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The presence of a kinin in inflammatory synovial effusion from arthritides of varying etiologies.

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The Presence of a Kinin in Inflammatory Synovial Effusion
from Arthritides of Varying Etiologies
By KENNETH L. MELMON,MARIONE. WEBSTER,
STEPHENE. GOLDFINGER
AND J. EDWINSEEGMILLER
I
certain
vasoactive polypeptides (bradykinin and
kallidin) may be invoIved in inflammatory
reactions by virtue of their ability to produce vasodilatation, increased capillary permeability, leukotaxis, and pain in mamm a k 3 AIthough the kinins have the necessary properties of a biological mediator of
inflammation, there is comparatively little
information concerning their presence in
various inflammatory e x ~ d a t e s .This
~
has
been due, in part, to the lack of methods
sufficiently sensitive and specific to detect
physiological, as well as pathological,
amounts of the kinins in biological fluids.
Using a recently described method for the
estimation of the kin in^,^ we have found
these polypeptides in synovial fluid obtained
from inflamed joints of patients with arthritides of various etiologies and from dogs
with induced “arthritis.” This report presents evidence that kinins are present in
synovial fluid of acutely inflamed joints.
T
HAS
BEEN
S U G G E S T E D ~ ~that
~
METHODS
Twenty-five samples of synovial fluid were taken
from 13 patients (7 men and 6 women. aqed
10 to 58 years) hospitalized at the Clinical Center.
National Institutes of Health. All had synovial
effusions of varying degrees of severity in the
knees; the etiologies of the arthritis (four with
gout, six with rheumatoid arthritis, one with
psoriatic arthritis, and two with arthritis of unknown etiology) were determined by standard
From the Experimental Therapeutics Branch
and Laboratory of Cardiovascular Physiology, National Heart Institute; the Arthritis and Rheumatism Branch, National Institute of Arthritis and
Metabolic Diseases; National Institutes of Health,
Bethesda, Md.; and the Department of Medicine
and Pharmacology and the Cardiovascular Re-
clinical and laboratory procedures. Prior to aspirations of synovial fluid, the degree of warmth,
swelling, and tenderness of each knee was evaluated by two different investigators. Each of these
indices of inflammation was graded arbitrarily from
0 to 4 f . Synovial fluid for determination of kinin
was aspirated from the knee shortly after the
patients were admitted to the hospital; samples
were taken again if the patient’s clinical course
changed considerably. Aspirations were performed
under sterile conditions without evidence of bleeding into the synovial fluid. The fluid (usually
5.0 ml.) was drawn within 15 seconds into a
sterile siliconized syringe containing 1.2 ml. of
1 M phosphoric acid and 0.2 ml. soybean trypsin
inhibitor (10 mg./ml., Worthington Biochemical
Corp.). The polypeptide present in the synovial
fluid was partially isolated and identified as previously described.5 This involved adsorption and
elution of the kinin from IRC-50 (H-t), bioassay
of the oxytocic activity on the isolated rat uterus.
and destruction of the polypeptide by guinea pig
plasma. Of the 25 samples assayed, 4 samples
(from 3 patients with different arthritides) showed
the presence of other unidentified oxytocic substancefs) which, unlike bradykinin, were not destroyed by the carboxypeptidase N in guinea pig
plasma. Added polypeptide was recovered (500
ng.) and examined periodically and fell within the
expected range (20 to 50 per cent). Samples of
synovial fluid were also analyzed for their content
of total protein and leukocytes and were always
cultured for pathogens.
Ailicrocrystalline Sodium Urate Injections
Three male patients with gout, who were experiencing a prolonged remission, volunteered to
omit medications and to permit injection of up to
search Institute, University of California Sun
Francisco Medical Center, Sun Francisco, Calif.
Supported in part by USPHS Grants HE-03964
and HE-06285.
+Present address: Massachusetts General Hospital, Boston, Massachusetts.
13
ARTHRITIS
AND RHEUMATI~M,
VVL. 10, No. 1 (FEBRUARY,
1967)
14
100 mg. of sterile microcrystalline sodium uratefi
suspended in 5 ml. saline into both knees. After
receiving the injection, one patient experienced
severe gouty effusions in both knees. Joint fluid
prior to the injection of sodium urate could not
be obtained since the knee was entirely quiescent,
but was obtained 6, 24, and 54 hours after injection. The 54 hour sample was taken after colchicine appeared to have exerted a beneficial affect.
Injection of Synthetic Bradykinin into the
Knees of Dogs
Synthetic hradykinin (500 kg in 1.0 ml. saline)
was injected intra-articularly into the synovial
space of one knee of pentobarbital-anesthetized
dogs (60 mg./Kg.). Saline was injected into the
other knee as a control. The skin temperature
medial to the patella was measured at brief intervals by a thermistor (Yellow Springs Instrument
Co.), and the circumference of the knee was
measured frequently. After 40 minutes, the animals
were killed and the joints opened and inspected
for the presence of fluid, as well as for the degree
of hyperemia of synovial tissue. In two dogs, both
knees were injected with 500 kg bradykinin, followed within 4 minutes by injection of 10 mg.
carboxypeptidase B in one knee and equivalent
volumes of saline in the other.
RESULTS
The Presence of Kinin in the Synovial
Fluid of Acute Arthritis
Detectable concentrations of kinin (Tables 1 and 2 ) were found in knee effusions
of all patients regardless of the etiology of
their arthritis. Synovial peptide ranged
from 1.6 ng./ml. (the lower level of sensitivity for the method used) to 58 ng./ml.
fluid. There was, however, little correlation
between the concentration of kinin and the
degree of warmth, pain and tenderness of
the joints. The kinin levels taken from a single patient (W. R., Table 1) may show such
a correlation. A similar correlation, however,
was not found in synovial effusions from
patient J. F., possibly due to the minimal
degree of inflammation of this joint. In any
event the data are too limited to resolve
these questions. The volume of synovial
fluid from quiescent joints was insufficient
for assay. Since normal kinin levels in human plasma have been less than 3.0 ng./
MELMON ET AL.
ml.5 and synovial fluid contains a lower
protein concentration, it is to be expected
that the kinin levels would be correspondingly less and possibly undetectable.
In two gouty patients, W. J. and R. J.,
an acute attack of arthritis resulted in an
increase in polypeptide 24 hours after the
onset of symptoms (Table 2). Patient W. J.
suffered a spontaneous attack of gout in
the left knee whereas the attack of patient
R. J. was induced by the injection of microcrystalline sodium urate into both knees.
Patient R. J. (Fig. 1) had such severe
reaction in both knees that he was unable
to walk 24 hours after the injections. Colchicine therapy was then initiated and
proved moderately effective in reducing the
clinical symptoms. As the symptoms receeded, the kinin levels were lowered. Although the acute process was rapidly ameliorated by colchicine, both knees of this
patient remained somewhat stiff for about
two weeks.
Induction of Arthritis by Injection of Synthetic Bradykinin into the Knees of Dogs
When synthetic bradykinin was injected
into the knees of four dogs, definite warmth
appeared within six minutes, followed by
swelling and, on two occasions, the knee
became so painful that the animal could
be roused from stage three pentobarbital
anesthesia by rather gentle manipulation
of the joint. The temperature was high
for about 20 minutes (Fig. 2) and then
began returning to control levels. Swelling,
however, (Table 3 ) persisted until the time
of sacrifice (40 minutes). All of the changes
produced by the polypeptide could be prevented or aborted if carboxypeptidase B,
which rapidly inactivates the kinins,7 was
injected shortly after the administration of
the polypeptide (Fig. 2, bottom, and Table
3 ) . The knees injected with bradykinin
showed increased synovial fluid and hyperemia of the synovial membrane when compared to saline-injected knees. No synovialfliuid leiikocytosis occurred in either group,
15
KININ I N INFLAMMATORY SYNOVIAL EFFUSION
Table 1.-The
Patient
W. R.
Presence of a Kinin in Injlummatory Synovial Effusions
of Acute Arthritis
Kinin'
ng./ml.
Arthritis
A. C.
Swelling
+
+
++
+
+
+
++
++
+
++
++
-++
++
++
4.9
12
Unknown
N. S.
v. s.
A . M.
A. E.
++
-+
++
0
+
0
0
++
4.0
3.1
4.3
3.8
-+-
0
-t-
Total
Protein
Leukocytes
Gm./100 ml. X l @ / m ~ n . ~
3.7
4.7
-t
++
*
+
+++
+
+
2.9
7.6
18
26
27
44
Rheumatoid
0
+
0
+
++
0
0
10
14
Psoriatic
*ZI'I
1). 13.
D. D.
Warmth
1.6
2.1
4.3
1.7
2.3
3.1
3.4
Gout
J. F.
C. F.
Tenderness
+++
+++
++
++
++
+++
++++
5.3
4.3
3.7
9.4
3.2
4.0
2.6
5.6
+
++
+
3.8
42
t++
3.5
2.2
19
24
11
26
16
.4
25
27
21
19
18
1.7
8.0
"Calculated as bradykinin (Sandoz Pharmaceuticals).
Table 2.-lncrease of Kinin Concentrations in Synovial Effusions
During Spontaneous and Induced Acilte Gouty Arthritis
Patient
Attack
W. J .
Spontaneous
R. J.
Induced
Time'
(hr.)
Knee?
Kinin
ng./ml.
6
LK
24
LK
58
6
LK
RK
LK
RK
8.4
12
19
43
4.0
7.3
24
54
LK
RK
Tenderness
3.5
Warmth
+++ +++
+
+++
+++ ++++
+++ ++++
++++ ++++
++++ ++++
+++
++
+++
++++
Swellinz
LeukoTotal
cytes
Protein
X loJ/
G m . i l 0 0 ml. mm:'
++
++
++
++
++++
+++
++
++I+
42
44
3.1
3.0
3.6
3.4
3.8
4.2
19
21
3.8
6.7
3.6
3.7
'Time in hours after initiation of symptoms (spontaneous attack) or after injection of 100 mg. microcrystalline sodium urate (induced attack). Colchicine therapy given to patient H. J. after 24 hours.
iLK-left knee; RK-right knee.
and routine sections showed no histologic
alteration of synovium.
DISCUSSION
The present studies show that kinins are
present in synovial effusions from patients
with arthritides of various etiology and that
in gout the levels of polypeptide increase
in response either to a spontaneous acute
attack of gouty arthritis or to the intraarticular injection of urate crystals. Also,
certain aspects of the acute inflammatory
process-such as warmth, tenderness, and
swelling-can be reproduced in dogs after
intra-articular injection of bradykinin. Activation of the plasma kallikrein-kininogenkinin system provides an attractive hypothesis to explain certain aspects of the acute
16
MELMON ET AL.
0
I
2
TIME (days)
Fig. 1.-Levels of kinin in knee joint fluid
following injection of urate crystals. The experiment was initiated by injection of 100 mg.
microcrystalline sodium urate in both knees
of it gouty volunteer. Right knee 0-0;left
knee 0 - 0 . After the development of symptoms in day 1, the patient was given intravenously 2 mg. colchicine; treatment was repeated after a 10 hour interval.
inflammatory response. Plasma kallikrein
is present in blood and lymph as an inactive precursor (prekallikrein). This kallikrein acts specifically on an a2 globulin in
human plasma (kininogen) to release the
nonapeptide bradykinin. The initial activator4 of the prekallikrein-kallikrein system
is thought to be Hageman factor, a clotpromoting substance activated by glass or
other negatively charged particles. These
observations, together with the recent revivals of Garrods original hypothesis that
sodium urate crystals initiate the typical
acute attack of gouty arthritis, provide a
mechanism for the activation of the prekallikrein-kallikrein system. Thus deposition
of monosodium urate microcrystals in the
joint could activate Hageman factor; activated Hageman factor, either directly or
through a series of intermediate enzymatic
steps, would activate prekallikrein; the active kallikrein would act on kininogen to
release the polypeptide. This hypothesis
is essentially identical to that recently proposed by Kellermeyer and Breckenridge.$
These authors, referring to a preliminary
report of these studies,l(’ have recently
established that the microcrystalline sodium
urate crystals are negatively charged; moreover, they have shown that these and other
negatively charged crystals are capable of
activating Hageman factor. The presence
of possible inhibitors to plasma kallikrein,
and of enzymes destructive to the kinins,
may lengthen the time required to produce
sufficient quantities of bradykinin to overcome the body’s defensive mechanisms.
Also, as the inflammatory reaction progresses, plasma proteins migrate into the
synovial space, thus increasing the substrate arid enzymes available for production of detectable concentrations of bradykinin. This could explain the latent period
of four or more hours before the inflammatory response is evident in humans following injection of urate crystals, as compared
to the prompt appearance of the inflammatory reaction following (f3-25 minutes) the
intra-articular injection of bradykinin in
dogs. I n related types of arthritis the production of kinins could be due to the deposition of other negatively charged particles, such as calcium pyrophosphate crystals, which have been foundll in the
synovial fluid of patients with chondrocalcinosis.12
The polypeptide found in the synovial
fluid has not yet been identified by chromatographic technics. It is presumed to be
bradykinin. If plasma leaks into the synovial
space during arthritis, the kinin released
by kallikrein would be bradykinin. This
would be in agreement with the findings
17
KININ IN INFLAMMATORY SYNOVIAL EFFUSION
36.5
-
36.0 -
Fig. 2.-Effect of
intra-articular injection of bradykinin
in the dog. Top
illustrates the temperature response to
the injection (1)of
0.5 mg. bradykinin
in 1.0 ml. ( 0 - 0 )
and 1.0 ml. saline
( 0-0).
Bottom illustrates the temperature response to the
injection of 0.5 mg.
bradykinin (J1) followed by injection
of 10 mg. carboxypeptidase B (12)
( 0 - 0 ) or saline
(0-0).
34.5
0
I
5
I
10
I
I
15
20
TIME (min)
of Melchiorri,l" who isolated a polypeptide
from pooled and dialyzed synovial fluid
and found it to be identical with bradykinin. Melchiorri also found the concentration of the polypeptide to be higher in
pooled and dialyzed synovial fluid from
normal subjects. However, he could not
have been measuring the concentration of
bradykinin in intact synovial fluid because
dialysis removes the polypeptide. Rather
the data would indicate increased levels
of substrate and enzymes in the synovial
effusion.
Although in the present studies a kinin
1
25
I
30
was found in synovial fluid, it might be
noted that intra-articular injection of synthetic bradykinin does not mimic all of
the events usually associated with arthritis.
No synovial fluid leukocytosis occurred, and
there was no histologic alteration of synovium. The lack of such events does not
exclude a role for the kinins in joint inflammation. Kinins may not be the only
stimulus to leukocyte accumulation, and although the high concentrations of kinin
injected into the joints in dogs produced
symptoms rapidly, time may have been
insufficient to allow granulocyte accumula-
18
MELMON ET AL.
Table 3.-SweZZing
articukw Injection
of
as the Result of ZntraBradykinin in the Dog
Substance
Time*
(min.)
0
Bradykinin
0
18
23
14.8
15.7
15.8
14.8
14.8
14.8
0
10
15
29
0
10
15
29
14.8
15.6
16.4
15.8
14.8
14.5
14.9
14.8
18
25
Saline
Bradykinin
+ Saline
Bradykinin
+ COB#
Cireumference
(cm.)
‘Time in minutes after iniection.
fCOB-carboxypeptidase
B ( Worthington Biochemical Gorp.)
tion. Because granulocytes are key to the
pathogenesis of crystal-induced acute arthritis,14 and because granulocytes may
themselves be an important source of kallikrein,15 other stimuli may be primarily
responsible for leukotaxis and histologic
changes in synovial tissue. The accumulated
leukocytes may then produce kinins which
contribute to further swelling, warmth, and
pain.
The exact contribution of the kinins to
the inflammatory reaction in humans remains to be determined. Changes in synovial fluid kinin concentration seemed to correlate well with the severity of symptoms
(tenderness, warmth, and swelling) in
selected individual patients. On the other
hand, the clinical severity of arthritis from
person to person could not have been predicted by knowing the kinin concentration
in synovial fluid. This seems to diminish
the importance of the role of kinin peptides
in the pathogenesis of the process. However, several factors must be considered
before such a conclusion can be supported:
1. Single determinations of kinin in synoviai
fluid do not reflect the activity of the kallikrein-
kininogen-kinin system in the synovial tissue or
accumulated granulocytes.
2. The determinations in this study reflect a
single point in time and neglect the rate and extent of synovial fluid accumulation, activity of the
kinin-destroying system, levels of substrate (kininogen) in the locale of kinin production, and accumulation or destruction of kallikrein or kininase
inhibitors.
3. Because the total volume of fluid could not
be easily assessed, no attempt was made to determine the absolute amounts of kinin in a synovial
space.
4. No specific antikinin agents are available for
use in human synovial spaces. PheIps e t al.16
recently reported that carboxypeptidase B failed
to block synovitis in dogs induced with microcrystalline monosodium urate. While it is true that
these crystals have been shown to activate Hageman factor on the synovial fluid of dogs,l7 there is
considerable evidence that the prekallikrein-kallikrein system in dogs is not activated by glass
beads and is thus quite different from humans.18
Whether the data obtained in the canine system
can be extrapolated to man remains to be determined.
Another unanswered question is how
various therapeutic agents work. In the
course of these studies, agents commonly
used in the treatment of arthritis, such as
phenylbutazone, hydrocortisone, colchicine,
and acetylsalicylic acid, were added to the
smooth muscle chamber in concentrations
of 10-7 to 10d3M. None of these agents
inhibited the response of the uterus used
for assay to a standard amount ( 4 to 10
ng.) of synthetic bradykinin. There can be
no conclusions, however, concerning the
ability of these agents to alter kinin concentrations in synovial effusions, for inhibitors to the polypeptide may be effective in
one biological system and not in another.lQ
Also, these agents might have no direct effect on the polypeptide but may alter the
rate of its formation, as has been suggested
by recent data,20 or increase its rate of
destruction or alter its peripheral activity.
ACKNOWLEDGMENTS
The technical assistance of W. Anderson, Jr.,
and D. England js gratefnll~ acknowledged. The
19
KININ I N INFLAMMATORY SYNOVIAL EFFUSION
authors are indebted to Dr. E. D. Nicolaides,
Parke, Davis and Co., Ann Arbor, Michigan, and
Sandoz Pharmaceuticals, Hanover, New Jersey,
for gifts of synthetic bradykinin and to Dr. L.
Sokoloff for histologic examination of synovial
tissue obtained from dogs.
SUMMARY
Detectable concentrations of a kinin (1.6-58 ng./ml. fluid) were found in effusions
from the knees of patients with arthritides of varying etiology. In two patients suffering
from gouty arthritis, a n acute attack (spontaneous or induced by microcrystalline
sodium urate) resulted in a n increase in polypeptide 24 hours after the onset of
symptoms.
Intra-articular injection of synthetic bradykinin in the dog produced warmth, swelling, and pain. It is tentatively suggested that the kinins may contribute to the inflammatory synovial reaction seen in arthritides.
SUMMARIO
IN INTERLINGUA
Detegibile concentrationes d e un kinina (1,6 a 58 ng/ml) esseva trovate in effusiones
de articulationes del genus in patientes con arthritis d e varie etiologias. I n duo patientes
con arthritis guttose, un attacco acute (spontanee o inducite per microcrystallin urato
de natrium) resultava in un augmentate concentration d e polypeptida 24 horas post
le declaration del symptomas. Le injection intra-articular de bradykinina synthetic in
canes produceva calor, tumescentia, e dolor. Es suggestionate que le kininas contribue
possibilemente a1 reaction synovio-inflammatori incontrate in certe arthritides.
REFERENCES
1. Collier, H. 0. J.: Kinins. Sci. Amer. 207:111,
1962.
2. Schachter, M.: Kinins-a group of active peptides. Ann. Rev. Pharmacol. 4:281, 1964.
3. Lewis, G. P.: Bradykinin. Nature (London)
1922596, 1961.
4. Webster, M. E., and Innerfield, J.: Interrelationship of human plasma kallikrein and
plasmin in inflammation. Enzymol. Biol.
Clin. 5: 129, 1965.
5. Webster, M. E., and Gilmore, J. P.: The estimation of the kallidins in blood and urine.
Biochem. Pharm. 14:1161, 1965.
6. Seegmiller, J. E., Howell, R. R., and Malawista,
S. E.: The inflammatory reaction to sodium
urate. J.A.M.A. 180:469, 1962.
7. Erdos, E. G., Wohler, J. R., and Levine, M. I.:
Blocking of the in V ~ V Oeffects of bradykinin
and kallidin with carboxypeptidase B. J.
Pharmacol. Exp. Ther. 142:327, 1963.
8. Seegmiller, J. E., Laster, L., and Howell, R. R.:
Biochemistry of uric acid and its relation to
gout. New Eng. J. Med. 268:712, 764, 821,
1963.
9. Kellermeyer, R. W., and Breckenridge, R. T.:
The inflammatory process in acute gouty
arthritis. I. Activation of Hageman factor by
sodium urate crystals. J. Lab. Clin. Med. 65:
307,1965.
10. Goldfinger, S., Melmon, K. L., Webster, M. E.,
Sjoerdsma, A., and Seegmiller, J. E.: The
presence of a kinin-peptide in inflammatory
synovial effusions. Arthritis Rheum. 7:311,
1964.
11. McCarty, D. J., Kohn. N. N., and Faires, J. S.:
Significance of calcium phosphate crystals in
synovial fluid of arthritic patients: “pseudogout syndrome.” I. Clinical Aspects. Ann.
Intern. Med. 56:711, 1962.
12. Zitnan, D., and Sitaj, S.: Calcifications multiples du cartilage articnlaire. 9th International Congress sur les hlaladies Rheumatismales 2:291, 1957.
13. Melchiorri, P.: Occurrence of bradykinin in the
synovia of subjects affected with rheumatoid
arthritis. In: Bradykinin and its Precursors.
F. Sicuteri, Ed., Rome, Centro Editoriale
Publicitario Italiano. 1963, p. 65.
14. Phelps, P., and RlcCarty. D. J., Jr.: Crystal
induced inflammation in canine joints. 11.
Importance of polymorphonudear leukocytes. J. Exp. Med. 124:115, 1966.
15. Melmon, K. L., and Cline, M. J. The interaction of plasma kinins and granulocytes.
Nature (London). In press.
16. Phelps, P., Prockop, D. J., and hlccarty, D. I.,
Jr.: Crystal induced inflammation in canine
joints. 111. Evidence against bradykinin as a
20
mediator of inflammation. J. Lab. Clin. Med.
58:433, 1966.
17. KellermeYer, R. w., and Breckenridge, R. T.
The inflammatory process in acute gouty
arthritis. 11. The presence of Hageman factor
and plasma thromboplastin antecedent in
synovial fluid. J. Lab. clin.~ ~ 67:455,
d .
1966.
18. Armstrong, D., Jepson, J. B., Keele, C. A., and
Stewart, J. W.: Pain-producing substance in
MELMON ET AL.
human inflammatory exudates and plasma. J.
Physiol. 135350, 1957.
19. Collier, H. 0. J.: The action and antagonism
of kinins on bronchioles. Ann. N. Y. Acad.
Sci. 104:290, 1963.
20. Cline, M. J., and Melmon, K. L. Plasma kinins
and cortisol: A possible explanation of the
anti-inflammatory action of cortisol. Science.
In press.
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