analyte was injected at a flow rate of

20 lLÆmin)1 for 120 s Background was subtracted from a

quenched (activated⁄ deactivated) surface showing an

absence of nonspecific binding The surface was fully

regen-erated using low concentrations of NaOH Kinetics were

determined using the software biaevaluation, version 3.1

(BIAcore)

Two independent experiments were performed with all

concentrations Data are reported for one representative

experiment

Culture of MM6 cells

MM6 was used as a monocytic cell line Cells were cultured

at 37C under 5% CO2in Excell medium (to avoid serum)

with the addition of 1% glutamine and 1% penstrep These

cells were stimulated twice with 10 ngÆmL)1 LPS with a

3-day interval and harvested on day 6 or 7

Flow cytometry analysis of MM6 cells

A sample containing 105 cells was centrifuged to remove

the culture medium and the cells were washed with

NaCl⁄ Pi The cell pellet was resuspended in 10 lL of

NaCl⁄ Pi and stained by adding 2 lL of F-C1q or

FITC-labelled HSA for 20 min at room temperature Staining

with CD91 antibody, CD14 antibody or isotype control

antibodies was performed in accordance with the manufac-turer’s instructions After staining, cells were washed with NaCl⁄ Pi and resuspended in 1 mL of the buffer Flow cytometry was performed using a Calibur instrument (BD Biosciences), counting 10 000 cells and using cellquest (BD Biosciences) and winmdi (http://facs.scripps.edu) for the data analysis

Flow cytometry analysis of whole blood Two microlitres of F-C1q, F-HSA or antibodies labelled with PE or flourescein were added to 12.5 lL of heparin-ized blood After incubation for 15 min, red blood cells were lysed by the addition of 1 mL of High-yield Lyse (Invitrogen) Cells were analyzed on a Calibur instrument (BD Biosciences), counting 5000 or 10 000 events

PBMC isolation and inhibition experiments Human peripheral blood was collected and mixed with NaCl⁄ Pi (1 : 1) The mixture was overlayed on Ficoll and centrifuged (800 g for 20 min at 4C) The interface of mononuclear cells was harvested and stained with 2 lL of F-C1q and the described antibodies for 10 lL of PBMC, with or without the addition of inhibitor, for 20 min at room temperature Cells were analyzed on a Calibur instru-ment (BD Biosciences), counting 10 000 events

Confocal scanning laser microscopy MM6 cells were grown on glass cover slips for 6 days and stimulated with LPS (10 ngÆmL)1) on days 1 and 3 The cells were fixed with 4% paraformaldehyde for 20 min at 37C and permeabilized with 0.1% Triton X-100 for 5 min after three washes with NaCl⁄ Pi After four washes, the cells were stained for 30 min with biotinylated mouse anti-CD91 (dilu-tion 1 : 25) in Hanks balanced salt solu(dilu-tion containing 5% inactivated fetal bovine serum and 40 mm Hepes After four washes with NaCl⁄ Pi, the cells were stained with F-C1q (10 lgÆmL)1) and streptavidin-Alexa 546 (dilution 1 : 50) in the same staining buffer The coverslips were mounted with 4¢,6¢-diamidino-2-phenylindole-containing mounting solu-tion and pictures were taken with a Leica, SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany) Pic-tures were processed using image j [53]

Acknowledgements

Dorthe Tange Olsen and Anne Mortensen are thanked for their excellent technical work Søren Kragh Moest-rup and Christian Jacobsen are thanked for providing purified CD91 as well as helpful suggestions Hans Jør-gen Jensen and Susana Aznar are thanked for help with the confocal microscopy

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