The role of kinins and cytokines in rheumatoid arthritis.
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Date
2001
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Abstract
Introduction: Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized
by inflammatory synovitis. The histopathological features include synovial hyperplasia, an
inflammatory cell infiltration, angiogenesis and an inflammatory exudate into the synovial
joint with progression to bone and joint destruction. While the exact aetiology of RA is
unknown, a number of inflammatory cells and mediators have been implicated in the
pathogenesis. Kinins are vasoactive peptides that have the capacity to induce the cardinal
features of inflammation and considerable evidence exists for a role for the kallikrein-kinin
cascade in inflammatory arthritis. The proinflarnmatory cytokines are also important
mediators in rheumatoid arthritis and there is evidence for a functional relationship
between the kallikrein-kinin and cytokine cascades in rheumatoid arthritis. Methods: Following approval from the Ethics Committee of the University of Natal,
synovial tissue samples were obtained at arthroscopy from patients with RA and at autopsy
(for controls). The tissue samples were processed for light microscopy and immunostained
by the immunoperoxidase method to detect tissue kallikrein and the kinin B 1 and B2
receptors. The intensity of the immunostaining was quantified by image analysis.
Blood and synovial fluid samples were obtained from patients with RA and blood from age
and sex matched healthy volunteers. The RA patients were assessed clinically to
determine the degree of disease activity and the presence or concomitant diseases. Disease
activity was determined by the duration of morning stiffness, the twenty eight tender and
swollen joint counts, pain on a visual analogue scale, patient's and physician's global
assessment of disease activity (Likert scale), a local activity index, the modified Health
Assessment Questionnaire (HAQ), disease activity score (DAS) and the erythrocyte
sedimentation rate (ESR) and C reactive protein (CRP). In the synovial fluid (SF) samples, the functional activity of tissue kallikrein (TK) was demonstrated using an amidolytic assay and the total amount of TK was measured in a sandwich enzyme linked immunosorbent assay (ELISA). The Pearson's correlation coefficient was used to correlate the TK levels with measures of disease activity. Further,
basal and generated kinins were measured in the SF by competitive ELISA, and the levels
correlated with measures of disease activity.
In the cytokine study, interleukin lP (IL IP) and tumour necrosis factor p (TNFP) were
measured in the synovial fluid samples by ELISA, and the relationship between the
cytokine levels and disease activity as well as TK, basal and generated kinins determined.
Neutrophils were isolated from the blood and synovial fluid samples from the rheumatoid
arthritis patients and from the blood samples from healthy volunteers. The circulating and
synovial fluid neutrophils were immunostained to detect tissue kallkrein, the kinin moiety
in the kininogen molecule and kinin BI and B2 receptors, and the immunofluorescence
visualized by confocal microscopy. The images were digitally analysed using the Analysis
2.1 Pro system. The Kruskal Wallis and one-way ANOVA tests were used to compare the
mean intensity of imrnunostaining in the control neutrophils with that present on the
circulating and SF neutrophils harvested from RA patients. The intensity of labeling for
these antigens was correlated with measures of disease activity.
Results:
1. Synovial tissue samples: Labeling for tissue kallikrein was observed in the
synovial lining and endothelial cells in control and rheumatoid tissue. There was a
significant increase in the intensity of TK labeling in the endothelial cells of the
rheumatoid tissue (p < 0.05). The kinin Bl and B2 receptor were visualized in the synovial
lining cells, endothelial cells and the subintimal fibroblasts and macrophages in the control and rheumatoid synovial samples, with a significant increase in B 1 receptor labeling in the
synovial lining cells in rhewnatoid synovial tissue (p < 0.01).
2. Tissue kallikrein activity and the total TK concentration was measured in the
synovial fluid obtained from 20 patients with RA. There was no direct correlation between
the between the enzymic and antigenic tissue kallikrein. There w
as a significant negative
correlation between the enzymic TK and the twenty eight swollen joint count (r = -0.464; p
<0.05).
3. There was a significant negative correlation between the basal kinin and generated
kinin levels (r= -0.454; p < 0.05). In addition, there was a negative correlation between the
basal kinin levels and the C
RP (r = -0.537; p < 0.05) and the disease activity score
(r = -0.458; p < 0.05). In contrast, there was a positive correlation between the generated
kinin levels and the twenty-eight tender and swollen joint counts (r = 0.536; p < 0.05 and r
= 0.509; p < 0.05 respectively), the ESR (r = 0.598; p < 0.01), CRP (r = 0.725; p < 0.01)
and the disease activity score (r = 0.676; p < 0.01).
There was a significant correlation between the SF levels of IL IP and pain (r = 0.462; p <
0.05), physician's global assessment of disease activity (r = 0.549; p < 0.05), 28 tender
joint count (r = 0.4 72; p < 0.05) and CRP (r = 0.530; p < 0.05). Although there appeared to
be a correlation between the IL 1 p and disease activity score, this was not significant
(r = 0.412; p = 0.07). In addition, the levels of synovial fluid lNF a correlated with the 28
tender joint (r = 0.458; p < 0.05) count and CRP (r = 0.653; p < 0.01).
4. There appeared to be a trend towards a negative correlation between the SF
amidase TK levels and IL 1 p, however this was not significant. While there was no direct
relationship between the SF levels of IL 1 P and the generated kinins, there was a positive
correlation between low to moderate levels of IL 1 p and the generated kinins (r = 0.51, p < 0.05). In contrast, there was a negative correlation with higher levels of IL Ip
(r = -0.5, p < 0.05).
5. Immunoreactive TK, kinin moiety and the Bl and B2 receptors were visualized on
the circulating neutrophils from the healthy volunteers and the circulating and SF
neutrophils from the RA patients. There was no statistically significant difference in the
mean intensity of TK labeling in the circulating neutrophils from healthy volunteers (n=8)
and the circulating and synovial fluid neutrophils of the RA patients n=8). However, when
the intensity of labeling of the SF neutrophils (n=80) from the RA patients was compared to
the circulating neutrophils (n=80) of healthy volunteers, there was a significant loss of TK
labeling in the SF neutrophils of the RA patients (1-Way ANOVA, p < 0.01). In the RA
patients, there was a loss of the kinin moiety from both the SF and circulating neutrophils
compared to controls (Kruskal-Wallis: p < 0.05 and < 0.01 respectively). Although there
was a clear increase in the intensity of labeling of the kinin BI receptor on the SF
neutrophils from RA patients (n=8), when compared to the circulating neutrophils from
healthy volunteers (n=8), the mean values did not reach significance (Kruskal Wallis; p >
0.05). However, a significant increase in Bl receptor labeling was observed on both the
circulating and SF neutrophils of the RA patients (n=80) when compared to circulating
neutrophils of the healthy volunteers (n=80) (1 Way ANOVA, p < 0.01 and < 0.05
respectively). In addition, there was a positive correlation between the immunoreactivity
for the BI kinin receptor on the circulating neutrophils from the RA patients and the local
activity index (r = 0.783; p < 0.05). Although there was a clear increase of the kinin B2
receptor on the circulating and SF neutrophils from the RA patients compared to circulating
neutrophils from healthy volunteers, this was only significant for the circulating neutrophils
from the RA patients (Kruskal-Wallis, p = 0.05). There was no correlation between the intensity of labeling of TK, the kinin moiety and the B2 receptor and measures of disease
activity.
Discussion and conclusions
1. This study provides the first evidence for the localization of TK and the kinin
receptors in control and rheumatoid synovial tissue using antibodies specific for each
protein and standard immunolabelling techniques. Synovial fibroblasts, macrophages and
endothelial cells through the release of enzymes and cytokines have the ability to mediate
the inflammatory changes and cartilage and bone destruction in RA. The presence of TK
and the kinin receptors in these cells therefore provides evidence for a pathogenetic role for
the kallikrein-kinin cascade in RA.
2. In addition, in RA there is an exudation of fluid into the joint space. Tissue
kallikrein has been previously reported in the synovial fluid obtained from RA patients,
however the correlation of TK levels and disease activity has not been previously studied.
The negative correlation between enzymic TK and the twenty-eight swollen joint count, an
indicator of disease activity suggests that there is a consumption of TK in inflammation,
presumably due to increased kininogenase activity. Similarly, the kinin generating capacity
of synovial fluid obtained from RA patients has been previously reported, however, this is
the first study demonstrating a link between kinin generating capacity and validated
markers of disease activity. The kinin generating capacity is a complex and dynamic
cascade involving the bioregulation of all the components of the kallikrein-kinin system and
is therefore more likely to accurately define the role of kinins in inflammatory arthritis than
are individual components of the kallikrein-kinin cascade. Measurement of tissue kallikrein
and basal kinins is affected by the presence of natural inhibitors and their short half-life in
biological fluids. In addition to the synovial fluid study a decrease in the urinary TK
activity and an increase in urine kinin generated kinins was demonstrated, suggesting that
there is a systemic activation of the kallikrein kinin cascade in RA.
3. Although there is evidence for an interaction between the kallikrein-kinin and
cytokine cascades in inflammation, in this study a direct correlation between the levels of
interleukin 1 J3 and tumour necrosis factor J3 in the synovial fluid and TK and generated
kinin levels was not found. This may be due to the wide variations in the levels of
cytokines, the presence of inhibitors and anti-inflammatory cytokines, or a complex and
dynamic relationship between the two cascades. However, the correlation of both
generated kinins and interleukin l J3 and tumour necrosis factor J3 with disease activity
provides circumstantial evidence for a synergistic role for these mediators in inflammatory
arthritis.
4. In the neutrophil study, loss of immunoreactive tissue kallkrein and kinin moiety
from the neutrophils obtained from RA patients was demonstrated. This finding supports
the hypothesis that kinins are released from the neutrophils by the enzymatic action of
tissue kallkrein and suggests that the kallkrein-kinin system is activated both locally and
systemically in patients with RA. Further, there was upregulation of the both the kinin B 1
and B2 receptors on the neutrophils from the RA patients. While the B2 receptor is
thought mediate most of the actions of kinins, the correlation of the intensity of B 1
receptors and the local activity index implies that the B 1 receptor may be important in
inflammation.
5. These findings provide convincing evidence for the role of the kallikrein-kinin
cascade in the pathogenesis of inflammation in RA. Further development of kinin receptor
antagonists may provide a novel therapeutic modality.
Description
Masters Degree. University of KwaZulu-Natal, Durban.
Keywords
Arthritis., Rheumatism., Cytokines., Kinins mediators.