STUDY OF PLASMA OSTEOPONTIN IN PATIENTS WITH RHEUMATOID
ARTHRITIS AND SYSTEMIC LUPUS ERYTHEMATOSUS WITH AND
WITHOUT EVIDENCE OF LUNG FIBROSIS
By
Mahmood Alsalahy*MD, Mohammad Hassan**MD, Refaat Altanawy***MD, Anas
Yosof****MD, Mohammad Abdelfatah*****MD
*Chest, ***Rheumatology, ****Clinical pathology, *****Clinical bacteriology and
immunology Departments, Benha Faculty of Medicine, **Internal Medicine
Department, Zagazig Faculty of Medicine
ABSTRACT:
To study the relation of the newly discovered proinflammatory cytokine osteopontin
(OPN) to lung fibrosis in patients with rheumatoid arthritis (RA) and systemic lupus
erythematosus (SLE), we selected 18 RA patients ( 7 ♂ and 11♀; 29.28 ± 10.42 years )
and 17 (6 ♂and 11♀, 37.7 ± 7.11 years) and 10 normal controls ( 27.3 ± 9.01 years). 7
of RA and 6 of SLE patients have clinical and radiological evidence of lung fibrosis.
FEV1 % predicted, ESR, serum CRP and plasma osteopontin were measured. FEV1 %
predicted was significantly lower in patients than controls and in those with fibrosis
than in those without in both groups (p< 0.001 and <0.05 respectively) while CRP and
OPN were significantly higher than in controls (p<0.001 & <0.05 for CRP and <0.001
for OPN respectively). No significant difference seen between RA and SLE patients in
all parameters (p>0.05 for all). OPN showed significant negative correlation to FEV1
% predicted and significant positive correlation to ESR and CRP in both patients
groups (r = -0.83 , = 0.93 and 0.87 in RA respectively, p<0.001for all, while in SLE, r
= -0.66, 0.47 and 0.51 respectively, P< 0.001 for FEV1 % predicted and <0.05 for ESR
and CRP). Conclusion : plasma OPN could be a useful marker for disease activity and
could predict those who at risk of developing lung fibrosis in RA and SLE patients.
INTRODUCTION:
Osteopontin, a newly discovered cytokine (OPN, also known as Eta-1) is a
phosphorylated acidic glycoprotein secreted by a variety of cells including osteoclasts,
activated T cells and activated macrophages(1). OPN contains an arginine–glycine–
aspartate (RGD)-binding motif that binds to the integrin family of adhesion molecules
and it functions as a proinflammatory cytokine that causes cellular adhesion and
chemotaxis of different cell types, such as inflammatory leukocytes and vascular
smooth muscle cells(2,3). The potential role of OPN in interstitial fibrosis in different
granulomatous lung diseases as well as drug induced alveolitis was elucidated by many
authors(4,5,6). We carried out this study to evaluate the relation between OPN and lung
fibrosis in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) which are
the two most common collagen vascular diseases associated with lung fibrosis.
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SUBJECTS AND METHODS:
We selected 18 (7♂ and 11♀) RA and 17 (6♂ and 11♀) SLE patients from
rheumatology, internal medicine and chest outpatient clinics. We selected patients
complaining of dyspnea especially on exertion and who did HRCT of chest to
investigate the cause of their dyspnea. Ten of both patients groups showed no evidence
of fibrosis (normal or near normal FEV1% predicted as well as absent evidence of
fibrosis (ground glass opacity, reticulations and micro nodules) on CT (7), while 8 of
RA and 7 of SLE patients showed evidence of fibrosis. 10 healthy subjects of the same
age group were selected from patients relatives as normal controls.
Full clinical history and examination, revision of CT chest and medications were
done to all patients. A recent X-ray chest also was done to all patients to exclude recent
lung events developed after CT imaging. Smokers, diabetics, cardiac and renal
impairment patients as well as those on steroids or cytotoxic immunosuppressant drugs
were excluded to avoid the effect of these conditions on plasma osteopontin levels as
well as on erythrocyte sedimentation rate (ESR) and serum C-reactive protein (CRP) the other inflammatory markers to be studied. Patients were only on their ordinary antiinflammatory therapy (8). The following were done to all patients and subjects:
1.Ventilatory function was measured using a pneumotachometer spirometer (MIR,
Italy) and FEV1 % predicted was used to measure any present ventilatory defects.
2.ESR measured by Westergren method as mm in the 1st hour.
3.CRP measured by latex agglutination test using Plasmatec UK kits as follows (9):
fresh serum was obtained by centrifugation of clotted fresh morning blood samples.
After warming of reagents to room temperature a drop of undiluted serum is then put
on the circle of the test slide using the disposable pipette provided. A drop of latex
reagent is then added next to the serum and both drops are then spread on the whole
circle by the other end of the pipette and the test slide is then tilted gently forwards and
backwards every 2 seconds for 2 minutes comparing with positive and negative
controls.
4.Plasma osteopontin measured by an enzyme-linked immunosorbent assay (ELIZA)(8)
using Promokine® Bioscience alive (UK) kits. The kit contains: human OPN microtiter
plate, one plate of 96 Wells; human OPN labeled antibody, 1 vial; assay buffer, 30 ml;
labeled antibody diluent, 10 ml; wash buffer concentrate, 50 ml; human OPN standard,
1 vial; substrate buffer, 5 ml; peroxide solution, 5.5 ml; TMB tablets, 2 tablets; stop
solution, 11 ml; human OPN assay layout sheet, 1 each; plate sealer, 2 each.
The plate washed by adding 400 µL of wash solution to each well. Wash repeated 1
more time for a total of 2 washes. After the final wash, wells were emptied or aspirated,
and the plate firmly tapped on a lint free paper towel to remove any remaining wash
buffer. 100 µL of assay buffer was pipeted into the S0 (0 ng/ml Standard) wells then
pipet 100 µL of standards #1 through #7 into the appropriate wells and 100 µL of
patient plasma (obtained by centrifugation of morning blood samples) into the
appropriate wells and the plate tapped gently to mix the contents. The plate then sealed
and incubated at 37 °C for 1 hour then the wells emptied and washed by adding 400 µL
of wash solution to every well. The wash repeated 6 more times for a total of 7 washes.
After the final wash, wells were emptied or aspirated, and the plate then firmly tapped
on a lint free paper towel to remove any remaining wash buffer. 100 µL of the labeled
antibody was then pipeted into each well, except the blank and the plate sealed and
incubated at 4 °C for 30 minutes. Substrate then prepared and the wells emptied and
washed by adding 400 µL of wash solution to every well and the wash repeated 8 more
times for a total of 9 washes. After the final wash, the wells were emptied or aspirated,
and the plate firmly tapped on a lint free paper towel to remove any remaining wash
buffer then 100 µL of the substrate solution was added to each well and the plate
2
incubated at room temperature for 30 minutes in the dark. 100 µL of stop solution was
then added to each well and the plate reader blanked against the blank wells. The
optical density then read at 450 nm, preferably with correction between 570 and 590 nm
or the mean optical density of the blank wells subtracted manually from all readings.
Finally results were read using the provided table and curve.
Results were tabulated and statistically analyzed using a statistical software (KyPlot,
Kioshi Yoshioka, Japan 1999-2001v2)
RESULTS:
Clinical characteristics of patients and control subjects are shown in table 1. The table
shows that patients of both RA and SLE as well as controls are comparable as regard
age and sex.
Comparison of FEV1% pred showed statistically highly significant difference between
patients as whole groups as well as those with and without fibrosis and controls in both
of RA and SLE (p < 0.001 for all) while the difference was non significant between RA
and SLE patients as a whole or in sub groups (p > 0.05). A significant difference was
found between patients with fibrosis and those without in both RA and SLE (P <0.05)
which reflects the restrictive ventilatory effect of lung fibrosis in these patients (table 2)
Comparison of CRP between patients as whole groups and controls showed a highly
significant difference in both of RA and SLE ( p< 0.001 for all) reflecting the active
systemic inflammation in these patients. Also, a significant difference was seen between
patients with fibrosis and those without in each disease group (p < 0.05) indicating a
more active inflammatory process in those with fibrosis but a non significant difference
was found between similar subgroups of the two diseases (p > 0.05) reflecting a
comparable degree of inflammation in the two patients groups ( tables 3).
Comparison of plasma OPN between patients and controls also showed highly
significant difference in both RA and SLE as well as in those with and without evidence
of fibrosis (p <0.001 for all). Moreover, a highly significant difference was found
between those with fibrosis and those without in both groups (p < 0.001). No
statistically significant difference seen between RA and SLE patients whether as a
whole group or in those with and without evidence of fibrosis (table 4), again reflecting
a comparable degree of inflammation in the two groups.
Correlation between OPN and FEV1% pred showed statistically highly significant
negative correlation in both RA and SLE patients (r = - 0.83 and r = - 0.66, p < 0.001
for both respectively)(table 5, fig 1&2). Correlation between OPN and ESR and CRP
showed highly significant positive correlation in RA patients (p <0.001, r = 0.93 and r =
0.87 respectively) (table 5, fig 3,5). In SLE patients the relation was only significant (p
< 0.05, r = 0.47 and 0.51 respectively) (table 5, fig 4,6). This means that higher levels of
plasma OPN is associated with higher inflammation and more restriction (i.e. fibrosis).
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Table (1): Clinical characteristics of all groups included in the study
Age in
years
Sex
Smoking
FEV1%
predicted
Rheumatoid arthritis
Systemic lupus
29.28 ± 10.05
No fibrosis
With fibrosis
29.3 ± 10.42
29.25 ± 10.3
7 ♂ , 11 ♀
No fibrosis
With fibrosis
4 ♂, 6 ♀
3 ♂, 5 ♀
No
68.5 ± 7.68
No fibrosis
With fibrosis
70 ± 4.85
63.63 ± 3.96
33.88 ± 7.88
No fibrosis
With fibrosis
37.7 ± 7.11
29.86 ± 6.89
6 ♂ , 11 ♀
No fibrosis
With fibrosis
3 ♂, 7 ♀
2 ♂, 5 ♀
No
66.94 ± 8.63
No fibrosis
With fibrosis
69.8 ± 3.76
58.57 ± 6.19
Controls
27.3 ± 9.01
6 ♂, 4 ♀
No
83.7 ± 3.35
40.833 ± 9.332
39.11 ± 11.385
ESR
No fibrosis
With fibrosis
34.9 ± 5.82
48.25 ± 10.57
31.11 ± 7.967
No fibrosis
With fibrosis
34.4 ± 10.57
45.857 ± 9.06
28.058 ± 7.277
CRP
No fibrosis
26.7 ± 6.429
Total
With fibrosis
36.625± 6.209
18
No fibrosis
24.45 ± 6.52
Total
With fibrosis
33.21 ± 4.965
17
3.75 ± 1.253
No fibrosis
10
No fibrosis
10
10
With fibrosis
8
With fibrosis
7
Number
8.31 ± 2.63
Table (2): Comparison of FEV1 % predicted between different groups
included in the study
Rheumatoid Arthritis patients
Controls
With
fibrosis
Whole
No
fibrosis
With
fibrosis
70.5
76
63.63
66.94
72.8
58.57
83.7
7.68
1.81
4.85
2.25
3.69
3.81
8.63
3.17
3.76
1.92
6.19
3.14
3.3
1.04
Versus
controls
Versus
controls
Versus
controls
Versus
controls
Versus
controls
Versus
controls
2.55
< 0.05
S
2.625
< 0.05
S
2.83
< 0.001
HS
2.106
< 0.05
S
2.207
< 0.05
S
2.886
< 0.001
HS
Whole
M
SD±
SE±
Systemic Lupus patients
No
fibrosis
No fibrosis versus fibrosis RA
Comparison
No fibrosis versus fibrosis SLE
t
p
Sign
t
p
Sign
2.459
< 0.05
S
2.601
< 0.05
S
Whole RA versus whole
SLE
t
P
Sign
No fibrosis RA versus no
fibrosis SLE
With fibrosis RA versus
with fibrosis SLE
t
P
Sign
t
P
Sign
t
P
Sign
1.290
> 0.05
NS
1.647
> 0.05
NS
1.409
> 0.05
NS
Sign = significance
NS = non significant
S = significant
HS = highly significant
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Table (3): Comparison of serum CRP in mg/L between different groups
included in the study
Rheumatoid Arthritis patients
M
SD±
SE±
Comparison
Systemic Lupus patients
Whole
No fibrosis
With
fibrosis
Whole
No
fibrosis
With
fibrosis
31.111
7.969
1.878
26.7
6.426
2.035
36.625
6.209
2.195
28.058
7.277
1.765
24.45
6.525
2.063
33.214
4.965
1.876
Versus
controls
Versus
controls
Versus
controls
Versus
controls
Versus
controls
Versus
controls
10.695
< 0.001
HS
11.078
< 0.001
HS
16.449
< 0.001
HS
10.390
< 0.001
HS
9.851
< 0.001
HS
18.189
< 0.001
HS
No fibrosis versus fibrosis RA
t
3.303
t
1.81
Sign = significance
P
> 0.05
t
2.988
p
< 0.01
No fibrosis RA versus no
fibrosis SLE
Whole RA versus whole SLE
Sign
NS
3.75
1.253
0.396
t
P
Sign
No fibrosis versus fibrosis SLE
Sign
S
p
< 0.01
Controls
t
0.776
NS = non significant
P
> 0.05
Sign
NS
S = significant
Sign
S
With fibrosis RA versus with
fibrosis SLE
t
1.162
P
> 0.05
Sign
NS
HS = highly significant
Table (4): Comparison of plasma osteopontin in ng/ml between different
groups included in the study
Rheumatoid Arthritis patients
M
SD±
SE±
Comparison
Systemic Lupus patients
Whole
No fibrosis
With
fibrosis
Whole
No
fibrosis
With
fibrosis
973
483.36
113.93
666.4
254.45
80.464
1356.25
426.62
150.833
873.111
404.18
98.013
667.7
276.43
87.346
1166.57
387.635
146.512
Versus
controls
Versus
controls
Versus
controls
Versus
controls
Versus
controls
Versus
controls
5.412
< 0.001
HS
6.474
< 0.001
HS
9.042
< 0.001
HS
5.694
< 0.001
HS
5.994
< 0.001
HS
8.745
< 0.001
HS
No fibrosis versus fibrosis RA
t
4.269
p
< 0.001
Whole RA versus whole SLE
t
0.661
Sign = significance
P
> 0.05
Sign
NS
Controls
136.6
46.987
14.858
t
P
Sign
No fibrosis versus fibrosis SLE
Sign
HS
t
3.111
p
< 0.001
No fibrosis RA versus no
fibrosis SLE
t
0.011
NS = non significant
P
> 0.05
Sign
NS
S = significant
Sign
HS
With fibrosis RA versus with
fibrosis SLE
t
0.896
P
> 0.05
Sign
NS
HS = highly significant
5
Table (5): Correlation between plasma osteopontin and FEV1% pred., ESR
and CRP in RA and SLE patients
Parameter
Osteopontin with
FEV1%
pred.
Osteopontin with
ESR
Osteopontin with
CRP
RA
SLE
RA
SLE
RA
SLE
FEV1%
OSP
FEV1%
OSP
ESR
OSP
ESR
OSP
CRP
OSP
CRP
OSP
Σ
Σ2
1269
17154
1138
13644
735
17154
655
13644
560
17154
477
13644
90467
21012959
77370
12654668
31493
21012954
28087
12654668
18502
21012954
14231.5
12654668
Reg Q
SE ±
Reg Q
r
- 0.8285
0.134
- 0.83
- 0.6622
0.193
- 0.66
0.9257
0.094
0.93
0.4732
0.247
0.47
0.8684
0.123
0.87
0.5091
0.245
0.51
P /Sign
<0.001
HS
<0.001
HS
<0.001
HS
<0.05
S
<0.001
HS
<0.05
S
6
DISCUSSION:
Osteopontin (OPN), a newly discovered proinflammatory cytokine, is now under
extensive evaluation for its rule in different inflammatory disorders within and outside
the lung (10). We carried out this study to find the relation of OSP to lung inflammation
and fibrosis in patients with rheumatoid arthritis (RA) and systemic lupus
erythematosus (SLE), the most common collagen vascular disorders.
We have found a highly significant difference in FEV1% of predicted between
patients of both groups and controls (p<0.001) reflecting the restrictive ventilatory
defect in these patients. Patients of both groups with evidence of fibrosis on CT showed
more reductions in FEV1% of predicted (table 2), a well known finding in such patients
(11). Also ESR and CRP were significantly higher in both groups of patients than
controls (p< 0.001) with no significant difference seen between them which reflects the
active inflammatory process in these patients. In RA patients plasma OPN was found to
be significantly higher than in controls (p<0.001) and to correlate significantly and
positively to plasma CRP levels and ESR and negatively to FEV1% of predicted.
This means that OPN almost has a role in the inflammatory process or at least
secreted by the activated inflammatory cells in these patients. Although after extensive
search we found no paper that studied OPN in RA patients with lung fibrosis, yet we
have found important data in papers that studied it in RA and idiopathic lung fibrosis
(IPF). Oshima et al (2002)(12) found increased production of OPN in plasma and
synovial fluid of RA patients than those with osteoarthritis and normal controls and
also found significant positive correlation to the severity to the joint affection. Because
lung lesions in rheumatoid arthritis have the same pathology as that in the synovial
membrane (13), osteopontin almost plays the same role in the lung. Also, the pathology
of lung fibrosis in RA is identical to and cannot be differentiated from that in IPF in
which plasma OPN levels were found by Kadota et al (2004)(8) to be significantly
elevated. These authors also showed by immunohistochemical staining of surgical lung
biopsy specimens obtained from 2 patients with IPF and 2 with NSIP (non specific
interstitial pneumonitis) that OSP is localized mainly in alveolar macrophages and
airway epithelium and concluded that this cytokine plays an important role in
development of lung fibrosis in these patients.
Using microarrays for gene expression, Prado et al (2005)(14) studied the effects of
OPN on lung fibroblasts and alveolar epithelial cells in 13 lung samples obtained from
IPF patients and found that it induced a significant increase in migration and
proliferation in both fibroblasts and epithelial cells. However, although the effect on
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fibroblast migration/proliferation was dependent mainly on integrins, in epithelial cells
proliferation was mainly dependent on CD44 and migration was dependent on CD44
and integrin signaling. Also they found OPN to have profibrotic-relevant effects on
molecules involved in extra cellular matrix remodeling as in fibroblasts it increased
TIMP-1 and type I collagen and inhibited MMP-1 expression, whereas in alveolar
epithelial cells it induced MMP-7.
In a murine model, Yamamoto et al (2003)(15) showed that antibodies to the
SLAYGLR epitope of OPN (the most active portion of the molecule named M5)
inhibited synovial membrane cell proliferation and inflammatory cell infiltration and
that M5 antibodies prevented the development of arthritis in pretreated mice which
constitutes a strong evidence of the involvement of OPN in the inflammatory process of
RA..
In SLE patients also we found plasma OPN levels to be significantly higher than in
controls and in patients with fibrosis than in those without. A significant positive
correlation between OPN and both of ESR and CRP and a significant negative
correlation with FEV1% of predicted was seen although correlation to ESR and CRP
was less stronger than in RA (table;5, figs; 3-6). These findings suggest a role for OPN
in lung fibrosis in these patients like in RA. This agrees with Wong et al ( 2005)(16)
who showed significantly higher concentrations of plasma OPN in SLE patients
especially those with renal affection and those levels positively correlated to disease
activity score and IL-18 which is an important marker of disease activity. The authors
also found excess production of OPN from monocytes ex-vivo and concluded that the
cytokine almost has a relation to inflammation in these patients and could serve as a
potential marker for disease activity.
Not only OPN was shown to be related to disease activity in collagen vascular
diseases but also to disease susceptibility. Using polymerase chain reaction and
restriction fragment length polymorphism (PCR-RFLP), An-Ping et al (2007)(17) found
OPN gene polymorphism to be associated with significantly higher incidence of RA in
Chinese people. In our study we found no significant difference in ESR, CRP or plasma
OPN levels between RA and SLE groups whether with or without fibrosis which
reflects a comparable degree of inflammation in these patients.
Finally, we can conclude that plasma OPN could be a good marker for disease activity
and could be used to predict those who at risk of developing lung fibrosis in RA and
SLE patients. We also recommend more studies that measure OPN in broncho-alveolar
lavage fluid and lung biopsy samples in these patients.
REFERENCES:
1. Rodan G. Osteopontin overview. Ann NY Acad Sci 1995;760:1–5.
2. Giachelli CM, Lombardi D, Johnson RJ, et al. Evidence for a role of osteopontin in
macrophage infiltration in response to pathological stimuli in vivo. Am J Pathol
1998;152: 353–8.
3. Okada H, Moriwaki K, Kalluri R, et al. Osteopontin expressed by renal tubular
epithelium mediates interstitial monocyte infiltration in rats. Am J Physiol Renal
Physiol 2000;278: F110–21.
4. Nau GJ, Guilfoil P, Chupp GL, et al. A chemoattractant cytokine associated with
granulomas in tuberculosis and silicosis. Proc Natl Acad Sci USA 1997;94:6414–9.
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