Hydrolysis of coagulation factors by circulating IgG is associated with a reduced risk for chronic allograft nephropathy in renal transplanted patients.
ABSTRACT Chronic allograft nephropathy (CAN), a major cause of late allograft failure, is characterized by a progressive decline in graft function correlated with tissue destruction. Uncontrolled activation of the coagulation cascade by the stressed endothelium of the graft is thought to play an important role in the pathophysiology of CAN. In this study, we demonstrate that circulating IgG from renal-transplanted patients are endowed with hydrolytic properties toward coagulation factors VIII and IX, but fail to hydrolyze factor VII and prothrombin. The hydrolytic activity of IgG was reliably quantified by the measure of the hydrolysis of a fluorescent synthetic substrate for serine proteases: proline-phenylalanine-arginine-methylcoumarinamide (PFR-MCA). A retrospective case-control study indicated that an elevated hydrolysis rate of PFR-MCA by circulating IgG correlated with the absence of CAN lesions on protocol graft biopsy performed 2 years posttransplantation. We propose that circulating hydrolytic IgG may counterbalance the procoagulation state conferred by the activated endothelium by disrupting the amplification loop of thrombin generation which is dependent on factors VIII and IX. Interestingly, low rates of PFR-MCA hydrolysis, measured 3 mo posttransplantation, were predictive of CAN at 2 years down the lane. These data suggest that PFR-MCA hydrolysis may be used as a prognosis marker for CAN in renal-transplanted patients.
- SourceAvailable from: Bharath Wootla[Show abstract] [Hide abstract]
ABSTRACT: Catalytic antibodies are immunoglobulins endowed with enzymatic activity. Catalytic IgG has been reported in several human autoimmune and inflammatory diseases. In particular, low levels of catalytic IgG have been proposed as a prognostic marker for chronic allograft rejection in patients undergoing kidney transplant. Kidney allograft is a treatment of choice for patients with end-stage renal failure. Intravenous immunoglobulins, a therapeutic pool of human IgG, is used in patients with donor-specific antibodies, alone or in conjunction with other immunosuppressive treatments, to desensitize the patients and prevent the development of acute graft rejection. Here, we followed for a period of 24 months the levels of catalytic IgG towards the synthetic peptide Pro-Phe-Arg-methylcoumarinimide in a large cohort of patients undergoing kidney transplantation. Twenty-four percent of the patients received IVIg at the time of transplantation. Our results demonstrate a marked reduction in levels of catalytic antibodies in all patients three months following kidney transplant. The decrease was significantly pronounced in patients receiving adjunct IVIg therapy. The results suggests that prevention of acute graft rejection using intravenous immunoglobulins induces a transient reduction in the levels of catalytic IgG, thus potentially jeopardizing the use of levels of catalytic antibodies as a prognosis marker for chronic allograft nephropathy.PLoS ONE 01/2013; 8(8):e70731. · 3.53 Impact Factor
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ABSTRACT: The major complication of the substitutive treatment of haemophilia A (HA) is the development of antifactor VIII (FVIII) antibodies. Most of these antibodies neutralize FVIII procoagulant activity, and are identified as FVIII inhibitor. A subgroup of these antibodies, 'catalytic antibodies', catalyses the FVIII hydrolysis. We investigated the frequency and the activity of catalytic antibodies, according to the phenotype of HA and the presence or absence of FVIII inhibitor. IgG from 16 patients with inhibitor and 17 patients without inhibitor were purified. Rates of FVIII hydrolysis and inhibitor titres were evaluated. Anti-FVIII catalytic antibodies were detected in 63.6% of patients with HA, irrespective of the HA phenotype and the presence of FVIII inhibitor. The frequency was significantly higher for severe HA patients (73.3%) and patients with inhibitor (87.5%), but their FVIII-proteolytic activity was not significantly different from patients with mild or moderate HA and patients without inhibitor. The evolution of both catalytic and inhibitory activities was studied for 11 patients with FVIII inhibitor. We observed two profiles. In the profile 1, 18.2% of patients, the catalytic activity and the inhibitor titre coevolved. In contrast, a dissociated evolution of these two parameters was observed in 72.8% patients in profile 2. These data confirm the importance of anti-FVIII catalytic activity in patients with severe, moderate and mild HA. Interestingly, most of the patients presented a dissociated profile, suggesting that anti-FVIII antibodies might not systematically act as FVIII inhibitors.Haemophilia 12/2012; · 3.17 Impact Factor
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ABSTRACT: Solid organs have been transplanted for decades. Since the improvement in graft selection and in medical and surgical procedures, the likelihood of graft function after 1 year is now close to 90%. Nonetheless even well-matched recipients continue to need medications for the rest of their lives hence adverse side effects and enhanced morbidity. Understanding Immune rejection mechanisms, is of increasing importance since the greater use of living-unrelated donors and genetically unmatched individuals. Chronic rejection is devoted to T-cells, however the role of B-cells in rejection has been appreciated recently by the observation that B-cell depletion improve graft survival. By contrast however, B-cells can be beneficial to the grafted tissue. This protective effect is secondary to either the secretion of protective antibodies or the induction of B-cells that restrain excessive inflammatory responses, chiefly by local provision of IL-10, or inhibit effector T-cells by direct cellular interactions. As a proof of concept B-cell-mediated infectious transplantation tolerance could be achieved in animal models, and evidence emerged that the presence of such B-cells in transplanted patients correlate with a favorable outcome. Among these populations, regulatory B-cells constitute a recently described population. These cells may develop as a feedback mechanism to prevent uncontrolled reactivity to antigens and inflammatory stimuli. The difficult task for the clinician, is to quantify the respective ratios and functions of "tolerant" vs. effector B-cells within a transplanted organ, at a given time point in order to modulate B-cell-directed therapy. Several receptors at the B-cell membrane as well as signaling molecules, can now be targeted for this purpose. Understanding the temporal expansion of regulatory B-cells in grafted patients and the stimuli that activate them will help in the future to implement specific strategies aimed at fighting chronic allograft rejection.Frontiers in Immunology 01/2013; 4:444.
Hydrolysis of Coagulation Factors by Circulating IgG Is
Associated with a Reduced Risk for Chronic Allograft
Nephropathy in Renal Transplanted Patients1
Bharath Wootla,* Antonino Nicoletti,* Natacha Patey,†Jordan D. Dimitrov,*
Christophe Legendre,‡Olivier D. Christophe,§Alain Friboulet,¶Srinivas V. Kaveri,*
Se ´bastien Lacroix-Desmazes,* and Olivier Thaunat2*?
Chronic allograft nephropathy (CAN), a major cause of late allograft failure, is characterized by a progressive decline in
graft function correlated with tissue destruction. Uncontrolled activation of the coagulation cascade by the stressed endo-
thelium of the graft is thought to play an important role in the pathophysiology of CAN. In this study, we demonstrate that
circulating IgG from renal-transplanted patients are endowed with hydrolytic properties toward coagulation factors VIII
and IX, but fail to hydrolyze factor VII and prothrombin. The hydrolytic activity of IgG was reliably quantified by the
measure of the hydrolysis of a fluorescent synthetic substrate for serine proteases: proline-phenylalanine-arginine-methyl-
coumarinamide (PFR-MCA). A retrospective case-control study indicated that an elevated hydrolysis rate of PFR-MCA by
circulating IgG correlated with the absence of CAN lesions on protocol graft biopsy performed 2 years posttransplantation.
We propose that circulating hydrolytic IgG may counterbalance the procoagulation state conferred by the activated endo-
thelium by disrupting the amplification loop of thrombin generation which is dependent on factors VIII and IX. Interestingly,
low rates of PFR-MCA hydrolysis, measured 3 mo posttransplantation, were predictive of CAN at 2 years down the lane.
These data suggest that PFR-MCA hydrolysis may be used as a prognosis marker for CAN in renal-transplanted
The Journal of Immunology, 2008, 180: 8455–8460.
nal graft survival. Contrasting with this progress, the half-life of
the transplants has remained the same due to chronic allograft
nephropathy (CAN),3which represents a major cause for graft loss
dvances during the last 20 years in immunosuppressive
therapy and surgical techniques have resulted in a reg-
ular improvement of short-term (i.e., 1-year) rate of re-
after the first year of transplantation (1). The pathophysiology of
CAN remains elusive. Accumulating evidence suggest however
that the uncontrolled activation of the coagulation cascade by the
activated endothelium of chronically rejected graft is an important
contributor to graft damages. Indeed, 1) extensive fibrin deposition
has been evidenced within the vessels of chronically rejected grafts
(1), 2) tissue factor expression is up-regulated in chronically re-
jected graft (2, 3), and 3) hirudin (a specific inhibitor of thrombin)
attenuates experimental rat cardiac chronic rejection (4).
Our group has recently demonstrated the presence in plasma of
IgG endowed with serine protease-like activity able to hydrolyze
certain coagulation factors (5). In the present study, we hypothe-
sized that hydrolyzing IgG may reduce the uncontrolled activation
of the coagulation cascade within rejected allografts and prevent
the development of CAN. To address this issue, we have investi-
gated whether hydrolyzing IgG are present in the serum of renal
graft recipients and whether the hydrolyzing activity of serum IgG
is associated with a decreased risk for CAN.
Materials and Methods
The present study relies on the Biobank established by the Renal Trans-
plantation Department of the Necker Hospital (Paris, France), where biop-
sies and serum samples are routinely collected at regular time points for all
grafted patients. For the first part of the study, we randomly selected 20
sera collected at various time points after renal transplantation. For the
case-control study, we retrospectively selected from the database 20 pa-
tients that 1) had received a first cadaveric kidney graft and 2) displayed
normal graft histology on the 3 mo posttransplantation protocol biopsy.
One group (n ? 10) of patients displayed chronic allograft nephropathy
defined as ?grade 2 (Banff classification) at the 2-year histological exam-
ination (group CAN), while the second group (n ? 10) displayed normal
biopsies (group without CAN, wCAN). Clinical characteristics of patients
from the two groups are presented in Table I.
*Centre de Recherche des Cordeliers, Universite ´ Pierre et Marie Curie-Paris 6, Unite ´
Mixte de Recherche S 872, Universite ´ Paris Descartes, Unite ´ Mixte de Recherche S
872, Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 872, Paris;
†Service d’anatomopathologie, Ho ˆpital Necker, Assistance Publique-Ho ˆpitaux de
Paris, Paris;‡Service de Transplantation Re ´nale et de Soins Intensifs, Ho ˆpital Necker,
Assistance Publique-Ho ˆpitaux de Paris, and Universite ´ Rene ´ Descartes, Paris;§Unite ´
770, Institut National de la Sante ´ et de la Recherche Me ´dicale, Universite ´ Paris-Sud,
Le Kremlin-Bice ˆtre;¶Centre National de la Recherche Scientifique Unite ´ Mixte de
Recherche 6022, Compie `gne Technological University, Compie `gne; and?Service de
Transplantation Re ´nale et d’Immunologie Clinique, Ho ˆpital Edouard Herriot, and
Universite ´ Claude Bernard Lyon I, Lyon, France
Received for publication October 9, 2007. Accepted for publication April 14, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Institut National de la Sante ´ et de la Recherche Me ´di-
cale, Centre National de la Recherche Scientifique, Universite ´ Pierre et Marie Curie,
and Agence Nationale de la Recherche (ANR-05-MRAR-012). O.T. is supported by
the Fondation pour la Recherche Me ´dicale and the Fondation du Rein; B.W. is the
recipient of a fellowship from Laboratoire Franc ¸ais du Fractionnement et des Bio-
technologies (Les Ulis, France).
2Address correspondence and reprint requests to Dr. Olivier Thaunat, Service de
Transplantation Re ´nale et d’Immunologie Clinique, Ho ˆpital Edouard Herriot, 5 place
d’arsonval, 69437 Lyon, Cedex 03, France. E-mail address: olivier.thaunatpastu@
3Abbreviations used in this paper: CAN, chronic allograft nephropathy; IVIg, intrave-
nous Ig; FVIII, factor VIII; FIX, factor IX; FVII, factor VII; PFR-MCA, proline-phenyl-
alanine-arginine-methylcoumarinamide; ROC, receiver operating characteristic; wCAN,
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
Blood tubes were allowed to clot for 1h at room temperature and centri-
fuged at 1500 ? g for 10 min at 20°C. Serum was immediately aliquoted
and stored at ?80°C until analysis.
The presence of antidonor alloantibodies was tested in each serum sam-
ple using the microlymphocytotoxicity assay according to the manufactur-
er’s instructions (Lambda Cell Trays; One Lamba).
Formalin-fixed, paraffin-embedded renal tissues were cut into 4-?m-thick
sections that were stained with H&E and Masson’s trichome. Chromophore
silver methenamine staining was performed to study glomerular basement
Immunohistochemical staining for C4d was performed on paraffin-em-
bedded renal tissues as previously described (6). Briefly, 4-?m-thick renal
sections were incubated with polyclonal rabbit antiserum to human C4d
(1/40; Biomedica Gruppe) and secondary goat anti-rabbit IgG Ab (1/360;
Jackson ImmunoResearch Laboratories). Detection was performed with
streptavidin/HRP (Jackson ImmunoResearch Laboratories) and developed
with stable DAB (DakoCytomation).
Purification of IgG
IgG were isolated from serum by affinity-chromatography on protein G-
Sepharose (Amersham Pharmacia Biotech). In brief, IgG was isolated from
plasma by 50% ammonium sulfate precipitation. Following dialysis, Ig was
incubated with protein G-Sepharose overnight at 4°C, eluted using 0.2M
glycine-HCl pH 2.8, dialyzed against PBS-0.02% NaN3for 3 h at 4°C, and
concentrated using Amicon (Millipore). A therapeutic preparation of
pooled normal human IgG (intravenous Ig (IVIg); Sandoglobulin) was
used as a source of control IgG. Size-exclusion chromatography of pa-
tients’ IgG and IVIg was performed on a Superose-12 column (GE Health-
care Europe) equilibrated with catalytic buffer (50 mM Tris pH 7.7, 8 M
urea and 0.02% NaN3), at a flow rate of 0.25 ml/min to exclude potentially
contaminating proteases. IgG-containing fractions were then pooled and
dialyzed against PBS-0.02% NaN3for 2 days at 4°C, followed by dialysis
against catalytic buffer containing 5 mM CaCl2(pH 7.7) for 2 days at 4°C.
The purity of IgG preparations was confirmed by SDS-PAGE and immu-
noblotting under nonreducing conditions, and by coincubation of the IgG
with a biotinylated suicide inhibitor for serine proteases (phosphonate di-
ester CRA, a gift from Prof. S. Paul, University of Texas, Houston, TX)
followed by detection in Western blot of the biotin-labeled material (data
not shown). IgG was quantified by OD measurements at 280 nm.
Biotinylation of Ags
Recombinant human factor VIII (FVIII, Kogenate FS; BayerHealthcare)
was reconstituted in distilled water to a final concentration of 600 ?g/ml,
desalted by dialyzing against borate buffer (100 mM borate (pH 7.0), 150
mM NaCl, and 5 mM CaCl2). Sulfo-NHS-LC-biotin (440 ?l at 25 ?g/ml)
was allowed to react with 600 ?g of FVIII with gentle agitation in the dark
for 2 h at 4°C. Biotinylated FVIII was dialyzed against catalytic buffer
containing 5 mM CaCl2for 3 h at 4°C, aliquoted, and stored at ?20°C until
use. The protocol was essentially identical for the biotinylation of human
proteins factor IX (FIX, BeneFIX; Baxter), activated factor VII (FVIIa,
NovoSeven; Novo Nordisk) and prothrombin (Kordia).
Hydrolysis of biotinylated Ags
Biotinylated FVIII, FIX, FVIIa, and prothrombin (185 nM) were incubated
in 40 ?l of catalytic buffer containing 5 mM CaCl2with the purified pa-
tients’ IgG (10 ?g/ml, 66.67 nM) in the dark for 24 h at 37°C. Samples
were mixed with Laemmli’s buffer without 2-ME (1:1, v/v) and 25 ?l of
each sample was subjected to 10% SDS-PAGE. Protein fragments were
then transferred onto nitrocellulose membranes (Schleicher & Schu ¨ll Mi-
croscience). Following overnight blocking in TBS containing 0.2% Tween
20 at 4°C, membranes were incubated with streptavidin-coupled alkaline
phosphatase (KPL) diluted 1:4000 in blocking buffer, for 60 min at room
temperature. After washing in TBS containing 0.1% tween-20 and TBS,
labeled proteins were revealed using the BCIP/NBT kit (Kirkegaard &
Perry Laboratories). Blots were scanned using a SnapScan 600 (Agfa)
scanner and rates of hydrolysis were calculated by densitometric analysis.
Hydrolysis of PFR-MCA
IgG (66.67 nM) were mixed with 100 ?M PFR-MCA (Peptide) in 40 ?l of
catalytic buffer containing 5 mM CaCl2(pH 7.7) in white 96-well U-bot-
tom plates and incubated in the dark for 24 h at 37°C. Hydrolysis of the
PFR-MCA substrate was determined by the fluorescence of the leaving
group (aminomethylcoumarin; ?em465 nm, ?ex360 nm) using a spec-
trofluorometer (GENios; Tecan Trading). Fluorescence values were com-
pared with a standard curve of free MCA and the corresponding quantities
of released MCA were computed. At each time point, background release
of MCA, measured in wells containing the substrate alone, was subtracted
from the value observed in the presence of the Abs. Data are expressed as
the quantity of released MCA computed at time 0 subtracted from the
quantity of released MCA computed at a given time point per amount of
time per amount of IgG. The significance of the increase in the hydrolytic
activity of patient IgG as compared with that of IVIg was assessed by
ANOVA and Fischer post hoc tests.
Circulating IgG of renal-transplanted patients display hydrolytic
activity against coagulation factors
We purified IgG from 20 randomly selected serum samples of
renal-transplanted patients and tested their ability to hydrolyze hu-
man recombinant FVIII, FIX, FVIIa, and prothrombin. The choice
for FVIII, FIX, and prothrombin as proteolytic targets for catalytic
IgG was motivated by their key role in the amplification loop of
thrombin generation in the coagulation cascade, while FVIIa is a
key molecule of the extrinsic pathway of coagulation. Absence of
contamination of the IgG samples by adventitious proteases was
ensured by the use of a double-step purification procedure that
involves a step of purification based on affinity and a step of pu-
rification based on protein size under denaturing conditions. IgG
from renal-transplanted patients were endowed with proteolytic
activity. The results demonstrate an heterogeneity in the hydrolytic
activity of circulating IgG. Four catalytic profiles were identified.
Representative examples of these four proteolytic behaviors are
provided in Fig. 1, A and B. IgG from some patients hydrolyzed
both FVIII and FIX (patients P1 and P2), while IgG from other
patients hydrolyzed FIX (patient P3), or FVIII (patient P4) or pre-
sented with no proteolytic activity (patient P5). IgG from trans-
planted patients did not display hydrolytic activity toward FVIIa
and prothrombin (data not shown).
Table I. Characteristics of the study populationa
(n ? 10)
(n ? 10)
Sex ratio (male/female)
Cause of end-stage renal
Cold ischemia time (h)
No. of HLA mismatches
% of panel-reactive Ab
No. of biopsy-proven
0.5933.42 ? 10.98 35.75 ? 7.84
17.86 ? 8.33 19.75 ? 11.21 0.68
1.33 ? 0.50
1.00 ? 0.71
1.00 ? 0.71
6.67 ? 13.46
0.82 ? 0.75
0.91 ? 0.83
0.82 ? 0.87
5.46 ? 18.09
0.56 ? 0.530.55 ? 0.690.97
and, Not determined. Statistic significance was assessed using one-way ANOVA.
8456 HYDROLYTIC Abs IN RENAL TRANSPLANTATION
Hydrolytic activity of circulating IgG of renal-transplanted
patients against PFR-MCA
The heterogeneous specificity of catalytic IgG from different pa-
tients precluded a systematic analysis with a single Ag hydrolysis
assay. We therefore relied on a generic substrate for serine pro-
tease-like activity (7): PFR-MCA.
Incubation of patients’ IgG with the peptide PFR-MCA resulted
in hydrolysis of the peptide and release of the fluorescent MCA
tag. The released fluorescence allowed for the calculation of
rates of hydrolysis. Hydrolysis of PFR-MCA was dose- and
time-dependent (data not shown). IVIg, used as a control, dem-
onstrated a marginal PFR-MCA-hydrolyzing activity of 0.06 ?
0.03 mmol/min per mol consistent with our previous observa-
tions (5) (Fig. 1C).
Patients with IgG hydrolyzing both FVIII and FIX demonstrated
the highest rates of hydrolysis of PFR-MCA (patients P1 and P2).
Rates of PFR-MCA hydrolysis were intermediate in the case of
IgG hydrolyzing either FVIII (patient P4) or FIX (patient P3) and
were low for IgG that were devoid of proteolytic activity (patient
P5; Fig. 1C).
Low rates of IgG-mediated hydrolysis of PFR-MCA correlate
We postulated that a high hydrolytic activity of circulating IgG
against PFR-MCA may be associated with a better control of the
coagulation cascade that could translate into a reduced risk
We undertook a pilot case-control study to determine whether
the level of hydrolytic activity against PFR-MCA is predictive of
the occurrence of CAN. The follow-up procedure in the Renal
Transplantation Department of Necker Hospital includes serum
collection and graft biopsies performed at regular time points. We
selected 10 patients that received a first cadaveric kidney graft and
who displayed potent (? grade 2 of the Banff classification (8))
CAN lesions on the biopsy performed 2 years after the transplan-
tation (group CAN; Fig. 2B). Importantly, the possibility that
histological lesions were already present in the grafts before the
from the plasma of patients with renal graft transplants. Biotinylated re-
combinant human FVIII (185 nM; A), biotinylated recombinant human FIX
(185 nM; B), and PFR-MCA (100 ?M; C) were incubated alone (control
(Ctl)) or in the presence of IgG (66.67 nM) from renal-transplanted patients
(P1, P2, P3, P4, and P5) for 24 h at 37°C. Immune globulin (IVIg) was used
as a source of normal IgG and as a control. In the case of FVIII and FIX,
samples were subjected to 10% SDS-PAGE and transferred onto a nitro-
cellulose membrane before revelation of biotinylated fragments. Rates of
hydrolysis of PFR-MCA were calculated as explained in Materials and
Methods and are shown as millimoles of substrate hydrolyzed per minute
per mole of IgG.
Hydrolysis of FVIII, FIX, and PFR-MCA by IgG purified
els, Representative features of graft biopsies obtained 2 years posttrans-
plantation (original magnification, ?200). Ten patients (group wCAN) dis-
played normal biopsies (shown in the case of one representative patient; A),
the remaining 10 patients (group CAN) presented with Banff grade ? II
CAN lesions (shown in the case of one representative patient; B). In the
latter group, Masson’s trichrome reveals tubular atrophy with interstitial
fibrosis (green) in ?50% of cortical area. Middle and lower panels, We
performed a careful histological analysis to evaluate the mechanisms in-
volved in the development of the lesions in the CAN group. Some patients
displayed features of chronic rejection that were either 1) cellular-medi-
ated, as assessed by the infiltration of immune effectors in the interstitium
(black arrows; Masson’s trichrome; original magnification, ?50; C) or 2)
Ab-mediated as assessed by a positive C4d staining of peritubular capillary
(Immunohistochemistry; original magnification, ?200; D). Features sug-
gestive of nephrotoxicity induced by calcineurin inhibitors were 1) beaded
medial hyaline deposits (black arrowheads; Masson’s trichrome; original
magnification, ?200; E) and 2) stripped fibrosis with tubular atrophy (Mas-
son’s trichrome; original magnification, ?20; F).
Histological features of renal allograft biopsies. Upper pan-
8457The Journal of Immunology
transplantation was ruled out by checking that the biopsy per-
formed 3 mo posttransplantation was normal. It is widely accepted
that both the immune response of the recipient (responsible for
chronic rejection) and various non-alloantigen-dependent factors
(including the direct nephrotoxicity of the drugs used as immuno-
suppressive therapy) are important contributors to CAN lesions. A
careful analysis relying on the criteria detailed in the recent Banff
2005 Meeting Report (9) was therefore performed to determine the
relative contributions of each mechanism in the development of
the lesions (Table II). Forty percent (4 of 10) of the patients in the
CAN group displayed features suggestive of chronic rejection that
was either cellular mediated (n ? 3, Fig. 2C) or Ab mediated as
assessed by the presence of circulating antidonor alloantibodies
and a positive C4d staining on the biopsy (n ? 1, Fig. 2D). Forty
percent (4 of 10) of the patients had developed CAN lesions due to
the nephrotoxicity of the calcineurin inhibitors (Fig. 2, E and F).
Pathological changes attributable to both processes were evi-
denced in the biopsy of the two remaining patients (20%), sug-
gesting a mixed pathophysiology.
Ten control patients with similar clinical characteristics (Table
I) but normal biopsy 2 years posttransplantation were then re-
trieved from the database (group wCAN, Fig. 2A).
Two years after transplantation, the hydrolytic rates of circulat-
ing IgG against PFR-MCA were higher in the group devoid of
lesions than in the group with CAN (6.17 ? 3.29 vs 2.52 ? 2.56
mmol/min per mol, respectively; p ? 0.006). Interestingly, this
difference was also observed 3 mo after transplantation (Fig. 2B;
8.94 ? 6.12 vs 3.80 ? 2.15 mmol/min per mol, respectively; p ?
0.008) at a time when all patients displayed normal biopsy. These
data suggest that a high hydrolytic activity of circulating IgG may
be protective against CAN and that the hydrolytic rate of circulat-
ing IgG may be used as a biological marker to predict the occur-
rence of CAN.
Hydrolytic activity of circulating IgG is a potential predictive
marker for CAN
The receiver operating characteristic (ROC) curve is a classical
tool to evaluate the accuracy of a diagnostic test. The percentage
of false-positive cases was plotted against the percentage of true-
positive cases for 10 possible cutoffs using the levels of IgG-me-
diated PFR-MCA hydrolysis measured at 3 mo posttransplantation
(Fig. 2C). Cases were patients with CAN lesions on the renal
biopsy performed 2 years posttransplantation. An area under the
PFR-MCA hydrolysis by circulating IgG in renal transplantation. A, Ret-
rospective longitudinal case-control comparison of the rate of PFR-MCA
hydrolysis by IgG from renal-transplanted patients with or without CAN.
Box plot representing the distribution of the rate of PFR-MCA hydrolysis
of circulating IgG collected 3 mo (left panel) or 2 years (right panel)
posttransplantation from renal-transplanted patients with (CAN) or without
(wCAN) CAN at 2 years. ??, p ? 0.01 CAN vs wCAN (Mann-Whitney U
nonparametric test). B, ROC curve for PFR-MCA hydrolysis by circulating
IgG was obtained by plotting the percentage of false-positive cases against
the percentage of true-positive cases for 10 possible cutoff values using the
rate of PFR-MCA hydrolysis of circulating IgG obtained 3 mo posttrans-
plantation. Cases were patients with CAN on renal biopsies 2 years post-
transplantation. The accuracy of the test indicated by the area under the
ROC curve (AUC). An area of 1 represents a perfect test and an area of 0.5
represents a worthless test. With an area under the ROC curve close to 0.9,
the rate of IgG-mediated PFR-MCA hydrolysis exhibits excellent accuracy
in predicting CAN. C, The glomerular filtration rate was estimated using
the MDRD formula (25) at 3 mo, 1 year, and 2 years posttransplantation.
?, p ? 0.05 CAN vs wCAN (one-way ANOVA). D, The proteinuria was
quantified in the 24-h urine collection at 3 mo, 1 year, and 2 years post-
transplantation. Log scale. ?, p ? 0.05 CAN vs wCAN (one-way
Pilot retrospective case-control study of the clinical value of
Table II. Histological analysis of chronically rejected allograftsa
Chronic Allograft Damages
Chronic Csa Nephrotoxicity CellularHumoral
aCsa, Cyclosporine; Csa NTox, cyclosporine nephrotoxicity; PTC, peritubular capillary; CR, chronic rejection.
8458HYDROLYTIC Abs IN RENAL TRANSPLANTATION
ROC curve of 0.9 demonstrated an excellent predictive value of
the level of hydrolytic activity of circulating IgG for CAN.
Several biological parameters have been shown to be predictive
for late renal graft failure. Among the most widely used are the
estimated glomerular filtration rate (10) and the proteinuria (11).
Interestingly, we found that the level of hydrolytic activity of cir-
culating IgG had a superior predictive value of CAN when com-
pared with these two established markers. Indeed, estimated glo-
merular filtration rate decline was only detectable at 2 years in the
CAN group when histological changes were already patent (Fig.
3C). Proteinuria was not different in the two groups at 3 mo after
transplantation and increased in the CAN group after 1year; how-
ever, the difference between the two groups (wCAN vs CAN)
failed to reach statistical significance (Fig. 3D).
In the present study, we demonstrate that 1) circulating IgG of
renal-transplanted patients are endowed with hydrolytic activity
against coagulation factors VIII and IX and that 2) a high hydro-
lytic rate of circulating IgG correlates with a lesser prevalence of
CAN. Interestingly, the difference in hydrolytic activities between
patients that will develop CAN and patients that will not 2 years
The presence of circulating hydrolytic IgG has been reported in
various pathological situations including inflammatory, autoim-
mune, and infectious diseases (12–15). Their deleterious role has
been suspected on the basis of their association with disease and
confirmed in the case of hemophilia (16), multiple sclerosis (17),
and HIV-1-related immune thrombocytopenia (18). In contrast, we
have recently demonstrated that a high hydrolytic activity of cir-
culating IgG is associated with a higher survival rate in severe
sepsis (5). The present data complements our previous observa-
tions by suggesting that hydrolytic IgG may carry a protective
effect against CAN.
Interestingly, although sepsis and CAN display obviously dif-
ferent pathophysiologies, the two situations share in common a
defective control of the coagulation cascade. Indeed, both sepsis
and CAN are characterized by an abnormal activation of the en-
dothelium, which is systemic in sepsis (19) and restricted to the
endothelium of the graft in transplantation (20). Activation of the
endothelium tilts the coagulation balance toward a hypercoagula-
tory state. Our study documents that hydrolytic activity of circu-
lating IgG of some renal-transplanted patients is directed toward
coagulation factors VIII and/or IX, an observation reminiscent of
our findings in severe sepsis (5). Interestingly, these two factors
play a central role in the amplification loop for the generation of
thrombin. We have previously demonstrated that IgG-mediated
FVIII hydrolysis results in inactivation of the procoagulant activity
of FVIII (21). It is thus tempting to speculate that hydrolytic cir-
culating IgG exert their preventive action against CAN by disrupt-
ing the amplification loop of the coagulation cascade, therefore
counterbalancing the abnormal activation of the endothelium.
Accumulating evidence suggests that Ab play a crucial role in
the pathophysiology of CAN (22). Consequently, current thera-
peutic strategies to treat or prevent alloantibody-mediated rejection
include B cell depletion therapy in combination with plasmaphere-
sis (23). Our data suggest that some IgG subpopulations may be
endowed with a beneficial effect, i.e., regulation of the coagulation
cascade by hydrolytic IgG. In the future, more efficient therapies
against CAN should thus rely on a targeted control of pathogenic
IgG rather than on the global elimination of circulating Ab. Inter-
estingly, patients are heterogeneous in their ability to generate hy-
drolytic IgG. Experimental studies aiming at the identification of
the B cells responsible for the production of hydrolytic IgG and
of the factors that influence their synthesis would pave the way to
As yet, no efficient therapy is available to treat CAN. Recent
encouraging evidence suggests however that graft failure might be
delayed through adequate management if initiated early (24), out-
lining the need for a reliable predictive marker to identify the pa-
tients prone to develop CAN. Our present results suggest that the
rate of IgG-mediated PFR-MCA hydrolysis at 3 mo posttransplan-
tation (i.e., at a time when graft biopsies are still normal) were
significantly lower in the case of patients with CAN on the 2-year
biopsies. The IgG hydrolytic activity could therefore represent a
very early marker, the decrease of which would be detectable be-
fore any tissue damage is apparent. The measurement of IgG-me-
diated PFR-MCA hydrolysis is an affordable, simple, noninvasive
test that can be done at large scale and that displays a satisfactory
predictive value. A large clinical prospective study shall allow
both to validate the test and to provide additional information in-
cluding the sensitivity and specificity, the subgroups of patients for
whom this test may be more specifically beneficial, and the optimal
We thank Sophie Lechaton for excellent research assistance. FVIII (Ko-
genate FS) and FIX (Benefix) were gifts from BayerPharma and Baxter,
The authors have no financial conflict of interest.
1. Matsuyama, M., R. Yoshimura, K. Akioka, M. Okamoto, H. Ushigome,
Y. Kadotani, T. Nakatani, and N. Yoshimura. 2003. Tissue factor antisense oli-
gonucleotides prevent renal ischemia-reperfusion injury. Transplantation 76:
2. Holschermann, H., R. M. Bohle, H. Zeller, H. Schmidt, U. Stahl, L. Fink,
H. Grimm, H. Tillmanns, and W. Haberbosch. 1999. In situ detection of tissue
factor within the coronary intima in rat cardiac allograft vasculopathy.
Am. J. Pathol. 154: 211–220.
3. Yen, M. H., G. Pilkington, R. C. Starling, N. B. Ratliff, P. M. McCarthy,
J. B. Young, G. M. Chisolm, and M. S. Penn. 2002. Increased tissue factor
expression predicts development of cardiac allograft vasculopathy. Circulation
4. Holschermann, H., R. M. Bohle, H. Schmidt, H. Zeller, L. Fink, U. Stahl,
H. Grimm, H. Tillmanns, and W. Haberbosch. 2000. Hirudin reduces tissue factor
expression and attenuates graft arteriosclerosis in rat cardiac allografts. Circula-
tion 102: 357–363.
5. Lacroix-Desmazes, S., J. Bayry, S. V. Kaveri, D. Hayon-Sonsino, N. Thorenoor,
J. Charpentier, C. E. Luyt, J. P. Mira, V. Nagaraja, M. D. Kazatchkine, et al.
2005. High levels of catalytic antibodies correlate with favorable outcome in
sepsis. Proc. Natl. Acad. Sci. USA 102: 4109–4113.
6. Troxell, M. L., L. A. Weintraub, J. P. Higgins, and N. Kambham. 2006. Com-
parison of C4d immunostaining methods in renal allograft biopsies. Clin. J. Am.
Soc. Nephrol. 1: 583–591.
7. Li, L., S. Paul, S. Tyutyulkova, M. D. Kazatchkine, and S. Kaveri. 1995. Cata-
lytic activity of anti-thyroglobulin antibodies. J. Immunol. 154: 3328–3332.
8. Racusen, L. C., K. Solez, R. B. Colvin, S. M. Bonsib, M. C. Castro, T. Cavallo,
B. P. Croker, A. J. Demetris, C. B. Drachenberg, A. B. Fogo, et al. 1999. The
Banff 97 working classification of renal allograft pathology. Kidney Int. 55:
9. Solez, K., R. B. Colvin, L. C. Racusen, B. Sis, P. F. Halloran, P. E. Birk,
P. M. Campbell, M. Cascalho, A. B. Collins, A. J. Demetris, et al. 2007. Banff ’05
Meeting Report: differential diagnosis of chronic allograft injury and elimination
of chronic allograft nephropathy (“CAN”). Am. J. Transplant. 7: 518–526.
10. Salvadori, M., A. Rosati, A. Bock, J. Chapman, B. Dussol, L. Fritsche, V. Kliem,
Y. Lebranchu, F. Oppenheimer, E. Pohanka, et al. 2006. Estimated one-year
glomerular filtration rate is the best predictor of long-term graft function follow-
ing renal transplant. Transplantation 81: 202–206.
11. Fernandez-Fresnedo, G., J. J. Plaza, J. Sanchez-Plumed, A. Sanz-Guajardo,
R. Palomar-Fontanet, and M. Arias. 2004. Proteinuria: a new marker of long-term
graft and patient survival in kidney transplantation. Nephrol. Dial. Transplant 19
(Suppl. 3): iii47–iii51.
12. Paul, S., D. J. Volle, C. M. Beach, D. R. Johnson, M. J. Powell, and R. J. Massey.
1989. Catalytic hydrolysis of vasoactive intestinal peptide by human autoanti-
body. Science 244: 1158–1162.
8459The Journal of Immunology
13. Shuster, A. M., G. V. Gololobov, O. A. Kvashuk, A. E. Bogomolova,
I. V. Smirnov, and A. G. Gabibov. 1992. DNA hydrolyzing autoantibodies. Sci-
ence 256: 665–667.
14. Thiagarajan, P., R. Dannenbring, K. Matssura, A. Tramontano, G. Gololobov,
and S. Paul. 2000. Monoclonal antibody light chain with prothrombinase activity.
Biochemistry 39: 6459–6465.
15. Vlassov, A., C. Florentz, M. Helm, V. Naumov, V. Buneva, G. Nevinsky, and
R. Giege. 1998. Characterization and selectivity of catalytic antibodies from hu-
man serum with RNase activity. Nucleic Acids Res. 26: 5243–5250.
16. Lacroix-Desmazes, S., A. Moreau, Sooryanarayana, C. Bonnemain, N. Stieltjes,
A. Pashov, Y. Sultan, J. Hoebeke, M. D. Kazatchkine, and S. V. Kaveri. 1999.
Catalytic activity of antibodies against factor VIII in patients with hemophilia A.
Nat. Med. 5: 1044–1047.
17. Ponomarenko, N. A., O. M. Durova, I. I. Vorobiev, A. A. Belogurov, Jr.,
I. N. Kurkova, A. G. Petrenko, G. B. Telegin, S. V. Suchkov, S. L. Kiselev,
M. A. Lagarkova, et al. 2006. Autoantibodies to myelin basic protein catalyze
site-specific degradation of their antigen. Proc. Natl. Acad. Sci. USA 103:
18. Nardi, M., S. Tomlinson, M. A. Greco, and S. Karpatkin. 2001. Complement-
independent, peroxide-induced antibody lysis of platelets in HIV-1-related im-
mune thrombocytopenia. Cell 106: 551–561.
19. Riewald, M., and W. Ruf. 2003. Science review: role of coagulation protease
cascades in sepsis. Crit. Care 7: 123–129.
20. Salom, R. N., J. A. Maguire, and W. W. Hancock. 1998. Endothelial activation
and cytokine expression in human acute cardiac allograft rejection. Pathology 30:
21. Lacroix-Desmazes, S., B. Wootla, S. Dasgupta, S. Delignat, J. Bayry, J. Reinbolt,
J. Hoebeke, E. Saenko, M. D. Kazatchkine, A. Friboulet, et al. 2006. Catalytic
IgG from patients with hemophilia A inactivate therapeutic factor VIII. J. Im-
munol. 177: 1355–1363.
22. Terasaki, P. I., and J. Cai. 2005. Humoral theory of transplantation: further ev-
idence. Curr. Opin. Immunol. 17: 541–545.
23. Pescovitz, M. D. 2006. B cells: a rational target in alloantibody-mediated solid
organ transplantation rejection. Clin. Transplant. 20: 48–54.
24. Afzali, B., A. L. Taylor, and D. J. Goldsmith. 2005. What we CAN do about
chronic allograft nephropathy: role of immunosuppressive modulations. Kidney
Int. 68: 2429–2443.
25. Poge, U., T. Gerhardt, H. Palmedo, H. U. Klehr, T. Sauerbruch, and R. P. Woitas.
2005. MDRD equations for estimation of GFR in renal transplant recipients.
Am. J. Transplant. 5: 1306–1311.
8460HYDROLYTIC Abs IN RENAL TRANSPLANTATION