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of June 18, 2017.
Alternative Pathway Activation of
Complement by Shiga Toxin Promotes
Exuberant C3a Formation That Triggers
Microvascular Thrombosis
Marina Morigi, Miriam Galbusera, Sara Gastoldi, Monica
Locatelli, Simona Buelli, Anna Pezzotta, Chiara Pagani,
Marina Noris, Marco Gobbi, Matteo Stravalaci, Daniela
Rottoli, Francesco Tedesco, Giuseppe Remuzzi and
Carlamaria Zoja
Supplementary
Material
http://www.jimmunol.org/content/suppl/2011/06/06/jimmunol.110049
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 3 June 2011
http://www.jimmunol.org/content/early/2011/06/03/jimmun
ol.1100491
Published June 3, 2011, doi:10.4049/jimmunol.1100491
The Journal of Immunology
Alternative Pathway Activation of Complement by Shiga
Toxin Promotes Exuberant C3a Formation That Triggers
Microvascular Thrombosis
Marina Morigi,*,1 Miriam Galbusera,*,1 Sara Gastoldi,* Monica Locatelli,* Simona Buelli,*
Anna Pezzotta,* Chiara Pagani,* Marina Noris,* Marco Gobbi,† Matteo Stravalaci,†
Daniela Rottoli,* Francesco Tedesco,‡ Giuseppe Remuzzi,*,x and Carlamaria Zoja*
S
higa toxin (Stx)-producing Escherichia coli (STEC) O157:
H7 is a foodborne or waterborne pathogen, responsible for
worldwide spread of hemorrhagic colitis complicated by
diarrhea-associated hemolytic uremic syndrome (D+HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia,
and acute renal failure that mainly affects infants and small children (1–3). Death or end-stage renal disease occurs in ∼12% of
patients with D+HUS, and 25% of survivors demonstrate long-
*Mario Negri Institute for Pharmacological Research, Centro Anna Maria Astori,
Science and Technology Park Kilometro Rosso, 24126 Bergamo, Italy; †Mario
Negri Institute for Pharmacological Research, 20156 Milan, Italy; ‡Department of Physiology and Pathology, University of Trieste, 34127 Trieste, Italy; and xUnità di Nefrologia
e Dialisi, Azienda Ospedaliera Ospedali Riuniti di Bergamo, 24128 Bergamo, Italy
1
M.M. and M.G. contributed equally to this work.
Received for publication February 16, 2011. Accepted for publication April 19, 2011.
This work was supported in part by a Genzyme Renal Innovation Program grant.
M.L. is the recipient of a fellowship from Fondazione Aiuti per la Ricerca sulle
Malattie Rare, Bergamo, Italy.
Address correspondence to Carlamaria Zoja, Mario Negri Institute for Pharmacological Research, Centro Anna Maria Astori, Science and Technology Park Kilometro
Rosso, Via Stezzano, 87, 24126 Bergamo, Italy. E-mail address: carlamaria.zoja@
marionegri.it
The online version of this article contains supplemental material.
Abbreviations used in this article: Bf2/2, factor B-deficient; BUN, blood urea nitrogen;
C, complement; D+HUS, diarrhea-associated hemolytic uremic syndrome; HMEC,
human microvascular endothelial cells; HS, human serum; KIU, kallikrein inhibitor
unit; pM, picomolar; RU, resonance units; sCR-1, soluble C receptor-1; SPR, surface
plasmon resonance; STEC, Shiga toxin-producing Escherichia coli; Stx, Shiga toxin;
TM, thrombomodulin; t-PA, tissue-plasminogen activator; VWF, von Willebrand factor.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100491
term renal sequelae (4). Over the last two decades, E. coli O157:
H7 has been the cause of multiple outbreaks, becoming a public
health problem in both developed and developing countries (1,
5–7). Apart from supportive therapy, there are presently no specific treatments for D+HUS (8), and strategies including STECcomponent vaccines, Stx receptor mimics, and Abs against Stx
are still under investigation (9). After STEC ingestion, Stx is
transported in the circulation to the capillary bed of target organs,
including the kidney (5, 6). Glomerular endothelium that expresses the receptor globotriaosyl ceramide (Gb3)/CD77 (10, 11)
is the main target of the toxic effects of Stx1 and 2 (12), which
activate a cascade of signals contributing to vascular dysfunction,
leukocyte recruitment, and thrombus formation (13–19). We previously reported that Stx promoted von Willebrand factor (VWF)dependent thrombus growth on microvascular endothelium under
flow via upregulation of P-selectin (17). In patients with D+HUS,
elevated plasma levels of P-selectin were measured during the
acute phase, which reflected increased P-selectin expression by
activated endothelial cells and platelets (20). P-selectin overexpressed on activated platelets and transfected Chinese hamster
ovary cells had the capacity to activate the complement (C) system
by acting as C3b binding protein, thereby generating the anaphylatoxins C3a and C5a and the membrane attack complex (C5b9) (21). C3a and C5a, through binding to G-protein coupled receptors, elicit inflammatory responses such as cytokine production, adhesive molecule expression, and increased vascular permeability, leading to endothelial cell activation (22–24).
Whether C activation at renal endothelial level may contribute to
microangiopathic lesions in D+HUS has never been addressed.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Shiga toxin (Stx)-producing E.coli O157:H7 has become a global threat to public health; it is a primary cause of diarrheaassociated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute
renal failure with thrombi occluding renal microcirculation. In this study, we explored whether Stx triggers complementdependent microvascular thrombosis in in vitro and in vivo experimental settings of HUS. Stx induced on human microvascular
endothelial cell surface the expression of P-selectin, which bound and activated C3 via the alternative pathway, leading to
thrombus formation under flow. In the search for mechanisms linking complement activation and thrombosis, we found that
exuberant complement activation in response to Stx generated an increased amount of C3a that caused further endothelial Pselectin expression, thrombomodulin (TM) loss, and thrombus formation. In a murine model of HUS obtained by coinjection of
Stx2 and LPS and characterized by thrombocytopenia and renal dysfunction, upregulation of glomerular endothelial P-selectin
was associated with C3 and fibrin(ogen) deposits, platelet clumps, and reduced TM expression. Treatment with anti–P-selectin Ab
limited glomerular C3 accumulation. Factor B-deficient mice after Stx2/LPS exhibited less thrombocytopenia and were protected
against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the
alternative pathway in the glomerular thrombotic process in HUS mice. The functional role of C3a was documented by data
showing that glomerular fibrin(ogen), platelet clumps, and TM loss were markedly decreased in HUS mice receiving C3aR
antagonist. These results identify Stx-induced complement activation, via P-selectin, as a key mechanism of C3a-dependent
microvascular thrombosis in diarrhea-associated HUS. The Journal of Immunology, 2011, 187: 000–000.
2
Materials and Methods
Endothelial cell culture and incubation
The human microvascular endothelial cell line of dermal origin (i.e.,
HMEC-1, from Dr. Edwin Ades and Francisco J. Candal [Centers for
Disease Control and Prevention] and Dr. Thomas Lawley [Emory University]) (31) was cultured as described (17). Purified Stx1 (provided by
Dr. Helge Karch and Dr. Martina Bielaszewska, Institute for Hygiene,
University of Münster, Germany) (32) and commercially available Stx1
and Stx2 (Toxin Technology) were used. Human serum (HS) from a pool
of four healthy volunteers was used as a source of C. To remove platelet
microparticles, serum was filtered with a 0.22-mm filter (Millipore) before
each experiment (33). Fifty percent of HS was chosen as the most appropriate nontoxic dose on the basis of preliminary experiments evaluating
C3 deposits.
To study the effect of Stxs on C3 accumulation and thrombus formation,
HMEC-1 were treated with medium (control), purified Stx1, or commercially available Stx1 or Stx2 (50 picomolar [pM]) for 24 h in static conditions. Next, cells were perfused or not with 50% HS (3 min) followed or
not followed by whole blood in a parallel plate flow chamber at a constant
flow rate of 1500 s21 (60 dynes/cm2) for 3 min (Supplemental Fig. 1).
Soluble C receptor-1 (sCR-1; 100 mg/ml; CellDex) was incubated for
20 min with HS before perfusion on HMEC-1. To evaluate whether Stxinduced C activation occurred via the alternative pathway, HS was pretreated for 30 min with MgCl2-EGTA (5 mM; Sigma), an inhibitor of the
classical pathway of C activation.
P-selectin expression was examined in HMEC-1 incubated with purified
Stx1 (24 h, static condition). Costaining of P-selectin and C3 was performed
on Stx-treated HMEC-1 perfused with HS. Functional blocking anti-Pselectin Ab (25 mg/ml; R&D Systems) or the irrelevant anti-CD44 Ab
(25 mg/ml; Abcam), which binds to the endothelial cell surface, were
added for 20 min to HMEC-1 at the end of Stx challenge and to serum
(Supplemental Fig. 1). P-selectin was also blocked by PSGL-1 (20 mg/ml;
R&D System), the soluble ligand of P-selectin, added to both Stx-treated
cells (20 min before perfusion) and serum. Involvement of C3a in thrombus formation on Stx-treated HMEC-1 was investigated by adding the
C3aR antagonist SB290157 (1 mM; Calbiochem) to the cells before perfusion and to serum and blood (Supplemental Fig. 1). Endothelial expression of thrombomodulin (TM) and P-selectin was evaluated after
incubation with purified C3a (1 mM, Calbiochem) in static condition. The
serine protease inhibitor aprotinin (200 kallikrein inhibitor unit [KIU]/ml;
Sigma) was added to medium alone or to C3a. TM expression on HMEC-1
was also investigated after 6 h exposure to tissue-plasminogen activator
(t-PA; 10 mg/ml; Actyplase, Boehringer Ingeheim Pharma).
mg/ml; Dako), anti-human C5b-9 (27.6 mg/ml; Calbiochem) followed by
FITC-conjugated secondary Ab (13.6 mg/ml; Jackson Immunoresearch
Laboratories), anti-human P-selectin (20 mg/ml, R&D Systems) followed
by Cy3-conjugated secondary Ab (13.6 mg/ml; Jackson Immunoresearch
Laboratories), and anti-human TM (1:50; R&D Systems) followed by Cy3conjugated secondary Ab. Isotype-matched irrelevant Abs were used as
a negative control. Coverslips were examined under confocal inverted laser
microscopy (LSM 510 Meta; Zeiss, Jena, Germany). Fifteen fields per
sample were acquired, and the area of staining was quantified (pixel2/field)
using Image J software (National Institutes of Health, Bethesda, MD). For
colocalization studies, samples were imaged by confocal microscopy using
a z-scan (eight images acquired at 1.5-mm intervals by vertical projection).
TM expression was measured by counting cells with moderate or strong
intensity staining in 20 fields per sample. Data were expressed as a percentage of fluorescent cells per total cells in each field.
Surface plasmon resonance analysis
Binding studies were performed using the ProteOn XPR36 Protein Interaction Array system (Bio-Rad Laboratories), based on surface plasmon
resonance (SPR) technology. Purified human C3b (Complement Technology) was covalently immobilized onto one flow channel surface of a Proteon
GLC sensor chip (Bio-Rad), using amine coupling chemistry. The amount
of C3b covalently immobilized onto the surface, expressed in resonance
units (RU; 1 RU = 1 pg protein/mm2), was ∼7500 RU. A reference channel,
with no ligand immobilized, was always prepared in parallel. Different
concentrations, ranging from 21 to 310 nM, of recombinant human soluble
P-selectin (R&D System) were then injected simultaneously over immobilized C3b (or over the reference surface in parallel) at a rate of 30 ml/min
for 2 min (association phase). The dissociation phase was evaluated in the
following 10 min. The running buffer, also used to dilute analytes, was
either phosphate-buffered saline pH 7.4, 0.005% Tween 20 (Bio-Rad) or
Tris HCl (10 mM, pH 7.4 containing 150 mM NaCl, 1.2 mM CaCl2 and 1.2
mM MgCl2). All these assays were done at 25˚C.
Platelet adhesion assay under flow condition
Perfusion of heparinized whole blood (10 UI/ml) obtained from healthy
subjects (prelabeled with the fluorescent dye mepacrine, 10 mM) was
performed in a flow chamber (17). After 3 min of perfusion, the endothelial
cell monolayer was fixed in acetone. Fifteen images per sample of platelet
thrombi on endothelial cell surface were acquired by confocal inverted
laser microscope, and areas occupied by thrombi were evaluated by using
Image J software.
Experimental model of HUS
Male C57BL/6 mice were obtained from Charles River Italia. Bf2/2 mice of
C57BL/6 genetic background (34) and C57BL/6 wild type mice were a gift
from Dr. Marina Botto (Imperial College, London). For this study, Bf2/2
mice and control wild type littermates were obtained by heterozygote 3
heterozygote matings at the Mario Negri Institute. Genotypes were determined by PCR (34). Animal care and treatment were performed in
accordance with institutional guidelines in compliance with national and
international laws and policies. Animal studies were approved by the institutional animal care and use committees of the Mario Negri Institute
(Milan, Italy). HUS was induced in mice (26–28 g body weight) by i.p.
injection of Stx2 (200 ng; Toxin Technology) plus LPS (75 mg, O111:B4,
Sigma). Mice injected with saline served as controls. In parallel, some
mice were injected with Stx2 or LPS alone. Stx2 was chosen because it is
associated with HUS clinical isolates more often than Stx1 (6) and because
of the evidence that Stx2 injected in mice is more toxic and accumulates in
the kidney to a greater extent than Stx1 (35).
The dosing protocol was based on previous studies (16, 36). Mice were
sacrificed at 3, 6, 24, or 48 h after injection. Blood platelet count and renal
function measured by serum blood urea nitrogen (BUN; Reflotron test,
Roche Diagnostics) were assessed. In separate experiments, C57BL/6 mice
were given a 50-mg dose i.v. of a function-blocking anti-mouse P-selectin
Ab RB40.34 or an isotype-matched control Ab A110.1 (BD Pharmingen),
1 h before Stx2/LPS. A second dose was administered 6 h later; mice were
sacrificed 24 h after Stx2/LPS. The dose of RB40.34 was chosen on the
basis of previous studies (37). Other experiments included treatment of
mice with the C3aR antagonist SB290157 (15 mg/kg i.p.) or an equal
volume of vehicle (saline and dimethyl sulfoxide) four times per day (1 h
before and 1, 4, and 8 h after Stx2/LPS injection).
Immunofluorescence analysis of endothelial cells
Renal ultrastructural analysis
HMEC-1 at the end of the perfusions were fixed in 3% paraformaldehyde
and stained with the following Abs: FITC-conjugated anti-human C3c (11.3
Fragments of kidney tissue were processed as described (16). Ultrathin
sections were stained with uranyl acetate and lead citrate and were ex-
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There are anecdotes on reduced C3 and augmented C3b, C3c, and
C3d serum levels in patients with active D+HUS (25–27). High
plasma levels of Bb and C5b-9 were recently measured in 17
children with D+HUS, indicating C activation via alternative
pathway during the onset of D+HUS (28). Another study reported
that Stx2 activated C in the fluid phase in vitro (29). However, the
use of huge amounts of Stx2, together with the observation of no
active Stx in plasma of patients with D+HUS (30), raises concerns
about the pathophysiologic meaning of these results.
In this study, we investigated in vitro whether Stx activated C via
the alternative pathway by promoting binding of C3 on microvascular endothelial P-selectin under flow conditions. The role of C
in favoring thrombus formation on endothelial cells in response to
Stx was evaluated. A novel mechanism by which C3a generated by
Stx-induced C activation impaired endothelial thromboresistance
was explored. To validate the in vitro findings, we assessed the
causal role of glomerular P-selectin on C3 deposition in a mouse
model of HUS obtained by coinjection of Stx2 and LPS. The
involvement of C activation via the alternative pathway in the
development of microvascular thrombosis was studied applying the
HUS model to factor B-deficient (Bf2/2) mice. Finally, the contribution of C3a in the thrombogenic process induced by Stx was
assessed by blocking C3a receptor in HUS mice.
COMPLEMENT ACTIVATION IN STX-ASSOCIATED HUS
The Journal of Immunology
amined using a transmission electron microscope (Morgagni 268D; Philips,
Brno, Czech Republic).
Immunofluorescence analysis of renal tissue
Statistical analysis
Results are expressed as means 6 SEM. Data were analyzed by the
nonparametric Mann–Whitney or Kruskal–Wallis tests, or by ANOVA as
appropriate. The p values ,0.05 were considered statistically significant.
Results
Stx promotes endothelial C3 activation and deposition via the
alternative pathway
To test whether C activation by Stxs involves C3 activation and
deposition, we evaluated C3 deposition on endothelial cell surface
in response to the toxin. HMEC-1 were incubated with purified
Stx1 (50 pM, 24 h) and then perfused in a parallel plate flow
chamber (60 dynes/cm2, 3 min) with 50% HS as a source of C
(Supplemental Fig. 1). Under these conditions, monolayer integrity was preserved (Supplemental Fig. 2A). A marked increase of
C3 deposits on Stx1-treated cells was observed with respect to
unstimulated cells (Fig. 1A, 1B), indicating that endothelial cells
activated by Stx fixed C. Similar results were obtained using nonpurified, commercially available Stx1 and Stx2 (Supplemental Fig.
2B). No C5b-9 deposits were detected on Stx-treated HMEC-1 after
the short HS perfusion (not shown).
When HS was incubated with the sCR-1—an inhibitor of
classical, alternative, and lectin pathways of C activation—endothelial C3 deposits induced by Stx1 were abolished, indicating
specificity of the C3 signal (Fig. 1A, 1B). The role of the alternative pathway of C activation was documented by the finding that
C3 deposition induced by Stx1 was not modified by inhibiting the
classical pathway with MgCl2-EGTA added to HS before cell
perfusion (Fig. 1A, 1B).
C3 binds with high affinity to P-selectin expressed on
Stx-activated endothelial cells
Because we showed previously that Stx1 induced endothelial expression of P-selectin (17), an adhesive protein that also acts as
a C3b receptor on platelets and Chinese hamster ovary cells (21),
experiments were performed to evaluate whether P-selectin had
a role in C activation and C3 deposition on Stx-activated endothelial cells. First, we documented by immunofluorescence the
increase of P-selectin as a granular pattern on the endothelial
apical side after exposure to purified Stx1 (Supplemental Fig. 3A).
We then evaluated whether P-selectin could interact with C3 on
HMEC-1 exposed to Stx and perfused with HS. By confocal microscopy, the majority of C3 deposits colocalized with P-selectin
(Fig. 1C). Such a colocalization was visible in a large number of
FIGURE 1. Stx1 induces C3 activation and deposition on HMEC-1 via endothelial P-selectin. A, Images of C3 deposition on HMEC-1 exposed or not to
Stx1 (24 h) and then perfused at high-shear stress with 50% HS for 3 min. C deposition is inhibited by pretreating HS with sCR-1 or with MgCl2-EGTA
before perfusion (original magnification 3200). B, Quantification of C3 deposits. Values (mean 6 SEM) are expressed as fold increase versus control +
HS = 1 (n = 6 experiments). C, C3 (green) colocalizes with P-selectin (red) resulting in yellow overlap (merge) on HMEC-1 incubated with Stx1 and then
perfused with HS. Nuclei are stained with DAPI (blue). D, High-affinity binding of soluble P-selectin (s P-sel) to C3b by SPR. The left and middle panels
show the sensorgrams of two independent sensors (time course of the SPR signal in RU) obtained by simultaneously injecting three concentrations of
soluble P-selectin [P-sel] over sensor surfaces immobilizing C3b. The right panel shows the saturation isotherm. E, C3 deposition on Stx-treated HMEC-1
perfused with HS is reduced by anti–P-selectin (anti-Psel) Ab or PSGL-1. An Ab recognizing cell surface Ag (CD44) is used as irrelevant (irrel). Values
(mean 6 SEM) are expressed as fold increase versus control + HS = 1 (n = 4 experiments). ˚p , 0.01 versus control + HS, #p , 0.01 versus Stx1 + irrel
Ab + HS, *p , 0.01 versus Stx1 + HS.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Periodate lysine paraformaldehyde (PLP)-fixed frozen sections (3 mm)
were incubated with FITC-conjugated goat anti-rat fibrin(ogen) Ab (1:40;
Nordic Immunology) and with rhodamine WGA lectin (Vector Laboratories) dissolved in DAPI. Fibrin(ogen) deposition in glomerular capillary
walls was assessed by confocal microscopy and scored from 0 to 3
(0, absent or trace; 1, weak granular, discontinuous staining; 2, strongly
staining; 3, occasional obliteration). P-selectin expression was evaluated in
frozen sections incubated with anti-mouse P-selectin Ab (3 mg/ml; BD
Pharmingen) or with isotype-matched irrelevant Ab, followed by FITCconjugated secondary Ab (1:150; Sigma-Aldrich). To determine the location of P-selectin in the glomerulus, adjacent sections were incubated
with rabbit anti-VWF (1:100; Dako) followed by Cy3-conjugated antirabbit Abs (60 mg/ml; Jackson Immunoresearch Laboratories). Negative
controls were obtained with secondary Ab alone. C3 deposits were evaluated by staining with FITC-conjugated anti-mouse C3 Ab (10 mg/ml;
Cappel). For C9 staining, rabbit anti-rat C9 (1:200; a gift from Paul
Morgan, Cardiff, U.K.) and Cy3-conjugated secondary Abs (30 mg/ml;
Jackson Immunoresearch Laboratories) were used. Isotype-matched irrelevant Abs were used as negative controls. Glomerular C3 and C9 staining
was scored from 0 to 3 (0, no staining or traces [,5%]; 1, staining
in ,25% of the glomerular tuft; 2, staining affecting 26 to 50%; 3, staining
in .50%). TM was detected in PLP-fixed sections using rabbit anti-TM
Ab (1:25; Santa Cruz) followed by Cy3-conjugated goat anti-rabbit IgG
(15mg/ml; Jackson Immunoresearch Laboratories). Glomerular TM expression was estimated using Image J software.
3
4
COMPLEMENT ACTIVATION IN STX-ASSOCIATED HUS
Complement activation favors thrombus formation on
Stx-treated endothelial cells
To establish whether C activation and deposition on HMEC-1 could
be instrumental to thrombus formation, Stx1-treated cells were
exposed to a double perfusion consisting of HS, followed by whole
human blood prelabeled with the fluorescent dye mepacrine, or to
a single perfusion with blood alone (Supplemental Fig. 1). When
Stx-1–treated cells were perfused with HS and blood, a more intense deposition of C3 and increased endothelial surface area
covered by thrombi were detected, as compared with unstimulated
cells (Table I). Notably, after double perfusion, C3 deposits were
followed by thrombi detected on the endothelial surface to a significantly greater extent than on Stx-treated cells subjected to
single blood perfusion (Table I). A representative image of organized thrombi formed on the endothelial cell monolayer is shown
in Supplemental Fig. 3B. Pretreatment of HS and blood with sCR1 significantly (p , 0.01) limited platelet thrombi on HMEC-1
exposed to the toxin, indicating a causal link between C3 deposition and thrombus formation (Fig. 2A, 2B).
We next examined whether P-selectin blockade could limit Cdependent thrombus formation on endothelial cells in response
to Stx. P-selectin inhibition in Stx1-treated cells exposed to double
perfusion of HS and blood resulted in decreased C3 accumulation
(Supplemental Fig. 3C), followed by a significant reduction in
platelet thrombi (Fig. 2A, 2B). Irrelevant Ab had no effect on
Stx-induced C3 deposition (Supplemental Fig. 3C) and thrombus
formation (Fig. 2A, 2B). These data indicate that endothelial Pselectin upregulated by Stx is instrumental for C activation and
C3-dependent thrombus formation on microvascular endothelium.
C3a generated by Stx-induced C activation impairs endothelial
thromboresistance
To discover how C activation triggers thrombus formation on
endothelial cells, we studied the effect of the anaphylatoxin
C3a—a breakdown product generated during C activation and
C3 deposition—on endothelial thromboresistance properties. We
focused on TM, a glycoprotein highly expressed on endothelial
cell surface, with cytoprotective and antithrombotic effects (40).
TM was constitutively expressed on HMEC-1 (percentage of
fluorescent cells with moderate and strong intensity: 54.2 6 4.3
and 6.7 6 1.1%). Treatment with Stx1 alone slightly reduced TM
expression, which further decreased after HS perfusion (Fig. 3A,
Supplemental Fig. 3D). Addition of the C3aR antagonist
SB290157 on Stx-treated HMEC-1 perfused with HS prevented
TM loss (Fig. 3A, Supplemental Fig. 3D). Providing further evidence for a role of this anaphylatoxin, we found a marked reduction of TM expression on the surface of HMEC-1 exposed to
exogenous purified C3a (Fig. 3B). Because the cleavage of TM is
thought to be dependent by serine protease activity, we examined tPA, a serine protease present in endothelial Weibel-Palade bodies
possibly released upon C3a stimulation (41). Blockade of t-PA
activity by treatment of HMEC-1 with the serine protease inhibitor aprotinin prevented TM shedding in response to exogenous
C3a (Fig. 3B). Aprotinin alone as a control did not affect the
constitutive expression of TM (percentage of fluorescent cells with
moderate and strong intensity: aprotinin, 67.7 6 5.3 and 7.6 6 0.6
versus control, 62.4 6 4.9 and 8.3 6 0.7). Consistently, the addition
of t-PA to HMEC-1 significantly (p < 0.01) reduced TM expression
on the cell surface (percentage of fluorescent cells with moderate
and strong intensity: t-PA, 51.3 6 2.1 and 1.7 6 0.3 versus control,
62.2 6 1.2 and 6.6 6 0.9). The finding that C3a may favor exocytosis of the Weibel-Palade body’s content was also supported by
the increased apical staining of P-selectin on C3a-treated HMEC-1
(Fig. 3C). The effects induced by C3a were critical for thrombus
formation, as evidenced by the significant (p , 0.01) reduction of
Table I. Stx1 induces complement deposition and thrombus formation
on HMEC-1 under flow condition
Control
Stx1 (50 pM)
Single Perfusion
—
+ Blood Perfusion
C3 deposits
Thrombi
1.0
1.0
2.9 6 0.7a
7.5 6 3.4a
Control
Stx1 (50 pM)
Double Perfusion
+50% HS
+ Blood Perfusion
2.5 6 0.8a 10.5 6 6.4a,b,c
5.2 6 1.5a 29.0 6 18.8a,b,d
Area covered by C3 deposits or thrombi is expressed as fold increase over control
(single perfusion) imposed as 1.0. Data are mean 6 SEM.
a
p , 0.01 versus control plus blood perfusion.
b
p , 0.01 versus control plus HS plus blood perfusion.
c
p , 0.01 versus Stx1 plus blood perfusion.
d
p , 0.05 versus Stx1 plus blood perfusion.
FIGURE 2. C3 bound to P-selectin favors thrombus formation on endothelial cells in response to Stx. A, Representative images of fluorescent
thrombi on control or Stx1-treated HMEC-1 perfused with HS and blood
pretreated or not with sCR-1 before perfusion. Stx-treated HMEC-1 are
also exposed to irrelevant (irrel) or anti-Psel Abs before double perfusion
(original magnification 3200). B, Quantification of fluorescent thrombi
(mean 6 SEM) expressed as pixel2 3 103 (n = 6 experiments). ˚p , 0.01
versus control + HS, *p , 0.01 versus Stx1 + HS, #p , 0.01 versus Stx1 +
irrel Ab + HS.
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Stx-treated endothelial cells. Moreover, binding studies with SPR
showed a specific interaction between C3b and P-selectin. Fig. 1D
(left and middle panels) shows the sensorgrams (time-course of
SPR signal) obtained when flowing different concentrations of
soluble P-selectin over C3b immobilized on the sensor chip. A
dose-dependent binding signal was detected. A clearly biphasic
dissociation curve did not fit the simplest 1:1 interaction. The
interaction is more complex, suggesting a conformational change
in C3b molecules once bound to P-selectin, or the presence of
cooperativity among the multiple binding sites (SCR domains)
present on P-selectin, as previously described for binding of C3b
to CR1 (38, 39). However, the plot of the equilibrium responses
could be fitted by a simple saturation isotherm, allowing the estimation of a Kd value of 77 6 28 nM (Fig. 1D, right panel).
Next, we documented that P-selectin mediated C3 binding on
the surface of Stx-treated HMEC-1. Cell exposure to functional
blocking anti–P-selectin Ab, before and during HS perfusion, led
to a significant inhibition of C3 deposits in response to Stx1 (Fig.
1E), whereas the addition of irrelevant Ab had no effect. Moreover, a strong decrease of C3 accumulation was observed when
PSGL-1, the soluble ligand of P-selectin, was added to HMEC-1
before and during HS perfusion (Fig. 1E).
The Journal of Immunology
platelet thrombi on Stx-treated HMEC-1 perfused with HS and
blood in the presence of the C3aR antagonist (Fig. 3D).
Glomerular C activation and deposition, via endothelial
P-selectin, in a murine model of HUS
We have recently (16) established a murine model of HUS induced
by coinjection of Stx2 and LPS on the basis of the evidence that
two virulence factors of enterohemorrhagic E. coli, Stx and LPS,
are required to induce cardinal signs of clinical HUS in mice (42).
C57BL/6 mice treated with Stx2/LPS showed a dramatic decrease
of the blood platelet count during time relative to the baseline
(Fig. 4A). Renal function was severely impaired, as indicated by
high serum BUN levels (Fig. 4B). Kidneys from Stx2/LPS-treated
mice showed, over time, abundant fibrin(ogen) deposition in the
glomerular capillary loops (Fig. 4C), as compared with controls
given saline (score: saline, 0.17 6 0.07 and Stx2/LPS, 6 h 0.17 6
0.03, 24 h 1.48 6 0.16, 48 h 2.08 6 0.06; p , 0.01 Stx2/LPS 24
and 48 h versus saline). Glomerular expression of TM was reduced by 31.1 6 4.3% in Stx2/LPS-treated mice compared with
control mice at 24 h (p , 0.01). These data are consistent with the
presence of intracapillary glomerular platelet clumps as revealed
FIGURE 4. C57BL/6 mice treated with Stx2/LPS develop thrombocytopenia, renal failure, and glomerular thrombosis. A, Platelet count
decreases during time in mice coinjected i.p. with Stx2 and LPS (n = 7
mice at each time point). Data are presented as mean 6 SEM. B, These
mice exhibit elevated BUN levels. ˚p , 0.01 versus basal. C, Fibrin(ogen)
deposits (green) increase during time in the glomeruli of mice treated with
Stx2/LPS. Nuclei and cell membranes are counteracted with DAPI (blue)
and rhodamine WGA-lectin (red), respectively (original magnification
3630). D, Representative electron micrographs of glomeruli from mice
given saline (control) or Stx2/LPS. Platelet (P) clumps in the capillary
loops, focal endothelial swelling (arrow), marked infiltration of polymorphonuclear cells (PMN) and rare monocytes (M) are evident in Stx2/
LPS-treated mice 24 h after injection (original magnification 314,000 and
33500).
by transmission electron microscopy (Fig. 4D). Other ultrastructural changes included focal endothelial swelling and polymorphonuclear cell infiltrates.
In animals injected with Stx2 alone, platelet count remained
within the normal range, whereas it decreased after LPS (16)
(Supplemental Fig. 4A), in accordance with the expression of TLR4
on platelets (43). Each agent caused slight increases in BUN
(Supplemental Fig. 4B), mild glomerular endothelial swelling, and
low polymorphonuclear cell accumulation (data not shown).
Next, we evaluated the expression of P-selectin in glomeruli of
Stx2/LPS-treated mice. Positive staining for P-selectin was observed in glomerular endothelial cells at 3 (Fig. 5A) and 6 h (not
shown) after Stx2/LPS injection. The endothelial localization of
P-selectin was documented by evidence of labeling of the endothelial marker VWF on the same cells in serial sections (Fig. 5A).
No P-selectin staining was present in the glomeruli of controls
(Fig. 5A) or after treatment with LPS alone (data not shown).
Excessive glomerular C3 (Fig. 5B) and C9 (Supplemental Fig. 4D)
deposits followed P-selectin upregulation, with an irregular distribution along the glomerular capillary wall, starting 6 h after
Stx2/LPS. In the kidneys of control mice, C3 staining was confined to a linear reactivity along the Bowman’s capsule (44, 45). In
the glomeruli of mice treated with LPS alone, C3 deposits were
absent or minimal (Supplemental Fig. 4C). The role of P-selectin
in favoring glomerular C deposits in response to Stx2/LPS was
documented by finding that treatment with blocking P-selectin Ab
effectively limited C3 deposits compared to mice given the irrelevant Ab (Fig. 5C).
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FIGURE 3. C3a generated by Stx-induced C activation impairs the
endothelial antithrombogenic properties. A, TM expression on HMEC-1
exposed to control medium or Stx1 (24 h) and then perfused with medium
or 50% HS. Addition of the C3aR antagonist (SB290157) prevents TM
loss in response to Stx1 on HMEC-1 surface. Values (mean 6 SEM) are
expressed as percentage of fluorescent cells per total cells in each field (n =
5 experiments). ˚p , 0.05, ˚˚p , 0.01 versus corresponding control; *p ,
0.05, **p , 0.01 versus corresponding Stx1 + HS. B, TM expression on
HMEC-1 decreases upon incubation with purified C3a (6 h). Treatment of
HMEC-1 with aprotinin (200 KIU/ml) prevents TM loss in response to
C3a. Values (mean 6 SEM) are expressed as a percentage of fluorescent
cells per total cells in each field (n = 5 experiments). ˚p , 0.01 versus
corresponding control; *p , 0.01 versus corresponding C3a. C, Representative images showing that C3a (3 h) increases P-selectin expression on
HMEC-1 surface with respect to unstimulated cells. Nuclei are stained
with DAPI (original magnification 3200). D, Thrombus formation on
HMEC-1 treated with Stx-1 and then perfused with whole blood is
markedly decreased by a C3aR antagonist. Area covered by thrombi is
expressed as pixel2 3 103 (n = 4 experiments). ˚p , 0.01 versus control, *p ,
0.01 versus Stx1.
5
6
FIGURE 6. Activation of C, via the alternative pathway, favors C3adependent glomerular prothrombotic state in mice with HUS. A, In Bf2/2
mice the fall in platelet count after Stx2/LPS treatment is significantly less
than in wild type littermates. B, Bf2/2 mice are protected from renal
function impairment, assessed as serum BUN, with respect to wild type
after Stx2/LPS (n = 6–10 mice per group). Data are mean 6 SEM. ˚p ,
0.05, ˚˚p , 0.01 versus wild type. C, Few glomerular fibrin(ogen) deposits
in Bf2/2 mice given Stx2/LPS are present versus wild type mice exhibiting
abundant fibrin(ogen) staining (green). Nuclei are stained with DAPI
(blue), and cell membranes are stained with rhodamine WGA-lectin (red;
original magnification 3630). D, Representative images of TM expression
in glomeruli of wild type and Bf2/2 mice evaluated 48 h after saline or
Stx2/LPS injection. TM expression decreases in wild type but not in Bf2/2
mice after Stx2/LPS injection (original magnification 3630).
that C3aR antagonist limited (p , 0.01) glomerular TM loss after
Stx2/LPS (Fig 7B). A scheme of the mechanisms suggested by the
present data is shown in Fig. 8.
Discussion
C3a derived by C activation, via the alternative pathway,
contributes to a glomerular prothrombotic state in HUS mice
To establish the involvement of C activation in the microvascular
thrombotic process, we used mice deficient for factor B, a zymogen
that carries the catalytic site of the alternative pathway C3 and C5
convertases (46). After Stx2/LPS treatment, Bf2/2 mice exhibited
less thrombocytopenia and were protected against renal function
impairment compared with wild type littermate mice (Fig. 6A,
6B). The blockade of the alternative pathway of C activation also
resulted in fewer fibrin(ogen) deposits (Fig. 6C; 48 h, score: saline, Bf2/2 0.19 6 0.04 versus wild type 0.22 6 0.06; Stx2/LPS,
Bf2/2 0.70 6 0.23 versus wild type 2.15 6 0.09; p , 0.01). No
C3 deposits or platelet clumps were found in the glomeruli of
Stx2/LPS-treated Bf2/2 mice. Moreover, while glomerular TM
expression was reduced by 42.8 6 7.3% at 48 h in wild type mice
given Stx2/LPS compared with saline-treated control mice (Fig.
6D), no changes were observed in Bf2/2 mice regarding corresponding controls (Fig. 6D), thereby suggesting the causal role of
C activation in the loss of thromboresistance.
The contribution of C3a in the thrombogenic process induced by
Stx was evaluated by blocking C3aR in HUS mice. Administration
of a C3aR antagonist to Stx2/LPS-treated C57BL/6 mice significantly (p , 0.05) limited glomerular fibrin(ogen) deposition and
platelet clumps (not shown) compared to mice receiving vehicle
(Fig. 7A). In this study, we also assessed TM expression and found
To our knowledge, this study is the first to demonstrate by multifaceted approaches that Stx triggers microvascular thrombosis
via the alternative pathway of C activation and indicates C3a as
the key factor in the development of the thrombotic processes
in HUS. Previous findings of low serum C3 levels (25, 27) and
augmented breakdown C products (27, 28) suggested the involve-
FIGURE 7. C3a contributes to the thrombogenic process in mice with
HUS. A, C3aR blockade limits fibrin(ogen) deposition in the glomeruli of
C57BL/6 HUS mice. Mice are treated with C3aR antagonist (SB290157)
or with vehicle (n = 4 mice per group), and sacrificed after 24 h. Control
mice receive saline (n = 4). Fibrin(ogen) deposits are assessed by semiquantitative score. Data are mean 6 SEM. B, Loss of glomerular TM
expression after Stx2/LPS injection is limited by treatment with a C3aR
antagonist. Data are mean 6 SEM of n = 4 mice per group. ˚˚p , 0.01
versus control, *p , 0.05, **p , 0.01 versus vehicle.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 5. Glomerular endothelial expression of P-selectin causes C3
deposits in C57BL/6 mice treated with Stx2/LPS. A, Staining of P-selectin
in glomeruli of mice injected with saline (control) or Stx2/LPS at 3 h.
VWF staining in an adjacent renal section of Stx2/LPS-treated mouse
reflects localization of P-selectin in glomerular endothelial cells (arrow).
Original magnification 3630. B, Glomerular C3 staining increases 6 h
after Stx2/LPS treatment with respect to saline (control) as assessed by
semiquantitative score. Data are mean 6 SEM (n = 7 mice for each time
point). Original magnification 3630. C, P-selectin blockade limits glomerular C3 deposits in C57BL/6 HUS mice. Mice are injected with the
anti-P-selectin (anti-Psel) or irrelevant (irrel) Abs and sacrificed after 24 h
(n = 5 mice for group). Control mice receive saline (n = 5). Data are mean 6
SEM. ˚p , 0.05, ˚˚p , 0.01 versus control, *p , 0.05 versus irrel Ab.
COMPLEMENT ACTIVATION IN STX-ASSOCIATED HUS
The Journal of Immunology
7
FIGURE 8. Stx alters endothelial thromboresistance by triggering exuberant C activation via C3a, leading to microvascular thrombosis. The proposed
scheme summarizes the sequence of events through which Stx binds to its specific endothelial receptor Gb3 and favors surface mobilization of P-selectin
and thrombus formation under flow by either interacting with platelets or by binding and/or activating C proteins. Excessive C3 activation (dependent on
increased C3 convertase assembly and activity) in response to Stx generates an increased amount of the anaphylatoxin C3a, which interacts with the specific
endothelial C3aR, thereby potentiating P-selectin expression and t-PA-dependent TM shedding with final thrombus formation.
However, we cannot exclude a contribution of C5a in the prothrombotic effects triggered by Stx, considering that C5a shares
with C3a various inflammatory activities on endothelial cells
(22–24). It is known that TM, a cofactor of thrombin in the activation of protein C, regulates hemostasis and prevents local fibrin
formation (40, 48, 49). Other properties of TM include antiinflammatory and cytoprotective activities (40). It is therefore
conceivable that shedding of TM from the endothelial surface can
contribute to an increased risk of thrombosis associated with inflammation. In this regard, increased levels of soluble TM have
been detected in the plasma of patients with disseminated intravascular coagulation syndrome, pulmonary thromboembolism, and
chronic renal disorders (50). Little is known about the mechanisms
underlying TM shedding and the proteases involved in its cleavage. Serine proteases were reported to be implicated in thiolinduced proteolytic cleavage of TM in association with damage
of endothelial cell membrane (51, 52). Based on the evidence that
t-PA is the serine protease stored in Weibel-Palade bodies of endothelial cells, we show that the inhibition of t-PA by aprotinin
markedly limited C3a-dependent TM loss on microvascular endothelial cells. This finding was also supported by the direct effect
of exogenously added t-PA on TM shedding in HMEC-1. Our
findings shed light on the nexus among endothelium, C, and
thrombosis, considering that previous knowledge of the prothrombotic effect of C was restricted to studies on platelets (53).
Proteins of the C system, including C3, C5, C6, C7, C8, and C9,
significantly enhanced thrombin-induced aggregation and secretion in human platelets. Furthermore, the anaphylatoxin C3a
was shown to directly induce aggregation of human platelets and
release of serotonin (53). That the mechanisms of thrombosis may
be related to C activation has been also suggested by studies in
patients with familial or sporadic forms of atypical HUS or paroxysmal nocturnal hemoglobinuria, both characterized by deficiency
in C-regulatory molecules, platelet hyperactivity, and thrombosis
(33, 54).
The mechanisms proposed in vitro have been validated in
a murine model of Stx-associated HUS, which closely mimics
human D+HUS. The development of a mouse model able to reproduce the features of human HUS has been problematic, and
studies using E. coli O157:H7, purified Stx1 or Stx2 with or
without LPS (16, 35, 36, 42, 55, 56), have given controversial
results. The role of E. coli LPS has been recognized in the molecular pathogenesis of D+HUS, based on the evidence of serum
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
ment of C in D+HUS, although the functional relevance of these
abnormalities to the thrombotic process was not addressed. Our
data provide evidence that Stx1 promotes C3 activation and deposition on microvascular endothelial cells via the alternative
pathway, because no inhibition was found by blocking the classical pathway. A relevant observation is that C3 deposition favors
thrombus growth under flow on the surface of Stx-treated HMEC1, which is prevented by the C inhibitor sCR-1. This mechanism
applies also in vivo, because Bf2/2 mice with impaired C activation via the alternative pathway were protected from renal
prothrombotic processes when injected with Stx2/LPS compared
to wild-type mice that showed intraglomerular C3 and fibrin(ogen)
deposition, TM loss, and platelet clumps associated with severe
thrombocytopenia.
In the search for mechanisms underlying C activation elicited
by Stx and ultimately leading to thrombus formation, we have
identified P-selectin as an endothelial binding site for C3. As shown
previously (17) and documented in this study, upon challenge with
Stx the microvascular endothelium expresses P-selectin, an adhesive molecule implicated in VWF-dependent platelet deposition (47). Rather than simply a marker of endothelial activation, Pselectin acts as a specific ligand for C3 through high-affinity
binding, based on the evidence that it colocalizes with the majority of C3 deposits on Stx-treated HMEC-1. P-selectin is crucial
in C-dependent thrombus formation because functional blocking
of P-selectin leads to a marked reduction of C3 accumulation and
limits thrombus extension on Stx-treated endothelial cells. The
processes by which P-selectin favors C activation on endothelial
cells are still poorly defined. It is possible that C3b bound to Pselectin may undergo conformational changes that either prevent
its inactivation by C regulatory molecules or favor C3b interaction
with factor B, thus leading to assembly of alternative pathway C3
convertase.
We have also provided a novel mechanism through which activated C proteins foster thrombosis on Stx-activated endothelium.
In our experimental setting, Stxs promoted C3 activation and deposition that resulted in C3a generation; therefore, we focused on
the endothelial effect of this anaphylatoxin. C3a powerfully increased P-selectin expression and reduced TM on the HMEC-1
surface, indicating a major role for C3a as a driving factor in altering endothelial thromboresistance. The proof of this concept
rests on data showing that TM loss and thrombus growth on Stxtreated endothelium were markedly reduced by C3aR antagonist.
8
Acknowledgments
We thank Dr. A. Benigni for helpful suggestions and criticism, D. Corna and
C. Zanchi for technical assistance, Dr. E. Ades and Dr. F.J. Candal (Centers
for Disease Control and Prevention, Atlanta, GA) and Dr. T. Lawley (Emory
University) for developing developed HMEC-1, Dr. H. Karch and Dr.
M. Bielaszewska (Institute for Hygiene, University of Münster, Germany)
for providing purified Stx1, Dr. M. Botto (Imperial College, London, U.K.)
for the gift of the Bf2/2 mice, and CellDex (Needham, MA) for providing
sCR-1.
Disclosures
The authors have no financial conflicts of interest.
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