STUDY OF THE CYCLICAL CITRULINATED PEPTIDE ANTIBODY
(anti-CCP) IN RHEUMATOID ARTHRITIS PATIENTS WITH EXTRA
ARTICULAR LUNG MANIFESTATIONS
Mahmoud M. Alsalahy*, Hamdy S. Nasser**, Manal M. Hashem***, Sahar M.
Elsayed****, Mohammed I. Abdelfattah*****
Chest* and Clinical Microbiology*****, Benha University, Rheumatology**,
Alazhar University, Internal Medicine***, Zagazig University, Clinical
Pathology****, Mansora University
ABSTRACT:
Anti- CCP antibody has recently gained much interest as a marker for early
detection and prediction of severe articular and extra-articular disease in
rheumatoid arthritis. To evaluate this in patients with active rheumatoid disease
and lung involvement, we studied 40 rheumatoid arthritis patients with active
disease: 10 patients (6 males and 4 females) have chronic disease with joint
effusion but no extra articular affection, 13 chronic patients (5 males and 8
females) with joint effusions and evidence of lung fibrosis, 9 chronic patients (4
males and 5 females) with both joint and pleural effusions but no evidence of lung
fibrosis and 8 patients (4 males and 4 females) with relatively recent onset disease
with only joint inflammation without effusion plus 8 normal control subjects.
Blood anti- CCP was found to be significantly much elevated in all patient groups
than controls (p<0.001 for all) and in patients with lung disease than those without
(p < 0.001 for both) with highest levels seen in patients with lung fibrosis. In
patients without lung disease, levels were slightly (but significantly) higher in
those with joint effusion than in those without (p <0.01). Joint and pleural fluids
showed significantly higher anti-CCP levels than blood (p<0.001for both). Also
levels in joint fluid were significantly higher than in pleural fluid (p<0.001). Blood
anti-CCP showed a strong positive correlation with both of ESR and blood
rheumatoid factor (r = 0.599, p<0.001 and r = 0.841, p< 0.001 respectively) and
with joint and pleural fluid levels (r = 0.786, p<0.001 and r = 0.522, p<0.05
1
respectively). A highly significant inverse relation was seen between this antibody
in blood and FEV1% (r = 0.593, p <0.001). We concluded that: anti-CCP antibody
could be a good marker for diagnosis of rheumatoid arthritis that can predict
patients with severe disease and the association with extra articular affection
especially in the lung.
INTRODUCTION:
Although discovered longtime ago as the anti-perinuclear factor (1,2), anti-CCP
antibody has recently gained much interest as a marker for activity, progression
and prediction of complications in rheumatoid disease (3). Higher blood and
synovial fluid levels of this antibody were found in patients with active disease
especially in those with erosive synovitis and these levels were found to drop
significantly after successful treatment(4). It is claimed by some authors that
progression of rheumatoid disease and association with extra- articular systemic
manifestations occur more in patients with higher blood levels of these antibodies
(5). Also others claimed that it has higher sensitivity and specificity than
rheumatoid factor in detection of active disease. We carried out this study to
evaluate this antibody as a marker for lung affection in patients with active
rheumatoid disease.
SUBJECTS AND METHODS:
40 rheumatoid arthritis patients with active disease - diagnosed according to
diagnostic criteria of American Collage of Rheumatology (1988) - were selected
from patients attending rheumatology, internal medicine and chest outpatient
clinics for joint, systemic or respiratory complaints. During one year and five
months period - from February 2007 to June 2008- we collected these patients as
follows: 10 patients (6 males and 4 females) with chronic disease and one or more
joints with effusion but have no extra articular affection, 13 chronic patients (5
males and 8 females) with one or more joints with effusions and evidence of lung
fibrosis on X- ray and CT of the chest and restrictive ventilatory defect on
spirometry, 9 chronic patients (4 males and 9 females) with one or more joints
with effusions and associated pleural effusion but no evidence of lung fibrosis and
8 patients (4 males and 4 females) with relatively recent onset disease with no
2
evidence of joint effusions or lung affection. Also, 8 age matched normal subjects
(5 males and 3 females) were taken as controls.
Patients with diabetes mellitus, chronic renal or liver disease, thyroid disease,
psoriasis or other extensive dermatosis and those on high doses of antiinflammatory or immune-suppressive therapy where excluded to avoid the effect
of these factors on blood, joint and pleural fluid anti-CCP or rheumatoid factor
levels(6). Smokers also were excluded as tobacco smoke affects disease
progression and facilitates auto-immunity (7,8)
All patients were subjected to full clinical evaluation (history, examination and
general lab) to confirm exclusion criteria. Lung function done with a dry
spirometer ( a pneumotachometer, MIR, Italy) and FEV1 ( expired volume in the
1st second of forced vital capacity) % of predicted used for comparison. Lung
fibrosis confirmed by HRCT of the chest and pleural effusion by X- ray or
ultrasound (US) and fluid aspiration. Joint effusion also confirmed by US and
aspiration. Aspiration of joint and pleural fluid done under aseptic conditions.
Rheumatoid factor in blood was measured using IgG ELISA kits from
International Biological Laboratory( IBL, Germany) and patient serum samples(9).
The Rheumatoid Factor IgG ELISA is based on the principle of the enzyme
immunoassay (EIA) where goat-IgG is bound on the surface of the microtiter
strips. Diluted patient serum or ready to use standards and controls are pipetted
into the wells of the microtiter plate. A binding between the rheumatoid factor of
the serum and the immobilized IgG takes place. After a one hour incubation at
room temperature, the plate is rinsed with diluted wash buffer in order to remove
unbound material. Then the anti-human IgG peroxidase conjugate is added and
incubated for 30 minutes. After a further washing step, the substrate (TMB)
solution is pipetted inducing the development of a blue dye in the wells. The color
development is terminated by the addition of a stop solution, which changes the
color from blue to yellow. The resulting dye is measured spectrophotometrically at
the wavelength of 450 nm. The concentration of IgG rheumatoid factor is directly
proportional to the intensity of the color. Levels less than 20U were considered
negative, 20- 39U: equivocal or weakly positive, 40- 59U: positive and 60U or
more were considered strongly positive. All the above were also done to control
subjects except those for CT chest, joint and pleural fluid analyses.
3
Anti-CCP measured in plasma or serum using also ELISA assay( DIASTAT® antiCCP kits , Axis-Shield Diagnostics Ltd., Dundee, UK)(10).
Sampling: serum or plasma (EDTA, lithium heparin, sodium citrate) samples;
grossly haemolysed or turbid samples were not used. Supernatant fluid after
centrifuge of 50 mL of joint and pleural fluids. Samples kept at 2-8ºC if tested
within 3 weeks and at – 20ºC if kept for longer time.
Preparation for the Assay : Allow all kit components, including the microtitre
strips, to warm up to 18-25 °C for 30-60 minutes before use. Mix reagents by
gentle inversion. The reference control is not diluted but the following solutions
were diluted as follows: Wash Buffer Concentrate 1 vial in 375 mL
distilled/deionised water, Sample Diluent Concentrate 1 vial in 100 mL
distilled/deionised water and Positive and Negative Controls/samples : 10µL in
1mL diluted Sample Diluent.
Steps: pipette 100 μL Reference Control/Calibrators in duplicate, pre-diluted
(1:100) Positive and Negative Controls, and pre-diluted (1:100) patient samples
into appropriate wells. This step should not exceed 15 minutes for any one set of
Calibrators /Controls/samples. Incubate 60 ± 10 minutes at 18-25 °C. Decant strip
contents by quick inversion over a sink suitable for the disposal of biological
materials, bearing in mind the potential infective hazard of the samples. Blot
inverted strips well with paper towels. Wash wells three times with a minimum of
200 μL diluted Wash Buffer. Decant and blot after each wash step. Add 100 μL
Conjugate to each well. Incubate 30 ± 5 minutes at 18-25 °C. Repeat steps 4 and 5.
Add 100 μL Substrate to each well. Incubate 30 ± 5 minutes at 18-25 °C. Do not
decant. Add 100 μL Stop Solution to each well, in the same order and rate as the
Substrate. Tap wells gently to mix. Read strips within 24 hours at 550 nm (540-565
nm).
Data were collected and statistically analyzed using the statistical software
KyPlot.2001, Kioshi, Japan.
RESULTS:
Clinical characteristics of patients and controls included in the study were seen in
table (1).
Highly significant rise in ESR was seen in all patient groups than normal controls
(p<0.001 for all). Also it was significantly higher in patients with chronic disease
4
associated with joint effusion than those with recent onset disease (p<0.01)
indicating higher degree of inflammation in chronic patients. No significant
difference seen between patients with lung fibrosis and pleural effusion (p>0.05)
indicating nearly the same degree of inflammation (table 2).
A restrictive ventilatory defect (as indicated by reduction in FEV 1%) was
significantly higher in all patient groups than controls(p<0.001 for all). Also
reduction in FEV1% was higher in patients with lung fibrosis than those with
pleural effusion(p<0.05), reflecting the effect of the inflammatory process and lung
fibrosis on lung function.(table 2).
Blood anti-CCP was much elevated in all patient groups than controls (p<0.001
for all). Also, patients with lung fibrosis and pleural effusion have higher levels
than those without (p<0.001 for all). Although patients with lung fibrosis have
modestly elevated blood levels of anti-CCP than those with pleural effusion
(19.661±2.13 U/mL and 18±1.175 U/mL), yet the difference is significant
(p<0.05). This means that IPF patients have the highest degree of inflammation
among the three groups (table 3). Joint effusions showed highly significant
elevation of anti-CCP levels than blood in all groups with joint effusions and also
levels were significantly higher in joint fluid than pleural fluid ( p<0.001 for all),
reflecting that anti-CCP is concentrated at the main sites of inflammation in
rheumatoid arthritis(table 4).
In the whole rheumatoid patients, highly significant positive correlation was seen
between blood anti-CCP and both of ESR and rheumatoid factor ( r = 0.599,
p<0.001 and r = 0.841, p< 0.001 respectively). With FEV1% the relation is highly
significant also but negative ( r = - 0.593, p<0.001)(table 5, figures 1,2,3). This
means that higher levels of anti-CCP are associated with higher degrees of
inflammation and more reduction in lung function i.e. more restriction. Also a
significant positive correlation was found between blood and both of joint and
pleural fluid anti-CCP, the relation was stronger with the former (r = 0.786,
p<0.001 and r = 0.522, p<0.05 respectively)(table 5, figures 4,5). A strong direct
relation was found between joint and pleural fluid anti-CCP (r = 0.811, p<0.001)
(table 5, figure 6) indicating that a higher degree of joint inflammation is also
associated with higher degree of extra articular inflammation.
5
Table (1): Clinical characteristics of patient groups and controls included in the study:
Group
RA (no JE or
lung affection)
RA with JE (no
lung affection)
RA + JE + IPF
RA + JE + PE
Normal controls
No.
8
10
13
9
8
Age(years)
29.25±8.276
30.2±8.66
42.846±7.034
36.777±7.677
34.375±10.35
4
4
6
4
5
8
4
5
5
3
Smoking
No
No
No
No
No
ESR (mm/hr)
55.875±10.30
70.8±9.67
73.154±14.56
74±8.916
12.75±19.82
FEV1% pred.
79±2.449
75.2±1.229
70.461±2.988
R F (units)
56.125±10.02
Interm. positive
73.7±9.477
Highly positive
85.538±12.265
Highly positive
72.222±9.038
Highly positive
13.875±9.038
Negative
Disease activity
Active
Active
Active
Active
No disease
Disease duration
1.5±0.235 yrs
4.34±2.112 yrs
10.231±2.331 yrs
5.376±1.233 yrs
No disease
Parameter
Sex
M
F
M = male
F = female
73.444±1.33
82.25±2.251
Interm. = intermediate
pred. = predicted
Table (2): Comparison of ESR in mm/hr and FEV1% predicted between
different groups included in the study
ESR
FEV1%
M
SD±
SE±
M
SD±
SE±
RA
55.875
10.301
3.642
79
2.449
0.866
RA + JE
70.8
9.67
3.057
77.211
1.229
0.338
RA + JE + IPF
73.153
14.559
4.038
73.461
2.989
0.829
RA +JE +PE
Controls
74.7
12.75
8.916
3.928
2.972
1.982
76.444
82.25
1.13
2.251
0.376
0.796
ESR
FEV1%
GROUPS
t
p
Sig
t
p
Sig
RA vs. controls
11.627
< 0.001
HS
2.762
‹ 0.01
S
RA + JE vs. controls
16.604
< 0.001
HS
6.077
‹ 0.001
HS
RA + JE + IPF vs. controls
11.555
< 0.001
HS
7.136
‹ 0.001
HS
RA +JE +PE vs. controls
18.952
< 0.001
HS
6.843
‹ 0.001
HS
RA vs. RA + JE
3.16
< 0.01
S
1.807
› 0.05
NS
RA vs. RA + JE + IPF
2.913
< 0.01
S
4.397
‹ 0.001
HS
RA vs. RA +JE +PE
3.89
< 0.01
S
2.818
‹ 0.05
S
RA + JE + IPF vs. RA +JE
+PE
0.154
> 0.05
NS
2.238
‹ 0.05
S
COMPARISON
RA= rheumatoid arthritis,
JE= joint effusion,
IPF= lung fibrosis,
PE= pleural effusion
6
Table (3): Comparison of anti-CCP in U/mL between different groups
included in the study
M
SD ±
SE ±
RA
RA + JE
RA + JE + IPF
RA +JE +PE
Controls
15.522
1.248
0.441
17.45
1.474
0.466
19.661
2.130
0.591
18
1.175
0.391
5.312
0.864
0.305
COMPARISON
t
p
Sig
RA vs. controls
20.391
< 0.001
HS
RA + JE vs. controls
20.551
< 0.001
HS
RA + JE + IPF vs. controls
18.011
< 0.001
HS
RA +JE +PE vs. controls
23.081
< 0.001
HS
RA vs. RA + JE
3.084
< 0.01
S
RA vs. RA + JE + IPF
4.077
< 0.001
HS
RA vs. RA +JE +PE
4.178
< 0.001
HS
RA + JE + IPF vs. RA +JE +PE
2.346
< 0.05
S
RA= rheumatoid arthritis, JE= joint effusion,
IPF= lung fibrosis,
PE= pleural effusion
Table(4): Comparison of anti-ccp in U/mL between, blood and joint fluid in RA
patients
BLOOD
RA + JE
RA + JE + IPF
RA +JE +PE
JOINT FLUID
PLEURAL FLUID
M
SD±
SE±
M
SD±
SE±
17.45
1.474
0.466
21.84
1.782
0.563
19.661
2.130
0.591
24.046
3.193
0.885
17
1.175
0.391
22.4
1.965
0.655
M
SD±
SE±
19.977
1.688
0.562
COMPARISON
t
p
Sig
Blood versus JF in RA with JE only
12.038
< 0.001
HS
Blood versus JF in RA with JE and IPF
10.773
< 0.001
HS
Blood versus JF in RA with JE and PE
7.563
< 0.001
HS
Blood versus PF in RA with JE and PE
4.495
< 0.001
HS
JF versus PF in RA with JE and PE
7.630
< 0.001
HS
RA= rheumatoid arthritis,
JE= joint effusion,
IPF= lung fibrosis,
PE= pleural effusion
7
Table(5): Correlation between blood anti-CCP and each of ESR, FEV1%
predicted, rheumatoid factor and anti-CCP in joint and pleural fluid in
rheumatoid patients
Whole RA patients
RA with pleural effusion
ESR
FEV1%
RF
Anti-CCP
Bl anti-ccp
JF anti-ccp
PF anti-ccp
M
SD±
Σ
69.3
76.175
73.475
17.83
17
22.4
19.97
13.028
2.968
14.823
2.068
1.175
1.965
1.688
2772
3047
2939
12883.3
153
201.6
179.8
Σ²
198720
232449
224513
12716.35
2612.06
4546.74
3614.8
CORRELATIONS
z- statistic
r
p
Sig
Blood anti-CCP with ESR
4.207
0.599
< 0.001
HS
Blood anti-CCP with FEV1%
4.155
- 0.593
< 0.001
HS
Blood anti-CCP with RF
7.452
0.841
< 0.001
HS
Blood with JF anti-ccp
6.966
0.786
< 0.001
HS
Blood with PF anti-CCP
1.418
0.522
< 0.05
S
JF with PF anti-CCP
2.774
0.811
< 0.001
HS
RA= rheumatoid arthritis,
JE= joint effusion,
IPF= lung fibrosis,
PE= pleural effusion
8
9
DISCUSSION:
Anti-CCP antibody has recently gained much interest as a novel marker for
diagnosis and prediction of severity in rheumatoid arthritis (3). It was also claimed
that extra articular disease is associated with high titer of this antibodies (11). We
carried out this study to evaluate this antibody in rheumatoid patients with lung
affection and its relation to the other two most important inflammatory markers of
the disease i.e. ESR and rheumatoid factor. As expected, ESR was significantly
much elevated in patients than controls and in patients with joint effusion than
those without. Also, it was significantly higher in patients with lung affection than
those without indicating a higher inflammatory burden in the formers (12).
FEV1% predicted was found to be significantly reduced in rheumatoid patients
than controls and in those with lung affection than in those without indicating the
restrictive ventilatory defect of the disease in these patients which was most
marked in those with lung fibrosis. Many factors contribute to this restrictive
ventilatory defect in rheumatoid disease including limited movement and pain in
inflamed joints, associated muscle inflammation besides reduced lung elasticity
due to fibrosis and the space occupying effect of pleural fluid (13).
Blood anti-CCP levels showed highly significant elevations in patients than
controls and in lung affected patients than in patients without reflecting a higher
inflammatory state in rheumatoid patients. This agrees with Kastbom et al
(2004)(14), Turesson et al (2005)(15), and Navarro et al (2006)(16). Kastbom et al
followed serum anti-CCP levels in rheumatoid patients with recent onset (< 1year
duration) for the next 3 years and found levels to be higher as the disease duration
increases. Only 3 of 242 patients have their levels dropped while 5 of the antiCCP negative control patients became positive. Turesson et al (2005) found blood
anti-CCP to be more than 50 U/mL in 77% of the 57 cases of rheumatoid arthritis
with severe extra-articular multi-system affection which was much higher than in
positive controls i.e. patients without extra-articular disease. These anti-CCP levels
seen with these authors are much higher than those in our study and this can be
explained by that our patients were selected only with lung affection while they
included more severe cases with more than 3 system affection. Navaro and coauthors studied anti- CCP in sera of patients with interstitial lung disease (ILD)
due to rheumatoid arthritis (17 patients) and those of patients with ILD due to other
10
causes(hypersensitivity pneumonitis - HP: 13patients, idiopathic fibrosis: 9
patients, and others like tuberculosis: 7 patients). All rheumatoid patients showed
high levels of anti-CCP while only one patient of ILD due to HP gave positive
result. In our study highest levels of this antibody were seen in patients with IPF
and pleural effusion. This implies that higher levels in blood of rheumatoid patients
can be a predictor of the occurrence of extra articular disease (especially in the
lung).
In the present study, joint fluid showed significantly higher anti-CCP levels than
both blood and pleural fluids and also pleural fluid levels were significantly higher
than in blood. This means that this antibody is concentrated at sites of maximum
inflammation in joints and extra-articular tissues. Wither due to participation in the
pathogenesis of the disease or as a product of the inflammatory process, still need
further evaluation, although some authors (17) showed after examination of
biopsies from rheumatoid nodules and transbronchial biopsies that citrulinnation of
tissue proteins makes them escape immune tolerance and get attacked by auto
immunity. van Oosterhout and his colleagues (2007)(18) examined arthroscopic
synovial biopsies taken from 57 rheumatoid patients with active disease and with
positive anti-CCP. They found that patients with higher anti-CCP levels had the
severest degree of joint inflammation and joint destruction and specifically
lymphocytic infiltration. In support for its pathogenitic role, Syversen et al.
(2007)(19) showed that anti-CCP positive rheumatoid patients had higher load of
inflammatory cytokines (TNF alpha, IL6, IL1 beta, IL-ra, GM CSF, MCP 1, MIP 1
alpha, IL7, IL12) than anti-CCP negative patients.
In our study a significant positive correlation was seen between anti-CCP and the
two most important inflammatory markers of the disease i.e. ESR and RF. This is
an important clue to the relation between this antibody and the inflammation in
rheumatoid arthritis. This positive relation was also shown by van Venrooij et al.
(2006)(20), Sullivan (2006)(21) and Zendman et al. (2006)(22).
The significant negative correlation we found in this work between anti-CCP and
FEV1% means that deterioration in lung function increases as anti-CCP increases
which more evident in the group with lung fibrosis. This agrees with Turesson et
al. (2005)(15) and Bongartz et al.(2007)(17) who showed more reduction in vital
capacity and FEV1% was associated with more citrulinnation of lung tissues.
11
In this study we found significant direct relation between anti-CCP in blood and
both of joint and pleural fluids meaning that blood levels of this antibody reflects
the degree of local inflammation in joints or extra articular sites. The same also
applies to the relation between joint and pleural fluids. van Oosterhout and coworkers (2007)(18) as mentioned above , showed that severe synovitis and joint
destructive changes were associated with high blood levels of this antibody. There
were some patients with negative anti-CCP but have a severe disease seen among
patients studied by these authors. Also some patients converted from anti-CCP
positives to negatives over 3years follow up in the study by de Rycke et al. (2004).
Finally we can conclude that anti-CCP antibody could be a good marker for
diagnosis of rheumatoid arthritis which also can predict patients with severe
disease and the association with extra articular affection especially in the lung. So
screening of patients with high levels of this antibody for lung and other extra
articular disease is worthy.
We recommend more studies to show wither anti-CCP can be useful in
differentiation between parenchymal, airway or vascular lun. disease in rheumatoid
patients as well as to show any relation to increased susceptibility to infection in
these patients.
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