Human CRP Defends against the Toxicity of
Circulating Histones
This information is current as
of June 18, 2017.
Simon T. Abrams, Nan Zhang, Caroline Dart, Susan Siyu
Wang, Jecko Thachil, Yunyan Guan, Guozheng Wang and
Cheng-Hock Toh
J Immunol 2013; 191:2495-2502; Prepublished online 26
July 2013;
doi: 10.4049/jimmunol.1203181
http://www.jimmunol.org/content/191/5/2495
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Supplementary
Material
The Journal of Immunology
Human CRP Defends against the Toxicity of Circulating
Histones
Simon T. Abrams,*,†,1 Nan Zhang,‡,1 Caroline Dart,x Susan Siyu Wang,{ Jecko Thachil,†
Yunyan Guan,‖ Guozheng Wang,*,† and Cheng-Hock Toh*,†
T
he pentraxin protein, C-reative protein (CRP), has long
been recognized as a major acute-phase protein involved
in both innate and adaptive immunity (1, 2). It commonly
exists in pentameric form (3), but denatured forms have also been
described with different functions (4). Earlier studies have demonstrated that CRP activates complement, opsonizes pathogens
such as Streptococcus pneumoniae, and facilitates their clearance
(5–7). CRP binds FcgRs and FcaRI receptors to activate neutrophils and phagocytosis (8–11). CRP also enhances uptake and
presentation of bacterial Ags by dendritic cells to stimulate
adaptive immunity (12). Overall, CRP plays a crucial role in host
defense against bacterial infection.
*Department of Blood Sciences, Royal Liverpool and Broadgreen University Hospitals National Health Service Trust, Liverpool L7 8XP, United Kingdom;
†
Institute of Infection and Global Health, University of Liverpool, Liverpool L69
3GA, United Kingdom; ‡The Medical School, Southeast University, Nanjing 210009,
China; xInstitute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB,
United Kingdom; {School of Clinical Medicine, University of Cambridge, Cambridge CB2 0SP, United Kingdom; and ‖Intensive Care Unit, Wuxi Traditional Chinese Medicine Hospital, Wuxi 214001, China
1
S.T.A. and N.Z. contributed equally to this study.
Received for publication November 19, 2012. Accepted for publication June 24,
2013.
This work was supported by the National Institute of Health Research and the North
West Development Agency.
Address correspondence and reprint requests to Dr. Guozheng Wang and Cheng-Hock
Toh, University of Liverpool, 216G Ronald Ross Building, Derby Street, Liverpool L69 7BE, U.K. E-mail addresses: [email protected] (G.W.) and [email protected].
uk (C.-H.T.)
The online version of this article contains supplemental material.
Abbreviations used in this article: ahscFv, antihistone single-chain variable fragment;
CRP, C-reactive protein; dCRP, denatured C-reactive protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, propidium iodide; PS, phosphatidylserine;
sTM, soluble thrombomodulin; TAT, thrombin–antithrombin.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203181
CRP is also significantly upregulated in many clinical scenarios
without infection, such as trauma, pancreatitis, myocardial infarction, neoplasia, and inflammatory disorders. However, its use in
clinical assessment is often as a nonspecific marker, and its role(s)
in these diseases is largely unknown. In addition to opsonizing
pathogens, CRP has also been found to opsonize damaged cells,
including nuclear breakdown products, such as chromatin and
histones to facilitate their clearance (7, 13–16). Because the presence of circulating nuclear breakdown products has been linked to
the development of autoimmune diseases (17), CRP has been
proposed as a therapeutic reagent in mouse models of these disorders (18–20).
Recently, histone toxicity has been demonstrated in vitro and in
mice (21–27), which prompted us to investigate the potential of
CRP in neutralizing these toxic effects. In this article, we demonstrate that CRP detoxifies histones both in vitro and in vivo with
the major protective mechanism being through CRP–histone complex formation to block histone–cell membrane integration and the
consequent increase in intracellular calcium.
Materials and Methods
Patients
Sera from patients on the Intensive Care Unit at the Royal Liverpool University Hospital, United Kingdom, with diagnoses of severe trauma, necrotizing
pancreatitis, and severe sepsis were collected in accordance with protocol
approved by the Local Research Ethics Committee and the Hospital Trust.
Animals
C57/BL6 male mice of average weight ∼22 g from the Shanghai Laboratory Animal Center (Shanghai, China) were housed and used at the
Research Center of Gene Modified Mice, State Education Ministry Laboratory of Developmental Genes & Human Diseases, Southeast University,
China. All procedures were performed according to state laws and monitored by local inspectors. These were also in compliance with British
Home Office laws. Histones and CRP were injected through the tail veins
with outcome monitored for up to 6 d in survival assays. To compare the
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C-reactive protein (CRP) is an acute-phase protein that plays an important defensive role in innate immunity against bacterial
infection, but it is also upregulated in many noninfectious diseases. The generic function of this highly conserved molecule in diseases
that range from infection, inflammation, trauma, and malignancy is not well understood. In this article, we demonstrate that CRP
defends the human body against the toxicity of histones released into the circulation after extensive cell death. In vitro, CRP significantly alleviates histone-induced endothelial cell damage, permeability increase, and platelet aggregation. In vivo, CRP rescues
mice challenged with lethal doses of histones by inhibiting endothelial damage, vascular permeability, and coagulation activation, as
reflected by significant reductions in lung edema, hemorrhage, and thrombosis. In patients, elevation of CRP significantly increases
the capacity to neutralize extracellular histones in the circulation. We have also confirmed that CRP interacts with individual histones in vitro and forms CRP–histone complexes in serum from patients with both elevated CRP and histones. CRP is able to
compete with phospholipid-containing liposomes for the binding to histones. This explains how CRP prevents histones from
integrating into cell membranes, which would otherwise induce calcium influx as the major mechanism of cytotoxicity caused by
extracellular histones. Because histone elevation occurs in the acute phase of numerous critical illnesses associated with extensive
cell death, CRP detoxification of circulating histones would be a generic host defense mechanism in humans. The Journal of
Immunology, 2013, 191: 2495–2502.
2496
pathological changes of different treatments, we euthanized mice at 1 or
4 h after injection. Blood was collected and serum isolated within 2 h and
stored at 280˚C. Organs were fixed in 4% (w/v) paraformaldehyde for
24 h and then stored in 70% (v/v) ethanol until embedded and sectioned.
Tissue culture
EAhy926 (human endothelial) cell line was cultured in DMEM (Sigma)
supplemented with 20% (v/v) FBS (Sigma). HUVECs were isolated as
described previously (28) and cultured in DMEM supplemented with 20%
(v/v) FBS and 5 ng/ml epidermal growth factor (Invitrogen) for use within
three passages.
Reagents
Human CRP (Merck) was extensively dialyzed and concentrated. After
confirmation of the pentamer form of each protein by HPLC gel filtration,
CRP at final concentrations of 1, 2, or 4 mg/ml was stored at 280˚C.
Antihistone single-chain variable fragment (ahscFv) was expressed in
E. coli and purified, as previously described, with demonstration of antihistone specificity (27). LPS contamination was monitored using E-Toxate
CRP REDUCES HISTONE TOXICITY IN VITRO AND IN VIVO
reagents (Sigma). Recombinant histones (New England Biolabs) and calf
thymus histones (Roche) were also monitored using E-Toxate.
Histone cytotoxicity assay
A propidium iodide (PI) method was used, as previously described (29). In
brief, EAhy926 cells were grown to 70–90% confluence and treated with
histones or histones preincubated with CRP or activated protein C for 30
min in DMEM supplemented with 2% (v/v) FBS. After 1h of incubation,
the medium was removed to a fresh tube, and the remaining cells were
detached using Versene and transferred to the same tube. The cells were
pelleted, washed, and fixed in 70% (v/v) ethanol for 30 min at 220˚C
before staining with 20 mg/ml PI. Flow cytometric analysis of PI-stained
damaged nuclei results in a broad peak of hypodiploid particles, clearly
separated from the distinct diploid DNA peak of viable cells.
Quantification of circulating histones, histone–CRP complexes,
CRP, soluble thrombomodulin, and thrombin–antithrombin
A Cell Death Detection ELISAPLUS kit (Roche Diagnostics) was modified
for measuring histone–CRP complexes in serum by replacing anti-DNA
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FIGURE 1. CRP reduces histone-induced endothelial cell damage and vascular hyperpermeability. (A) The toxic effects of different histone concentrations
on EAhy926 cells in the presence of 2% (v/v) FBS. Means 6 SD are shown. *p , 0.05 compared with untreated (ANOVA). (B) Dose response of CRP in
protecting EAhy926 cells treated with 40 mg/ml histones in 2% (v/v) FBS medium. Means 6 SD are shown, and ANOVA test showed CRP from 50 mg/ml
significantly increased cell survival rates (p , 0.05). (C) Comparison of histone (40 mg/ml) toxicity with both primary (HUVEC) and immortalized
(EAhy926) human endothelial cells in 2% (v/v) FBS medium with activated protein C as positive control. *p , 0.05 compared with other groups (ANOVA).
(D) sTM in the blood of mice 4 h after i.v. injection of saline (control), 10 mg/kg CRP, 50 mg/kg histones and combined histones and CRP (Hist+CRP).
Means 6 SD of circulating sTM in mice (n = 10 per group) are shown; *p , 0.05 compared with other groups (ANOVA). (E) Inhibitory effects of CRP (250
mg/ml) on 50 mg/ml histone-induced endothelial permeability (Materials and Methods). Mean 6 SD of fold changes to untreated (UT) controls are shown.
*p , 0.05 compared with other groups (ANOVA). (F) Wet/dry weight ratios of lungs from mice (n = 10 per group) euthanized 4 h after i.v. injection of saline
(control), 50 mg/kg histones, or histones + 10 mg/kg CRP (Hist+CRP). *p , 0.05 compared with control (ANOVA).
The Journal of Immunology
Ab with HRP-conjugated anti-CRP mAb (Abcam). ELISA kits were also
used to determine CRP (Diamed), soluble thrombomodulin (sTM), and
thrombin–antithrombin (TAT; Cusabio Biotech) levels in serum. Each
sample was performed in duplicate. Histone H3 in plasma were detected
by Western blotting using anti-histone H3 Ab (Abcam) and calculated
using human recombinant histone H3 protein as standard.
Permeability assay
The in vitro permeability was analyzed in a dual-chamber system using
Evans blue–labeled BSA, as described previously (30). In brief, EAhy926
cells were grown on the upper chamber of Transwell polycarbonate
membranes (Corning) to a confluent monolayer and treated with histones
in DMEM with 2% (v/v) FBS for 1 h. Permeability was then assessed by
replacing the media in the upper chamber with 100 ml Evans blue–BSA
and in the lower chamber with 500 ml DMEM with 4% (w/v) BSA. After
10 min, a 100 ml aliquot was taken from the lower chamber, and absorbance was measured at 650 nm using a spectrometer. In vivo permeability
assay was carried out as follows: wild type C57BL/6 male mice of 22 6
0.5 g body weight were challenged with 50 mg/kg histones (nonlethal
dose; i.v.) or histones + 10 mg/kg CRP for 4 h. Pulmonary edema was
quantified by measuring the wet/dry weight ratio of the right lung. Wet
weight was obtained immediately after euthanasia and dry weight after
4 d of drying at 60˚C.
Whole-cell currents were recorded using the perforated patch configuration from single EAhy926 cells using an Axopatch 200B amplifier (Axon
Instruments) as previously described (31). Recorded membrane currents
were filtered at 5 kHz, digitized using a Digidata 1320A interface (Axon
Instruments), and analyzed using pCLAMP software. Histones (20 mg/ml),
CRP (250 mg/ml), or histones preincubated with CRP for 30 min were
added to the extracellular solution and applied to the cell by bath superfusion. All experiments were performed at room temperature (18–22˚C).
FIGURE 2. CRP inhibits histone-induced platelet aggregation and coagulation activation. (A) Effect of human recombinant histones on washed
platelets. Platelet aggregation was measured after 40 mg/ml individual
histone H3 or H4 was added to freshly isolated platelets from healthy
donors in the absence or presence of 250 mg/ml CRP. A typical curve of
light transmission is shown. (B) H3-induced platelet aggregation was set
up as 100%. Means 6 SD of relative percentages in platelet aggregation
from four independent measurements using platelets isolated from different donors are shown. *p , 0.05 compared with control (ANOVA), ‡p ,
0.05 compared with histone alone (Student t test). (C) Mice (10 per group)
were injected i.v. with saline (control), 60 mg/kg histones (LD50) preincubated without or with 10 mg/kg CRP. Blood was taken 15 min after
injection, and platelet count normalized against hematocrit (HCT) is
shown. *p , 0.05 compared with control (ANOVA), ‡p = 0.012 compared
with histones alone (Student t test). (D) Mean 6 SD of circulating TAT
levels in mice (n = 10 per group) 4 h after injection of 50 mg/kg histones
(nonlethal dose) preincubated without or with 10 mg/kg CRP (Hist+CRP);
*p , 0.05 compared with other groups (ANOVA).
Measurement of intracellular calcium
Intracellular Ca2+ concentration was determined by measuring fluorescence emission at 510 nm during excitation at 340 and 380 nm according
to published protocols (32) with fura 2-AM as the fluorescent probe.
Fluorescence was monitored continuously using a Hitachi F-7000 fluorescence spectrometer, and intracellular Ca2+ concentration was calculated
using the software provided. Ca2+ influx was stimulated by adding calf
thymus histones to the fura 2–loaded EAhy926 cells.
Fluorescent staining
ahscFv and histones were labeled using a FluoroTag FITC conjugation kit
(Sigma), and CRP was conjugated with Cy5 (GE Healthcare) separately.
Free FITC or Cy5 was removed using a Sephadex G25 column. EAhy926
cells were fixed with 4% (w/v) paraformaldehyde, permeabilized with 1%
(v/v) Triton X-100, and blocked with 5% (w/v) defatted milk before the
costaining with 10 mg/ml FITC-ahscFv and Cy5-CRP for 1 h. Images were
taken using LSM 710 confocal microscope. For histone–plasma membrane
interactions, EAhy926 cells were seeded in glass-bottom dishes for 24 h
and incubated with FITC-histones (10 mg/ml) alone or with CRP, denatured CRP (dCRP) (250 mg/ml), and liposomes (100 mM) made of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine
(PS). The images were taken after 10-min incubation using LSM 710 confocal microscope.
Binding assays
Gel overlay was used to determine which histones bind CRP. In brief, equal
amounts of proteins were subjected to SDS-PAGE. One gel was stained with
Coomassie brilliant blue to ensure equal loading and the other electroblotted
onto polyvinylidence difluoride membranes, which were incubated at 4˚C
overnight with 3 mg/ml CRP. Bound CRP was detected using anti–CRPHRP (Sigma). In addition, the competitive binding of histones to CRP or
FIGURE 3. CRP protects mice challenged with lethal doses of histones
by reducing lung edema, hemorrhage, and thrombosis. (A) Survival curves
representing survival fractions of mice injected i.v. with 75 mg/kg histones
(100% lethal dose) without (n = 10) or with preincubation with CRP at 1.6
(n = 7), 2.5 (n = 7), or 10 mg/kg (n = 7). (B) Pathological changes of lungs
from mice. H&E stained (a–c) and immunohistochemically stained (d–f)
with anti-fibrin Ab to demonstrate thrombosis. Lung sections from mice
were injected with saline (a, d), 75 mg/kg histones (b, e), or histones
preincubated with 10 mg/kg CRP (c, f). Black and blue arrows in (b) indicate lung edema and hemorrhage, respectively. Black arrow in (c)
indicates the increased thickness of the alveolar wall. Red arrows indicate
thrombosis (e) and fibrin deposition (f). Scale bar, 50 mm.
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Electrophysiology
2497
2498
phospholipids was examined by a competitive ELISA. In brief, histones
were coated on ELISA plates and incubated with different doses of liposomes mixed with 50 mg/ml CRP. The bound CRP was detected with
anti–CRP-HRP, and the absorbance at 450 nM was used to represent the
relative amount of CRP that bound to coated histones. The liposomes were
prepared as described previously (33) using PS and PE at a ratio 1:2 to
reflect the ratio in plasma membrane. PC, another major phospholipid in
cell membranes, was omitted to avoid the possibility of its interaction with
CRP (34).
Immunohistochemical staining
Paraffin-embedded lung sections were dewaxed and rehydrated followed
by Ag retrieval using DAKO PT-Pre-Treatment link system. Anti-fibrin Ab
(Abcam) and EnVision+ kit (DAKO) was used to probe fibrin. Images were
taken using Olympus Microscopy and Nikon ACT-1 software.
Platelet aggregation assay
Blood was withdrawn from healthy donors, and freshly isolated washed
platelets (35) were treated with recombinant human histone H3 and H4
(New England Biolabs), preincubated with or without CRP. Platelet aggregation was conducted using a Born Aggregometer (pap 8, Bio-DATA).
Statistical analysis
Results
CRP reduces histone-induced endothelial cell damage and
permeability increase
Extracellular histones have been demonstrated to be toxic to endothelial cells (24). Fig. 1A showed that calf thymus histones in
culture medium with 2% (v/v) FBS are significantly toxic to
cultured EAhy926 cells, a human endothelial cell line. The toxicity of 40 mg/ml histones to EAhy926 cells can be significantly
reduced when CRP was .50 mg/ml (Fig. 1B). BSA, as control,
was unable to block histone toxicity to these cells (data not
shown). Primary HUVECs isolated from cords were also used and
showed similar responses to indicate that histone toxicity and CRP
FIGURE 4. Acute CRP elevation in patients
reduces histone-induced toxicity to endothelial
cells. (A) Sera of healthy individuals (normal) and of
patients at different time points after severe trauma
(,4, 24, and 72 h) were incubated with EAhy926
cells and expressed in relation to circulating histone
and endogenous (Endo) CRP levels. Survival rate of
untreated cells was set as 100%. Means 6 SD of
normalized cell survival rates from three independent
experiments are presented; *p , 0.05 compared with
normal (ANOVA). (B) CRP–histone complex levels
in sera from seven patients at different time points
(,4, 24, and 72 h) after severe trauma were detected
using modified Cell Death ELISA kit (Roche) by
replacing anti–DNA-HRP with anti–CRP-HRP.
Means 6 SD are presented; *p , 0.05 compared
with first time point (ANOVA). (C and D) The
toxic effect of sera from patients with necrotizing
pancreatitis (C) or severe sepsis (D) on the survival
rate of EAhy926 cells in relation to CRP levels (endogenous [Endo] concentration and/or exogenous
[Exo] CRP addition). Means 6 SD of normalized cell
survival rates from three repeated experiments using
the same serum are presented; *p , 0.05 compared
with other groups (ANOVA).
protection are not cell-line–specific effects (Fig. 1C). Activated
protein C, which can cleave histones (24), was also used as positive control and could reduce histone toxicity in this assay.
In vivo translation of these data are shown in Fig. 1D–F. sTM,
a circulating marker of endothelial damage, increased significantly
in mice injected with a nonlethal infusion of histones at 50 mg/kg.
When this was coinjected with 10 mg/kg CRP, sTM levels decreased significantly (Fig. 1D). Because the immediate consequence
of endothelial damage is an increase in vascular permeability, preincubation with CRP was able to reduce significantly histoneinduced permeability in vitro (Fig. 1E) and in vivo (Fig. 1F). Collectively, these data indicate that CRP can inhibit extracellular
histone-induced endothelial damage and its consequences.
CRP inhibits histone-induced platelet aggregation and
coagulation activation
CRP significantly inhibited histone-induced platelet aggregation
by up to 50% but did not block it completely even at a very high
concentration of 250 mg/ml (Fig. 2A, 2B). In mice, coinfusion of
CRP (10 mg/kg) partially alleviated thrombocytopenia induced by
injection of 60 mg/kg histones (Fig. 2C). Activation of coagulation, as measured by TAT complexes, was also significantly reduced in mice injected with 50 mg/kg histones + 10 mg/kg CRP
compared with histone treatment alone (Fig. 2D). These data
strongly indicate that CRP is able to attenuate histone-induced
platelet and thrombin activation. The significant but incomplete
blockage of these effects by CRP might serve to retain vital hemostatic processes whereas limiting excessive histone-induced
damage at sites of vascular injury.
CRP protects mice challenged with lethal doses of histones by
reducing lung edema, hemorrhage, and thrombosis
A total of 75 mg/kg histones killed all mice within an hour. Survival
times were prolonged with one in seven mice surviving for .6 d
when low-dose CRP (1.6 mg/kg) was coinfused. When CRP level
was increased to 5 mg/kg, 3 of 7 mice survived for .6 d and 4 of 7
mice died between 7 and 11 h. At 10 mg/kg CRP, all seven mice
survived .6 d (Fig. 3A). A log-rank test showed significant differ-
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Intergroup differences were analyzed using ANOVA followed by the
Student–Newman–Keuls test. Two-group comparison before and after
treatment used Student t test with animal survival time analyzed using a
log-rank test. Association analysis used simple linear correlation. Mean 6
SD are from at least three independent experiments.
CRP REDUCES HISTONE TOXICITY IN VITRO AND IN VIVO
The Journal of Immunology
2499
ences in survival times among the four groups (p , 0.001). Histological examination showed that mice that died at 1 h after injection of 75 mg/kg histones alone had obvious lung edema and
hemorrhage (Fig. 3Bb), but mice euthanized 1 h after injection of
75 mg/kg histones + 10 mg/kg CRP had less edema and hemorrhage, although congestion and increased alveolar wall thickness
were observed (Fig. 3Bc). Immunohistochemical staining for fibrin
showed obvious thrombosis in lungs from mice injected with 75
mg/kg histones alone (Fig. 3Be), but not in mice injected with
histones preincubated with 10 mg/kg CRP. However, fibrin deposition was clearly observed in lungs (Fig. 2Bf). These pathological
changes support the finding that CRP reduces histone-induced
endothelial damage and coagulation activation.
Acute CRP elevation in patients reduces histone toxicity to
endothelial cells
The mechanism of CRP detoxification is through preventing
histone–cell membrane interaction by complex formation
We previously showed that histone-induced toxicity relied on
membrane interaction and calcium influx (27). Preincubation of
histones with CRP significantly reduced cell membrane association of FITC-labeled histones (Fig. 5A), as well as histone-induced
inward currents (Fig. 5B, 5C) and calcium influx (Fig. 5D, 5E).
These data strongly indicate that masking the membrane binding
sites of histones is the mechanism of CRP detoxification.
The proposed mechanism of histone–cell membrane interaction
is their affinity to phosphate groups of major phospholipids in the
cell membrane, such as PC, PS, and PE (27, 36, 37), because
phosphate groups exist in both DNA and bilayer lipid membranes.
Preincubation of FITC-labeled histones with liposomes made of
PC, PS, and PE significantly reduced the binding of histones to the
cell membrane (Fig. 6A). Functionally, this significantly reduced
the toxic effects of histones on endothelial cells (Fig. 6B). These
data support the hypothesis that phospholipids on cell membranes
are the major targets of histones.
It is also known that CRP binds to the phosphocholine group of
lysoPC (34). However, lysoPC is exposed only after the lipid bilayer of cell membranes has been disturbed, such as during necrosis and apoptosis (38). Therefore, CRP does not bind to normal
cell membranes. In this study, we found that incubating CRP with
EAhy926 cells for 1–4 h did not reduce histone-induced toxicity
if CRP were washed away before histone treatment (data not
shown). This experiment also indicates that CRP prevention of
FIGURE 5. CRP prevents histone–cell membrane interactions. (A)
Confocal images of living EAhy926 cells incubated with FITC-histones
(10 mg/ml, left panel), FITC-histones + CRP (100 mg/ml, middle panel)
and an equal amount of inactivated FITC alone (right panel) for 10 min.
Scale bar, 20 mm. Arrows in left panel indicate membrane binding of
FITC-histones. Arrows in middle panel indicate aggregation of FITChistones. (B) Typical whole-cell current change of EAhy926 cells treated
with 40 mg/ml histones preincubated with 250 mg/ml CRP followed by
histones alone, after washing. (C) Graphic representation of the density of
inward currents (pA/pF) from three independent experiments with mean 6
SD, *p , 0.001 compared with other groups (ANOVA). (D) Typical curves
of intracellular calcium ([Ca2+]i) changes of EAhy926 cells treated with
histones (50 mg/ml) preincubated with or without 250 mg/ml CRP. (E)
Intracellular calcium levels with mean 6 SD from three independent
experiments; *p , 0.05 compared with other groups (ANOVA).
histone–membrane interaction is not due to CRP association with
cell membranes.
In line with our findings of the existence of circulating CRP–
histone complexes, the interaction between CRP and histone has
been reported previously but with some discrepant findings (13, 14,
39–41). To clarify these discrepancies, we immunofluorescently
stained EAhy926 cells with Cy5-CRP and FITC-ahscFv to avoid
any nonspecific staining of IgGs. Their colocalization observed in
cell nuclei strongly indicates that CRP binds to histones (Fig. 6C).
Furthermore, a gel overlay assay showed that all histones could
bind CRP (Fig. 6D) and that CRP could protect endothelial cells
treated with the individual histones (Fig. 6E). Using competitive
ELISA, we found that CRP competed with liposomes in binding
histones (Fig. 6F, 6G) to explain how CRP blocks histone–plasma
membrane integration (Fig. 5A).
Discussion
The acute-phase response is a complex series of reactions initiated in response to infection, inflammation, injury, or malignancy. Elevation in CRP levels has long been recognized in this
context, but despite many reports into its properties, clear understanding of its generic function has been lacking (42). In part,
this is because no mutation of this phylogenetically ancient and
well-conserved molecule has been described, although a few
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When serum samples from patients with severe trauma were incubated with endothelial cells, those collected within 4 h of injury
could induce significant cell death (Fig. 4A). However, this effect
was not observed at later time points of 24 and 72 h despite histone levels remaining elevated. The functional difference could be
because of endogenous CRP levels, which were not raised within
4 h but were .150 mg/ml at 24 and 72 h after trauma. Indeed, this
corresponded to significant elevation of CRP–histone complex
levels, but only at 24 and 72 h (Fig. 4B). These data suggest that
CRP is a major factor in combating histone-induced toxicity in
patients.
To confirm this, we collected sera from patients with necrotizing pancreatitis (Fig. 4C) and severe sepsis (Fig. 4D), which are
conditions also associated with extensive cell damage. Sera that
contained high histone levels but low CRP concentrations were
toxic to endothelial cells, whereas sera that were high in both CRP
and histones were much less toxic. If exogenous CRP was added
to sera with high histone levels, their toxic effects could be attenuated. These data indicate that elevated CRP is able to reduce
clinically toxicity of circulating histones.
2500
CRP REDUCES HISTONE TOXICITY IN VITRO AND IN VIVO
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FIGURE 6. CRP–histone complex formation reduces histone-induced cell damage. (A) Confocal images of living EAhy926 cells incubated with FITChistones (10 mg/ml, left panel) or FITC-histones + liposomes made of PC, PS, and PE (right panel). Scale bar, 50 mm. Arrow in left panel indicates membrane binding of FITC-histones. (B) The protective effects of different concentrations of liposomes on histone-induced toxicity to EAhy926 cells in the
presence of 2% (v/v) FBS. Means 6 SD are shown. *p , 0.05 compared with histones alone (ANOVA). (C) Dual staining of EAhy926 cells with FITCahscFv (a), Cy5-CRP (b), phase contrast (c), and superimposed images (d) to demonstrate the colocalization of CRP with histones. All images were taken
using a Zeiss LSM710 confocal microscope. (D) Gel overlay assay to study CRP interaction with immobilized histones. A total of 1 mg of each type of
human recombinant histones (H1, H2A, H2B, H3.1, H4) and 6 mg S100P protein (negative control) were subjected to a gel overlay assay with 3 mg/ml CRP.
Coomassie blue stained gel (upper panel) shows the gel loading (n = S100P multimer, m = S100P monomer). Overlaid with CRP and detected with antiCRP Ab (lower panel). (E) CRP protection of EAhy926 cells treated with 40 mg/ml individual human recombinant histones in the presence (white)
or absence (black) of 250 mg/ml CRP. Means 6 SD from four independent experiments are shown; *p , 0.05 in cell survival compared with untreated
(UT), #p # 0.035 in relation to CRP addition (Student t test). (F) Competitive ELISA assay to demonstrate that CRP and phospholipids (PE+PS) competitively interact with histones. ELISA plates were coated with histones or buffer alone and incubated with 2 mM liposomes made of PS+PE (1:2) mixed
with 50 mg/ml CRP. After washing, the bound CRP was detected using anti–CRP-HRP. Means 6 SD of the absorbance from three independent experiments
are shown. *p , 0.001 compared with other groups (ANOVA), #p = 0.007 compared with group 1 (Student t test). (G) A typical dose–response curve of the
competitive ELISA with 0–200 mM liposomes. The bound CRP was detected using anti–CRP-HRP, and the means of absorbance from three independent
experiments are presented.
nucleotide polymorphisms that do not alter the protein sequence
have been identified (43). This would suggest a critical functional
role for CRP. Although the concept of CRP acting to detoxify and
clear damaging products of cell destruction was hypothesized
back in 1981 (44), this study demonstrates for the first time, to
our knowledge, that high levels of CRP in the acute clinical
phase plays a major role in combating the toxicity of circulating
histones.
The Journal of Immunology
Histones can bind different phospholipids (36, 37, 47, 48), and
as these are major components of the cell membrane, we speculate
that histones bind to the phospholipid bilayer in the plasma membrane in a manner similar to the interaction between the DNA
phosphodiester backbone and the histone cores (49). In this study,
we demonstrate that artificial liposomes made of phospholipids
compete with the cell membranes for histone binding and significantly reduce histone-induced toxicity. CRP is also able to compete
with phospholipids for binding to histones, and this indicates that
the major mechanism underlying the protective effects of CRP
in vitro and in vivo is in its interference with the histone association to cell membranes. Therefore, CRP binds histones, as in its
role in opsonizing pathogens, to block their toxic effect on cells
and to possibly facilitate their clearance. This action defends the
human body from the toxicity of circulating histones, especially in
the acute phase of many critical illnesses.
Acknowledgments
We are indebted to participating patients and their attending staff. We thank
Prof. Mark Pepys for constructive discussion and advice.
Disclosures
The authors have no financial conflicts of interest.
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CRP REDUCES HISTONE TOXICITY IN VITRO AND IN VIVO