The tissue pentraxin PTX3 limits C1q

The tissue pentraxin PTX3 limits C1q-mediated complement
activation and phagocytosis of apoptotic cells
by dendritic cells
Paramita Baruah,* Ingrid E. Dumitriu,* Giuseppe Peri,† Vincenzo Russo,‡ Alberto Mantovani,†,§
Angelo A. Manfredi,*,¶ and Patrizia Rovere-Querini*,1
*Clinical Immunology Unit and ‡Cancer Gene Therapy Unit, Cancer Immunotherapy & Gene Therapy Program, H.
San Raffaele Scientific Institute, Milano, Italy; †Istituto Clinico Humanitas, Rozzano, Italy; §Centro di Eccellenza
per l’Innovazione Diagnostica e Terapeutica (IDET), Institute of General Pathology, University of Milan, Italy; and
¶
Vita-Salute San Raffaele University, Milano, Italy
Abstract: Pentraxins (PTX) and complement belong to the humoral arm of the innate immune
system and have essential functions in immune defense to microbes and in scavenging cellular debris. The prototypic long PTX, PTX3, and the first
component of the classical complement pathway,
C1q, are innate opsonins involved in the disposal of
dying cells by phagocytes. Whether the interaction
between various innate opsonins impacts on their
function is not fully understood. We show here that
characterized Toll-like receptor (TLR) ligands
elicit the production of C1q and PTX3 by immature dendritic cells (DC). Moreover, these molecules bind to dying cells with similar kinetics, although they recognize different domains on the cell
membranes. PTX3 binds in the fluid phase to C1q,
decreasing C1q deposition and subsequent complement activation on apoptotic cells. C1q increases
the phagocytosis of apoptotic cells by DC and the
release of interleukin-12 in the presence of TLR4
ligands and apoptotic cells; PTX3 inhibits both
events. Moreover, PTX3 inhibited the cross-presentation of the MELAN-A/melanoma antigen-reactive T cell 1 (MART-1) tumor antigen expressed by
dying cells, even in the presence of C1q. These
results suggest that interaction of C1q and PTX3
influences the clearance of apoptotic cells by DC.
The coordinated induction by primary, proinflammatory signals of C1q and PTX3 and their reciprocal regulation during inflammation influences the
clearance of apoptotic cells by antigen-presenting
cells and possibly plays a role in immune
homeostasis. J. Leukoc. Biol. 80: 87–95; 2006.
Key Words: Toll-like receptor 䡠 systemic lupus erythematosus 䡠 lipopolysaccharide 䡠 apoptosis 䡠 inflammation
INTRODUCTION
Soluble factors of the innate immune system have well-described roles in host defense to microbes. Recently, comple0741-5400/06/0080-0087 © Society for Leukocyte Biology
ment factors and pentraxins (PTX) have come into focus as
important regulators in the clearance of dying cells [1– 6]. C1q
(the first component of the classical pathway of complement
activation) and PTX3 (the prototypic, long PTX) are soluble
pattern recognition receptors capable of binding to apoptotic
cells and modulating their uptake by phagocytes [2, 7, 8].
Deficiency of C1q has been shown to lead to a systemic lupus
erythematosus (SLE)-like syndrome in mouse models and in
humans [1]. PTX3 has been demonstrated to play a nonredundant role in immune defense to Aspergillus [9].
Interaction between soluble, innate factors and the reciprocal influences on their functions is not well understood. The
classical PTX, such as C-reactive protein (CRP) and serum
amyloid protein (SAP), are known to bind to C1q and activate
the complement cascade [10]. A similar role has been attributed to PTX3, which shares structural homology in the PTX
domain with the classical PTX. Immobilized PTX3 binds C1q
and activates the complement cascade [11]. Classical PTX and
components of the complement pathways are predominantly
produced in the liver [12]. In contrast, C1q and PTX3 are
produced by cells of the innate immune system, such as
macrophages and dendritic cells (DC) [13, 14]. Immature DC
are a major source of C1q [14], and PTX3 production by DC
and macrophages is induced in response to microbial components such as lipopolysaccharide (LPS) [13] and proinflammatory stimuli—tumor necrosis factor ␣ (TNF-␣). In addition,
C1q and PTX3 have differing effects on the phagocytosis of
apoptotic cells by professional phagocytes. C1q enhances
phagocytosis of apoptotic cells [14], and PTX3 has been described to inhibit phagocytosis by DC and macrophages [2, 15].
C1q and PTX3 are present at the sites of tissue inflammation
and necrosis. Their cross-talk possibly has connotations for the
safe clearance of dying tissue cells. In this study, we show that
C1q and PTX3, produced from DC, together contribute to
regulate the clearance of bystander dying cells.
1
Correspondence: Cancer Immunotherapy & Gene Therapy Program, Clinical Immunology Unit, H. San Raffaele Scientific Institute, via Olgettina 58,
Milano 20132, Italy. E-mail: [email protected]
Received August 10, 2005; revised February 24, 2006; accepted March 8,
2006; doi: 10.1189/jlb.0805445.
Journal of Leukocyte Biology Volume 80, July 2006 87
MATERIALS AND METHODS
Reagents
Purified human C1q isolated by affinity chromatography from human serum
and C1q-depleted human serum were purchased from Calbiochem (EMD
Biosciences, La Jolla, CA) and Quidel Corp. (San Diego, CA), respectively.
PTX3 was obtained by a combination of anion exchange chromatography and
gel filtration from the supernatant of Chinese hamster ovary cells stably and
constitutively expressing the protein. Biotinylation of recombinant human
PTX3 was performed as described previously [11]. ␤2-Glycoprotein 1 (␤2GPI) was purchased from Research Diagnostics (Flanders, NJ; Fitzgerald
Industries, Concord, MA).
Cells
Peripheral blood mononuclear cells were obtained from buffy coats of healthy
donors by sedimentation over Ficoll Hypaque as described [16]. Monocytes
were differentiated into DC by culture in RPMI 1640 (Gibco, Life Technologies, Italy) supplemented with 100 U/ml penicillin, 100 ␮g/ml streptomycin,
1.5 mM L-glutamine, and 10% heat-inactivated fetal calf serum (FCS) in the
presence of granulocyte macrophage-colony stimulating factor (800 U/ml) and
interleukin (IL)-4 (800 U/ml; BD Biosciences PharMingen, San Diego, CA)
[17]. The phenotype of the DC was monitored by flow cytometry, using
fluorochrome-labeled monoclonal antibodies (mAb) against CD1a, CD14,
CD40, CD83, CD86, human leukocyte antigen (HLA)-DR, and HLA-ABC
surface markers (BD Biosciences PharMingen). Maturation of DC was induced
by stimulation with LPS (10 –1000 ng/ml; Sigma Chemical Co., St. Louis, CA),
lipoteichoic acid (LTA; 10 –1000 ng/ml, Invivogen, San Diego, CA), and
peptidoglycan (PGN; 10 ␮g/ml, Fluka, Switzerland). HeLa and Jurkat cell lines
were purchased from American Type Culture Collection (Manassas, VA) and
maintained in culture in RPMI with 10% FCS.
Assessment of C3b deposition on apoptotic cells
Apoptotic HeLa cells were incubated with C1q, PTX3, or both for 1 h, followed
by incubation with C1q-depleted serum supplemented with 10% heat-inactivated FCS at 37°C for 1 h. Detection of C3b on apoptotic cells was done using
a purified mouse antibody anti-C3b (Research Diagnostics) and subsequent
incubation with a FITC-conjugated goat anti-mouse antibody (BD Biosciences
PharMingen). Assessment of C3b deposition on apoptotic cells was evaluated
using flow cytometry.
Phagocytosis assay
Apoptotic HeLa cells were labeled with carboxyfluorescein succinimidyl ester
(CFSE) as described [8]. Labeled apoptotic cells (5⫻105) were incubated with
2 ⫻ 105 DC in medium containing heat-inactivated serum alone or in the
presence of C1q and PTX3 for varying time periods. Then, the cells were
harvested, stained with PE-conjugated mAb anti-CD11c to label DC, and
analyzed by flow cytometry. DC were gated according to forward-scatter (FSC)
properties and CD11c positivity. The DC, which bound or phagocytosed
apoptotic cells, were identified as double-positive cells [3].
DC maturation
Maturation of DC was induced by stimulation with LPS (10 –1000 ng/ml; Sigma
Chemical Co.). C1q and/or PTX3 were added at a final concentration of
50 –100 ␮M. Apoptotic HeLa cells (1⫻106) were added to a final DC:apoptotic
cell ratio of 1:4. After 24 – 48 h, the cells were harvested, and the expression
of HLA-ABC, HLA-DR, and CD86 was assessed by flow cytometry using
fluorochrome-conjugated mAb (BD Biosciences PharMingen). The supernatants were collected and stored frozen until cytokine quantification by enzymelinked immunosorbent assay (ELISA).
Induction of apoptosis
Cross-presentation
Apoptosis was triggered by C-band ultraviolet light (UVC) irradiation (254 nm
wavelength) at the dose of 3.5 mW/cm2/s for 60 s (HeLa cells) or of 1.8
mW/cm2/s for 45 s (Jurkat cells) followed by incubation of irradiated cells for
up to 24 h. Induction and phase of apoptosis were verified by flow cytometry
using fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium
iodide (PI; Bender MedSystem, GmbH, Austria) as per the manufacturer’s
instructions. Confocal microscopy analysis was done using a Leica microscope
in selected experiments, as described previously [2].
Cross-presentation experiments were performed as described previously [18].
Briefly, NIH-3T3 fibroblasts, transduced or not with MELAN-A/melanoma
antigen-reactive T cell 1 (MART-1; 3T3M and 3T3, respectively), were committed to apoptosis by UVC irradiation (254 nm wavelength) at the dose of 1.8
mW/cm2/s for 45 s. Induction and phase of apoptosis were verified by flow
cytometry using FITC-conjugated annexin V and PI (Bender MedSystem,
GmbH). HLA-A2⫹ immature DC (2⫻104) were incubated with 5 ⫻ 104
apoptotic 3T3 and 3T3M fibroblasts per well in Iscoves’ modified Dulbecco’s
medium (Gibco, Invitrogen SRL) with 10% inactivated human serum and IL-2
(50 Cetus units/ml) and challenged with 1 ⫻ 104 MT27–-35 MELAN-A/
MART-1 epitope-specific CD8⫹ T cells for 24 h. LPS (10 ng/ml) was added for
the last 16 h of culture. Parallel cross-presentation assays were run in the
presence of C1q (50 ␮g/ml) and/or PTX3 (50 ␮g/ml). The supernatants were
collected after 24 h and assessed for interferon-␥ (IFN-␥) by ELISA.
Interaction of C1q and PTX3 with living and
apoptotic cells
Apoptotic or viable HeLa and Jurkat cells (1⫻105) were incubated with
various concentrations of C1q (5–100 ␮g/ml) or biotinylated human PTX3
(2.5–50 ␮g/ml) in a final volume of 50 ␮l phosphate-buffered saline (PBS) for
30 min at 4°C. C1q binding to cells was detected by staining with FITCconjugated anti-C1q antibody (Dako, Denmark), and PTX3 was detected by
subsequent incubation with streptavidin-FITC (BD Biosciences PharMingen)
prior to flow cytometric analysis. When indicated, cells were incubated with
C1q or PTX3 as above, washed, and chased for different time periods (0 –24 h
at 4°C or 37°C) before analysis. Moreover, in selected experiments, 1 ⫻ 105
apoptotic HeLa cells were incubated with PTX3 (10 or 50 ␮g/ml) or buffer
alone for 30 min at 4°C. Cells were then washed with PBS containing bovine
serum albumin (1%) and incubated with or without C1q (10 ␮g/ml) for 30 min
at 4°C. Alternatively, for the coincubation experiments, C1q (10 ␮g/ml) and
PTX3 (10 or 50 ␮g/ml) were added together to the apoptotic cells. C1q was
then revealed with FITC-conjugated anti-C1q antibody. For confocal microscopy, apoptotic cells were incubated with C1q (10 ␮g/ml) and/or biotinylated
PTX3 (50 ␮g/ml) simultaneously for 30 min at 4°C. Cells were then stained
with FITC-conjugated anti-C1q antibody or streptavidin-FITC or -phycoerythrin (PE). Cells were adhered to polylysinated glass coverslips, washed,
and fixed with 4% paraformaldehyde in PBS. Nuclei were counterstained with
Hoechst 33342 (Molecular Probes, Invitrogen SRL, Italy). The coverslips were
mounted on glass slides using Moviol and stored at 4°C until analysis.
88
Journal of Leukocyte Biology Volume 80, July 2006
ELISA
C1q, in culture supernatants, was measured by ELISA using mouse mAb
(Quidel, San Diego, CA) for coating and rabbit polyclonal antibodies anti-C1q
(Dako) for detection. Samples were analyzed in duplicates, and C1q concentration was determined by reference to standard curves. To measure PTX3
concentrations in the culture supernatants, a sandwich ELISA (Alexis Italia,
Vinci) was used as per the manufacturer’s instructions. This assay is highly
sensitive and specific; no cross-reactions were observed with other PTX and in
particular, with the CRP or SAP [19]. IL-12, IFN-␥, TNF-␣, and IL-10 levels
in culture supernatants were measured using a commercial Duotech ELISA kit
(R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions
Statistical analysis
Statistical analysis was performed using the two-tailed Student’s t-test for
unpaired samples with unequal variance. P values less than 0.05 were considered statistically significant.
http://www.jleukbio.org
RESULTS
C1q and PTX3 interact stably with definite,
distinct domains of apoptotic cells
C1q and PTX3 bind to apoptotic cells in a dose-dependent
manner (Fig. 1, A and B). These bindings are specific, as
viable cells did not bind PTX3 and bound only low levels of
C1q (Fig. 1, A and B). We found that C1q and PTX3
associate stably to the apoptotic cells, remaining detectable
over long periods of time (Fig. 1, C and D). On confocal
microscopy, we observed that C1q binding has a patchy
distribution on apoptotic HeLa cells (Fig. 2A) and apoptotic
Jurkat cells (data not shown), and PTX3 bound to apoptotic
HeLa cells in a diffuse, reticular pattern (Fig. 2B). C1q and
PTX3 did not colocalize on apoptotic cells (Fig. 2C), suggesting that they bind to distinct sites. These data suggest
that although the two molecules bind to each other upon
immobilization to solid amorphous substrates, they interact
at different sites on the apoptotic cells.
Fig. 2. C1q and PTX3 exhibit different patterns of binding to apoptotic cells.
Viable and apoptotic cells were incubated with C1q and biotinylated PTX3 and
analyzed by confocal microscopy. Bound C1q and PTX3 were detected using
FITC-conjugated polyclonal rabbit anti-C1q antibodies and FITC-conjugated
streptavidin, respectively. The apoptotic nuclei were counter-stained with
Hoechst 33342 (blue). The binding of C1q (green) to the apoptotic cells
displayed a patchy distribution (A, right panel). PTX3 (green) bound to the
apoptotic cells in a diffuse, reticular pattern (B, right panel). Viable cells are
also shown (A, B, left panels). Apoptotic cells were incubated with C1q and
biotinylated PTX3 simultaneously, and detection of the binding was done using
FITC-conjugated polyclonal rabbit anti-C1q antibodies and PE-conjugated
streptavidin, respectively. C1q and PTX3 bound to different sites on the
apoptotic cells (C). Results are from representative, routine experiments.
Fig. 1. C1q and PTX3 bind stably to apoptotic cells. Apoptotic HeLa cells
were incubated with C1q and biotinylated PTX3 (x-axis) for 1 h at 4°C.
Bound C1q and PTX3 were detected by using FITC-conjugated polyclonal
rabbit anti-C1q antibodies and FITC-conjugated streptavidin, respectively.
C1q and PTX3 were found to bind to apoptotic cells in a dose-dependent
manner (filled symbols) (A, B). Viable cells bound low amounts of C1q and
no PTX3 (open symbols). Apoptotic cells were incubated with C1q and
PTX3 for 1 h at 4°C and 37°C, washed, and chased for the indicated
periods of time. Bound C1q and PTX3 were detected using FITC-conjugated polyclonal rabbit anti-C1q antibodies and FITC-conjugated streptavidin, respectively. Binding of C1q and PTX3 was higher at 37°C than at
4°C (C, D). Results are representative of three independent experiments.
MFI, Mean fluorescence intensity.
The interaction in the fluid phase with PTX3
prevents C1q binding on apoptotic cells
We next analyzed if the binding of C1q to apoptotic cells is
influenced by PTX3. For this, we incubated apoptotic cells
with C1q alone or in the presence of PTX3. We found that the
binding of C1q to apoptotic cells was reduced significantly
when PTX3 was present along with C1q in solution (Fig. 3A).
Similar results were obtained with apoptotic Jurkat cells (data
not shown). In contrast, the coincubation of apoptotic cells with
an irrelevant plasma factor known to associate to their membranes [20], the ␤2-GPI, did not have any effect on C1q
binding (Fig. 3A). We found that preincubating apoptotic cells
Baruah et al. PTX3 modulates C1q effects on apoptotic cells
89
Fig. 3. PTX3 inhibits C1q binding on apoptotic
cells. Apoptotic HeLa cells were incubated with
C1q alone (10 ␮g/ml) or in the presence of PTX3
(10 ␮g/ml and 50 ␮g/ml) or ␤2-GPI (60 ␮g/ml), and
C1q binding was assessed. C1q binding to apoptotic
cells was inhibited significantly in the presence of
PTX3 (P⬍0.05), and ␤2-GPI did not change C1q
deposition (A). No change in C1q binding was seen
when the apoptotic cells were preincubated with
PTX3 (A). ELISA plates were coated with anti-C1q
mAb followed by addition of C1q, biotinylated
PTX3, or a mixture of both. After washing, plates
were incubated with streptavidin-FITC to detect
PTX3 (P⬍0.01), which was detected only in the
wells where the mixture of C1q and PTX3 was
added (B). No signal was elicited in the wells where
either protein was added alone (B). Samples were
analyzed in duplicates. Results are representative
of three independent experiments. Asterisks refer to the statistical significance versus controls: *, P ⬍ 0.05; **, P ⬍ 0.01. O.D. (a.u.), Optical density (arbitrary
units); w/o, untreated.
with PTX3 before adding C1q did not alter the binding of C1q
to the apoptotic cells (Fig. 3A). This observation was in keeping with the absence of colocalization between PTX3 and C1q
observed on the confocal microscopy and suggested that the
interaction between C1q and PTX3 may occur in the soluble
phase. To verify whether this was indeed the case, we coincubated C1q and biotinylated PTX3 in solution and then added
this mixture to ELISA plates coated with mAb anti-C1q. We
next verified the presence of C1q/PTX3 complexes by using
streptavidin-horseradish peroxidase. A positive reaction occurred in the presence of C1q and PTX3, suggesting the
formation and presence of the C1q-PTX3 complex (Fig. 3B). In
contrast, C1q or PTX3 alone did not elicit any signal (Fig. 3B).
Regulated production of C1q and PTX3 by DC
We then examined the secretion of C1q and PTX3 by DC
elicited with different ligands for Toll-like receptors (TLR). Up
to 5.8 ⫾ 0.3 ng/ml C1q was detectable in the supernatants of
immature DC when left undisturbed in culture for up to 48 h
(Fig. 4A), and the concentration of PTX3 in the same conditions changed from 0.232 ⫾ 0.005 to 0.89 ⫾ 0.042 ng/ml (Fig.
4B). Stimulation of DC with the TLR ligands, LPS that triggers
TLR4, or LTA and PGN, which trigger TLR2 substantially
increased the production of C1q (up to 30⫾5.1 ng/ml with LPS;
Fig. 4A). Furthermore, we found substantial PTX3 production
by DC upon treatment with TLR ligands (Fig. 4B). It is inter-
esting that PTX3 appeared in the medium as early as 6 – 8 h
following stimulation with TLR ligands (Fig. 4B), and C1q
appeared after a longer latency (24 – 48 h; Fig. 4A). These data
suggest that DC produce C1q and PTX3 in response to various
microbial stimuli over a common temporal window, allowing
the interaction between these two soluble innate factors.
PTX3 inhibits C1q-induced complement
activation on apoptotic cells and their
phagocytosis by DC
Apoptotic endothelial cells have been demonstrated to induce
complement deposition by activating the classical pathway
[21]. We incubated apoptotic cells with C1q alone or in the
presence of PTX3 and assessed the C3b deposition by further
incubation with C1q-depleted serum. Preincubation with C1q
resulted in significant C3b deposition on the apoptotic cells
(Fig. 5A). When the cells were incubated concomitantly with
C1q and PTX3, a significant decrease in C3b deposition was
observed (Fig. 5A). Incubation of apoptotic cells with C1qdepleted serum, alone or in the presence of PTX3, did not lead
to C3b deposition (Fig. 5A). These results are in keeping with
the decreased binding of C1q to apoptotic cells induced by the
presence of PTX3 (Fig. 3A).
Apoptotic cells are a preferential source of autoantigens, and
prompt clearance of dying cells is essential to prevent autoimmune phenomena [22]. We evaluated the interaction of CFSE-
Fig. 4. DC produce C1q and PTX3. Immature DC
were left alone or stimulated with LPS (10 ng/ml),
LTA (10 ng/ml), and PGN (10 ␮g/ml) for up to 48 h.
Supernatants were collected at the indicated timepoints, and C1q and PTX3 were measured by
ELISA. Production of C1q by immature DC was
increased upon stimulation with LPS, LTA, or PGN
(A). Secretion of PTX3 by DC was triggered by LPS,
LTA, and PGN (B). Immature DC did not produce
significant PTX3 (B). Samples were analyzed in
duplicates. Results representative of three independent experiments are depicted as mean ⫾ SD of
duplicate samples.
90
Journal of Leukocyte Biology Volume 80, July 2006
http://www.jleukbio.org
Fig. 5. C1q-induced C3b deposition and phagocytosis of apoptotic cells are inhibited by PTX3. Apoptotic cells were treated with C1q alone or in the
presence of PTX3 followed by incubation with C1qdepleted serum. mAb to C3b were used to detect
C3b deposition on the apoptotic cells. C1q treatment induced C3b deposition on apoptotic cells (A).
The C3b deposition was inhibited significantly in
the presence of PTX3 (A). DC were incubated with
CFSE-labeled apoptotic cells alone (–) or in the
presence of C1q, PTX3, or both, and phagocytosis/
binding was assessed by flow cytometry. DC were
gated as CD11c⫹ cells. A dot-plot of fluorescence 1
(FL-1) versus FSC was used to quantify the phagocytosis/binding of apoptotic cells (Apo) identified as
FL-1-positive DC. C1q induced a dose-dependent
increase in the phagocytosis/binding of apoptotic
cells by DC (B). The uptake of apoptotic cells
observed with C1q was inhibited significantly in the
presence of PTX3 (C, D). Results are presented as
mean ⫾ SD and are representative of three independent experiments performed with DC from different
donors. Asterisks refer to the statistical significance
versus controls: *, P ⬍ 0.05, **, P ⬍ 0.01.
labeled apoptotic cells and DC using flow cytometry, as described in Materials and Methods. PE-labeled CD11c⫹ DC
were gated, and the percentage of DC positive for CFSE (which
bound or phagocytosed apoptotic cells) was measured. C1q
substantially increased the uptake of apoptotic cells by DC.
This increase was dose-dependent (Fig. 5B). PTX3 decreased
the phagocytosis of apoptotic cells (Fig. 5C). Moreover, PTX3
hindered the C1q-elicited phagocytosis of apoptotic cells by
immature DC (P⬍0.05).
Effects of C1q and PTX3 on the crosspresentation of antigens from apoptotic cells
by DC
DC are known to acquire antigens from apoptotic cells and
activate antigen-specific CD8⫹ T cell responses, a process
known as cross-presentation. DC that phagocytosed apoptotic
murine fibroblasts expressing MELAN-A/MART-1 antigen
(3T3M) activated MELAN-A/MART-1-specific CD8⫹ T cells,
as evaluated measuring the production of IFN-␥ (Fig. 6A). In
agreement with our recent data [18], PTX3 significantly inhibits the cross-presentation of MELAN-A/MART-1 antigens derived from apoptotic 3T3M fibroblasts by DC, even in the
presence of C1q (Fig. 6A). In contrast, C1q did not change the
cross-presentation of apoptotic 3T3M fibroblast antigens (Fig.
6A). The coculture of MELAN-A/MART-1-specific CD8⫹ T
cells with DC, which phagocytosed wild-type murine fibroblasts (3T3), did not result in production of IFN-␥ (Fig. 6A). In
addition, the production of IFN-␥ by T cells stimulated with
DC loaded with MELAN-A/MART-1 MT25–37 peptide did not
change in the presence of C1q or PTX3, indicating that they do
not interfere with the presentation of a synthetic, soluble
sequence, which does not depend on internalization or intracellular processing for major histocompatibility complex-restricted presentation (Fig. 6B).
Effects of C1q and PTX3 on the cytokine
production by DC challenged with LPS and
apoptotic cells
We analyzed the effect of innate factors on the production of
cytokines by DC stimulated with LPS alone or in the presence
of apoptotic cells. PTX3 inhibits the production of TNF-␣ and
IL-10 by DC treated with LPS alone [18]. C1q alone did not
elicit any IL-12 p70 or TNF-␣ production from DC (Fig. 7, A
and B). However, C1q significantly increased the secretion of
IL-12 p70 by DC stimulated with LPS (Fig. 7, A and C), and
TNF-␣ was not altered (Fig. 7B). PTX3 alone did not influence
the IL-12 p70 production by DC stimulated with LPS (Fig. 7C).
However, PTX3 significantly inhibited the increased production of IL-12 p70 induced by C1q (Fig. 7C). Apoptotic cells
suppress the IL-12 p70 production by maturing DC (Fig. 7D).
The inhibitory effect of apoptotic cells on IL-12 p70 production
by maturing DC was partially reversed in the presence of C1q;
PTX3 abrogated this effect (Fig. 7D). C1q did not have a
detectable effect on the production of TNF-␣ and IL-10 by DC
Baruah et al. PTX3 modulates C1q effects on apoptotic cells
91
Fig. 6. Effects of C1q and PTX3 on cross-presentation of apoptotic cell-associated antigens by DC. Wild-type NIH-3T3 fibroblasts (3T3) and fibroblast transduced
with MELAN-A/MART-1 (3T3M) were induced to apoptosis by UVC irradiation. HLA-A2⫹ DC (2⫻104) were incubated with 1 ⫻ 104 antigen-specific CD8⫹ T
cells and 5 ⫻ 104 apoptotic 3T3 and 3T3M fibroblasts (apo) for 24 h. C1q (50 ␮g/ml), PTX3 (50 ␮g/ml), and the MELAN-A/MART-1 peptide MT27–35 (3 ␮M)
were added as indicated. The supernatants were collected after 24 h and assessed for IFN-␥ by ELISA. MELAN-A/MART-1-specific, HLA-A2-restricted CD8⫹
T cells secrete significant amounts of IFN-␥ when challenged with DC, which phagocytosed and processed apoptotic fibroblasts expressing the MELAN-A/MART-1
antigen (3T3M; A). Stimulation of MELAN-A/MART-1-specific, HLA-A2-restricted CD8⫹ T cells with DC, which phagocytosed apoptotic, wild-type fibroblasts
(3T3), did not alter the baseline production of IFN-␥ elicited by unchallenged DC (A). Induction of IFN-␥ by T cells challenged with DC, which phagocytosed
apoptotic 3T3M fibroblast, did not change in the presence of C1q and was decreased by PTX3 (A). Production of IFN-␥ by T cells stimulated with DC loaded with
MT27–35 peptide was not altered by C1q or PTX3. Results are represented as mean ⫾ SD of samples analyzed in triplicate. *, P ⬍ 0.05, statistically significant
values.
stimulated with LPS alone or in the presence of apoptotic cells
(Fig. 7, E and F, and data not shown). PTX3 increased the
production of IL-10 by DC stimulated with LPS, apoptotic
cells, and C1q (Fig. 7E), and TNF-␣ levels were unaffected
(Fig. 7F).
DISCUSSION
Microbial stimuli induce maturation of DC and production of
proinflammatory factors such as IL-1, IL-12, and TNF-␣ [23].
They also produce key factors of the innate immune system
(Fig. 4 and refs. [13, 14]). C1q and the complement cascade in
particular have nonredundant functions in the recognition and
immune response to microorganisms. PTX3 has an important
role in immune defense to Aspergillus, and elevated levels of
PTX3 have been reported in a number of inflammatory and
infectious conditions [24 –26].
In addition to the antimicrobial functions, complement factors and PTX have a protective effect against autoimmunity in
vivo. Deficiencies of the early components of the classical
complement pathway (C1q, C1r, C1s, C4, and C2) are associated with development of SLE. C1q-deficient mice develop
SLE and accumulate apoptotic cells in renal glomeruli, which
emphasizes the importance of C1q in the clearance of cellular
debris [1]. Moreover, patients with SLE have a well-characterized defect in the production of PTX during active phases of
92
Journal of Leukocyte Biology Volume 80, July 2006
the disease [27]. Administration of CRP is reported to have an
ameliorating effect on autoimmune nephritis in mouse models
[28, 29], suggesting a direct, protective role on tissue damage.
It has been demonstrated earlier that C1q and PTX3 bind to
apoptotic cells [2, 30 –32]. In this study, we show that C1q and
PTX3 bind to apoptotic cells, remaining stably associated to
the apoptotic cell membrane (Figs. 1 and 2). C1q has been
reported to bind PTX3 immobilized on plastic [11]. However,
we did not observe any colocalization of the two molecules on
apoptotic cells (Fig. 2C).
It has been proposed that C1q and other opsonins prevent
autoimmunity and maintain self-tolerance by supporting the
efficient clearance of apoptotic material [8, 33]. The in vivo
relevance of the complement system in maintaining immune
tolerance is well-established, as homozygous deficiency of any
of the classical pathway proteins (C1q, C4, C2) is strongly
associated with the development of SLE [34]. Deficiencies of
these factors are, however, exceedingly rare, and complement
deposition in inflamed tissues suggests a possibly harmful role
of complement; indeed, complement activation is regulated
tightly in physiological conditions to prevent immune-mediated
tissue damage [34]. C1q associates to apoptotic cells, even in
the absence of other innate factors [21, 31], but this event is
highly enhanced by the presence of natural immunoglobulin M
antibodies [35, 36]. Mevorach and co-workers [37] have shown
that complement opsonization of apoptotic cells, besides targeting them to DC for phagocytosis, induces tolerant, immature
http://www.jleukbio.org
Fig. 7. PTX3 inhibits the C1q-induced increase in
IL-12 p70 production by DC, which were stimulated with LPS alone (100 ng/ml) or in the presence
of apoptotic HeLa cells (apo) or left untreated for
24 h. C1q (50 ␮g/ml) and PTX3 (50 ␮g/ml) were
added as indicated. Supernatants were evaluated
for the production of TNF-␣, IL-10, and IL-12 by
ELISA. C1q increased the production of IL-12 p70
by LPS-stimulated DC (A), and TNF-␣ production
did not change (B). PTX3 inhibited the effect of
C1q on the production of IL-12 by DC stimulated
with LPS alone (C) or in presence of apoptotic cells
(D). Apoptotic cells decrease the production of
IL-10 (E) and TNF-␣ (F) by LPS-stimulated DC.
C1q did not alter the effect induced by apoptotic
cells (E, F). PTX3 increased the production of
IL-10 by DC stimulated with LPS, apoptotic cells,
and C1q (E), and the TNF-␣ did not change (F).
Results are represented as mean ⫾ SD of three
independent experiments performed with DC from
different donors. *, P ⬍ 0.05; **, P ⬍ 0.01, statistically significant values.
DC, which are able to migrate to lymph nodes. The amount of
complement, in particular, of iC3b [37], which associates to
dying cells, is crucial to determine the final outcome of the
phagocytic clearance in vivo. Dying cells are a source of
autoantigens, which in predisposed, genetic backgrounds, is
sufficient to induce and maintain systemic autoimmunity. Disease induction in vivo depends on the presence of environmental factors, including proinflammatory signals, in addition
to dying cells [38, 39].
Here, we show that C1q increases phagocytosis by DC (Fig.
5B) but does not increase cross-presentation of apoptotic cellassociated antigens (Fig. 6A). In contrast, PTX3 decreases
phagocytosis of apoptotic cells (Fig. 5C and ref. [2]) and the
cross-presentation of apoptotic cell-associated antigens (Fig.
6A, and ref. [18]). Preincubation of apoptotic cells with PTX3
has been reported to enhance C1q deposition and complement
activation on apoptotic cells [40]. Here, we observe that when
both factors are present simultaneously in the microenvironment, possibly mimicking what happens in the close proximity
of DC or macrophages upon recognition of pathogen-associated
molecular patterns, PTX3 can sequester soluble C1q, decreasing the activation of C3 on apoptotic cells (Fig. 5A) and the
C1q-mediated phagocytosis (Fig. 5C). These events can represent an additional level of protection against autoimmunity. In
addition, apoptotic cells inhibited the IL-12 p70 production by
DC stimulated with LPS. This inhibition was reversed in the
presence of C1q, but this effect of C1q abated in the presence
of PTX3 (Fig. 7D).
In conclusion, apoptotic cells in the steady-state are phagocytosed by macrophages and immature DC, which clear dying
cells and induce tolerance to autoantigens [41]. When microbial components are present, DC initiate their maturation pro-
Baruah et al. PTX3 modulates C1q effects on apoptotic cells
93
gram. This event may favor the loss of tolerance against peripheral antigens, especially if dying cells are present in the
microenvironment [42, 43]. We show that in physiological
conditions, maturing DC also secrete and produce innate factors in the microenvironment, including C1q and PTX3. Both
factors per se are capable of anchoring to ligands in the
microenvironment such as the ones exposed on apoptotic cells.
Furthermore, PTX3, produced by DC, will interact with the
C1q in the DC milieu, inhibiting complement activation on
apoptotic cells and phagocytosis by maturing DC and crosspresentation of self-antigens. Thus, the interaction of C1q and
PTX3 could have important implications for the safe disposal
of cell debris under inflammatory conditions.
12.
13.
14.
15.
16.
ACKNOWLEDGMENTS
This work was supported by the Associazione Italiana per la
Ricerca sul Cancro (AIRC; to A. A. M., P. R-Q., and A. M.), by
the Fondazione Berlucchi (to A. A. M.), by the Ministero della
Salute (to A. A. M. and A. M.), by Telethon (to A. M.), and by
the E. C. (APOCLEAR Project to P. R-Q.; DC-THERA and
MUGEN to A. M.).
17.
18.
19.
REFERENCES
1. Botto, M., Dell’Agnola, C., Bygrave, A. E., Thompson, E. M., Cook, H. T.,
Petry, F., Loos, M., Pandolfi, P. P., Walport, M. J. (1998) Homozygous C1q
deficiency causes glomerulonephritis associated with multiple apoptotic
bodies. Nat. Genet. 19, 56 –59.
2. Rovere, P., Peri, G., Fazzini, F., Bottazzi, B., Doni, A., Bondanza, A.,
Zimmermann, V. S., Garlanda, C., Fascio, U., Sabbadini, M. G., Rugarli,
C., Mantovani, A., Manfredi, A. A. (2000) The long pentraxin PTX3 binds
to apoptotic cells and regulates their clearance by antigen-presenting
dendritic cells. Blood 96, 4300 – 4306.
3. Gershov, D., Kim, S., Brot, N., Elkon, K. B. (2000) C-Reactive protein
binds to apoptotic cells, protects the cells from assembly of the terminal
complement components, and sustains an antiinflammatory innate immune
response: implications for systemic autoimmunity. J. Exp. Med. 192,
1353–1364.
4. Botto, M., Walport, M. J. (2002) C1q, autoimmunity and apoptosis. Immunobiology 205, 395– 406.
5. Vandivier, R. W., Ogden, C. A., Fadok, V. A., Hoffmann, P. R., Brown,
K. K., Botto, M., Walport, M. J., Fisher, J. H., Henson, P. M., Greene,
K. E. (2002) Role of surfactant proteins A, D, and C1q in the clearance of
apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common
collectin receptor complex. J. Immunol. 169, 3978 –3986.
6. Garlanda, C., Bottazzi, B., Bastone, A., Mantovani, A. (2005) Pentraxins at
the crossroads between innate immunity, inflammation, matrix deposition
and female fertility. Annu. Rev. Immunol. 23, 337–366.
7. Kim, S. J., Gershov, D., Ma, X., Brot, N., Elkon, K. B. (2003) Opsonization
of apoptotic cells and its effect on macrophage and T cell immune
responses. Ann. N. Y. Acad. Sci. 987, 68 –78.
8. Nauta, A. J., Castellano, G., Xu, W., Woltman, A. M., Borrias, M. C.,
Daha, M. R., van Kooten, C., Roos, A. (2004) Opsonization with C1q and
mannose-binding lectin targets apoptotic cells to dendritic cells. J. Immunol. 173, 3044 –3050.
9. Garlanda, C., Hirsch, E., Bozza, S., Salustri, A., De Acetis, M., Nota, R.,
Maccagno, A., Riva, F., Bottazzi, B., Peri, G., Doni, A., Vago, L., Botto,
M., De Santis, R., Carminati, P., Siracusa, G., Altruda, F., Vecchi, A.,
Romani, L., Manovani, A. (2002) Non-redundant role of the long pentraxin
PTX3 in anti-fungal innate immune response. Nature 420, 182–186.
10. Hicks, P. S., Saunero-Nava, L., Du Clos, T. W., Mold, C. (1992) Serum
amyloid P component binds to histones and activates the classical complement pathway. J. Immunol. 149, 3689 –3694.
11. Bottazzi, B., Vouret-Craviari, V., Bastone, A., De Gioia, L., Matteucci, C.,
Peri, G., Spreafico, F., Pausa, M., D’Ettorre, C., Gianazza, E., Tagliabue,
A., Salmona, M., Tedesco, F., Introna, M., Mantovani, A. (1997) Multimer
94
Journal of Leukocyte Biology Volume 80, July 2006
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
formation and ligand recognition by the long pentraxin PTX3. Similarities
and differences with the short pentraxins C-reactive protein and serum
amyloid P component. J. Biol. Chem. 272, 32817–32823.
Baumann, H., Gauldie, J. (1994) The acute phase response. Immunol.
Today 15, 74 – 80.
Doni, A., Peri, G., Chieppa, M., Allavena, P., Pasqualini, F., Vago, L.,
Romani, L., Garlanda, C., Mantovani, A. (2003) Production of the soluble
pattern recognition receptor PTX3 by myeloid, but not plasmacytoid,
dendritic cells. Eur. J. Immunol. 33, 2886 –2893.
Castellano, G., Woltman, A. M., Nauta, A. J., Roos, A., Trouw, L. A.,
Seelen, M. A., Schena, F. P., Daha, M. R., van Kooten, C. (2004)
Maturation of dendritic cells abrogates C1q production in vivo and in vitro.
Blood 103, 3813–3820.
van Rossum, A. P., Fazzini, F., Limburg, P. C., Manfredi, A. A., RovereQuerini, P., Mantovani, A., Kallenberg, C. G. (2004) The prototypic tissue
pentraxin PTX3, in contrast to the short pentraxin serum amyloid P,
inhibits phagocytosis of late apoptotic neutrophils by macrophages. Arthritis Rheum. 50, 2667–2674.
Rovere, P., Sabbadini, M. G., Fazzini, F., Bondanza, A., Zimmermann,
V. S., Rugarli, C., Manfredi, A. A. (2000) Remnants of suicidal cells
fostering systemic autoaggression. Apoptosis in the origin and maintenance of autoimmunity. Arthritis Rheum. 43, 1663–1672.
Sallusto, F., Lanzavecchia, A. (1994) Efficient presentation of soluble
antigen by cultured human dendritic cells is maintained by granulocyte/
macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor ␣. J. Exp. Med. 179, 1109 –1118.
Baruah, P., Propato, A., Dumitriu, I. E., Rovere-Querini, P., Russo, V.,
Fontana, R., Accapezzato, D., Peri, G., Mantovani, A., Barnaba, V.,
Manfredi, A. A. (2005) The pattern recognition receptor PTX3 is recruited
at the synapse between dying and dendritic cells and edits the crosspresentation of self, viral and tumor antigens. Blood 107, 151–158.
Vouret-Craviari, V., Cenzuales, S., Poli, G., Mantovani, A. (1997) Expression of monocyte chemotactic protein-3 in human monocytes exposed to
the mycobacterial cell wall component lipoarabinomannan. Cytokine 9,
992–998.
Manfredi, A. A., Rovere, P., Heltai, S., Galati, G., Nebbia, G., Tincani, A.,
Balestrieri, G., Sabbadini, M. G. (1998) Apoptotic cell clearance in
systemic lupus erythematosus. II. Role of ␤2-glycoprotein I. Arthritis
Rheum. 41, 215–223.
Mevorach, D., Mascarenhas, J. O., Gershov, D., Elkon, K. B. (1998)
Complement-dependent clearance of apoptotic cells by human macrophages. J. Exp. Med. 188, 2313–2320.
Casciola-Rosen, L. A., Anhalt, G., Rosen, A. (1994) Autoantigens targeted
in systemic lupus erythematosus are clustered in two populations of
surface structures on apoptotic keratinocytes. J. Exp. Med. 179, 1317–
1330.
Banchereau, J., Steinman, R. M. (1998) Dendritic cells and the control of
immunity. Nature 392, 245–252.
Luchetti, M. M., Piccinini, G., Mantovani, A., Peri, G., Matteucci, C.,
Pomponio, G., Fratini, M., Fraticelli, P., Sambo, P., Di Loreto, C., Doni,
A., Introna, M., Gabrielli, A. (2000) Expression and production of the long
pentraxin PTX3 in rheumatoid arthritis (RA). Clin. Exp. Immunol. 119,
196 –202.
Fazzini, F., Peri, G., Doni, A., Dell’Antonio, G., Dal Cin, E., Bozzolo, E.,
D’Auria, F., Praderio, L., Ciboddo, G., Sabbadini, M. G., Manfredi, A. A.,
Mantovani, A., Querini, P. R. (2001) PTX3 in small-vessel vasculitides: an
independent indicator of disease activity produced at sites of inflammation. Arthritis Rheum. 44, 2841–2850.
Muller, B., Peri, G., Doni, A., Torri, V., Landmann, R., Bottazzi, B.,
Mantovani, A. (2001) Circulating levels of the long pentraxin PTX3
correlate with severity of infection in critically ill patients. Crit. Care Med.
29, 1404 –1407.
Hind, C. R., Ng, S. C., Feng, P. H., Pepys, M. B. (1985) Serum C-reactive
protein measurement in the detection of intercurrent infection in Oriental
patients with systemic lupus erythematosus. Ann. Rheum. Dis. 44, 260 –
261.
Rodriguez, W., Mold, C., Kataranovski, M., Hutt, J., Marnell, L. L., Du
Clos, T. W. (2005) Reversal of ongoing proteinuria in autoimmune mice by
treatment with C-reactive protein. Arthritis Rheum. 52, 642– 650.
Ogden, C. A., Elkon, K. B. (2005) Single-dose therapy for lupus nephritis:
C-reactive protein, nature’s own dual scavenger and immunosuppressant.
Arthritis Rheum. 52, 378 –381.
Navratil, J. S., Watkins, S. C., Wisnieski, J. J., Ahearn, J. M. (2001) The
globular heads of C1q specifically recognize surface blebs of apoptotic
vascular endothelial cells. J. Immunol. 166, 3231–3239.
Gaipl, U. S., Kuenkele, S., Voll, R. E., Beyer, T. D., Kolowos, W., Heyder,
P., Kalden, J. R., Herrmann, M. (2001) Complement binding is an early
http://www.jleukbio.org
32.
33.
34.
35.
36.
37.
feature of necrotic and a rather late event during apoptotic cell death. Cell
Death Differ. 8, 327–334.
Manfredi, A. A., Iannacone, M., D’Auria, F., Rovere-Querini, P. (2002)
The disposal of dying cells in living tissues. Apoptosis 7, 153–161.
Roos, A., Xu, W., Castellano, G., Nauta, A. J., Garred, P., Daha, M. R.,
van Kooten, C. (2004) Mini-review: a pivotal role for innate immunity in
the clearance of apoptotic cells. Eur. J. Immunol. 34, 921–929.
Manderson, A. P., Botto, M., Walport, M. J. (2004) The role of complement
in the development of systemic lupus erythematosus. Annu. Rev. Immunol.
22, 431– 456.
Quartier, P., Potter, P. K., Ehrenstein, M. R., Walport, M. J., Botto, M.
(2005) Predominant role of IgM-dependent activation of the classical
pathway in the clearance of dying cells by murine bone marrow-derived
macrophages in vitro. Eur. J. Immunol. 35, 252–260.
Peng, Y., Kowalewski, R., Kim, S., Elkon, K. B. (2005) The role of IgM
antibodies in the recognition and clearance of apoptotic cells. Mol. Immunol. 42, 781–787.
Verbovetski, I., Bychkov, H., Trahtemberg, U., Shapira, I., Hareuveni, M.,
Ben-Tal, O., Kutikov, I., Gill, O., Mevorach, D. (2002) Opsonization of
apoptotic cells by autologous iC3b facilitates clearance by immature
dendritic cells, down-regulates DR and CD86, and up-regulates CC chemokine receptor 7. J. Exp. Med. 196, 1553–1561.
38. Bondanza, A., Zimmermann, V. S., Dell’Antonio, G., Dal Cin, E., Capobianco, A., Sabbadini, M. G., Manfredi, A. A., Rovere-Querini, P. (2003)
Cutting edge: dissociation between autoimmune response and clinical
disease after vaccination with dendritic cells. J. Immunol. 170, 24 –27.
39. Bondanza, A., Zimmermann, V. S., Dell’Antonio, G., Cin, E. D., Balestrieri, G., Tincani, A., Amoura, Z., Piette, J. C., Sabbadini, M. G., RovereQuerini, P., Manfredi, A. A. (2004) Requirement of dying cells and
environmental adjuvants for the induction of autoimmunity. Arthritis
Rheum. 50, 1549 –1560.
40. Nauta, A. J., Bottazzi, B., Mantovani, A., Salvatori, G., Kishore, U.,
Schwaeble, W. J., Gingras, A. R., Tzima, S., Vivanco, F., Egido, J., Tijsma,
O., Hack, E. C., Daha, M. R., Roos, A. (2003) Biochemical and functional
characterization of the interaction between pentraxin 3 and C1q. Eur.
J. Immunol. 33, 465– 473.
41. Steinman, R. M., Hawiger, D., Nussenzweig, M. C. (2003) Tolerogenic
dendritic cells. Annu. Rev. Immunol. 21, 685–711.
42. Georgiev, M., Agle, L. M., Chu, J. L., Elkon, K. B., Ashany, D. (2005)
Mature dendritic cells readily break tolerance in normal mice but do not
lead to disease expression. Arthritis Rheum. 52, 225–238.
43. Manfredi, A. A., Sabbadini, M. G., Rovere-Querini, P. (2005) Dendritic
cells and the shadow line between autoimmunity and disease. Arthritis
Rheum. 52, 11–15.
Baruah et al. PTX3 modulates C1q effects on apoptotic cells
95