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Therapy in Rheumatoid Arthritis1
*
Department of Rheumatology, Charing Cross Hospital, London, United Kingdom;
The Kennedy Institute of Rheumatology, London, United Kingdom;
Institute of Clinical Immunology and Rheumatology, Erlangen, Germany;
Department of Rheumatology, University Hospital, Leiden, The Netherlands;
¶
University Klinik fur Innere Medizin III, Vienna, Austria; and
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Centocor, Malvern, PA 19335
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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has been shown to suppress
inflammation and improve patient well-being in rheumatoid arthritis
(RA), but the mechanisms of action of such treatment have not been
fully explored. Here we show that in vivo administration of
anti-TNF- Ab, using a longitudinal analysis, results in the
rapid down-regulation of a spectrum of cytokines, cytokine inhibitors,
and acute-phase proteins. Marked diurnal variation in the serum levels
of some of these were detected. These results were consistent with the
concept of a cytokine-dependent cytokine cascade, and the degree of
clinical benefit noted after anti-TNF- therapy is probably due
to the reduction in many proinflammatory mediators apart from TNF-,
such as IL-6, which reached normal levels within 24 h. Serum
levels of cytokine inhibitors such as soluble p75 and p55 TNFR were
reduced as was IL-1 receptor antagonist. Reductions in acute-phase
proteins occurred after serum IL-6 fell and included serum amyloid A,
haptoglobin, and fibrinogen. The latter reduction could be of
importance, as it is a risk factor for atherosclerosis, which is
augmented in RA patients. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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at the heart of the inflammatory process in RA are particularly
compelling. TNF- and its two receptors (p55 and p75 TNFR) are
expressed at many sites within the synovial membrane, including the
cartilage/pannus junction (3, 4). Tissue expression of
these molecules is reflected in the synovial fluid, where elevated
levels of TNF- and soluble forms of the receptors (sTNFR) are seen
(5, 6, 7). Using an RA synovial cell culture system in which
there is spontaneous production of many cytokines (8), we
showed that neutralizing TNF- down-regulates the production of IL-1,
IL-6, IL-8, and GM-CSF (9, 10, 11), and these findings led us
to propose TNF- as a therapeutic target in RA.
Direct demonstrations of the importance of TNF- in inflammatory
arthritis in vivo was first provided by animal studies. In experiments
aiming to block the endogenous TNF production in arthritis, we and
others showed that systemic administration of blocking Abs to TNF or of
sTNFR:Fc fusion proteins after disease onset led to amelioration
of joint disease in murine collagen-induced arthritis
(12, 13, 14). Transgenic mice carrying a 3'-modified human
TNF- transgene showed dysregulated TNF- expression and the
development of a chronic inflammatory polyarthritis, preventable with
monoclonal anti-human-TNF- (15). Evidence that
TNF- exerted its pathogenic effect via IL-1 in this model was
provided by the therapeutic benefit observed with an anti-IL-1
receptor Ab (16), reflecting the hierarchy we observed in
RA cultures.
These findings led to the conclusion that TNF- was a prime
therapeutic target in RA (17, 18) and provided the
rationale for clinical trials of a specific, TNF-blocking chimeric
(human/mouse) mAb (cA2) in patients with RA (19, 20, 21). In
the most definitive of these studies, cA2 was compared with placebo in
a multicenter, randomized, double-blind trial. The outcome was
unequivocal, with the induction of large and highly significant
improvements in a wide range of clinical and laboratory measures of
disease activity in active treatment groups (20).
There are not many opportunities to investigate the pathogenesis of
human disease in vivo. The clear outcomes of cA2 therapy provides us
with an opportunity to probe deeper into the role of TNF- in the
pathogenesis of RA. We have previously described the effect of cA2
administration on leukocyte migration (22), documenting a
reduction in the expression of adhesion molecules and relating these
changes to circulating leukocyte numbers. Here, we explore the effects
of cA2 on circulating cytokines and cytokine inhibitors, and test the
relationship between these mediators and their major recognized
systemic effect in RA, the production of hepatic acute-phase proteins.
The results confirm the central role of TNF- in the pathogenesis
of RA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The selection of patients for this study and their treatment have been described in detail elsewhere (20). In brief, 73 patients meeting the revised American College of Rheumatology (ACR) criteria for the diagnosis of RA (23) were recruited from the clinics of four cooperating trial centers. All patients had active RA and evidence of erosive disease on x-rays of hands or feet. Patients taking disease modifying antirheumatic drugs were withdrawn from their therapy at least 4 wk before study entry, but were permitted to continue taking low dose oral corticosteroids or nonsteroidal antiinflammatory drugs at stable dosage.
cA2 (Remicade, Infliximab; Centocor, Malvern, PA) is a human/murine
chimeric mAb of IgG1
isotype, with specificity for recombinant and
natural human TNF- (24). At entry to the study,
patients were randomized to receive a single 2 h infusion of
either placebo (0.1% human serum albumin, 24 patients), low-dose cA2
(1 mg/kg, 25 patients), or high-dose cA2 (10 mg/kg, 24 patients) as an
outpatient procedure. Patients were then followed using clinical and
laboratory parameters for a period of 4 wk. Patients, investigators,
and laboratory personnel were blinded as to the treatment
administered.
Blood samples for laboratory measurements were drawn before the infusion on day 0 and at the following times after completion of the infusion: 1 and 8 h, 1 and 3 days, 1, 2, 3, and 4 wk. This resulted in the following mean collection times: 0830, 1300, 2000 (on day 0); 1200, 1100 (on days 1 and 3); 1030, 1100, 1045, 1045 (on weeks 1–4). Blood was collected into sterile tubes, allowed to clot for 30 min, and spun at room temperature for 20 min at 2500 rpm. Serum was aliquoted into plastic tubes and stored at -70°C until assayed. Plasma was prepared from EDTA blood and handled similarly.
Laboratory measurements
Laboratory measurements were made using commercially available assays where available according to the manufacturers’ directions. All samples from a given patient were assayed together to reduce interassay variability.
Cytokines and inhibitors.
TNF-, IL-6, and IL-10 were measured using enzyme-amplified
sensitivity immunoassays, each based on an oligoclonal detection system
(Biosource, Fleurus, Belgium). The sensitivity of the assays was 10
pg/ml (TNF- and IL-6) and 4 pg/ml (IL-10). IL-1 receptor antagonist
(IL-1ra) was measured using a quantitative sandwich enzyme immunoassay
(R&D Systems Europe, Abingdon, U.K.). The sensitivity of the assay was
94 pg/ml.
IL-1
is difficult to measure in serum, but we have previously
investigated a number of different assays for its measurement. IL-1
was measured using a solid phase ELISA. Monoclonal anti-IL-1
58.121.08 (Biosource) was coated onto a microtiter plate overnight at
4°C. Free binding sites were blocked using 3% BSA in PBS (0.15 M, pH
7.2). Excess blocking solution was washed away using 0.1% Tween in
0.8% saline. then, 200 µl of samples or standards were added in
duplicate to the wells together with 50 µl of biotin conjugated mAb
58.121.03 (Biosource). The plates were then incubated for 2 h at
room temperature. Unbound material was washed away using 0.1% Tween in
0.8% saline. Avidin-peroxidase (Boehringer Mannheim, Lewes, U.K.) was
added to the wells and incubated at room temperature for 30 min.
Unbound conjugate was then washed away. The reaction sites were
amplified using Biotynal tyramide (DuPont, Stevenage, U.K.) at room
temperature for 15 min, and then unbound material was washed away.
Avidin-peroxidase (Boehringer Mannheim) was added to the wells and
incubated at room temperature for 30 min. Unbound conjugate was then
washed away, tetramethylbenzidine (Sigma, Poole, U.K.) was added to
visualise the reaction, and the resulting color following addition of
50 µl 0.1 M sulfuric acid to each well was read at 450 nm. The OD
obtained were compared with those obtained from a standard curve made
from dilutions of rIL-1 (Biosource), and the concentration of
IL-1 in each sample calculated. The sensitivity of this assay was
found to be 0.5 pg/ml. Analysis was restricted to the placebo and 10
mg/kg cA2-treated groups to conserve on samples and reagents.
Soluble p55 and p75 TNFR were measured using an in house immunoassay, according to the method previously described (7, 25). Briefly, mAb to either p55 or p75 receptor were coated onto microtiter plates and incubated overnight at 4°C. Free binding sites were blocked with 3% BSA/PBS. Diluted standard or serum was added to each well and incubated for 2 h at room temperature. Unbound material was then washed away. Bound receptor was detected using complementary mAbs conjugated to biotin. After washing, incubation with streptavidin-peroxidase, further washing, and incubation with tetramethylbenzidine, the reaction was stopped with sulfuric acid. OD values obtained at 450 nm were compared with those obtained from a standard curve constructed using recombinant p55 and p75 TNFR. The standard curve covered a range of values from 200 pg/ml to 12.5 ng/ml. The sensitivity of each assay was 50 pg/ml. The normal values obtained from a panel of blood donors was <1000 pg/ml for p55 TNFR and <2000 pg/ml for p75 TNFR.
Acute-phase proteins. C-reactive protein (CRP) was measured by fluorescent polarization immunoassay using the TDX system (Abbot Diagnostics, Maidenhead, U.K.). The system works by comparing the polarization value obtained for a given sample to a precalibrated standard value. Each assay was validated by the inclusion of control sera containing known quantities of CRP. Serum amyloid A (SAA) was measured by a solid phase ELISA (Biosource).
Haptoglobin and fibrinogen were measured using radial immunodiffusion (Behring, Hounslow, U.K.). EDTA plasma was placed into a well cut into a gel containing Abs to haptoglobin or to fibrinogen. After 48 h (haptoglobin) or 18 h (fibrinogen), the diameter of the resulting precipitin rings was measured and the concentration compared with a predetermined concentration table. The assays were validated by the inclusion of control sera of known concentration. These assays were confined to high-dose (10 mg/kg cA2) and placebo-treated groups to conserve samples and reagents. CRP results for all treated groups have been reported previously (20).
Statistics
Data are expressed as median, interquartile range. Diurnal
variation in sTNFR, IL-1ra, and IL-6 was assessed in the placebo group
using the Wilcoxon signed rank test. ANOVA on the van der Waerden
normal scores was used to compare baseline values of TNF-, IL-6,
IL-1, sTNFR, IL-1ra, CRP, and SAA as well as for comparison of
changes from baseline at each posttreatment point. The model included
terms for both investigational site and treatment group. Significant
differences were further tested by Dunnett’s comparison to the placebo
group. The Mann-Whitney U test was used to compare the
IL-10, haptoglobin, and fibrinogen data for the placebo and high-dose
cA2 groups. Comparison between the percent reductions in IL-6, CRP, and
SAA in the high-dose cA2 group was made using the Kruskal-Wallis test.
Associations between parameters were defined using Spearman’s rank
correlation coefficient (
). No adjustment was made for multiplicity
of time points or laboratory parameters. Analyses were performed on a
VAX computer using SAS (SAS Software, NC) and on a PowerMacintosh
computer using Minitab (Minitab, PA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and sTNFR
Our first aim was to examine the effect of cA2 on the systemic
homeostasis of TNF-. We could detect circulating immunoreactive
TNF- in baseline serum samples in 39 of 72 patients tested (54%).
Median, interquartile range baseline circulating TNF- levels in the
placebo group were 15.5, 5–23 pg/ml (n = 24), for the
1 mg/kg-treated group were 5, 5–19 pg/ml (n = 24), and
for 10 mg/kg-treated group were 12, 5–28 pg/ml (n =
24). The normal range is 10 pg/ml. Patients treated with placebo showed
no change in circulating immunoreactive TNF- over the
28-day course of this study (Fig. 1
). In
contrast, patients treated with cA2 showed a rapid and highly
significant increase in median circulating TNF-, with evidence of a
clear dose response relationship (Fig. 1). In patients treated with
low-dose cA2, median circulating TNF- levels peaked at day 3 and
thereafter showed a gradual decline, with a return to baseline values
by day 21. Peak median values in the high-dose cA2 group were seen at
day 7 and showed a more gradual decline, with continuing significant
elevations relative to baseline at day 28. To determine whether the
circulating TNF- was biologically active, we tested samples from
several patients with high circulating immunoreactive TNF- in the
WEHI 164 TNF- bioassay. No patient showed biologically active
TNF- either before or after treatment with cA2 (data not shown).
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on levels of these TNF inhibitors. p55 sTNFR was
detectable in baseline serum samples from all 73 patients, with 67
(92%) showing values above the normal range (<1000 pg/ml). Median,
interquartile range circulating p55 sTNFR levels at baseline for
the placebo, low-dose, and high-dose cA2 groups were 2050, 1360–3683
pg/ml (n = 24), 2050, 1710–3130 pg/ml
(n = 25), and 1910, 1345–2670 pg/ml (n
= 24), respectively. The changes in circulating p55 sTNFR are shown in
Fig. 2. p55 levels fell in the placebo
group during the course of day 0, with trough values reported at
1300 h (p = 0.038 compared with baseline).
By 1200 h on day 1, p55 levels in the placebo group had returned
to baseline values. Median p55 levels in the two cA2-treated groups
showed a similar rapid decline over the morning of day 0 but in
contrast to the placebo group, remained below baseline values on day 1.
Overall, patients treated with placebo showed no significant change
over this period, while patients treated with low- or high-dose cA2
showed modest reductions in circulating p55, which were maximal by day
3 and showed significance (p < 0.05) compared
with placebo at day 14 and 28. However, median values remained above
the upper limit of normal in all treatment groups.
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) in the placebo group, but show a
reduction in both cA2 treatment groups, although the changes failed to
reach statistical significance compared with placebo.
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While IL-1 is clearly produced in the joints in RA, there is not
much present in the serum (26). To establish the normal
range for serum IL-1 using our assay, we tested serum samples
obtained from 36 normal individuals. IL-1 was detectable (>0.5
pg/ml) in 11 of 36 samples (31%), with values ranging from 1 to 128
pg/ml. The median value and interquartile range were <0.5, <0.5–48.0
pg/ml.
Using the same assay, we measured IL-1 in serum samples from
placebo-treated patients (n = 24) and patients treated
with high-dose cA2 (n = 22) only. IL-1 was
detectable in 33 of 46 patients at baseline (72%) with median,
interquartile range values of 5.8, <0.5–23.1 pg/ml and 8.4,
<0.5–28.1 pg/ml in the placebo and high-dose cA2 groups,
respectively. Changes in serum IL-1 levels in the two RA treatment
groups during the course of the study are shown in Fig. 4. The median values throughout the study
remained well within the range established earlier for normal
individuals. However, there was no statistically significant difference
between the two groups, and no clear cut reduction over the 28 days
analyzed.
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The changes in circulating IL-1ra occurring during day 0 and day 1 are
shown in Fig. 5. IL-1ra levels fell in
the placebo group during the course of day 0, with trough values
recorded at 2000 h, (p = 0.026 compared
with pretreatment). By 1200 h on day 1, IL-1ra levels had
recovered in the placebo group, with median values similar to those
observed pretreatment. The early changes seen in the two cA2 groups
were similar in kinetics, but greater in magnitude than in the placebo
group and failed to show recovery on day 1. The reduction from
pretreatment values in the high-dose cA2 group was statistically
significant compared with the reduction in placebo patients at this
time (p < 0.01).
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. Overall, patients treated with placebo
showed no significant change over this period, while patients treated
with low- or high-dose cA2 showed rapid and substantial reductions in
circulating IL-1ra, which were maximal by day 3 and highly significant
compared with placebo (p < 0.001). The
reduction in IL-1ra remained significant until day 14 in both groups.
Although the maximal fall seen with the treatment dose groups was
similar, patients treated with low-dose cA2 showed a more rapid loss of
effect, with a return to pretreatment values by week 4. The changes in median IL-1ra values were reflected in the individual patient responses for IL-1ra. Of the 23 low-dose cA2 patients tested, 16 had elevated circulating IL-1ra levels before treatment, and, following treatment, 7 of these achieved a serum value the normal range. Similarly, 14 of the 21 high-dose cA2 patients tested had elevated pretreatment IL-1ra levels, and, following treatment, 10 achieved normal values.
Effect of cA2 on IL-6
IL-6 was detectable in all but 4 of the 72 pretreatment sera tested. The median, interquartile range pretreatment circulating IL-6 levels for the three treatment groups were 125, 56–209 pg/ml, n = 24; 130, 57–225 pg/ml, n = 24; 114, 78–188 pg/ml, n = 24 (normal range, <10 pg/ml) (placebo, low-, and high-dose cA2, respectively, p < 0.05 for each group v/s normal).
The changes in circulating IL-6 following treatment are shown in Fig. 6. IL-6 levels showed significant
reductions in the placebo group at the 1300 and 2000 h time points
on day 0 (p < 0.001, p =
0.002, respectively), with partial recovery by day 1 (Fig. 6). Patients
treated with cA2 showed even more marked reductions in circulating IL-6
at 1300 h on day 0 and continuing decline thereafter, reaching
significance compared with placebo by day 1 (p
< 0.01, p < 0.001, low- and high-dose cA2,
respectively). In Fig. 6, changes in serum IL-6 over the longer term
are displayed. The highly significant falls in serum IL-6 seen at day 1
were maintained for the duration of the study in patients receiving
high-dose cA2, but there was a partial loss of effect in patients
treated with low-dose cA2 by week 4.
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Effect of cA2 on IL-10
While IL-10 is abundant in synovium, the levels reported in serum
are lower (27). IL-10 was measured in patients receiving
placebo, (n = 18) and in those receiving high-dose cA2
(n = 18) only. Of these, nine patients (24%) had
detectable IL-10 at baseline (>4 pg/ml). Median, interquartile range
circulating IL-10 levels at baseline were 2, 2–10.5 pg/ml and 2,
2–3.5 pg/ml in the placebo and high-dose cA2 groups, respectively
(normal range, <8.8 pg/ml). There was no significant change in values
in either treatment group during the course of the study (Table I).
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The changes in circulating CRP values in this study have been
reported previously (20). In brief, patients treated with
placebo showed no significant change in CRP values, while those treated
with high-dose cA2 showed a large and highly significant reduction,
evident as early as day 1 posttreatment and reaching maximal extent by
day 7 (Table II).
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),
while those patients treated with high-dose cA2 showed a large and
highly significant reduction.
Pretreatment haptoglobin and fibrinogen levels were similar in the
placebo and high-dose cA2 treatment groups (Table II). Placebo-treated
patients showed no significant change in either measure, while the
high-dose cA2 group showed a significant reduction in both measures by
week 4 (p < 0.001, p < 0.002,
haptoglobin and fibrinogen, respectively).
Association between circulating IL-6 and acute-phase proteins
Because IL-6 is the principal regulator of hepatic acute-phase
protein synthesis in vitro (28, 29) and in vivo in IL-6
gene-targeted mice (30), we tested the association between
IL-6, CRP, and SAA in our patients. A comparison of the kinetics of
change in each of these mediators following treatment with high-dose
cA2 showed falls in IL-6 preceding those of CRP or SAA. By day 1,
median circulating IL-6 values had fallen compared with pretreatment
values by 95% and exceed the reductions in CRP (20%) and SAA (5%)
(p < 0.001). Although the median values for
the two acute-phase proteins had fallen further by day 3, thereby
narrowing the gap with IL-6, the reduction in IL-6 at this time was
still significantly greater than for the acute-phase proteins
(p < 0.001). A scatter graph comparing
pretreatment IL-6 and CRP levels in all 73 patients is shown in Fig. 7A, indicating a moderate
association between these variables ( = 0.55, p
< 0.002). A similar association was found when comparing the reduction
in circulating IL-6 by day 3 with the reduction in CRP over the same
time period (Fig. 7B; = 0.59, p <
0.002). Less impressive, but still statistically significant,
associations were seen between circulating IL-6 and SAA (pretreatment
comparison: = 0.44, p < 0.002; reduction by
day 3 comparison: = 0.48, p < 0.002). The
strongest associations observed were between CRP and SAA (pretreatment
comparison: = 0.73, p < 0.002; reduction by
day 3 comparison: = 0.76, p < 0.002).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. It is blockade of the latter that has had the most
success, with reproducible clinical data using mAbs (cA2, Remicade,
Centocor; CDP571, Celltech, Berkshire, U.K.) and fusion proteins
(p75Fc, Enbrel, Immunex, Seattle, WA; p55Fc, Lenercept, Roche,
Gipf-Oberfrick, Switzerland).
The precise molecular targeting of biological agents that remain
outside the cell and do not have multiple targets makes it possible to
analyze and interpret the changes occurring after the therapy to shed
new insights into the pathogenesis of disease. The large and
reproducible effects of cA2 provided an opportunity to investigate the
consequences of neutralizing TNF- and other cytokines and cytokine
inhibitors. In particular, we wanted to know if the interactions we
found using in vitro joint cell cultures also applied in vivo. Although
the most important cytokine actions in RA are likely to be within the
joint, the relative inaccessibility of synovial tissue during clinical
trials makes direct study of this compartment difficult and the
assays that can be used in synovium such as immunohistochemistry or PCR
are not very quantitative. Here, we report an analysis of the effects
of cA2 on cytokines within the circulation and relate changes in their
levels to a key cytokine-dependant biological effect, the production of
acute-phase proteins (31).
Our first aim in undertaking this study was to analyze changes in
systemic TNF homeostasis following infusion of cA2. Approximately half
of the patients had detectable circulating TNF- at baseline, but
levels were generally low. The changes that occurred following infusion
of cA2 were remarkable, with a rapid and dose-dependent increase in
immunoreactive, but not biologically active, TNF-, evident as early
as 8–24 h and peaking by day 7. While the mechanism of this increase
in immunoreactive TNF- is still under investigation, preliminary
evidence suggest that the TNF- is present in the form of a high m.w.
complex, presumably with cA2 (our unpublished observations). Similar
increases in circulating immunoreactive IL-6 were noted following
treatment with a mAb to IL-6 in patients with RA (32) and
plasma cell leukaemia (33). Therefore, the rapid, but
transiently delayed (not present at 4 h) rise in immunoreactive
TNF- in our patients is likely to represent trapping of the TNF-
overproduced in the disease.
Changes in sTNFR following cA2 administration were in the opposite
direction of those seen for TNF- itself. Both p55 and p75 sTNFR were
rapidly diminished following infusion of the mAb, reaching statistical
significance compared with placebo for p55 sTNFR at several time
points. Interestingly, although levels of sTNFR were markedly reduced
by 1300 h on day 0 in both low- and high-dose cA2 groups, a
similar reduction was observed for the placebo group throughout day 0.
These findings strongly suggest a diurnal variation for sTNFR, although
the changes might also have resulted from some unknown factor inherent
in the infusion procedure with either cA2 or placebo, and further
studies are warranted. The observed reductions in sTNFR following
administration of cA2 occurred in parallel with improvement in disease
activity in this patient group (20) and support our
earlier findings showing a relationship between levels of sTNFR and
disease activity in RA (7).
The investigation of changes in IL-1 and its competitive antagonist,
IL-1ra, was a second important aim of this study. IL-1 shares many
biological functions with TNF- and is particularly potent in the
induction of damage to cartilage and bone, which led some to consider
it the prime therapeutic target in RA (34). On the basis
of in vitro experiments (9), we predicted that effective
TNF- neutralization in vivo would lead to a down-regulation of IL-1
production. However, although we saw a downward trend in circulating
IL-1 in patients treated with high-dose cA2 at 7 days, the changes
were not significant compared with placebo and took place entirely
within the previously established normal range. IL-1 is clearly
expressed within the synovial membrane (35, 36, 37) and in
blood mononuclear cells in RA (38, 39), but has been
difficult to detect in the circulation (26) which led us
to investigate a number of assays. Our data is in accord with these
findings and suggest that the great majority of synovial IL-1
production is consumed or degraded locally or is rapidly complexed with
natural inhibitors such as soluble IL-1R either within or following
release from the joint. A prior study on a subset of the same patients
has been reported by Lorenz et al. (40), who found a
reduction in serum IL-1 levels at days 1 and 7. We do not know why
there is a discrepancy; it may be due to the assay used (i.e., whether
it is receptive to soluble IL-1R) or the patients. Further work may
clarify this.
The biological effect of IL-1 depends upon the ratio of this cytokine
to its competitive antagonist, IL-1ra (41). Analysis of
IL-1ra in serum samples in our study showed elevated pretreatment
levels in the majority of patients. Treatment with cA2 lead to
substantial falls in circulating IL-1ra, reaching statistical
significance compared with placebo by day 1. The data support the
findings of Van der Poll and colleagues, who showed reduced circulating
IL-1ra after treatment of experimental endotoxemia with mAb to TNF-
(42). The rapidity and timing of this change suggest that
TNF- has an important role in the regulation of IL-1ra release.
Analysis of changes in IL-1ra at the early time points was complicated
by the significant reduction in circulating IL-1ra seen in the placebo
group on day 0. The pattern was similar to that seen for sTNFR and
suggests a diurnal rhythm in levels of cytokine antagonists in RA,
although as discussed previously, other factors inherent in the
infusion process cannot be ruled out. Although the differences between
the placebo and cA2 groups were not statistically significant at the
early time points on day 0, the greater falls in cA2-treated patients
and the trend toward a dose-response relationship (Fig. 5) suggest that
regulation of IL-1ra expression by cA2 is very rapid. It has been
reported that IL-1ra is made by hepatocytes and by cells within the
joint and behaves as an acute-phase protein.
The detection of elevated circulating IL-6 in our patients is
consistent with previous reports that showed the presence of IL-6 in
the majority of RA sera, although at lower concentrations than found in
matched synovial fluid samples (42, 43, 44, 45, 46). The reductions in
circulating IL-6 in placebo-treated patients during the course of days
0 and 1 are consistent with the recognized diurnal variation in this
cytokine in patients with RA (45). The reductions in
circulating IL-6 in cA2-treated patients were even more marked than in
the placebo group, reaching significance from day 1. These findings
support our earlier, preliminary report of reductions in circulating
IL-6 in patients treated in the open label trial of cA2
(19) and concur with the results of an open trial of cA2
in active, refractory Crohn’s disease (47). The data
provide in vivo confirmation of earlier in vitro findings that TNF-
is regulatory for IL-6 production in RA synovial tissue
(11). The difference in kinetics, more rapid in vivo, is
likely to be due to clearance mechanisms including excretion in
vivo.
In view of ample in vitro and animal data linking IL-6 with acute-phase protein synthesis in inflammation (28, 29), it was of interest to relate IL-6 levels in our patients to the cA2-induced falls in serum acute-phase proteins (19, 20, 21). Although IL-6, CRP, and SAA all fell markedly by day 3 and showed significant correlations with each other, the rate of fall for IL-6 was clearly much more rapid than for CRP or SAA. This temporal relationship is consistent with regulation of CRP and SAA production by IL-6, but interpretation of the data is complicated by marked differences in the circulating half times of these molecules (48, 49). An association between circulating IL-6 levels and CRP in patients with inflammatory arthritis has been noted previously (45).
The impressive falls in circulating levels of the rapid response
elements, CRP and SAA, following treatment with cA2 were accompanied by
significant reductions in the slower response proteins, haptoglobin and
fibrinogen. Although these proteins are normally measured as markers of
disease activity, rather than as pathophysiological agents in their own
right, acute-phase proteins may directly contribute to disease outcomes
in RA. Prolonged, high-level elevation in circulating SAA is associated
with the development of secondary amyloidosis, a cause of renal failure
and premature death in a small proportion of RA patients
(50). It has also been suggested that persistently
elevated SAA may be a risk factor for cardiovascular disease
(51). Potentially more important is the strong association
between elevation of plasma fibrinogen levels and the development of
vascular disease (52). Although other factors, including
high platelet counts (19, 20), undoubtedly also contribute
to the excess cardiovascular and cerebrovascular mortality seen in RA,
persistently elevated fibrinogen levels may be major contributors to
these adverse disease outcomes. Our data clearly show that TNF-
neutralization in the short term leads to normalization of SAA and
fibrinogen levels in many patients. Whether effective long-term TNF
neutralization will maintain control of these proteins and as a result
lead to a reduction in the development of amyloidosis and vascular
disease remains to be seen. The normalization of thrombocytosis, which
we have previously reported following treatment of RA patients with cA2
(19, 20), also has the potential to lower cardiovascular
risk, because platelets play an important role in both early and late
events in the development of the atherosclerotic lesion
(53).
The findings presented here support the notion of a cytokine cascade in
RA patients in vivo, with regulation by TNF- of IL-6 and of the
important antiinflammatory molecules, IL-1ra, soluble p55 TNFR, and
soluble p75 TNFR. The establishment of a relationship between TNF and
the regulation of the acute-phase response adds biological significance
to these findings, provides a model that may explain other beneficial
clinical effects of TNF neutralization in this disease, and helps
provide a rationale for the use of CRP in monitoring the activity
of RA.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ravinder N. Maini at his current address: The Kennedy Institute of Rheumatology, 1 Aspenlea Road, Hammersmith, London W6 8LH, U.K. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; sTNFR, soluble TNFR; IL-1ra, IL-1 receptor antagonist; CRP, C-reactive protein; SAA, serum amyloid A.
Received for publication October 30, 1998. Accepted for publication May 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), 1
mg/kg cA2 (
), or 10 mg/kg cA2 (
). Each point represents the
median circulating immunoreactive TNF-
