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Drug efflux transporters in rheumatoid arthritisComment on the article by Kremer.

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Vol. 52, No. 2, February 2005, pp 670–678
© 2005, American College of Rheumatology
is also capable of transporting di- and triglutamate forms of
MTX (9). This is important, as Kremer (1) highlights, since the
polyglutamated forms of MTX are critical to its intracellular
activity and efficacy. Further studies are required to clarify the
relative importance of the different cellular efflux proteins in
mediating an individual’s response to MTX in rheumatoid
DOI 10.1002/art.20770
Drug efflux transporters in rheumatoid arthritis:
comment on the article by Kremer
To the Editor:
We read with interest the article by Kremer (1) reviewing the mechanisms of action of methotrexate (MTX) and
potential mechanisms of MTX resistance. One facet that
Kremer highlighted is the role of drug efflux transporters in
mediating resistance to MTX. Although he highlights the role
of multidrug resistance–associated proteins (MRPs) in mediating MTX transport, he also emphasizes a study examining
permeable glycoprotein (P-gp) expression and response to
MTX (2,3). Although it is now established that MRP-1 transports MTX (4), whether P-gp transports MTX is less clear.
In order to clarify this issue, we undertook a drug
accumulation study using radiolabeled MTX in an in vitro T
lymphoblastoid cell line (CEM cells). Parent CEM cells do not
express any efflux transporters, but CEM cells pretreated with
vinblastine (VBL) constitutively express the efflux transporter
P-gp. CEM cells pretreated with epirubicin (E1000) constitutively express the efflux transporter MRP-1. This in vitro
system is well characterized and has been used to determine
the impact of the multidrug resistant transporters on the
cellular accumulation of other compounds (5). We incubated
1 ⫻ 106 cells/ml with radiolabeled MTX for 18 hours at 37°C
across a range of drug concentrations. The excess supernatant
was discarded and the cells were extracted in methanol. The
intracellular drug concentrations were calculated for the 3 cell
lines and results compared using the Kruskal-Wallis test for
multiple comparisons. The extent of accumulation of MTX in
the MRP-1–expressing cells was significantly reduced (P ⬍
0.001), compared with the CEM and VBL cells (Figure 1).
These data support the balance of published evidence
suggesting that MTX is a substrate for MRP-1 (4) but is not a
substrate for P-gp (6). This is also consistent with the observation that P-gp preferentially transports neutral hydrophobic
compounds, whereas MRP-1 preferentially transports anionic
compounds such as MTX (6). Instead of P-gp it is likely that
the transporters MRPs 1–4 (4,6,7), together with breast cancer
resistance protein (8), are more important in mediating MTX
resistance. Studies suggest that breast cancer resistance protein
Supported in part by the UK Arthritis Research Campaign.
Samantha L. Hider, MRCP, MSc, BM, BS
University of Manchester
Manchester, UK
Patrick Hoggard, PhD
Saye Khoo, MD, MRCP
David Back, PhD
University of Liverpool
Liverpool, UK
Ian N. Bruce, MD, FRCP
University of Manchester
Manchester, UK
1. Kremer JM. Toward a better understanding of methotrexate [review]. Arthritis Rheum 2004;50:1370–82.
2. Llorente L, Richaud-Patin Y, Diaz-Borjon A, Alvarado dlB, JakezOcampo J, De La FH, et al. Multidrug resistance-1 (MDR-1) in
rheumatic autoimmune disorders. Part I: Increased P-glycoprotein
activity in lymphocytes from rheumatoid arthritis patients might
influence disease outcome. Joint Bone Spine 2000;67:30–9.
3. Ranganathan P, Eisen S, Yokoyama WM, McLeod HL. Will
pharmacogenetics allow better prediction of methotrexate toxicity
and efficacy in patients with rheumatoid arthritis? Ann Rheum Dis
4. Hooijberg JH, Broxterman HJ, Kool M, Assaraf YG, Peters GJ,
Noordhuis P, et al. Antifolate resistance mediated by the multidrug
resistance proteins MRP1 and MRP2. Cancer Res 1999;59:2532–5.
5. Davey RA, Longhurst TJ, Davey MW, Belov L, Harvie RM,
Hancox D, et al. Drug resistance mechanisms and MRP expression
in response to epirubicin treatment in a human leukaemia cell line.
Leuk Res 1995;19:275–82.
6. Jansen G, Scheper RJ, Dijkmans BA. Multidrug resistance proteins
in rheumatoid arthritis, role in disease-modifying antirheumatic
drug efficacy and inflammatory processes: an overview. Scand
J Rheumatol 2003;32:325–36.
7. Zeng H, Chen ZS, Belinsky MG, Rea PA, Kruh GD. Transport of
methotrexate (MTX) and folates by multidrug resistance protein
(MRP) 3 and MRP1: effect of polyglutamylation on MTX transport. Cancer Research 2001;61:7225–32.
8. Volk EL, Farley KM, Wu Y, Li F, Robey RW, Schneider E.
Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance. Cancer Res 2002;62:5035–40.
9. Chen ZS, Robey RW, Belinsky MG, Shchaveleva I, Ren XQ,
Sugimoto Y, et al. Transport of methotrexate, methotrexate polyglutamates, and 17␤-estradiol 17-(␤-D-glucuronide) by ABCG2:
effects of acquired mutations at R482 on methotrexate transport.
Cancer Res 2003;63:4048–54.
DOI 10.1002/art.20744
Methotrexate and long-term treatment of rheumatic
disease: comment on the article by Kremer
Figure 1. Intracellular accumulation of methotrexate (MTX). Values
are the mean and SD (n ⫽ 4). ⴱ ⫽ P ⬍ 0.001 in multidrug
resistance–associated protein-1–expressing cells (E1000) versus CEM
and vinblastine (VBL) cells.
To the Editor:
We read with interest the excellent review on methotrexate (MTX) by Kremer (1). The author discusses drug
Figure 1. Competition of multidrug resistance-associated proteins
(MRPs) and folylpolyglutamate synthetase (FPGS) for methotrexate
(MTX). MTX is transported into the cell by the reduced folate carrier
(RFC). MRPs can transport unmodified MTX out of the cell, but not
polyglutamated MTX (MTX-Glu2–6) which inhibits dihyrofolate reductase (DHFR). Reproduced, with permission, from Borst P, Evers
R, Kool M, Wijnholds J. A family of drug transporters: the multidrug
resistance-associated proteins. J Natl Cancer Inst 2000;92:1295–302.
transporters in MTX metabolism, including those which cause
drug efflux, and their role in leading to the plateau in clinical
response, which is often seen when MTX is used in the
long-term treatment of rheumatic diseases. We would like to
highlight some of the controversies surrounding the role of
these transporters in mediating resistance to MTX.
As noted by Kremer, MTX transport into the cell is
mediated by members of the solute carrier family of transporters, specifically SLC19A1, which is also called the reduced
folate carrier. It is increasingly recognized that genetic variations in this transporter can lead to differential responses to
MTX (2,3). After entry into the cell, MTX is converted into
the polyglutamated form by the enzyme folylpolyglutamate
synthetase (FPGS). The multidrug resistant transporter
MDR1/P-glycoprotein (P-gp) and the multidrug resistance–
associated proteins (MRPs), both members of the ATPbinding cassette family of transporters, have been implicated in
MTX efflux from the cell. Whether MDR1/P-gp includes MTX
among its several substrates remains controversial. Norris et al
showed that leukemic cell lines resistant to MTX had increased
expression and function of the MDR1 gene, and MTX resistance was partially reversed by a P-gp–specific monoclonal
antibody (4). Similarly, it has been demonstrated that the
peripheral blood lymphocytes (PBLs) of patients with rheumatoid arthritis (RA) who are refractory to treatment with MTX
express higher levels of P-gp compared with responders (5). In
contrast to this, MDR1-transgenic mice overexpressing P-gp in
their bone marrow cells develop bone marrow suppression
when treated with MTX (6). Another study showed no difference in the expression of P-gp on PBLs between RA patients
who were MTX responders and those who were nonresponders (7).
The MRP family has seven members, MRP-1–MRP-7.
Of these, MRPs 1, 2, 3, and 4 have been mainly implicated in
MTX resistance. Interestingly, it has been found that MRPs 1,
2, and 3 cause MTX resistance in cells during a 4-hour
exposure to MTX at high concentrations, but not during a
96-hour continuous exposure to low-dose MTX (8, 9). This
may be because, although both FPGS and MRPs compete for
MTX, MTX polyglutamates cannot be transported by MRPs
(Figure 1). Cells with chronic exposure to low-dose MTX may
slowly accumulate these polyglutamates, and remain susceptible to MTX, whereas such a phenomenon does not occur with
short-term exposure to high concentrations of MTX. Such
resistance to high-dose MTX can be partially overcome by
inhibitors of MRP-1, such as sulfinpyrazone and probenecid.
We agree with Kremer that drug efflux transporters
such as MDR1/(P-gp) and MRPs contribute significantly to the
loss of response to MTX. However, although these transporters are crucial in determining resistance to high-dose MTX, as
used in cancer treatment, their role in mediating resistance to
low-dose MTX, as used in the treatment of rheumatic diseases,
remains quite controversial. Nevertheless, concomitant use of
inhibitors of these transporters with MTX may be a promising
strategy to improve the efficacy of the drug in the future.
Prabha Ranganathan, MD
Howard L. McLeod, PharmD
Washington University School of Medicine
St. Louis, MO
1. Kremer JM. Toward a better understanding of methotrexate [review]. Arthritis Rheum 2004;50:1370–82.
2. Rothern L, Aronheim A, Assaraf YG. Alterations in the expression
of transcription factors and the reduced folate carrier as a novel
mechanism of antifolate resistance in human leukemia cells. J Biol
Chem 2003;278:8935–41.
3. Dervieux T, Orentas Lein D, Park G, Barham R, Smith K, Walsh
M, et al. Single nucleotide polymorphisms (SNPs) in the folate/
purine synthesis pathway predict methotrexate’s effects in rheumatoid arthritis [abstract]. Arthritis Rheum 2003;48 Suppl 9:S438.
4. Norris MD, de Graaf D, Haber M, Kavallaris M, Madafiglio J,
Gilbert J, et al. Involvement of MDR1 P-glycoprotein in multifactorial resistance to methotrexate. Int J Cancer 1996;65:613–9.
5. Yudoh K, Matsuno H, Nakazawa F, Yonezawa T, Kimura T.
Increased expression of multidrug resistance of P-glycoprotein on
Th1 cells correlates with drug resistance in rheumatoid arthritis.
Arthritis Rheum 1999;42:2014–5.
6. Mickisch GH, Merlino GT, Galski H, Gottesman MM, Pastan I.
Transgenic mice that express the human multidrug-resistance gene
in bone marrow enable a rapid identification of agents that reverse
drug resistance. Proc Natl Acad Sci U S A 1991;88:547–51.
7. Hider SL, Morgan C, Bell E, Bruce IN. Methotrexate is not a
substrate for P-glycoprotein in patients with rheumatoid arthritis
[abstract]. Ann Rheum Dis 2002;61 Suppl 1:199.
8. Hooijberg JH, Broxterman HJ, Kool M, Assaraf YG, Peters GJ,
Noordhuis P, et al. Antifolate resistance mediated by the multidrug
resistance proteins MRP1 and MRP2. Cancer Res 1999;59:2532–5.
9. Kool M, van der Linden M, de Haas M, Scheffer GL, de Vree JM,
Smith AJ, et al. MRP3, an organic anion transporter able to
transport anti-cancer drugs. Proc Natl Acad Sci U S A 1999;96:
DOI 10.1002/art.20893
To the Editor:
I would like to thank Dr. Hider and colleagues for
describing their interesting study, which demonstrates that
there is no effect on MTX efflux from the cellular transport
protein P-gp in an in vitro T lymphoblastoid cell line. The
scientific and clinical communities will hopefully learn more in
the coming years about the mechanisms of resistance to MTX.
These potential advances in our understanding of MTX,
together with new insights into the very real effects of singlenucleotide polymorphisms on several enzymes in the folate
metabolic pathways, could lead to more effective and tailored
treatment strategies.
I would also like to thank Drs. Ranganathan and
McLeod for their thoughtful comments regarding the possible
role of MRPs in the phenomenon of MTX resistance. Their
summary of the possible differential effects of MRPs with
short- and long-term exposure to MTX, as well as the potential
role of sulfinpyrazone and probenecid, is again noted as a rich
area for future clinical investigation. I also agree with their
observation that the role of MRPs in the dose range of MTX
commonly used to treat RA is still controversial. Hopefully, we
will see more attention and research focused on the role of
MRPs, as well as their inhibitors, and how manipulation of
their activity could affect the clinical response to MTX, when
used to treat patients with RA. Our patients will benefit as
more is learned about the mechanisms of this old, but excellent, drug.
Joel M. Kremer, MD
Albany Medical College
Albany, NY
DOI 10.1002/art.20812
Study of individual joint pathology in rheumatoid
arthritis suggests a single pathology: comment on the
editorial by Kirwan
To the Editor:
We read with interest the recent editorial promoting
the concept that there are at least 2 pathologies involved in
rheumatoid arthritis (RA), suggesting that there are separate
processes for joint inflammation and direct joint destruction
(1). This is an interesting concept, but it is predominantly
based on studies that used conventional radiographic imaging
for determination of joint destruction. The concept of 2 or
more pathologies in RA stems from the radiographic observations that erosions progress despite the suppression of synovitis
and that these processes are therefore uncoupled. However,
we wish to highlight another interpretation of the data based
on analysis of individual joint damage, and in particular to cite
information from recent magnetic resonance imaging (MRI)
studies which enable imaging of both synovitis and erosions.
Most studies on RA therapies have evaluated summed
groups of joints (tender and swollen joint counts or cumulative
Sharp scores) or systemic markers of inflammation, such as
erythrocyte sedimentation rate or C-reactive protein levels.
Very few studies have examined inflammation at the individual
joint level. Boers and colleagues (2) analyzed data from the
COBRA (Combinatietherapie Bij Reumatoı̈de Artritis) study
using clinical measurements of individual joints and radiographic progression of the same joints. That study demonstrated that local signs of RA at baseline and at 1-year followup
strongly predicted radiographic progression of joint damage in
the individual joint.
It is now accepted that clinical examination may be
relatively insensitive and that imaging studies have confirmed a
discrepancy between clinical examination and MRI-detected
synovitis (3). Even in a careful analysis of clinical examination
and radiographic joint damage there is bound to be some
degree of mismatch, and it is therefore conceivable that
synovitis may be present at a subclinical level and may cause
subsequent bone erosion. Indeed, the majority of RA patients
with apparent clinical remission who are evaluated using
sensitive imaging techniques have been demonstrated to have
measurable synovitis (4), explaining the apparent contradiction
of erosion still occurring in patients whose disease is clinically
in remission (5).
The best way to visualize the relationship between
synovium and bone is to use the most sensitive imaging
technique (MRI) to image individual joints (such as the
metacarpophalangeal joints), rather than a complex structure
(such as the wrist), and follow the relationship over time.
Compounding the insensitivity of clinical examination is the
number of time points at which synovitis is measured; the
measurement of any potential fluctuation in synovitis is limited
by the number of study assessments. It would also seem
appropriate to concentrate on short-duration RA, in which
secondary osteoarthritis is less likely to confound pathologic
We have previously reported a study of 40 patients with
early RA (⬍12 months duration) who had baseline, 3-month,
and 12-month MRI scans performed in the course of a
randomized trial (6). In this study, no mismatch between
MRI-detected synovitis and subsequent bone damage was
observed. Where there was no synovitis, no subsequent MRI
erosions developed. The number of new erosions developing at
the individual joint level was proportional to the amount of
synovitis in a given joint, even given the possible limitations of
semiquantitative synovitis measurement. Certainly mechanical
factors, such as the location of collateral ligaments, are important at the site of erosions (7), but synovitis is the predictor of
erosion development.
We do not intend to suggest that MRI should be
used as an outcome measure in every RA trial, but we do
think that MRI provides the best available tool for studying
in vivo the intrajoint pathologic relationships in RA. Simultaneous imaging of the synovium and bone, at enough time
points to understand the total synovial “load” on a given
joint over time, demonstrates only a single process in RA
subjects. In conclusion, we believe rheumatologists would be
well served by focusing research and therapy on inflammation suppression as the mechanism for controlling joint
damage in RA.
Philip G. Conaghan, MBBS, FRACP, FRCP
Dennis McGonagle, MB, PhD, FRCPI
Ai Lyn Tan, MRCP
Paul Emery, MA, MD, FRCP
University of Leeds
Leeds, UK
1. Kirwan JR. The synovium in rheumatoid arthritis: evidence for (at
least) two pathologies [editorial]. Arthritis Rheum 2004;50:1–4.
2. Boers M, Kostense PJ, Verhoeven AC, van der Linden S, for the
COBRA Trial Group. Inflammation and damage in an individual
joint predict further damage in that joint in patients with early
rheumatoid arthritis. Arthritis Rheum 2001;44:2242–6.
3. Goupille P, Roulot B, Akoka S, Avimadje AM, Garaud P,
Naccache L, et al. Magnetic resonance imaging: a valuable method
for the detection of synovial inflammation in rheumatoid arthritis.
J Rheumatol 2001;28:35–40.
4. Brown AK, Quinn MA, Karim Z, Wakefield RJ, Conaghan PG,
Pollard AS, et al. Neither the ACR remission criteria nor the
Disease Activity Score accurately define true remission in rheumatoid arthritis [abstract]. Arthritis Rheum 2002;46 Suppl 9:S243.
5. Molenaar ET, Voskuyl AE, Dinant HJ, Bezemer PD, Boers M,
Dijkmans BA. Progression of radiologic damage in patients with
rheumatoid arthritis in clinical remission. Arthritis Rheum 2004;
6. Conaghan PG, O’Connor P, McGonagle D, Astin P, Wakefield
RJ, Gibbon WW, et al. Elucidation of the relationship between
synovitis and bone damage: a randomized magnetic resonance
imaging study of individual joints in patients with early rheumatoid
arthritis. Arthritis Rheum 2003;48:64–71.
7. Tan AL, Tanner SF, Conaghan PG, Radjenovic A, O’Connor P,
Brown AK, et al. Role of metacarpophalangeal joint anatomic
factors in the distribution of synovitis and bone erosion in early
rheumatoid arthritis. Arthritis Rheum 2003;48:1214–22.
DOI 10.1002/art.20894
To the Editor:
I thank Dr. Conaghan and colleagues for pointing out
some of the evidence against the thesis that there are at least
2 pathologic processes underway in parallel in the joints of
patients with RA. The definitions of “synovitis” and “erosions”
are crucial to this debate.
Conaghan et al take the view that findings seen on
MRI, which they call “synovitis” and “erosions,” are the same
as the pathologic activities of the same names. In their
excellent and important work on MRI of joints in early
arthritis, the Leeds group led by Dr. Emery has consistently
found many more MRI “erosions” than radiographic erosions,
and has argued that the large majority of patients with early
RA have erosions when examined by MRI. In spite of this,
many longitudinal studies have shown that a substantial proportion of hospital patients with RA never develop joint
damage visible on conventional radiography. Nonerosive RA is
well recognized. This calls into question the validity of labeling
MRI “erosions” as the same pathology as radiographic erosions.
Further, showing that there is a broad correlation
between 2 aspects of RA, the presence of synovial inflammation and bony erosions, does not prove one causes the other.
For example, we would not accept an assertion that pain in the
toes causes erosions in the fingers—and yet the 2 are manifestly correlated in patients with RA.
The argument that there are at least 2 parallel pathologic processes in the joints of patients with erosive RA is not
watertight, but neither is it based on a single observation about
controlling clinical signs while allowing radiographic progression. A second relevant observation is that glucocorticoids
have the ability to almost stop the progression of joint erosions, even when given in low doses, which adds little in the
long run to current antiinflammatory symptom control. A third
observation is that RA synovium exhibits varying histology,
and the different histologic abnormalities may relate to the
subsequent progression or nonprogression of erosions in different ways. This wider range of evidence is reviewed in the
original editorial.
John R. Kirwan, BSc, MD, FRCP
University of Bristol
Bristol, UK
DOI 10.1002/art.20866
Careful attention to blood sampling as a preanalytical
determinant of circulating matrix metalloproteinase 9
to avoid misinterpretations: comment on the article by
Ainiala et al
To the Editor:
Ainiala et al (1) described increased serum matrix
metalloproteinase 9 (MMP-9) concentrations in systemic lupus
erythematosus patients with neuropsychiatric complications
compared with patients without neuropsychiatric manifestions.
The authors concluded that elevated serum MMP-9 concentrations might reflect small-vessel cerebral vasculopathy in
these patients. Although I do not have experience in that
special field, I suggest that preanalytical problems of MMP
determinations due to blood sampling should also be considered to avoid misinterpretation of the study results. Because
blood sampling markedly determines the concentration of
circulating MMP-9, I would direct the attention of Ainiala et al
and that of the interested reader to this issue. The significance
of blood collection as a preanalytical determinant appears to
have escaped the attention of Ainiala et al, because these facts
have been discussed primarily in analytical journals (2,3).
A report comprising the results obtained in my laboratory is shown in Figure 1. Briefly, blood samples obtained
from 8 healthy subjects were prepared in blood collection
devices (Sarstedt, Numbrecht, Germany) by centrifugation at
1,600g for 15 minutes, within 30 minutes after venipuncture.
Plastic tubes were used either without additives (S-Monovette
system 01.1728; Sarstedt) to obtain pure serum (serum⫺) or
with kaolin-coated plastic granulate (S-Monovette system
01.1601; Sarstedt) to obtain serum after enhanced clotting
(serum⫹). Plasma samples were collected as citrate, heparin,
or EDTA plasma in S-Monovette tubes coated with sodium
Figure 1. Matrix metalloproteinase 9 (MMP-9) concentrations in
serum and plasma. MMP-9 was measured in serum (left) and plasma
(right) derived from blood samples obtained from 8 healthy adults.
Serum was collected in Monovette tubes without additive (serum⫺) or
tubes containing kaolin-coated granulates as clot activator (serum⫹).
Plasma was collected in tubes coated with sodium citrate, lithium
heparin, or potassium EDTA. Significance levels were calculated by
repeated-measures analysis of variance with Tukey’s posterior test.
Individual and median values are shown. a ⫽ P ⬍ 0.05 versus serum⫺;
b ⫽ P ⬍ 0.05 versus serum⫹; c ⫽ P ⬍ 0.05 versus plasma citrate; d ⫽
P ⬍ 0.05 versus plasma heparin; e ⫽ P ⬍ 0.05 versus plasma EDTA.
citrate, lithium heparin, or potassium EDTA. Measurements
of MMP-9 were performed with an enzyme-linked immunosorbent assay (MP2211; Medac Diagnostika, Wedel, Germany).
MMP-9 concentrations in serum samples collected in tubes
with clot activator (serum⫹) were ⬃3-fold higher than those in
pure serum samples (serum⫺) and essentially higher than the
concentrations observed in plasma samples.
Because platelets and leukocytes contain high concentrations of MMP-9, the varying release of MMP-9 from blood
cells during the platelet activation or sampling process could
cause these differences (3). In addition, changes in white blood
cell count and increased apoptotic neutrophils in these patients
are well known (4) and could also generate different serum
MMP-9 concentrations. Therefore, the direct association of
the changed MMP-9 concentration with the cerebral vasculopathy as postulated by Ainilia et al could be only detected
with a blood collection method that avoids the preanalytical
interferences. Serum samples collected neither with nor without clot activator seem to be appropriate to fulfill that precondition.
In summary, in order to ascertain a relationship between MMPs in the peripheral blood and pathologic processes
in tissue/organs and to use MMPs as diagnostic markers, the
influence of blood sampling must be considered. Recently, the
use of blood samples collected with sodium citrate was suggested to avoid the detrimental effect of other anticoagulants
or serum and to optimize the diagnostic validity of MMPs in
peripheral blood (3).
Klaus Jung, MD, EurClinChem
University Hospital Charité
Berlin, Germany
1. Ainiala H, Hietaharju A, Dastidar P, Loukkola J, Lehtimaki T,
Peltola J, et al. Increased serum matrix metalloproteinase 9 levels in
systemic lupus erythematosus patients with neuropsychiatric manifestations and brain magnetic resonance imaging abnormalities.
Arthritis Rheum 2004;50:858–65.
2. Jung K, Laube C, Lein M, Lichtinghagen R, Tschesche H, Schnorr
D, et al. Kind of sample as preanalytical determinant of matrix
metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinase
2 in blood. Clin Chem 1998;44:1060–2.
3. Mannello F, Luchetti F, Canonico B, Papa S. Effect of anticoagulants and cell separation media as preanalytical determinants on
zymographic analysis of plasma matrix metalloproteinases. Clin
Chem 2003;49:1956–7.
4. Ren Y, Tang J, Mok MY, Chan AW, Wu A, Lau CS. Increased
apoptotic neutrophils and macrophages and impaired macrophage
phagocytic clearance of apoptotic neutrophils in systemic lupus
erythematosus. Arthritis Rheum 2003;48:2888–97.
DOI 10.1002/art.20895
To the Editor:
Dr. Jung points out that preanalytical handling of
blood samples used for the determination of MMP-9 should be
considered in the evaluation of the results of our study. Dr.
Jung himself used plastic blood collection vessels with or
without additives and measured serum or plasma MMP-9 by
enzyme-linked immunosorbent assay. MMP-9 content was
highest in serum from the tubes containing added beads, while
serum obtained without beads gave lower values. Heparin or
citrate plasma contained less MMP-9 than did serum, and
EDTA plasma contained no MMP-9. Dr. Jung suggests that
differences between the differently treated samples might be
caused by variable release of MMP-9 from blood cells during
the sampling process or platelet activation, because platelets
and leukocytes contain MMP-9.
In our study, blood was drawn into glass tubes (Vacutainer; Becton Dickinson, Oxford, UK) without additive, and
the tubes were centrifuged within an hour after venipuncture
for the assessment of serum MMP-9. Aliquots of the sera were
stored at ⫺70°C until the time of analysis, in a freezer that was
not used daily. All samples were processed in the same way,
which makes us believe that the results are comparable. The
absence of MMP-9 in Dr. Jung’s EDTA plasmas may be
attributable to degradation of MMP-9 in the absence of
calcium (Makowski GS, Ramsby ML. Use of citrate to minimize neutrophil matrix metalloproteinase-9 in human plasma.
Anal Biochem 2003;322:283–6). Citrate will also bind ionized
calcium, and heparin might affect the stability of MMP-9 in
some way. It is apparent that procedures for the measurement
of MMP-9 require standardization. We thank Dr. Jung for his
valuable comments.
Hanna Ainiala, MD
Aki Hietaharju, MD, PhD
Tampere University Hospital
Tampere, Finland
Seppo T. Nikkari, MD, PhD
University of Tampere
Tampere, Finland
DOI 10.1002/art.20813
Accuracy of haplotype association studies is enhanced
by increasing number of polymorphic loci examined:
comment on the article by Meulenbelt et al
To the Editor:
We read with interest the article by Meulenbelt et al
regarding haplotypes of the interleukin-1 (IL-1) gene cluster
and osteoarthritis (OA) (1). These authors identified 8 haplotypes from 3 polymorphisms: IL1B ⫹3953, IL1B ⫺511, and the
IL1RN variable-number tandem repeat (VNTR). They describe the frequency of 2 haplotypes (1-1-2 and 1-2-1) that
confer an increased risk for the development of radiographic
OA of the hip and 1 protective haplotype (2-1-1). We commend the study for its investigation of combined polymorphisms across both IL1B and IL1RN, 2 genes that are likely to
have equal significance in the development of OA.
However, the 3 polymorphisms examined represent
only a limited sample of the known extended haplotypes within
this region. We previously investigated the association between
extended haplotypes within the IL-1 gene cluster and radiographic OA of the knee (2). Our study examined 8 loci across
the IL1A, IL1B, and IL1RN region. Although single polymorphic markers revealed no association, we identified a common
haplotype associated with significant increased risk of knee
OA in 2 separate patient cohorts.
In a smaller study, we also examined IL-1 haplotypes
from UK patients with severe hip OA who underwent primary
or revision hip replacement surgery. We reexamined the
haplotypes (generated with the PHASE 2.0 program) (3,4)
representing the 3 loci analyzed by Meulenbelt et al, and their
predominant risk haplotype (1-1-2) was replicated in our data
(odds ratio [OR] 3.87, 95% confidence interval [95% CI]
1.79–8.30, P ⫽ 0.00007). However, when our 8-locus extended
haplotype was examined, we identified a common risk haplotype, 2CCTC2CT, (OR 24.81, 95% CI 6.53–101.72, P ⫽
0.0000003) within this population that was extremely rare in
healthy controls. This 2CCTC2CT haplotype includes the 3
alleles of the 1-1-2 risk haplotype (Table 1). We identified
several other 8-locus haplotypes that included the 1-1-2 alleles,
but these bore no significant risk for OA.
The accuracy of haplotype association studies is en-
Table 1. Association of IL-1 gene cluster with hip OA using 3-locus
haplotype compared with 8-locus haplotype*
Hip OA
* Values are the number. IL-1 ⫽ interleukin-1; OA ⫽ osteoarthritis.
† IL1B ⫹3953, IL1B ⫺511, IL1RN variable-number tandem repeat
(VNTR); 1 ⫽ most frequent alleles.
‡ Alleles arranged in the following order: IL1A TTA repeat, IL1A
⫺889, IL1B ⫹3953, IL1B ⫺31, IL1B ⫺511, IL1RN VNTR, IL1RN
⫹8006, IL1RN ⫹11100. Underlined alleles are equivalent to the 1-1-2
hanced by increasing the number of polymorphic loci examined, particularly in genomic regions where linkage disequilibrium varies widely, as within the IL-1 gene cluster. We believe
that the 2CCTC2CT haplotype we describe here represents a
significant risk for the development of severe hip OA. To verify
these findings, it would be of great interest to see the study by
Meulenbelt et al extended by employing 8-locus haplotype
Andrew J. P. Smith
Christopher J. Elson, PhD
Mark J. Perry, PhD
Jeffrey L. Bidwell, PhD, FRCPath
University of Bristol
Bristol, UK
1. Meulenbelt I, Seymour AB, Nieuwland M, Huizinga TW, van Duijn
CM, Slagboom PE. Association of interleukin-1 gene cluster with
radiographic signs of osteoarthritis of the hip. Arthritis Rheum
2. Smith AJ, Keen LJ, Billingham ME, Perry MJ, Elson CJ, Kirwan
JR, et al. Extended haplotypes and linkage disequilibrium in the
IL1R1-IL1A-IL1B-IL1RN gene cluster: association with knee osteoarthritis. Genes Immun 2004;5:451–60.
3. Stephens M, Smith NJ, Donnelly P. A new statistical method for
haplotype reconstruction from population data. Am J Hum Genet
4. Stephens M, Donnelly P. A comparison of Bayesian methods for
haplotype reconstruction from population genotype data. Am J
Hum Genet 2003;73:1162–9.
DOI 10.1002/art.20896
To the Editor:
We thank Smith and his colleagues for their interest in
our observed predisposing association of 2 haplotypes (1-1-2
and 1-2-1), formed by polymorphisms IL1B ⫹3953, IL1B
⫺511, and IL1RN VNTR, with radiographic OA (ROA) of the
hip. Interestingly, Smith et al not only confirmed the association of the 1-1-2 haplotype with severe, symptomatic hip OA
among patients in the UK but also indicated an extended
8-locus haplotype (2CCTC2CT) across the IL1A, IL1B, and
IL1RN genes that was extremely rare in controls and conferred
a very high risk for symptomatic hip OA (OR 24.81, 95% CI
6.53–101.72). This 2CCTC2CT haplotype includes the 3 alleles
of the 1-1-2 risk haplotype.
To verify these findings, we genotyped 4 of the 5
additional polymorphisms suggested by Smith et al in our
55–65–year-old cohort from the population-based Rotterdam
study (n ⫽ 791). Using the THESIAS 2.0 program (Tregouet
DA, Escolano S. Tiret L, Mallet A, Golmard JL. A new
algorithm for haplotype-based association analysis: the
Stochastic-EM algorithm. Ann Hum Genet 2004;68:165–77),
we tested the effect of the original haplotype (IL1B ⫹3953,
IL1B ⫺511, and IL1RN VNTR, 1-1-2) and the extended
haplotype (IL1A ⫺889, IL1B ⫹3953, IL1B ⫺31, IL1B ⫺511,
and IL1RN VNTR, IL1RN ⫹8006, IL1RN ⫹11100, 1-1-1-1-22-1) in patients with hip ROA (n ⫽ 70) as compared with the
population we studied, excluding patients with hip ROA (n ⫽
721). For the original 3-locus haplotype (1-1-2), we observed
an OR of 3.2 (95% CI 1.5–6.8); however, with the 7-locus
extended haplotype (1-1-1-1-2-2-1, identical to the CCTC2CT
haplotype identified in the UK) the risk for hip ROA increased
to an OR of 7.3 (95% CI 2.7–19.5, P ⫽ 0.00008).
We agree with Smith et al that increasing the number
of polymorphic loci at this gene cluster enhanced the accuracy
of our haplotype association study. The extended haplotype
1-1-1-1-2-2-1 encompassing the IL1A ⫺889, IL1B ⫹3953, IL1B
⫺31, IL1B ⫺511, and IL1RN VNTR, IL1RN ⫹8006, IL1RN
⫹11100 polymorphisms does, indeed, confer a significant risk
for the development of both severe hip OA (in patients in the
UK) and the more common hip ROA (in the Dutch
population-based sample). The extended haplotype analysis
with our data showed increased effects not only of the original
1-1-2 haplotype that was the focus of Smith and colleagues but
also of the original 1-2-1 haplotype. This implies that both
extended haplotypes harbor functionally relevant variation.
Again, this gene cluster associates to hip OA. It would be
relevant to know whether other OA phenotypes could also
develop as a consequence of genetic variation at this locus.
Ingrid Meulenbelt, PhD
P. Eline Slagboom, PhD
Leiden University Medical Center
Leiden, The Netherlands
Cornelia M. van Duijn, PhD
Erasmus University Medical School
Rotterdam, The Netherlands
DOI 10.1002/art.20814
Detailing ethnicity and phenotypes is critical for
pooling association studies: comment on the article by
Huizinga et al
To the Editor:
In a recent special article, Dr. Huizinga and colleagues
presented recommendations for genetic association studies
in Arthritis & Rheumatism (1). Proper evaluation of genetic
association studies is critical to our understanding and interpretation of such results, when reported. Thus, this article is
timely and important, addressing some of the pitfalls encountered in genetic association studies and providing sound,
logical guidelines to minimize the plethora of false-positive
associations that have hampered this approach (2).
A strategy that may provide a more accurate representation of the presence of genetic association is the pooling or
meta-analysis of all results (published or not). For such an
approach to be feasible, all articles must provide enough
information to confidently ascertain the ethnicity, the mode of
ascertainment of subjects, the criterian used to diagnose the
phenotype, and various disease manifestations. Whenever
possible, standard or widely accepted definitions should be
used to increase uniformity among various studies. With the
growing confidence in genotyping methodology, the greatest
variability in pooling results will now likely occur at the
population and phenotype levels. Thus, to effectively perform
meta-analysis, detailed information regarding the heritage/
ethnicity of the population being studied and assurances
regarding the quality of the phenotypic data are critical. Such
data are best ascertained by clinicians who have considerable
experience with the diagnosis of interest (3). Thus, articles
being considered for publication should ensure that the phenotyping is of utmost quality, and that standard criteria have
been used to define phenotypes whenever possible.
Proton Rahman, MD, MSc, FRCPC
Memorial University of Newfoundland
St. John’s, Newfoundland, Canada
1. Huizinga TW, Pisetsky DS, Kimberly RP. Associations, populations, and the truth: recommendations for genetic association
studies in Arthritis & Rheumatism. Arthritis Rheum 2004;50:
2. Hirschhorn JN, Lohmueller K, Byrne E, Hirschhorn K. A comprehensive review of genetic association studies. Genet Med 2002;4:
3. Endicott J. Good diagnoses require good diagnosticians: collecting
and integrating the data. Am J Med Genet 2001;105:48–9.
DOI 10.1002/art.20897
To the Editor:
We thank Dr. Rahman for his positive comments. For
meta-analysis, it is necessary that data on putative variation on
the population level are given, hence our recommendation to
avoid hidden population stratification. One of the major steps
forward in the science of rheumatology has been the correct
classification of patients by well-studied criteria, so we have not
explicitly stressed the relevance of correct phenotypic classification. Nevertheless, we completely agree with Dr. Rahman
that the quality of phenotypic information is a key factor in
determining the quality of articles on the phenotype–genotype
Tom W. J. Huizinga, MD, PhD
Leiden University Medical Center
Leiden, The Netherlands
David S. Pisetsky, MD, PhD
Durham VA Hospital
and Duke University Medical Center
Durham, North Carolina
Robert P. Kimberly, MD
University of Alabama at Birmingham
DOI 10.1002/art.20845
Corrected QT interval in anti-SSA–positive adults
with connective tissue disease: comment on the article
by Lazzerini et al
To the Editor:
Because previous reports have shown a prolongation of
the corrected QT (QTc) interval in anti-SSA–positive children
without congenital heart block (CHB) (1,2), Lazzerini et al
recently investigated the same issue in adults (3). They reported a significant prolongation of the mean QTc interval in
adult patients with anti-SSA–positive connective tissue diseases (CTDs) compared with controls (anti-SSA–negative
patients with CTDs): the mean ⫾ SD QTc interval was 445 ⫾
21 in 31 anti-SSA–positive patients versus 419 ⫾ 17 msec in 26
anti-SSA–negative patients (P ⫽ 0.000005 by Student’s t-test).
This result suggests that anti-SSA–positive patients may be at
increased risk for cardiovascular mortality.
We have studied electrocardiograms of 89 adults with
CTD. QTc interval duration was compared in patients with and
patients without anti-SSA antibodies. The anti-SSA–positive
group consisted of 32 patients (29 women and 3 men; mean ⫾
SD age 37 ⫾ 11 years); 15 were positive for both anti–60-kd
SSA and anti–52-kd SSA, 15 were positive for anti–60-kd SSA,
and 2 were positive for anti–52-kd SSA. Six patients were also
positive for anti-SSB antibodies. Twenty-eight of these patients
had systemic lupus erythematosus (SLE) and 4 had primary
Sjögren’s syndrome (SS). The anti-SSA–negative group (controls) consisted of 57 patients (54 women and 3 men; age 38 ⫾
12 years) who were negative for both anti-SSA and anti-SSB
antibodies. Forty-nine had SLE, 4 had undifferentiated CTD,
and 4 had mixed CTD. The QT interval was measured
manually with a digitizing pad (SummaSketch II Professional
MMII 1812; SummaGraphics, Seymour, CT) connected to a
PC computer, by an investigator who was blinded to anti-SSA
status. QT interval was corrected for heart rate by the Bazett
formula, to yield the QTc value.
The anti-SSA–positive and anti-SSA–negative groups
were very similar with regard to age and CTD. All of the
patients were treated with hydroxychloroquine. None was
taking other drugs that could influence the QTc interval (3).
The mean ⫾ SD QTc was 409 ⫾ 30 msec in the anti-SSA–
positive group and 409 ⫾ 28 msec in the anti-SSA–negative
group (P ⫽ 0.78 by Mann-Whitney U test). Five of the
anti-SSA–positive patients (16%) and 6 of the anti-SSA–
negative patients (11%) had QTc values above the upper limit
of normal (440 msec) (P ⫽ 0.35 by Fisher’s exact test).
Our data thus do not confirm the findings of Lazzerini
et al. They are, however, in accordance with those reported by
Gordon et al (4). Those authors studied electrocardiograms in
adults with anti-SSA antibodies. No statistically significant
difference in QTc was found between a group of 49 anti-SSA–
positive patients (mean ⫾ SD 411 ⫾ 19 msec), a group of 62
anti-SSA–negative patients (403 ⫾ 24 msec), and a group of 19
anti-SSA–positive mothers of children with CHB (408 ⫾ 19
msec) (4).
It should be noted that our study focused only on
surface electrocardiography whereas Lazzerini et al performed
other tests more appropriate for determining possible autonomic dysfunction. Nevertheless, our findings were not consistent with theirs but were in accordance with those reported by
Gordon et al (4). Another limitation of our study is the marked
preponderance of SLE patients in both groups, whereas in the
study by Lazzerini et al, 48% of the patients in the anti-SSA–
positive group had SS and 65% of those in the anti-SSA–
negative group had systemic sclerosis. It remains to be confirmed whether patients with SS and anti-SSA antibodies might
indeed have prolonged QTc compared with anti-SSA–positive
patients with other CTDs.
Nathalie Costedoat-Chalumeau, MD
Zahir Amoura, MD
Jean-Sébastien Hulot, MD
Pascale Ghillani, MD
Philippe Lechat, MD
Centre Hospitalier Universitaire Pitié-Salpêtrière
Christian Funck-Brentano, MD
Centre Hospitalier Universitaire Saint Antoine
Jean-Charles Piette, MD
Centre Hospitalier Universitaire Pitié-Salpêtrière
Paris, France
1. Cimaz R, Stramba-Badiale M, Brucato A, Catelli L, Panzeri P,
Meroni PL. QT interval prolongation in asymptomatic anti-SSA/
Ro–positive infants without congenital heart block. Arthritis
Rheum 2000;43:1049–53.
2. Gordon PA, Khamashta MA, Hughes GR, Rosenthal E. Increase in
the heart rate–corrected QT interval in children of anti-Ro–positive
mothers, with a further increase in those with siblings with congenital heart block: comment on the article by Cimaz et al [letter].
Arthritis Rheum 2001;44:242.
3. Lazzerini PE, Acampa M, Guideri F, Capecchi PL, Campanella V,
Morozzi G, et al. Prolongation of the corrected QT interval in adult
patients with anti-Ro/SSA–positive connective tissue diseases. Arthritis Rheum 2004;50:1248–52.
4. Gordon PA, Rosenthal E, Khamashta MA, Hughes GR. Absence of
conduction defects in the electrocardiograms of mothers with
children with congenital complete heart block. J Rheumatol 2001;
DOI 10.1002/art.20898
To the Editor:
Costedoat-Chalumeau et al describe their study of
QTc interval duration in 89 adults with CTD (32 anti-SSA–
positive and 57 anti-SSA–negative), in which they found no
significant differences between the 2 groups. As they note,
these data do not seem to confirm the results of our study, in
which we showed significant prolongation of the mean QTc
interval in 31 anti-SSA–positive patients compared with 26
anti-SSA–negative patients. Possible explanations for the differing results in the 2 studies may arise from the following
Costedoat-Chalumeau et al studied a very selected
cohort of CTD patients. Their study population consisted
almost exclusively of patients with SLE (close to 90% in each
group). Similarly, in the study by Gordon et al that CostedoatChalumeau and colleagues cite (Gordon PA, Rosenthal E,
Khamashta MA, Hughes GR. Absence of conduction defects
in the electrocardiograms of mothers with children with congenital complete heart block. J Rheumatol 2001;28:366–9),
there was a less extreme but still significant preponderance of
SLE patients (⬃70% overall). As a consequence, the absence
of significant differences in the QTc interval duration between
the anti-SSA–positive and anti-SSA–negative subjects may be
related to a peculiar “resistance” of SLE patients to the
hypothesized electrophysiologic effects of these antibodies.
Indeed, it should be noted that in Gordon and colleagues’
study, in which the prevalence of SLE was not as high as in the
study by Costedoat-Chalumeau et al, the QTc in the antiSSA–positive group was reported to be longer than in the
anti-SSA–negative group and this difference, although nonsignificant, approached significance (P ⫽ 0.063).
In our study there was also a prevalence, albeit less
marked and partially related to epidemiologic factors, of
specific forms of CTD, i.e., SS in the anti-SSA–positive group
(48%) and systemic sclerosis (SSc) in the anti-SSA–negative
group (65%). Costedoat-Chalumeau et al hypothesize that SS
patients may have peculiar characteristics compared with
patients who have other CTD, more frequently developing
QTc interval prolongation in the presence of anti-SSA antibodies. This is a plausible hypothesis, especially considering
the results of a recent study by Pirildar et al (Pirildar T, Sekuri
C, Utuk O, Kemal Tezcan UK. QT dispersion in rheumatoid
arthritis patients with and without Sjögren’s syndrome. Clin
Rheumatol 2003;22:225–8), in which the authors showed that
QT dispersion and corrected QT dispersion values were significantly longer in rheumatoid arthritis (RA) patients with
secondary SS than in RA patients without secondary SS. In
that report, the behavior of the QTc interval was not mentioned and the number of anti-SSA–positive patients was very
small (7 of 58 [12%]). Nevertheless, these data may reflect an
unusual “vulnerability” of cardiac repolarization in patients
with SS, possibly leading to a prolongation of the QTc in the
presence of anti-SSA positivity.
However, when we divided the anti-SSA–positive patients in our study into those with SS (n ⫽ 15) and those with
other CTD (n ⫽ 16 [6 with SLE, 5 with undifferentiated CTD,
4 with SSc, and 1 with mixed CTD]), we found that the QTc
interval was prolonged in both groups, with no significant
difference (mean ⫾ SD 446 ⫾ 24 msec versus 444 ⫾ 17 msec,
respectively; P ⫽ 0.77 by Student’s t-test for unpaired data);
there also was no significant difference in the proportion of
patients with QTc intervals above the upper limit of normal
(i.e., 440 msec) in the SS group versus the non-SS group (75%
versus 50%).
In conclusion, based on the present data, we cannot
clearly define the role of anti-SSA antibodies in ventricular
repolarization in patients with CTD. However, it is likely that
the clinical setting in which these antibodies act may be of
relevance, with a possible peculiar “vulnerability” of SS patients or a particular “resistance” of SLE patients, or both. In
order to further clarify this, the need for study of a large cohort
of patients, including a sufficient number of comparable
anti-SSA–positive patients with different forms of CTD, seems
crucial. Such a study is now in progress at our institution.
Pietro Enea Lazzerini, MD
Maurizio Acampa, MD
Mauro Galeazzi, MD
Franco Laghi-Pasini, MD
University of Siena
Siena, Italy
DOI 10.1002/art.20899
In the article by Palao et al published in the September 2004 issue of Arthritis & Rheumatism (pp 2803–2810),
there was an error in the labeling of the lanes in Figure 5b. The lane labeled “AS-FLIP” should have been
labeled “NS” and vice versa.
DOI 10.1002/art.20929
In the article by del Rincón et al published in the December 2004 issue of Arthritis & Rheumatism
(pp 3813–3822), there was an error in the text on page 3818. The last full sentence on that page should have
read, “Arterial incompressibility was present in 75 (12%) of these patients, and obstruction or [not ‘and’]
incompressibility occurred in 155 (24%) of the patients.”
We regret the errors.
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