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Distribution of TNFA haplotypes in healthy CaucasiansComment on the articles by Newton et al and Zeggini et al.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 6, June 2004, pp 2034-2040
© 2004, American College of Rheumatology
group of genotyped individuals, without the need for family
data (10). Although the EM algorithm aims at finding haplotype frequencies that yield a maximum likelihood given the
input genotypes, the procedure is such that alternative haplotype distributions with high likelihood estimations may exist in
addition to the reported one. Thus, one can speculate that the
discussed discrepancies are caused by different results (but still
consistent with the data) of the procedure estimating the
haplotypic frequencies. However, one fact argues against this
possibility. Without performing haplotype estimations, our
analysis confirmed that ⫺1031C and ⫺863A are in very strong
(virtually complete) linkage disequilibrium (8). The strong
association between ⫺863A and ⫺1031C (but not ⫺1031T)
could be detected in our data by simple 2 ⫻ 2 contingency
tables (P ⬍ 10⫺10), i.e., an approach free from the errors that
are possible with the EM algorithm. In accordance with this
notion, Zeggini et al (2) showed that the ⫺863A allele is found
exclusively on haplotypes encoding ⫺1031C (Table 1). Conversely, Newton et al (1) did not find such a haplotype but instead
reported a relatively high frequency of the haplotype positive for
⫺1031T and ⫺863A (Table 1). Thus, the discrepancies with the
results of Newton et al (1) cannot be caused just by different
solutions to the problem of the haplotypic associations.
Whereas the distribution of TNF␣ promoter singlenucleotide polymorphisms has been shown to vary considerably in different ethnic groups (11), it is interesting that similar
frequencies of TNF haplotypes were found in an Anglo-Saxon
(UK) (2) and a Slavic (Polish) population. At the same time, it
is not clear why considerably different results were obtained in
the Caucasian UK population studied by Newton et al (1).
DOI 10.1002/art.20283
Distribution of TNFA haplotypes in healthy
Caucasians: comment on the articles by Newton et al
and Zeggini et al
To the Editor:
We read with interest the articles by Newton et al (1)
and Zeggini et al (2), who found evidence that a gene(s)
located in the region of the tumor necrosis factor (TNF) locus
may contribute to the susceptibility to rheumatoid arthritis
(RA) and juvenile idiopathic arthritis (JIA), respectively.
Studies analyzing the association of non-HLA genes encoded
in the major histocompatibility complex (MHC) with susceptibility to HLA-linked diseases such as RA and JIA are
important; however, they are complicated by the relatively
large number of potentially relevant polymorphisms as well as
the complex patterns of linkage disequilibrium occurring in the
MHC region (3,4).
A way to approach this problem, which was adopted by
both groups of investigators, relies on the analysis of haplotypes rather than individual markers. The analysis of haplotypes may reveal biologically relevant associations that are not
apparent on analysis of individual markers. Further, analysis of
large numbers of individual markers can be considerably
simplified when the haplotypic associations among them are
elucidated (5). In accordance with this notion, both studies
showed that the majority of genetic variation in the TNF locus
can be explained by 4 (1) or 5 (2) haplotypes with frequencies
of ⬎5% in the healthy population.
However, it is surprising that although both studies
were performed in Caucasians living in the UK there are
considerable differences between the frequencies of TNF
haplotypes reported as representative of a normal population.
For example, the TNF haplotype defined by the presence of
nucleotides ⫺1031T, ⫺863C, ⫺308G, 851A, and 1304A (numbering of positions relative to the start of transcription) had a
frequency of 56% in the population studied by Zeggini et al but
only 5% in the population investigated by Newton et al (␹2 ⫽
108, 1df, P ⬍ 10⫺9). A number of other differences between
the results of the 2 studies (1,2) are shown in Table 1.
To further study the frequencies of TNF haplotypes in
Caucasians, we analyzed the distribution of 5 polymorphisms
in the promoter region of the TNF␣ gene (⫺1031T/C,
⫺863C/A, ⫺857C/T, ⫺308G/A, ⫺238G/A) among 248 healthy,
unrelated Polish adults from previously described cohorts
(6,7). Polymorphisms in the 5⬘ flanking region of the TNF␣
gene were identified by dot-blot hybridization with sequencespecific oligonucleotide probes, as previously described (8),
and the haplotypes were derived using expectationmaximization (EM) algorithm implemented using the Arlequin software package (9). As can be seen in Table 1, we found
evidence for the presence in a Polish population of 5 TNFA
haplotypes with frequencies exceeding 5%, which together
accounted for 99% of all haplotypes. The frequencies of
individual haplotypes were similar to those reported by Zeggini et al (2) but were considerably different from those found
by Newton et al (1) (Table 1).
The EM algorithm implemented with the Arlequin
software package allows, in a typical situation, an estimation
with ⬎95% accuracy of the distribution of haplotypes in a
Rafal Ploski, MD, PhD
Warsaw Medical University
Warsaw, Poland
Tomasz Bednarczuk, MD, PhD
Polish Academy of Science
Warsaw, Poland
Yuji Hiromatsu, MD, PhD
Kurume University School of Medicine
Kurume, Japan
1. Newton J, Brown MA, Milicic A, Ackerman H, Darke C, Wilson
JN, et al. The effect of HLA–DR on susceptibility to rheumatoid
arthritis is influenced by the associated lymphotoxin ␣–tumor
necrosis factor haplotype. Arthritis Rheum 2003;48:90–6.
2. Zeggini E, Thomson W, Kwiatkowski D, Richardson A, Ollier W,
Donn R. Linkage and association studies of single-nucleotide
polymorphism–tagged tumor necrosis factor haplotypes in juvenile
oligoarthritis. Arthritis Rheum 2002;46:3304–11.
3. Smerdel A, Lie BA, Ploski R, Koeleman BP, Førre O, Thorsby E,
et al. A gene in the telomeric HLA complex distinct from HLA–A
is involved in predisposition to juvenile idiopathic arthritis. Arthritis Rheum 2002;46:1614–9.
4. Ploski R, Vinje O, Rønningen KS, Spurkland A, Sørskaar D,
Vartdal F, et al. HLA class II alleles and heterogeneity of juvenile
rheumatoid arthritis: DRB1ⴱ0101 may define a novel subset of the
disease. Arthritis Rheum 1993;36:465–72.
5. Schork NJ, Fallin D, Lanchbury JS. Single-nucleotide polymorphisms and the future of genetic epidemiology. Clin Genet 2000;
58:250–64.
6. Bednarczuk T, Hiromatsu Y, Fukutani T, Jazdzewski K,
Miskiewicz P, Osikowska M, et al. Association of Cytotoxic
T-lymphocyte-associated antigen-4 (CTLA-4) gene polymorphism
2034
LETTERS
2035
Table 1. Comparison of distribution of TNFA haplotypes among healthy Polish subjects with data reported as being representative of UK
Caucasian controls*
Population (ref.)
TNFA haplotype
⫺1031
T
T
T
T
C
C
C
C
T
Sum
⫺863
C
C
C
C
A
C
C
C
A
⫺857
C
T
C
T
C
C
C
⫺308
G
G
A
A
G
G
G
A
G
⫺238
G
G
G
G
G
G
A
Polish Caucasians
(n ⫽ 496)
UK Caucasians (2)
(n ⫽ 162)
51 ⫾ 2
16 ⫾ 2
16 ⫾ 2
0
12 ⫾ 1
0
5⫾1
0
0
99
UK Caucasians (1)
(n ⫽ 200)
51
12
13
2
11
6
4
NR
NR
99
P1†
P2‡
8
⫺6
⬍10
⬍10⫺6
2
⬍10⫺5
⬍10⫺3
NR
55
⬍10⫺4
⬍10⫺6
⬍10⫺3
⬍10⫺6
15
15
95
⬍10⫺6
⬍10⫺6
⬍10⫺4
⬍10⫺4
* Values are mean ⫾ SD percentage or the percentage. Positions ⫺857 and ⫺238 were not analyzed by Newton et al (1). There were no statistically
significant differences in the distribution of these haplotypes between Polish Caucasians and the UK Caucasians studied by Zeggini et al (2). TNF ⫽
tumor necrosis factor; n ⫽ number of chromosomes; NR ⫽ not reported.
† UK Caucasians, Newton et al versus Polish Caucasians.
‡ UK Caucasians, Newton et al versus UK Caucasians, Zeggini et al.
7.
8.
9.
10.
11.
and non-genetic factors with Graves’ ophthalmopathy in European
and Japanese populations. Eur J Endocrinol 2003;148:13–8.
Ploski R, Ronningen KS, Thorsby E. HLA class II profile of a
Polish population: frequencies of DRB1, DQA1, DQB1, and
DPB1 alleles and DRB1-DQA1-DQB1 haplotypes. Transplant
Proc 1996;28:3431–2.
Higuchi T, Seki N, Kamizono S, Yamada A, Kimura A, Kato H, et
al. Polymorphism of the 5⬘-flanking region of the human tumor
necrosis factor (TNF)-␣ gene in Japanese. Tissue Antigens 1998;
51:605–12.
Schneider S, Roessli D, Excoffier L. Arlequin: a software for
population genetics data analysis: version 2.000. Geneva: Genetics
and Biometry Lab, Department of Anthropology, University of
Geneva; 2000.
Fallin D, Schork NJ. Accuracy of haplotype frequency estimation
for biallelic loci, via the expectation-maximization algorithm for
unphased diploid genotype data. Am J Hum Genet 2000;67:
947–59.
Baena A, Leung JY, Sullivan AD, Landires I, Vasquez-Luna N,
Quinones-Berrocal J, et al. TNF-␣ promoter single nucleotide
polymorphisms are markers of human ancestry. Genes Immun
2002;3:482–7.
DOI 10.1002/art.20455
Reply
To the Editor:
We thank Ploski and colleagues for their interest in our
study. The explanation for the difference in our findings is a
typographic error in Table 2 of our article, whereby the alleles
for marker TNF ⫺1031 were labeled incorrectly. The correct
frequencies are shown here in Table 1. Using the correct allele
frequencies, there is little difference between the 2 studies of
UK Caucasians (P ⫽ 0.03) (1), but quite marked differences
are apparent when comparing either of the UK studies with the
Polish study (for Newton et al, P ⫽ 7 ⫻ 10⫺9; for Zeggini et al,
P ⫽ 5 ⫻ 10⫺7).
The 2 probable explanations for the difference between the findings of Ploski et al and those of the 2 UK studies
are that the UK studies were family studies, whereas the Polish
study involved only unrelated individuals, and ethnic differences in haplotype frequencies. Family-based methods are
more accurate in predicting haplotypes than are methods in
which unrelated individuals are used, but it is reassuring that
for common haplotypes, relatively little difference was observed using the different methods.
Our study used both the PHAMILY and PHASE (2)
programs to determine haplotypes, whereas Zeggini et al used
the GeneHunter program (3). We also analyzed our data using
TRANSMIT (4) and found no significant difference between
Table 1.
TNFA haplotype frequencies in British and Polish subjects*
Population, author
TNFA haplotype
Polish
UK
UK
Caucasians, Caucasians, Caucasians,
Plotski
Zeggini
Newton
et al
et al
et al
⫺1031 ⫺863 ⫺857 ⫺308 ⫺238 (n ⫽ 496) (n ⫽ 162) (n ⫽ 200)
T
T
T
T
C
C
C
C
T
C
C
C
C
A
C
C
C
A
C
T
C
T
C
C
C
G
G
A
A
G
G
G
A
G
G
G
G
G
G
G
A
51
16
16
0
12
0
5
0
0
51
12
13
1
11
6
4
NR
NR
54
4
15
0
15
4.4
1
0
0
* Values are the percentage. n ⫽ number of chromosomes; NR ⫽ not
reported.
2036
LETTERS
the results obtained with TRANSMIT and the PHAMILY/
PHASE results. In situations such as that of the MHC where
there is strong linkage disequilibrium, haplotyping programs
such as the EM algorithm or PHASE are likely to perform well
in unrelated individuals. EM algorithm–based programs such
as Arlequin (5) are attractively simple to use. However,
because they do not report posterior haplotype probabilities,
they cannot be generally recommended, because in the absence of significant linkage disequilibrium the predictions of all
haplotyping programs are insecure. In this setting, Bayesian
programs such as PHASE have the major advantage that,
unlike Arlequin and other EM algorithm programs, the accuracy of the haplotype prediction is reported.
Julia Newton, MRCP
Dominic Kwiatkowski, FRCP
Wellcome Trust Centre for Human Genetics
Oxford, UK
Paul Wordsworth, FRCP
Matthew A. Brown, MD, FRACP
University of Oxford
Oxford, UK
1. Zeggini E, Thomson W, Kwiatkowski D, Richardson A, Ollier W,
Donn R. Linkage and association studies of single-nucleotide
polymorphism–tagged tumor necrosis factor haplotypes in juvenile
oligoarthritis. Arthritis Rheum 2002;46:3304–11.
2. Stephens M, Smith NJ, Donnelly P. A new statistical method for
haplotype reconstruction from population data. Am J Hum Genet
2001;68:978–89.
3. Pratt SC, Daly MJ, Kruglyak L. Exact multipoint quantitative-trait
linkage analysis in pedigrees by variance components. Am J Hum
Genet 2000;66:1153–7.
4. Clayton D. A generalization of the transmission/disequilibrium test
for uncertain-haplotype transmission. Am J Hum Genet 1999;65:
1170–7.
5. Schneider S, Roessli D, Excoffier L. Arlequin: a software for
population genetics data analysis: version 2.000. Geneva: Genetics
and Biometry Lab, Department of Anthropology, University of
Geneva; 2000.
DOI 10.1002/art.20282
Bone mass in adolescents with early-onset juvenile
idiopathic arthritis: comment on the article by
Lien et al
To the Editor:
The issue of skeletal integrity and achievement of peak
bone mass is crucial in a chronic disease such as juvenile
idiopathic arthritis, as noted by Lien et al in their recent report
(1). It is therefore very important to conduct long-term
outcome studies as was done by Lien and colleagues. In
addition, in a very detailed examination protocol, they addressed many factors that might influence skeletal development.
The conclusions drawn are nevertheless hampered by
the fact that height and bone geometry have a strong effect on
bone mineral density (BMD) and bone mineral content
(BMC) measured by dual x-ray absorptiometry (DXA). The
authors refer to this problem in their Patients and Methods
section, where they state that in children, total-body measurements are preferable and BMC is preferable to BMD, but both
parameters will still be reduced in a growth-retarded child
whose BMD and bone geometry might be perfectly normal for
body size. The adolescents with low total-body BMC in their
study were significantly smaller and weighed less than those
with normal total-body BMC. The only other parameters with
significant differences at the same P value (⬍0.001) were lean
mass, physician’s global assessment, number of actively involved joints, and number of erosions (Table 3 in ref. 1). It is
not stated whether the lower height and weight represented
reduced values as compared with age-matched reference data,
but the impression from the data presented is that the adolescents with low BMC had a longer and more severe disease
course, less physical activity, were smaller, and had less muscle
(lean mass).
One could therefore conclude that low BMD and BMC
are explained at least in part by less bone due to smaller body
size compared with age-matched controls. The skeleton of
these patients might actually be perfectly normal for their body
size. The conclusion of Lien et al that the future fracture risk
will be increased cannot be drawn from these data. If these
patients remain smaller than normal but their bones are
adapted to their body size and muscle mass, there is not
necessarily an increased fracture risk.
The interaction of muscle and bone is not addressed in
the Discussion section of Lien and colleagues’ report. Muscle
mass/muscle force is the single most important factor influencing bone growth and maintenance (2), and in healthy children,
adolescents, and adults, muscle mass and bone mass are
strongly correlated (3). The reduced bone mass in the study by
Lien et al might actually be the result of the reduced lean mass
which, in turn is very well explained by body size, longer
disease duration, and a higher number of actively involved
joints, which might lead to inactivity.
Whether there are true abnormalities in bone mass in
these patients cannot be determined without proper correction
for body/bone size (4), and whether there are true abnormalities in cortical density or cortical thickness cannot be determined by DXA. The only method to answer this question
would be a true 3-dimensional measurement with quantitative
computed tomography (5).
Johannes Roth, MD
Charité Virchow Klinikum
Berlin, Germany
1. Lien G, Flato B, Haugen M, Vinje O, Sorskaar D, Dale K, et al.
Frequency of osteopenia in adolescents with early-onset juvenile
idiopathic arthritis: a long-term outcome study of one hundred five
patients. Arthritis Rheum 2003;48:2214–23.
2. Frost HM, Schoenau E. The “muscle bone unit” in children and
adolescents: a 2000 overview. J Pediatr Endocrinol Metab 2000;
13:571–90.
3. Schiessl H, Frost HM, Jee WS. Estrogen and bone-muscle strength
and mass relationships. Bone 1998;22:1–6.
4. Molgaard C, Thomsen BL, Prentice A, Cole TJ, Michaelsen KF.
Whole body bone mineral content in healthy children and adolescents. Arch Dis Child 1997;76:9–15.
5. Schoenau E. Problems of bone analysis in childhood. Pediatr
Nephrol 1998;12:420–9.
LETTERS
2037
DOI 10.1002/art.20281
Concerns in reporting of serum vascular endothelial
growth factor levels: comment on the article by
Nakahara et al
To the Editor:
I read with great interest the article by Nakahara et al
(1) reporting a reduction in serum levels of vascular endothelial growth factor (VEGF) after anti–interleukin-6 receptor
monoclonal antibody (anti–IL-6R mAb) therapy in patients
with rheumatoid arthritis. I would like to call attention to some
methodologic concerns. When VEGF is measured, the conditions of processing (e.g., length of time and force of centrifugation, interval between sample collection and processing) are
relevant; they should be standardized and specified.
First, concentrations of VEGF measured in serum
samples may reflect blood platelet degranulation in vitro
rather than synthesis of VEGF by peripheral tissues (2–4). In
plasma, platelet degranulation is minimized by adding anticoagulants to the blood samples, and as a consequence, plasma
VEGF concentrations are up to 20 times lower than matched
serum VEGF concentrations (4). Citrated, EDTA-treated, or
heparinized plasma processed in glass tubes is the material of
choice for measurement of circulating extracellular VEGF.
Second, the serum VEGF concentration changes according to clotting duration (2,3). Hormbrey et al found that
there is no significant difference in VEGF levels in serum
samples processed between 30–60 minutes after collection
versus immediate processing, but the difference becomes
significant when the samples are processed after 2–6 hours (3).
In a clinical situation, where blood samples are kept for
variable amounts of time before processing, the contribution of
the clotting processing may interfere with the measurement of
circulating VEGF levels at the time of sampling.
Third, even if a uniform clotting time could be applied
to all samples, between-subject variation in generation of
VEGF in clotted samples may make the interpretation of any
observed difference between disease and control groups very
difficult and may invalidate the results (4).
Finally, when using serum for VEGF measurement,
it may be advisable to correct the measurements to platelet
count (5).
In conclusion, it would be of interest to confirm the
effect of anti–IL-6R mAb therapy on extracellular circulating
VEGF levels by performing the measurements on plasma
samples.
Simone Ferrero, MD
San Martino Hospital
University of Genoa
Genoa, Italy
1. Nakahara H, Song J, Sugimoto M, Hagihara K, Kishimoto T,
Yoshizaki K, et al. Anti–interleukin-6 receptor antibody therapy
reduces vascular endothelial growth factor production in rheumatoid arthritis. Arthritis Rheum 2003;48:1521–9.
2. Webb NJ, Bottomley MJ, Watson CJ, Brenchley PE. Vascular
endothelial growth factor (VEGF) is released from platelets during
blood clotting: implications for measurement of circulating VEGF
levels in clinical disease. Clin Sci 1998;94:395–404.
3. Hormbrey E, Gillespie P, Turner K, Han C, Roberts A, McGrouther D, et al. A critical review of vascular endothelial growth
factor (VEGF) analysis in peripheral blood: is the current literature
meaningful? Clin Exp Metastasis 2002;19:651–63.
4. Banks RE, Forbes MA, Kinsey SE, Stanley A, Ingham E, Walters C,
et al. Release of the angiogenic cytokine vascular endothelial
growth factor (VEGF) from platelets: significance for VEGF
measurements and cancer biology. Br J Cancer 1988;77:956–64.
5. Verheul HM, Hoekman K, Luykx-de Bakker S, Eekman CA,
Folman CC, Broxterman HJ, et al. Platelet: transporter of vascular
endothelial growth factor. Clin Cancer Res 1997;3:2187–90.
DOI 10.1002/art.20305
Intraarticular injection of anti–tumor necrosis factor:
comment on the letter by Arnold et al
To the Editor:
The report by Arnold et al of a case of a presumed
adverse reaction to etanercept injected into the affected knee
of a patient with psoriatic arthritis (1) may, as the authors
suggest, be an example of a local immune/allergic reaction to
etanercept, by analogy to the injection site reactions seen fairly
frequently during the administration of etanercept by the usual
subcutaneous route. Arnold and colleagues’ patient received
high-dose antibiotics immediately after the reaction occurred.
In spite of negative findings on cultures of fluid later drawn
from the joint, there may have been an infection that was
resolved with the antibiotic treatment. There is a risk, albeit
small, of low-grade subclinical infection at the time of a second
injection, and it would seem appropriate to wait a reasonable
interval of time between injections.
In a series of 25 patients who received 1 injection of
etanercept, my colleagues and I did not observe any troublesome reactions (2). More than 10 of our patients have received
further injections of etanercept in the same or other joints,
with no local reaction. However, in all cases, there was an
interval of ⬎3 months between the first and second injections.
In some of our patients, smaller joints were injected,
with varying effect, including some cases in which the treatment was highly successful (3). This is in contrast to the
negative results reported by Arnold et al (1). Similar conflicting results have been observed with infliximab treatment (4,5).
It is likely that a number of factors influence the effect,
including the type of arthritis and the joint treated, and
previous lack of response to steroid injection may be a
predictor of lack of response to anti–tumor necrosis factor
injection.
Finally, injections should be administered with ultrasound guidance for positioning, because even after knee
puncture with fluid aspiration, it is possible that the subsequent
injection may be inadvertently administered outside the joint
(6). This can be easily avoided with ultrasonographic monitoring (7). For scientific assessment of the value of anti–tumor
necrosis factor injection into joints, investigators must be
assured that the treatment indeed reached the intended site.
Henning Bliddal, MD
Parker Institute
Frederiksberg, Denmark
2038
1. Arnold EL, Khanna D, Paulus H, Goodman MP. Acute injection
site reaction to intraarticular etanercept administration [letter].
Arthritis Rheum 2003;48:2078–9.
2. Bliddal H, Terslev L, Qvistgaard E, Kristoffersen H, Torp-Pedersen
S, Danneskiold-Samsoe B. Injection of etanercept into arthritic
joints. I. Safety. Ann Rheum Dis 2002; 61 Suppl 1:169.
3. Bliddal H, Qvistgaard E, Terslev L, Savnik A, Holm CC, Danneskiold-Samsoe B, et al. Injection of etanercept into arthritis joints:
dose-response and efficacy [abstract]. Arthritis Rheum 2002;46
Suppl 9:S518.
4. Bokarewa M, Tarkowski A. Local infusion of infliximab for the
treatment of acute joint inflammation. Ann Rheum Dis 2003;62:
783–4.
5. Kellner H, Kroetz M, Schattenkirchner M, Kellner W. Successful
therapy of sacroileitis in AS patients by intraarticular injection of
infliximab [abstract]. Arthritis Rheum 2002;46 Suppl 9:S431.
6. Jones A, Regan M, Ledingham J, Pattrick M, Manhire A, Doherty
M. Importance of placement of intra-articular steroid injections.
BMJ 1993;307:1329–30.
7. Bliddal H, Torp-Pedersen S. Use of small amounts of ultrasound
guided air for injections. Ann Rheum Dis 2000;59:926–7.
DOI 10.1002/art.20456
LETTERS
Figure 1. Interleukin-1␤ (IL-1␤)–induced expression of aggrecanase
1 (ADAMTS-4) RNA and protein in bovine chondrocytes and human
synovial fibroblasts. A, Analysis of untreated (control) and IL-1–
treated bovine chondrocyte RNA by Northern hybridization with
ADAMTS-4 and 28S ribosomal RNA (rRNA) probes or protein by
Western blotting of conditioned media. B, Reverse transcriptase–
polymerase chain reaction (RT-PCR) analysis of RNA from control
and IL-1–treated human synovial fibroblasts. Results are representative of studies of fibroblasts from 4 different patients.
Induction of ADAMTS-4 by interleukin-1: comment on
the article by Pratta et al
To the Editor:
We read with great interest the article by Pratta et al
(1), who attributed increased interleukin-1 (IL-1)–stimulated
aggrecanase 1 (ADAMTS-4) activity to activation of the
constitutively produced enzyme by an activator, and not to its
increased production in bovine nasal cartilage, chondrocytes,
and capsular fibroblasts. Results from several laboratories
support the notion of increased enzyme activity in response to
IL-1 stimulation. However, the results of various studies do not
support the conclusion about lack of increased ADAMTS-4
production. We therefore conducted experiments to further
investigate the inducibility of ADAMTS-4 expression by IL-1.
Confluent bovine articular chondrocytes and normal human
synovial fibroblasts were maintained in serum-deficient medium for 24 hours and stimulated with recombinant human
IL-␤ for 24 hours. ADAMTS-4 RNA was analyzed by reverse
transcriptase–polymerase chain reaction (RT-PCR) with specific primers as reported previously (2), or by Northern hybridization with an antisense probe after cloning and sequencing of
the RT-PCR product.
In contrast to the findings reported by Pratta et al (1),
we consistently observed IL-1–inducible expression of the
4.2-kb ADAMTS-4 RNA by Northern blotting (Figure 1A)
and RT-PCR analysis (results not shown) in bovine chondrocytes with similar levels of 28S ribosomal or GAPDH RNA. By
Western blotting of the conditioned media with an antibody
against ADAMTS-4 (Triplepoint Biologics, Forest Grove,
OR), we also detected IL-1–inducible 98 kd (zymogen form)
and 64-kd (processed form) ADAMTS-4 protein bands (Figure 1A). These results are consistent with those reported by
Tortorella et al (3), Little et al (2), and Sztrolovics et al (4).
ADAMTS-4 RNA (692-bp complementary DNA
[cDNA]) was also shown to be induced by IL-1␤ in RT-PCR
analysis of several human synovial fibroblast cell lines from
different patients, while control GAPDH RNA levels (226-bp
cDNA) remained constant (Figure 1B). Yamanishi and colleagues (5) reported similar IL-1␤–inducible expression of
ADAMTS-4 in human synovial fibroblasts. Findings have
varied with regard to inducibility in human chondrocytes or
cartilage. For example, Flannery et al (6) found no
ADAMTS-4 RNA induction by IL-1␤, tumor necrosis factor ␣,
or retinoic acid, but the latter increased aggrecanase activity,
suggesting a posttranscriptional activation mechanism as supported by Pratta et al (1). In a real-time PCR analysis of
human T/C28a4 chondrocytes, Koshy and colleagues (7) did
not observe any induction of ADAMTS-4 RNA by IL-1␣.
Similarly, Bau et al (8) reported only slight induction of
ADAMTS-4 RNA by IL-1␤ in normal human chondrocytes or
cells grown in alginate beads. Aggrecanase activity was induced
by retinoic acid but not by IL-1␤ in human adult cartilage (4).
Interestingly, induction depended upon the type of stimulus,
species, and age of the cartilage.
Thus, most of the available evidence from studies of
bovine chondrocytes supports the notion that ADAMTS-4
induction by IL-1 occurs via both increased production and
enhanced activity, in contrast to the findings reported by Pratta
et al (1). Basal (constitutive) aggrecanase 1 RNA and protein
expression is inducible by IL-1 in bovine articular chondrocytes, cartilage explants, and human synovial fibroblasts. Its
inducibility in the arthritis-pertinent model of human adult
chondrocytes is controversial and requires additional research.
Abdelhamid Liacini, MSc
Muhammad Zafarullah, PhD
CHUM-Notre-Dame Hospital
Montreal, Quebec, Canada
LETTERS
1. Pratta MA, Scherle PA, Yang G, Liu RQ, Newton RC. Induction of
aggrecanase 1 (ADAM-TS4) by interleukin-1 occurs through activation of constitutively produced protein. Arthritis Rheum 2003;48:
119–33.
2. Little CB, Hughes CE, Curtis CL, Jones SA, Caterson B, Flannery
CR. Cyclosporin A inhibition of aggrecanase-mediated proteoglycan catabolism in articular cartilage. Arthritis Rheum 2002;46:
124–9.
3. Tortorella MD, Malfait AM, Deccico C, Arner E. The role of
ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a
model of cartilage degradation. Osteoarthritis Cartilage 2001;9:
539–52.
4. Sztrolovics R, White RJ, Roughley PJ, Mort JS. The mechanism of
aggrecan release from cartilage differs with tissue origin and the
agent used to stimulate catabolism. Biochem J 2002;362:465–72.
5. Yamanishi Y, Boyle DL, Clark M, Maki RA, Tortorella MD, Arner
EC, et al. Expression and regulation of aggrecanase in arthritis: the
role of TGF-␤. J Immunol 2002;168:1405–12.
6. Flannery CR, Little CB, Hughes CE, Caterson B. Expression of
ADAMTS homologues in articular cartilage. Biochem Biophys Res
Commun 1999;260:318–22.
7. Koshy PJ, Lundy CJ, Rowan AD, Porter S, Edwards DR, Hogan A,
et al. The modulation of matrix metalloproteinase and ADAM gene
expression in human chondrocytes by interleukin-1 and oncostatin
M: a time-course study using real-time quantitative reverse
transcription–polymerase chain reaction. Arthritis Rheum 2002;46:
961–7.
8. Bau B, Gebhard PM, Haag J, Knorr T, Bartnik E, Aigner T.
Relative messenger RNA expression profiling of collagenases and
aggrecanases in human articular chondrocytes in vivo and in vitro.
Arthritis Rheum 2002;46:2648–57.
DOI 10.1002/art.20457
Reply
To the Editor:
I appreciate the opportunity to discuss the points
raised by Liacini and Zafarullah. In our article, we reported
that induction of aggrecanase activity involved activation of
constituitively expressed protein. We based this conclusion on
the observations that 1) unstimulated chondrocytes, isolated
articular and nasal cartilage, and capsular fibroblasts contain
relatively high levels of cell-associated ADAMTS-4 based on
immunocytochemistry studies; and 2) there was no detectable
difference in the total ADAMTS-4–immunoreactive proteins
present in unstimulated and IL-1–stimulated articular chondrocytes (based on immunofluorescence studies) and cell
lysates (based on Western blot analysis). Consistent with
conversion from zymogen to active ADAMTS-4, there was an
apparent “shift” in the molecular weight of immunoreactive
ADAMTS-4 proteins upon IL-1 stimulation, and activity was
confirmed in a novel cell-based assay using an immobilized
peptide substrate. Specificity of the anti–ADAMTS-4 (antiVMAH) used in the immunofluorescence studies and the
Western blot analysis was confirmed by the demonstration that
both staining and immunoblotting could be blocked by preincubation of the antibody with immunizing peptide, but not with
an irrelevant peptide. Antibody specificity was also confirmed
by the demonstration of immunofluorescence of transfected
2039
Drosophila S2 cells, but not of untransfected cells or transfected cells stained in the presence of immunizing peptide.
Although we didn’t specifically measure effects on
ADAMTS-4 expression in these studies, we are aware that
IL-1 can cause an increase in ADAMTS-4 expression, and we
described the findings of others in demonstrating this effect.
Based on studies reported by others, as well as studies that we
performed following publication of our report, we would agree
that IL-1 induces a marginal increase in ADAMTS-4 expression, and, as suggested by others including Liacini and Zafarullah, we agree that the increase depends on species and age
of the chondrocytes. However, none of these data address the
presence of cell-associated ADAMTS-4 in chondrocytes and
capsular fibroblasts, and there is no simple way to distinguish
the origin of the active enzyme present in our cell-based assay
or in IL-1–induced cartilage explants. While it is reasonable to
assume that IL-1 induces ADAMTS-4 expression and this
leads to a corresponding increase in protein and activity as
suggested by Liacini and Zafarullah, the results of our Western
blot and immunofluorescence studies do not support this
conclusion. Their hypothesis also ignores the fate of the
cell-associated ADAMTS-4, and it is reasonable to assume
that this population of enzyme could contribute to the activity
measured in the cell-based assay and to aggrecan degradation
in IL-1–stimulated cartilage explants through induction of an
as-yet-unidentified activator that is produced in response to
IL-1 stimulation.
It is intriguing that Liacini and Zafarullah were able
to generate presumably “active” ADAMTS-4 from IL-1–
stimulated bovine chondrocytes. We, as well as many others,
have never observed a soluble form of the enzyme released
from monolayer chondrocytes. Liacini and Zafarullah are
incorrect in concluding that their observation with isolated
chondrocytes is supported by others (Tortorella et al, Little et
al, Sztrolovics et al) since those authors reported induction of
ADAMTS-4 in response to IL-1–stimulated cartilage explants,
and not by monolayer chondrocytes (1–3). We noted in our
report that conditioned medium from IL-1–stimulated bovine
chondrocytes was assayed for aggrecanase activity using both
the peptide substrate described as well as isolated bovine
aggrecan monomers, and we were unable to demonstrate
aggrecanase activity in the conditioned medium. Consistent
with these results, we were unable to detect the presence of
ADAMTS-4 in the conditioned medium by Western blot
analysis. For these reasons, we designed the cell-based assay
for aggrecanase and were able to detect cell-associated aggrecanase activity, specifically ADAMTS-4 based on the use of
selective inhibitors, from monolayer chondrocytes and capsular fibroblasts.
Liacini and Zafarullah conclude that the immunoreactive proteins observed in their Western blot analyses represent
active ADAMTS-4, based presumably on the size of the
proteins. I have several questions regarding this conclusion.
Have they demonstrated enzymatic activity of the conditioned
medium that contains the proposed active ADAMTS-4? Does
this medium digest aggrecan monomers and give rise to the
characteristic cleavage products attributed to ADAMTS-4?
Have they demonstrated specificity of the antibody used in
these studies? Without these appropriate controls, it is difficult
to draw any conclusions regarding the identity of the proteins
detected by Western blot analysis.
2040
LETTERS
These studies continue to raise questions regarding the
identity of the activator involved in activation of constituently
expressed ADAMTS-4. It has been reported that ADAMTS-4,
as well as other ADAM family members, contain a furin
activation site, and it is possible that a furin-like protease may
be contributing to activation of ADAMTS-4 in these studies. It
has also been suggested that a glycosylphosphatidyl inositol–
anchored protease, possibly membrane type 4 matrix metalloproteinase, may contribute to ADAMTS-4 activation (4).
Identification of the activators will require further investigation and may provide an alternative target for therapeutic
intervention in rheumatic diseases.
Michael A. Pratta, MS
GlaxoSmithKline
King of Prussia, PA
1. Tortorella MD, Malfait AM, Deccico C, Arner E. The role of
ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a
model of cartilage degradation. Osteoarthritis Cartilage 2001;9:
539–52.
2. Little CB, Hughes CE, Curtis CL, Jones SA, Caterson B, Flannery
CR. Cyclosporin A inhibition of aggrecanase-mediated proteoglycan catabolism in articular cartilage. Arthritis Rheum 2002;46:
124–9.
3. Sztrolovics R, White RJ, Roughley PJ, Mort JS. The mechanism of
aggrecan release from cartilage differs with tissue origin and the
agent used to stimulate catabolism. Biochem J 2002;362:465–72.
4. Gao G, Plaas A, Thompson VP, Jin S, Zuo F, Sandy JD. ADAMTS4 (aggrecanase-1) activation on the cell surface involves
C-terminal cleavage by glycosylphosphatidyl inositol-anchored
membrane type 4-matrix metalloproteinase and binding of the
activated proteinase to chondroitin sulfate and heparan sulfate on
syndecan-1. J Biol Chem 2004;279:10042–51.
DOI 10.1002/art.579
Erratum
In the article by Eekhoff et al published in the May 2004 issue of Arthritis & Rheumatism (pp 1650–1654), there
was an error in the second-to-last sentence of the fourth paragraph of the Results section. The sentence
should have read, “The third mutation was a T⬎C conversion at position ⫹1251 [not ‘⫹1250’], resulting in
a methionine–threonine substitution at codon 404 (M404T).”
We regret the error.
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