Cyclophilin A Is a Damage-Associated
Molecular Pattern Molecule That Mediates
Acetaminophen-Induced Liver Injury
This information is current as
of June 16, 2017.
James W. Dear, Kenneth J. Simpson, Melianthe P. J.
Nicolai, James H. Catterson, Jonathan Street, Tineke
Huizinga, Darren G. Craig, Kevin Dhaliwal, Sheila Webb,
D. Nicholas Bateman and David J. Webb
References
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Copyright © 2011 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2011; 187:3347-3352; Prepublished online 8
August 2011;
doi: 10.4049/jimmunol.1100165
http://www.jimmunol.org/content/187/6/3347
The Journal of Immunology
Cyclophilin A Is a Damage-Associated Molecular Pattern
Molecule That Mediates Acetaminophen-Induced Liver
Injury
James W. Dear,*,† Kenneth J. Simpson,‡ Melianthe P. J. Nicolai,* James H. Catterson,*
Jonathan Street,* Tineke Huizinga,* Darren G. Craig,‡ Kevin Dhaliwal,‡
Sheila Webb,x D. Nicholas Bateman,† and David J. Webb*
C
ell death induces a complex inflammatory response that is
vital for tissue repair but, in excess, worsens organ injury
(1). The immune system is alerted to cell death when
intracellular molecules are released into the extracellular space
as a result of damage to the cell membrane. These molecules,
referred to as damage-associated molecular patterns (DAMPs) (1,
2), which include urate microcrystals (3), high-mobility group box
1 protein (4), and mitochondrial DNA (5), initiate and perpetuate
the inflammatory response.
Cyclophilin A (CypA; also known as peptidyl-prolyl isomerase
A [Ppia]) is an abundant cytosolic protein (∼0.25% of cellular
protein) (6). It is a member of the immunophilin class of proteins
that mediates protein folding (7). Intracellular CypA regulates
T lymphocyte receptor signaling by binding to the tyrosine kinase,
Itk (8). When intracellular CypA is bound by the immunosup-
*University/British Heart Foundation Centre for Cardiovascular Science, Edinburgh
University, Edinburgh, EH16 4TJ United Kingdom; †National Poisons Information
Service, Edinburgh, EH16 4SA United Kingdom; ‡Centre for Inflammation Research,
Edinburgh University, Edinburgh, EH16 4TJ United Kingdom; and xMedical Research Council Human Genetics Unit, Edinburgh University, Edinburgh, EH4 2XU
United Kingdom
Received for publication January 19, 2011. Accepted for publication July 6, 2011.
This work was supported by Medical Research Scotland. This work was also supported by National Health Service Research Scotland, through National Health Service Lothian.
Address correspondence and reprint requests to Dr. James W. Dear, University/
British Heart Foundation Centre for Cardiovascular Science, Edinburgh University,
Queen’s Medical Research Institute, Room E3.23, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. E-mail address: [email protected]
Abbreviations used in this article: ALT, alanine transferase; CypA, cyclophilin A; CypD,
cyclophilin D; DAMP, damage-associated molecular pattern; KC, keratinocyte-derived
chemokine; MPO, myeloperoxidase; ND, not detected; Ppia, peptidyl-prolyl isomerase
A; WT, wild-type.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100165
pressive drug cyclosporine, the CypA–cyclosporine complex binds
calcineurin and inhibits T lymphocyte receptor signaling (9, 10).
CypA is actively released into the extracellular milieu by
a number of cell types, including macrophages (11), vascular
endothelial cells (12), and vascular smooth muscle cells (13).
In vivo, extracellular CypA concentrations are elevated in rodent
models of asthma (14) and acute lung injury (15). In blood vessels,
concentrations are increased during vascular remodeling (16), and
mice lacking the gene for CypA, commonly referred to as Ppia2/2
mice, are protected from aortic aneurysm formation (17). In
humans, CypA is elevated in the synovial fluid of patients with
active rheumatoid arthritis (18) and in the serum of patients with
severe sepsis (19). Once released from the cell, CypA is a chemotactic agent for a range of inflammatory cells (11, 14) and
directly stimulates the release of a variety of inflammatory
mediators (20). CD147 is a receptor that mediates the actions of
extracellular CypA [e.g., an anti-CD147 Ab completely prevents
CypA-induced chemotaxis of neutrophils (15)]. In vivo, inhibition
of CD147 reduces allergic inflammation in mouse models of
asthma (14), collagen-induced arthritis (21), and sepsis-induced
acute kidney injury (22). Although these in vivo effects have
been attributed to CypA antagonism, there are other ligands that
activate CD147, such as E-selectin (23). Therefore, in addition to
CD147 antagonism, to clearly define the role of CypA/CD147 in
an experimental model, it is necessary to remove CypA using
techniques such as gene deletion.
Acetaminophen-induced liver injury in rodents is used as a
model of sterile cell death and has demonstrated that DAMPs
mediate organ injury in vivo (24). Because CypA is an abundant
intracellular protein and is proinflammatory when released into
the extracellular space, we hypothesized that CypA is released
from necrotic liver cells and acts as a DAMP to worsen
acetaminophen-induced liver injury.
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The immune system is alerted to cell death by molecules known as damage-associated molecular patterns (DAMPs). These molecules
partly mediate acetaminophen-induced liver injury, an archetypal experimental model of sterile cell death and the commonest cause
of acute liver failure in the western world. Cyclophilin A (CypA) is an intracellular protein that is proinflammatory when released
by cells. We hypothesized that CypA is released from necrotic liver cells and acts as a DAMP to mediate acetaminophen-induced
liver injury. Our data demonstrated that mice lacking CypA (Ppia2/2) were resistant to acetaminophen toxicity. Antagonism of the
extracellular receptor for CypA (CD147) also reduced acetaminophen-induced liver injury. When injected into a wild-type mouse,
necrotic liver from Ppia2/2 mice induced less of an inflammatory response than did wild-type liver. Conversely, the host
inflammatory response was increased when CypA was injected with necrotic liver. Antagonism of CD147 also reduced the
inflammatory response to necrotic liver. In humans, urinary CypA concentration was significantly increased in patients
with acetaminophen-induced liver injury. In summary, CypA is a DAMP that mediates acetaminophen poisoning. This mechanistic insight presents an opportunity for a new therapeutic approach to a disease that currently has inadequate treatment
options. The Journal of Immunology, 2011, 187: 3347–3352.
3348
CYCLOPHILIN A IS A DAMAGE-ASSOCIATED MOLECULAR PATTERN
Materials and Methods
Animals
Ppia2/2 mice were purchased as cryopreserved embryos from The Jackson
Laboratory (Bar Harbor, ME) (strain name: 129.Cg-Ppiatm1Lubn/J; stock
number 5320). All mice were genotyped by PCR using tissue from ear
clips. The genotyping results were validated by Western blotting (Ab:
rabbit anti-CypA; Cell Signaling Technology, Beverly, MA). In separate
studies, commercial C57BL/6 mice (Harlan) were used. All investigations
conformed with the Guide for the Care and Use of Laboratory Animals
(National Institutes of Health Publication No. 82-23, revised 1996) and
were performed with appropriate Home Office (U.K.) licenses.
Model of acetaminophen poisoning
After a 6-h fast, mice were injected i.p. with acetaminophen dissolved in
sterile saline (350 mg/kg) or sterile saline vehicle control. After 6–24 h,
blood was collected by cardiac puncture, and tissue was collected for
histology and glutathione assay. Histology was assessed by a reviewer
blinded to the mouse genotype. Glutathione was measured by colorimetric
assay, per the manufacturer’s instructions (Sigma-Aldrich, Gillingham,
U.K.). In some studies, C57BL/6 mice were treated with a rat anti-CD147
Ab or isotype control Ab (25 mg, i.p.) 2 h prior to acetaminophen injection.
FIGURE 2. Histological liver injury was less in Ppia2/2 mice (KO;
lower panel) than in sex-matched littermate controls (WT; upper panel)
24 h after acetaminophen injection. Cell vacuolation is reduced in the
KO mouse. H&E staining. Scale bar, 200 mm.
FIGURE 3. A, Serum IL-6 concentrations were lower in Ppia2/2 mice
(KO) than in littermate controls (WT) 24 h after acetaminophen injection.
B, Necrotic liver cells from Ppia2/2 mice (WT) lacking the gene for CypA
induced a lower serum IL-6 concentration than did WT liver cells when
injected into WT mice. (n = 10 mice/group.) Bar graphs represent mean 6
SEM. *p , 0.05.
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FIGURE 1. Serum ALT concentrations were lower in Ppia2/2 mice
(KO) lacking the gene for CypA
than in littermate controls (WT) 24 h
after acetaminophen injection. Serum ALT concentration after vehicle
injection is also presented. A, These
data are presented as individual
mice. B, Liver glutathione concentration (GSH) is reduced 6 h after
acetaminophen injection in both KO
and WT mice compared with after
vehicle injection (n = 5 per group,
graphs represent mean 6 SEM). C,
Pretreatment with an anti-CD147
Ab significantly reduced serum ALT
24 h after acetaminophen injection
compared with an isotype Ab (n =
10 per group; graphs represent
mean 6 SEM). *p , 0.05, compared with WT or control.
The Journal of Immunology
3349
FIGURE 4. A and B, In WT mice,
peritoneal lavage fluid concentrations of
KC and MPO were lower 6 h after injection of necrotic liver from Ppia2/2
mice (KO) lacking the gene for CypA
than 6 h after injection of WT liver. C
and D, In WT mice 6 h after injection
of WT necrotic liver, peritoneal lavage
fluid concentrations of KC and MPO
were lower following pretreatment with
an anti-CD147 Ab compared with isotype control. (n = 10 mice/group.) Bar
graphs represent mean 6 SEM. *p ,
0.05.
Model of the inflammatory response to necrotic liver
To determine whether CypA acts as a DAMP, we followed the protocol used
by Chen et al. (24). Livers were removed from wild-type (WT) and Ppia2/2
mice. After homogenization in sterile saline and centrifugation, the
resulting supernatant was freeze/thawed five times. Mice were then
injected i.p. with the necrotic liver cells (36 mg protein). After 6 h, the
peritoneal cavity was lavaged with 5 ml warmed sterile saline, and blood
was taken by cardiac puncture. In a second study, necrotic liver from
C57BL/6 mice (36 mg protein) was injected i.p. into C57BL/6 mice that
had been treated 2 h before with either anti-CD147 Ab or isotype control
Ab (25 mg, i.p.). After 6 h, the peritoneal cavity was lavaged with 5 ml
warmed sterile saline. In a third study, necrotic liver from C57BL/6 mice
(20 mg protein) was injected i.p. into C57BL/6 mice together with 100 mg
purified CypA (a kind gift from Prof. M. Walkinshaw, Edinburgh University) or vehicle (saline). After 6 h, the peritoneal cavity was lavaged
with 5 ml warmed sterile saline.
Concentrations of myeloperoxidase (MPO) and keratinocyte-derived
chemokine (KC) were measured in the lavage fluid by ELISA (Calbiochem, La Jolla, CA), whereas IL-6 concentration was measured in blood by
ELISA (R&D Systems).
Human studies
Urine samples were collected from patients at the Royal Infirmary of
Edinburgh (Edinburgh, U.K.). The study was prospectively approved by
the Scotland ‘A’ Research Ethics Committee. Informed consent or assent
was obtained from all patients or the patient’s nominated next of kin before
study inclusion. The inclusion criterion for this study was an adult patient
with a clear history of excess acetaminophen ingestion. Exclusion criteria
were patients detained under the Mental Health Act; patients with known
permanent cognitive impairment, an acute life-threatening illness, or an
unreliable history of overdose; and patients who take anticoagulants (e.g.,
warfarin) therapeutically or have taken an overdose of anticoagulants.
Patients were classified into four groups based on routine blood results:
not-detected (ND) group, acetaminophen not detected in the blood 4 h after
ingestion; below-line group, acetaminophen was detected in the blood, but
the concentration was below the treatment line on the Rumack–Matthew
nomogram; above-line group, acetaminophen concentration was above the
treatment line, but no liver injury occurred subsequent to the overdose; and
organ-injury group, ALT 103 the upper limit of normal (.500 U/l) and
a clear history of acetaminophen ingestion. All urine samples were collected within the first 24 h of hospital admission and were stored at 280˚C.
The urinary CypA concentration was determined by Western blotting,
as previously described (22). The team member performing the Western
blotting was blinded to which of the four groups the urine sample
belonged. Each gel contained at least one sample from each patient group
and a positive control (purified human CypA). The primary Ab was a
rabbit anti-human CypA Ab (USBiological, Swampscott, MA), and the
secondary Ab was an HRP-conjugated anti-rabbit Ab (Santa Cruz Biotechnology, Santa Cruz, CA). After visualization of the secondary Ab’s
binding using ECL, the photographic films were scanned using the VersaDoc imaging system (Bio-Rad, Hercules, CA), and relative band densities were measured using Quantity One software (Bio-Rad). The relative
band densities were normalized by urinary creatinine concentration.
In a substudy, one urine sample was separated into the exosomal and
nonexosomal fraction to determine the localization of urinary CypA. The
sample was centrifuged at 15,000 3 g for 10 min to pellet any shed cells,
large membrane fragments, and other debris. The supernatant was then
centrifuged at 200,000 3 g for 1 h to pellet the exosomes (25). The pellet
and supernatant were probed by Western blotting for CypA and the urine
exosome marker CD24 (26) (anti-CD24 Ab, a kind gift of Dr. P. Altevogt,
German Cancer Research Center, Heidelberg, Germany).
Statistical analysis
Pilot studies with WT mice demonstrated that, 24 h after acetaminophen
injection, the mean serum ALT concentration was ∼4000 U/l, with an SD
∼2500 U/l. Therefore, detection of a 2000-U/l difference between groups,
with 80% power at a two-sided 5% significance level, required ∼50 mice.
FIGURE 5. The peritoneal lavage fluid concentration
of KC (A) and MPO (B) was increased following i.p.
injection of necrotic liver from WT mice (20 mg) plus
CypA (100 mg) compared with vehicle (saline) plus
necrotic liver. The lavage fluid concentration of KC and
MPO after vehicle and CypA injection without liver is
also presented. (n = 10 mice/group.) Bar graphs represent mean 6 SEM. *p , 0.05, versus vehicle plus
liver.
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The anti-CD147 Ab (from RL73.2 hybridoma) and isotype control Ab
were kind gifts from Dr. Stephanie Constant (George Washington University, Washington, D.C.). Serum alanine transaminase (ALT) was measured on a Cobas Fara centrifugal analyzer (Roche Diagnostics, Welwyn
Garden City, U.K.), and serum IL-6 concentration was measured by
ELISA (R&D Systems, Minneapolis, MN).
3350
CYCLOPHILIN A IS A DAMAGE-ASSOCIATED MOLECULAR PATTERN
FIGURE 6. Urinary CypA concentration was significantly elevated in patients with organ injury compared with patients assigned to the ND group, the
below-line group, or the above-line group. Urinary
CypA concentration is presented as the mass/14 ml of
urine (A) and normalized to urinary creatinine (B).
*p , 0.05.
The significance of differences between experimental groups was determined by the Student t test. A nominal significance of p , 0.05 was
considered significant.
Results
Acetaminophen injection (350/mg, i.p.) produced a significant
increase in serum ALT 24 h postinjection in mice (Fig. 1).
Compared with sex-matched, littermate WT controls, Ppia2/2
mice had significantly lower serum ALT concentrations postacetaminophen injection (Fig. 1). Six of 50 mice studied died
within 24 h of acetaminophen injection and, therefore, were not
included in the analysis of serum ALT concentration. All of the
dead mice were of WT genotype, consistent with Ppia2/2 mice
being comparatively resistant to acetaminophen poisoning. Liver
glutathione concentration was reduced by acetaminophen in both
Ppia2/2 mice and WT controls (Fig. 1). Pretreatment with an antiCD147 Ab significantly reduced the serum ALT value compared
with a control isotype Ab (Fig. 1). Acetaminophen injection
produced liver cell vacuolation and necrosis that was greater in
WT mice than in Ppia2/2 mice (Fig. 2).
Extracellular CypA signals necrosis and initiates an
inflammatory response
CypA stimulates inflammatory cells to release IL-6 (data not
shown). Postacetaminophen injection, serum IL-6 concentrations
were significantly lower in Ppia2/2 mice than in controls (Fig. 3).
To investigate whether this is due to CypA being a DAMP, we
injected necrotic liver from Ppia2/2 mice and littermate controls
into WT mice. Necrotic liver from Ppia2/2 mice induced significantly lower serum IL-6 concentrations 6 h after injection compared with WT cells (Fig. 3). Published studies demonstrated that
CypA induces neutrophil chemotaxis (11). To explore whether
CypA released from liver cells is chemotactic for neutrophils
in vivo, we measured the MPO and KC concentration in peritoneal
lavage fluid 6 h after injection of necrotic liver. Both MPO and KC
concentrations were reduced in the peritoneal lavage fluid when
CypA is released in humans with acetaminophen-induced liver
injury
Using Western blot, we could not detect CypA in blood from
patients with acetaminophen-induced liver injury (data not shown).
However, urinary CypA concentration was significantly elevated
in patients with acetaminophen-induced liver injury (organ-injury
group) compared with the other three patient groups (Fig. 6). This
finding remained statistically significant when the CypA concentration was normalized for differences in urinary concentration (urinary creatinine). Human urine contains exosomes,
protein-rich lipid vesicles that contain CypA (27). We separated
the exosomal fraction from the soluble fraction to determine the
location of the CypA released into the urine with acetaminophen
poisoning (Fig. 7). CypA was present in both the soluble fraction
and exosomes. Western blotting of the exosomes revealed CypA
and additional immunoreactive proteins not identified in whole
urine or the soluble fraction. These may represent other cyclophilins that are known to be contained in human urinary exosomes
(27).
Discussion
This study demonstrated that CypA is a key mediator of
acetaminophen-induced liver injury and suggests that it acts as
a DAMP. Consistent with this, in humans, CypA is released from
cells following acetaminophen-induced liver injury.
Inflammation plays an important, but incompletely defined, role
in the pathophysiology of acetaminophen-induced liver injury. It
is well-established that hepatotoxicity is dependent on the intracellular formation of the reactive acetaminophen metabolite
FIGURE 7. Urine from a patient with organ injury
was separated into exosomal (EXO) and soluble fractions (SUP). A, CypA was present in the whole urine
(U), as well as in the EXO and SUP fractions. Purified
CypA was used as a positive control (STD). B, CD24
was only present in the EXO fraction, confirming the
isolation of exosomes.
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CypA/CD147 mediates acetaminophen toxicity
Ppia2/2 necrotic liver cells were injected (compared with necrotic
liver cells from littermate WT controls) and with anti-CD147 Ab
pretreatment (compared with isotype Ab pretreatment) (Fig. 4).
The concentration of KC in necrotic cells was similar in WT and
Ppia2/2 mice (11 and 15 ng/g protein, respectively). Conversely,
the concentration of MPO and KC was increased in the lavage
fluid when CypA was coinjected with necrotic liver cells (Fig. 5).
The Journal of Immunology
assays to determine whether CypA can be accurately and reproducibly measured in blood and further develop this protein as
a potential clinical biomarker. The key point is that these human
data complement our mouse results by demonstrating that, with
liver injury, CypA is released into the extracellular space (a prerequisite for a DAMP). The increase in urinary CypA is due to an
increased amount of soluble protein and CypA contained within
exosomes. Previous studies reported that human urinary exosomes
contain CypA (27). The increase in CypA in both the soluble and
exosomal compartments of the urine is consistent with the release
of intracellular exosomes and cytoplasmic proteins secondary to
necrotic cell death.
Cyclosporine binds CypA to prevent its intracellular and extracellular actions (9). Analogs of cyclosporine that do not cross
the cell membrane promise to further define the contribution of
extracellular CypA to disease (38). However, in the context of
acetaminophen poisoning, intracellular cyclosporine provides an
additional pathway to prevent injury because it binds to cyclophilin D (CypD) in mitochondria. CypD is a key regulator of the
mitochondrial permeability transition pore, the opening of which
leads to mitochondrial disruption and cell death (39, 40). The
direct cellular toxicity induced by acetaminophen is largely due to
mitochondrial permeability transition pore opening (28), with
a significant role for CypD (41). Consistent with this, cyclosporine
was demonstrated to prevent acetaminophen injury (42). Therefore, there are two mechanisms by which cyclosporine may prevent acetaminophen-induced liver injury in humans: the prevention of mitochondrial injury inside the cell and inhibition of
the actions of CypA outside the cell. Both of these mechanisms
are independent of the well-established immunosuppressive action
of cyclosporine. Nonimmunosuppressive cyclophilin inhibitors that
bind to CypA and CypD are in human trials as treatments for
hepatitis C infection (43). Future studies will be needed to confirm
that these agents inhibit acetaminophen-induced liver injury, but
the absence of immunosuppression has the potential to improve
the drug’s risk/benefit profile by reducing the risk for sepsis in
these critically ill patients.
In summary, our data demonstrated that CypA is a DAMP that
mediates acetaminophen poisoning. A proof-of-concept safety
and efficacy study of cyclosporine in patients with acetaminophen-induced liver injury is now warranted.
Acknowledgments
We acknowledge the contribution of core facilities, which are supported
by the British Heart Foundation Research Excellence Award, to this study.
Disclosures
The authors have no financial conflicts of interest.
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However, there are other ligands for CD147 (23), and we cannot
fully exclude that these, in addition to CypA, are activating
CD147 in our experimental models.
Rodent models often do not faithfully reflect human disease, and
this can lead to blocks in the translation of experimental findings to
improved patient care. We found that CypA was significantly elevated in the urine of patients with acetaminophen-induced liver
injury but not in our control groups. CypA is an intracellular
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from necrotic cells injured directly by acetaminophen or release
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may be detecting subclinical kidney injury. An alternative explanation is that the urine reflects an increase in the blood CypA
concentration. However, we could not detect CypA in blood
samples from patients with acetaminophen-induced liver injury.
Future studies will focus on the development of more sensitive
3351
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CYCLOPHILIN A IS A DAMAGE-ASSOCIATED MOLECULAR PATTERN
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