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Questionable role of leukotriene B4 in monosodium urate (MSU)-induced synovitis in the dog
PROSTAGLANDINS
QUESTIONABLE ROLE OF LEUKOTRIENE B 4 IN MONOSODIUM URATE (MSU)-INDUCED SYNOVITIS IN THE DOG
Richard P. Carlson*, Joseph Chang, Louis J. Datko, and Alan J. Lewis
Department of Experimental Therapeutics Wyeth Laboratories, Inc. P.O. Box 8299 Philadelphia, PA 19101 * Author to whom correspondence should be addressed
ABSTRACT Monosodium urate (MSU)-induced synovitis in the dog's stifle (knee joint) is similar to an acute gouty attack in man in which a loss of function of the joint correlates with massive influx of neutrophils and the release of an assortment of inflammatory mediators (e.g. histamine, bradykinin, lysosomal enzymes, complement and eicosanoids) into the synovial space. We found in the urate-induced inflammatory exudates 3 hr post MSU the following: 88 million leukocytes/ml (,,w95% neutrophils) and eicosanoid concentrations of LTB., LTC4, and PGE^ of < 0.I, 1.4 and 20 ng/ml, respectively. Isotonic sa~ine injected knee joints at 3 hr contained 5 million leukocytes/ml (--,95% neutrophils) and concentrations of LTB4, LTC&, and PGE 2 of < 0.i, 0.7 and 0.2 ng/ml, respectively. Intrasynovial injections of I u g LTB&, i0 ug PGE 2 or the combination of LTBA and PGE 2 produced no reduction of paw pressure for up to 3 hr. Leukocyte concentrations measured at 3 hr in joints injected with these arachidonic acids metabolites were similar to saline controls. These results question the role of L T B as a chemotactic and inflammatory mediator in urate-induced synovitis in the dog but confirm the importance of PGE 2 and possibly LTC 4 in this model.
INTRODUCTION Monosodlum urate (MSU) crystals are identified in joints of patients with acute and chronic gout; moreover, MSU crystals elicit an acute, painful inflammatory response when injected into the synovial space of the hind knee joint of dogs (1,2). The response is characterized by a massive influx of neutrophils into the synovial space. Numerous inflammatory mediators are thought to contribute to this inflammatory response including Hageman factor, the kallikrein-kinin system, complement, histamine and the prostaglandins. Recently, MSU crystals were shown to stimulate the production of arachidonic acid metabolites, including LTB4, by human neutrophils and platelets (3). Since LTBA is a potent chemotactic factor (4), we performed the following studies in an attempt to establish the presence of LTBA in the synovial fluid of dogs with urate-induced synovitis and to examine the effects of exogenous LTB 4 injected into the synovial space of normal dogs.
OCTOBER 1986 VOL. 32 NO. 4
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PROSTAGLANDINS
METHODS Healthy, mongrel dogs (Hazleton Dutchland Labs, Denver, PA) of either sex weighing 13-20 kg were adapted to an automated canine paw pressure apparatus as described by Carlson et. al (5). Control left hind paw pressures were obtained by using a load cell under the platform during a one-minute interval. A synovitis was induced by injecting 5 mg of MSU crystals (5-15 ~m length) in a volume of i ml (0.9% pyrogen free saline) into the synovial space of the left stifle (knee joint). Post urate readings were taken at i min and at 30 min intervals up to 5 hr. Synovial exudate was aspirated at 3 hr and leukocyte and differential counts were made from the exudate. An aliquot of the supernatant fluid (cell free synovial exudate) after centrifugation was examined for levels of LTB., LTC 4 and PGEp by radioimmunoassay, as previously described (6)~ In some studies, exudate samples were assayed for the 20-OH and 20-COOH metabolites of LTB 4 using reversed phase HPLC after Sep-Pak extraction. The column was eluted at a flow rate of i ml/min with the solvent system consisting of methanol: water: phosphoric acid (65:35:0.001%). The column effluent was monitored for absorbance at 280 nm. Normal synovial fluid at zero time (i.e. just prior to urate) was also assessed for the above parameters. Percent of control paw pressures were calculated and recorded by a HP 85 computer as described previously (5). In separate experiments, I ~g of LTBA (Biomol Research Labs, Philadelphia, PA) or i0 ~g PGE~ (Nu-Chek, Elysian, MN) in I ml of pyrogen free saline or in 0.25% ethanol in saline and the combination of I ~g LTB~ and 10 ~g PGE9 in 0.5 ml of saline or ethanol in saline were injected'(i.e. LTB 4 was"injected just prior to PGEp). Paw pressures were assessed at i min and at 30 min intervals up ~o 3 hr. Synovial samples were removed to determine leukocyte concentration and cell type prior to injection of eicosanoids and at 3 hr. Prior to injection, LTB 4 was purified by HPLC and shown to have chemotactic activity against human neutrophils. RESULTS Monosodium urate (5 mg) intrasynovially produced a 3 legged gait (zero paw pressure) by 2 hr (Fig 1) which did not change over a 3 hr period (2 to 5 hr post urate). Synovial fluid cells prior to injection of MSU numbered 0.9 million leukocytes/ml (n=13) and PMNs made up1~..l% of the total (Fig I). At 3 hr after MSU, the leukocyte count had risen to 88.2 million/ml and PMNs made up 94-96% of the cells. Only 2% of cells from the 3 hr exudate were monocytes or macrophages. In Fig. 2 ) concentrations of PGE2, LTB, and LTC4/ml in synovial fluid before injection of MSU and at 3 hr after 0.9% saline or 5 mg of MSU are shown. LTCA concentration (1.8 ng/ml) was higher in prelnjected synovial fluid than in synovial fluid obtained from saline or MSU injected at 3 hr. Only the PGEp concentration at 3 hr (20 ng/ml) showed a significant change over normal synovial fluid and aspirate obtained from 0.9% saline injected joints. The LTB level was found to be less 4 than 0.125 ng/ml in synovial fluid before and after MSU injection. Also, the metabolites of LTB4, 20-OH LTB 4 and 20-COOH LTB.,4 were found in concentrations of less than 5 ng/ml in synovial fluid obtained from MSU injected joints at 3 hr.
580
OCTOBER 1986 VOL. 32 NO. 4
PROSTAGLANDINS
100
-
-
i
90 o
3
tOO
(12)
90
8O
80 70 60
60
~
"~ 50
50 ~
~
40
40
~ -×
e g
30
30
~
~
20
2O
tO
I0
O~
0.5
Preinjection
I
1.5
2
2.5
3.5
4
o~
4.5
Time after intrasynovial injection Ihr)
Control Figure
i.
Time course of left paw pressures after 5 mg MSU-induced inflammation in the synovial space of the dog's knee and leukocyte counts at 0 and 3 hr post urate. Number of dogs used are indicated in the parenthesis.
PGE2
LTB4
LTC4
~
25 +I
20
(a) 20-OHLTB4 | 20-COOHLTB41
=o
)
y////A
3
/ / / / / / A
ol-r ..(3.!.11111111111il
(5)
K//~//g . . . . . . . . . .
Preinjection control
(3)[]]]t]]]]11]U -,IIIIIIIUIHI
3 hr Saline control
.............}.3.!.1111111111111
3 hr Na urate
(a)None of the major LTB4metabolites were detected by RP-HPLC (28Onto). Figure
2.
Concentrations of PGE2, LTB 4 and LTC 4 in the synovial fluid before intrasynovial injection of MSU and at 3 hr post MSU in the synovial exudate. Number of dogs used are indicated in the parenthesis.
OCTOBER 1986 VOL. 32 NO. 4
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The effects of 1 ~g LTB&, I0 ~g PGE2, the combination of 1 ~g LTB 4 and i0 ~g PGEp, and 5 mg MSU on paw pressure are shown as a time/effect curve in Fig. 3. No effect on hind paw pressure was observed after injection of these eicosanoids. In contrast, MSU produced a complete loss of paw pressure by 2 hr which continued for another 3 hr (Fig i). In Fig. 4, the mean leukocyte count in the synovial space at 3 hr is shown after the injection of eicosanoids and MSU. Only MSU injected joints showed a major influx of leukocytes (88 million/ml, 94-96% neutrophils). Saline injected joints had a mean leukocyte count of 4.4 million/ml + 2.1 (S.E.); the eicosanoids when injected intrasynovially produced similar leukocyte counts (3.2-6.6 million/ml). DISCUSSION LTB& has been identified in human joints during inflammation, especially during an acute gouty attack (7,8) and has been shown to be a potent chemotactic agent and to degranulate lysosomes (9,10). The instability of LTB& (as shown by the rapid formation of the metabolites 20-OH and 20-COOH LTB 4 by u-oxidation in inflammatory exudates) may, however, preclude a critical role for this mediator in the inflammatory process (~,12). Nevertheless, Taylor and Sun (13) have noted that in vivo [ C] LTB h disappeared slowly (half-life of 46 min) from inflammatory exudate o~tained in the rat carrageenan pleurisy model. Our studies indicate that intrasynovial injection of 1 ~g of LTB 4, i0 ~g PGE or the combination of these two eicosanoids did not induce an inflammatory response over a 3 hr period. In contrast, MSU injected intrasynovially produced a rapid onset of inflammation which resulted in a complete loss of paw pressure by 2 hr. We found only trace amounts of LTB 4 (< 0.125 ng/ml) in the 3 hr post-MSU exudate which consisted of 90 million neutrophils/ml; this trace amount of LTB 4 is not due to rapid metabolism since barely detectable amounts of 20-OH and 20-COOH LTB 4 (< 5 ng/ml by HPLC) were present. Interestingly, Serhan et. al (3) showed that I00 million human leukocytes incubated in vitro for 15 min with sodium urate produced only 4 ng of LTB&, and Dr. D. C. Henke (U. of North Carolina, Dept. of Medicine-pers6nal communication) also showed no observable production of LTB. from calcium ionophore stimulated 4 peripheral blood neutrophils from dogs. In agreement with Moncada et. al (14) we found that PGE_ levels were z elevated by a 100 fold (20 ng/ml) in the dog synovitis model at 3 hr post MSU. Reduction of the synovitis by nonsteroidal antiinflammatory drugs in this model supports the importance of PGE 2 in this MSU-Induced inflammatory response (2,5). Bradykinin has been shown to induce pain in a dose related manner in the dog knee joint (15), and it was observed that 0.05 ~g bradykinin, a subliminal dose, produced no pain. In preliminary experiments, LTB (i ~g) when injected intrasynovially before bradykinin did not produce 4 significant hyperalgesia in response to a subthreshold dose of bradykinin (0.05 ~g). Under identical conditions, PGE 2 (i ug) was effective in inducing hyperalgesia (14). These data support the importance of PGEp in pai~ production during MSU inflammation and imply that LTB 4 does nor play a critical and contributory role in this model
582
OCTOBER 1986 VOL. 32 NO. 4
PROSTAGLANDINS
LTB4(Iug)
120
100
Q.
80
Q
6o
~ ao
~ i
0
0.5
I
1
Q
Q
i
i
i
i
1.5
2
2.5
3
Time after intrasynovial injection (hr) Figure 3.
Time course of left paw pressure after the injection of LTB 4, PGE2, combination of LTB 4 and PGE 2 and MSU into the dog's synovial space (n = 3-5 dogs/point).
100 90 q~ E
80
,=
70 x
o=
60 50
30 20
10 0
Figure 4.
PGE2 (101~g)
LTB4 (Ipg)
PGE2 + LTB4 (10Mg) (iMg)
NA URATE(Smg)
Leukocyte concentrations at 3 hrs after the injection of LTB4, PGE2, combination of LTB 4 and PGE2, and urateo Number of dogs used are indicated in the parenthesis.
O C T O B E R 1986 V O L . 32 NO. 4
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of inflammation. It should be noted, however, that LTB. has been shown to produce slight to moderate hyperalgesia in normal an~ yeast injected rat paws (16, 17). The concentration of LTC. at 3 hr in the urate-induced inflammatory 4 exudate dropped slightly compared to preinjection values (1.8 versus 1.4 ng/ml). However, this concentration of LTC A at 3 hr post urate is probably sufficient to produce with the higfily elevated PGEp a synergistic increase in capillary permeability as demonstrated by Williams (18,19) in rabbit skin. In conclusion, urate-induced synovitis in the dog produced augmented levels of PGEp in synovlal exudate but there is no evidence for the presence of LTB. or its metabolites. Furthermore, LTB. injected alone into the dog's ~nee Joint was not inflammatory. Together, these observations argue against a role for LTB 4 in MSU-induced synovitls in the dog. ACKNOWLEDGMENTS The authors wish to thank Laura Marlnari for determining eicosanoid concentrations, Paul Freimuth for the graphic arts and Bernadette Reczek for preparing the manuscript. REFERENCES
584
i.
McCarty, D.J., Phelps, P. and J. Pyenson. Crystal-lndueed inflammation in canine Joints. I. An experimental model with quantification of the host response. J. Exp. Med. 124: 99-114, 1966.
2.
Van Arman C.G., Carlson, R.P., Risley, E.A., Thomas, R.H. and G.W. Nuss. Inhibitory effects of indomethacin, aspirin and certain other drugs on inflammation induced in rat and dog by carrageenan, sodium urate and ellagic acid. J. Pharmacol. Exp. Ther. 175: 459-468, 1970.
3.
Serhan, C.N., Lundberg, U., Weissman, G. and B. Samuelsson. Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate. Prostaglandins 27: 563-581, 1984.
4.
Ford-Hutchinson, A.W., Bray, M.A., Dolg, M.V., Shipley, M.E. and M.J.H. Smith. Leukotriene B: A potent chemokinetlc and aggregating substance released from polymorphonuclear leucocytes. Nature (London) 286: 264-265, 1980.
5.
Carlson, R.P., Datko, L.J., Welch, T.M., Durvis, W.F., Shaw, G.W., Thompson, J.C. and T.R. Brunner. An automated mlcrocomputer-based system for determining canine paw pressure quantitatively in the dog synovitis model. J. Pharmacol. Methods. 15: 95-104, 1986.
6.
Chang, J., Lamb, B., Marinari, L., Kreft, A.F. and A.J. Lewis. Modulation by hydroxy eicosatetraenoic acids (HETEs) of arachldonic acid metabolism in mouse resident peritoneal macrophages. Eur. J. Pharmacol. 107: 215-222, 1985.
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7.
Davidson, E.M., Rae, S.A. and M.J.H. Smith. Leukotriens B., a mediator of inflammation present in synovial fluid in rheumatoid arthritis. Ann. Rheum. Dis. 42: 677-679, 1983.
8.
Rae, S.A., Davidson, E.M. and M.J.H. Smith. Leukotriene B4, an inflammatory mediator in gout. Lancet ~: 1122-1124, 1982.
9.
A.W. Ford-Hutchinson. Leukotrienes: their formation and role as inflammatory mediators. Fed. Proc. 44: 25-29, 1985.
10.
M.A. Bray. The pharmaco]ogy and pathophysiology of leukotriene B 4. Br. Med. Bull. 39: 249-254, 1983.
II.
Ueno, A., Tanaka, K. and M. Kaferi. Detection of leukotriene C 4 and DA in the exudate of rat carrageenin induced pleurisy. Prost~glandins 26: 493-504, 1983.
12.
Shak, S. and I.M. Goldstein. The major pathway for leukotriene B 4 catabolism in human polymorphonuclear leukocytes involves -oxidation by a cytochrome P-450 enzyme. In, Prostaglandins, Leukotrienes, and Lipoxins. (Ed. J.M. Bailey), Plenum Press, New York, 1985, pp. 97-109.
13.
Taylor, B.M. and F.F. Sun. Disappearance and metabolism of leukotriene B during carrageenan-induced pleurisy. Biochem. Pharmacol. 349 3495-3498, 1985.
14.
Moncada, S., Ferreira, S. and J.R. Vane. Inhibition of prostaglandin biosynthesis as the mechanism of analgesia of aspirin-llke drugs in the dog knee joint. Eur. J. Pharmacol. 31: 250-260, 1975.
15.
Van Arman, C.G. and R.P. Carlson. The two distinct phases of inflammatory response in the dog's knee joint. In, Bradykinin and Related Kinins: Cardiovascular Biochemical and Neural Actions (Ed. F. Sicuteri, M. Rocha e Silva and Nathan Back), Plenum Press, New York, 1970, pp. 525-533.
16.
Rackham, A. and A.W. Ford-Hutchinson. Inflammation and pain sensitivity: Effect of leukotrienes D., B. and prostaglandin E in the rat paw. Prostaglandins 25: 193-2~3, 9983.
17.
Levine, J.D., Lau, W. and G. Kwiat. Leukotriene B 4 produces hyperalgesia that is dependent on polymerphonuclear leukocytes. Science 225: 743-745, 1984.
18.
Williams, T.J. Interactions between prostaglandlns, leukotrienes and other mediators of inflammation. Br. Med. Bull. 39: 239-242, 1983.
19.
Williams, T.J. Prostaglandins E2, prostaglandin Ip and the vascular changes of inflammation. Br. J. Pharmacol. 65: 517-524, 1979.
Editor:
h. R a i s z
OCTOBER 1986 VOL. 32 NO. 4
R e c e i v e d :5 - 1 - 8 6
A c c e p t e d :9-4-86
585
QUESTIONABLE ROLE OF LEUKOTRIENE B 4 IN MONOSODIUM URATE (MSU)-INDUCED SYNOVITIS IN THE DOG
Richard P. Carlson*, Joseph Chang, Louis J. Datko, and Alan J. Lewis
Department of Experimental Therapeutics Wyeth Laboratories, Inc. P.O. Box 8299 Philadelphia, PA 19101 * Author to whom correspondence should be addressed
ABSTRACT Monosodium urate (MSU)-induced synovitis in the dog's stifle (knee joint) is similar to an acute gouty attack in man in which a loss of function of the joint correlates with massive influx of neutrophils and the release of an assortment of inflammatory mediators (e.g. histamine, bradykinin, lysosomal enzymes, complement and eicosanoids) into the synovial space. We found in the urate-induced inflammatory exudates 3 hr post MSU the following: 88 million leukocytes/ml (,,w95% neutrophils) and eicosanoid concentrations of LTB., LTC4, and PGE^ of < 0.I, 1.4 and 20 ng/ml, respectively. Isotonic sa~ine injected knee joints at 3 hr contained 5 million leukocytes/ml (--,95% neutrophils) and concentrations of LTB4, LTC&, and PGE 2 of < 0.i, 0.7 and 0.2 ng/ml, respectively. Intrasynovial injections of I u g LTB&, i0 ug PGE 2 or the combination of LTBA and PGE 2 produced no reduction of paw pressure for up to 3 hr. Leukocyte concentrations measured at 3 hr in joints injected with these arachidonic acids metabolites were similar to saline controls. These results question the role of L T B as a chemotactic and inflammatory mediator in urate-induced synovitis in the dog but confirm the importance of PGE 2 and possibly LTC 4 in this model.
INTRODUCTION Monosodlum urate (MSU) crystals are identified in joints of patients with acute and chronic gout; moreover, MSU crystals elicit an acute, painful inflammatory response when injected into the synovial space of the hind knee joint of dogs (1,2). The response is characterized by a massive influx of neutrophils into the synovial space. Numerous inflammatory mediators are thought to contribute to this inflammatory response including Hageman factor, the kallikrein-kinin system, complement, histamine and the prostaglandins. Recently, MSU crystals were shown to stimulate the production of arachidonic acid metabolites, including LTB4, by human neutrophils and platelets (3). Since LTBA is a potent chemotactic factor (4), we performed the following studies in an attempt to establish the presence of LTBA in the synovial fluid of dogs with urate-induced synovitis and to examine the effects of exogenous LTB 4 injected into the synovial space of normal dogs.
OCTOBER 1986 VOL. 32 NO. 4
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PROSTAGLANDINS
METHODS Healthy, mongrel dogs (Hazleton Dutchland Labs, Denver, PA) of either sex weighing 13-20 kg were adapted to an automated canine paw pressure apparatus as described by Carlson et. al (5). Control left hind paw pressures were obtained by using a load cell under the platform during a one-minute interval. A synovitis was induced by injecting 5 mg of MSU crystals (5-15 ~m length) in a volume of i ml (0.9% pyrogen free saline) into the synovial space of the left stifle (knee joint). Post urate readings were taken at i min and at 30 min intervals up to 5 hr. Synovial exudate was aspirated at 3 hr and leukocyte and differential counts were made from the exudate. An aliquot of the supernatant fluid (cell free synovial exudate) after centrifugation was examined for levels of LTB., LTC 4 and PGEp by radioimmunoassay, as previously described (6)~ In some studies, exudate samples were assayed for the 20-OH and 20-COOH metabolites of LTB 4 using reversed phase HPLC after Sep-Pak extraction. The column was eluted at a flow rate of i ml/min with the solvent system consisting of methanol: water: phosphoric acid (65:35:0.001%). The column effluent was monitored for absorbance at 280 nm. Normal synovial fluid at zero time (i.e. just prior to urate) was also assessed for the above parameters. Percent of control paw pressures were calculated and recorded by a HP 85 computer as described previously (5). In separate experiments, I ~g of LTBA (Biomol Research Labs, Philadelphia, PA) or i0 ~g PGE~ (Nu-Chek, Elysian, MN) in I ml of pyrogen free saline or in 0.25% ethanol in saline and the combination of I ~g LTB~ and 10 ~g PGE9 in 0.5 ml of saline or ethanol in saline were injected'(i.e. LTB 4 was"injected just prior to PGEp). Paw pressures were assessed at i min and at 30 min intervals up ~o 3 hr. Synovial samples were removed to determine leukocyte concentration and cell type prior to injection of eicosanoids and at 3 hr. Prior to injection, LTB 4 was purified by HPLC and shown to have chemotactic activity against human neutrophils. RESULTS Monosodium urate (5 mg) intrasynovially produced a 3 legged gait (zero paw pressure) by 2 hr (Fig 1) which did not change over a 3 hr period (2 to 5 hr post urate). Synovial fluid cells prior to injection of MSU numbered 0.9 million leukocytes/ml (n=13) and PMNs made up1~..l% of the total (Fig I). At 3 hr after MSU, the leukocyte count had risen to 88.2 million/ml and PMNs made up 94-96% of the cells. Only 2% of cells from the 3 hr exudate were monocytes or macrophages. In Fig. 2 ) concentrations of PGE2, LTB, and LTC4/ml in synovial fluid before injection of MSU and at 3 hr after 0.9% saline or 5 mg of MSU are shown. LTCA concentration (1.8 ng/ml) was higher in prelnjected synovial fluid than in synovial fluid obtained from saline or MSU injected at 3 hr. Only the PGEp concentration at 3 hr (20 ng/ml) showed a significant change over normal synovial fluid and aspirate obtained from 0.9% saline injected joints. The LTB level was found to be less 4 than 0.125 ng/ml in synovial fluid before and after MSU injection. Also, the metabolites of LTB4, 20-OH LTB 4 and 20-COOH LTB.,4 were found in concentrations of less than 5 ng/ml in synovial fluid obtained from MSU injected joints at 3 hr.
580
OCTOBER 1986 VOL. 32 NO. 4
PROSTAGLANDINS
100
-
-
i
90 o
3
tOO
(12)
90
8O
80 70 60
60
~
"~ 50
50 ~
~
40
40
~ -×
e g
30
30
~
~
20
2O
tO
I0
O~
0.5
Preinjection
I
1.5
2
2.5
3.5
4
o~
4.5
Time after intrasynovial injection Ihr)
Control Figure
i.
Time course of left paw pressures after 5 mg MSU-induced inflammation in the synovial space of the dog's knee and leukocyte counts at 0 and 3 hr post urate. Number of dogs used are indicated in the parenthesis.
PGE2
LTB4
LTC4
~
25 +I
20
(a) 20-OHLTB4 | 20-COOHLTB41
=o
)
y////A
3
/ / / / / / A
ol-r ..(3.!.11111111111il
(5)
K//~//g . . . . . . . . . .
Preinjection control
(3)[]]]t]]]]11]U -,IIIIIIIUIHI
3 hr Saline control
.............}.3.!.1111111111111
3 hr Na urate
(a)None of the major LTB4metabolites were detected by RP-HPLC (28Onto). Figure
2.
Concentrations of PGE2, LTB 4 and LTC 4 in the synovial fluid before intrasynovial injection of MSU and at 3 hr post MSU in the synovial exudate. Number of dogs used are indicated in the parenthesis.
OCTOBER 1986 VOL. 32 NO. 4
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PROSTAGLANDINS
The effects of 1 ~g LTB&, I0 ~g PGE2, the combination of 1 ~g LTB 4 and i0 ~g PGEp, and 5 mg MSU on paw pressure are shown as a time/effect curve in Fig. 3. No effect on hind paw pressure was observed after injection of these eicosanoids. In contrast, MSU produced a complete loss of paw pressure by 2 hr which continued for another 3 hr (Fig i). In Fig. 4, the mean leukocyte count in the synovial space at 3 hr is shown after the injection of eicosanoids and MSU. Only MSU injected joints showed a major influx of leukocytes (88 million/ml, 94-96% neutrophils). Saline injected joints had a mean leukocyte count of 4.4 million/ml + 2.1 (S.E.); the eicosanoids when injected intrasynovially produced similar leukocyte counts (3.2-6.6 million/ml). DISCUSSION LTB& has been identified in human joints during inflammation, especially during an acute gouty attack (7,8) and has been shown to be a potent chemotactic agent and to degranulate lysosomes (9,10). The instability of LTB& (as shown by the rapid formation of the metabolites 20-OH and 20-COOH LTB 4 by u-oxidation in inflammatory exudates) may, however, preclude a critical role for this mediator in the inflammatory process (~,12). Nevertheless, Taylor and Sun (13) have noted that in vivo [ C] LTB h disappeared slowly (half-life of 46 min) from inflammatory exudate o~tained in the rat carrageenan pleurisy model. Our studies indicate that intrasynovial injection of 1 ~g of LTB 4, i0 ~g PGE or the combination of these two eicosanoids did not induce an inflammatory response over a 3 hr period. In contrast, MSU injected intrasynovially produced a rapid onset of inflammation which resulted in a complete loss of paw pressure by 2 hr. We found only trace amounts of LTB 4 (< 0.125 ng/ml) in the 3 hr post-MSU exudate which consisted of 90 million neutrophils/ml; this trace amount of LTB 4 is not due to rapid metabolism since barely detectable amounts of 20-OH and 20-COOH LTB 4 (< 5 ng/ml by HPLC) were present. Interestingly, Serhan et. al (3) showed that I00 million human leukocytes incubated in vitro for 15 min with sodium urate produced only 4 ng of LTB&, and Dr. D. C. Henke (U. of North Carolina, Dept. of Medicine-pers6nal communication) also showed no observable production of LTB. from calcium ionophore stimulated 4 peripheral blood neutrophils from dogs. In agreement with Moncada et. al (14) we found that PGE_ levels were z elevated by a 100 fold (20 ng/ml) in the dog synovitis model at 3 hr post MSU. Reduction of the synovitis by nonsteroidal antiinflammatory drugs in this model supports the importance of PGE 2 in this MSU-Induced inflammatory response (2,5). Bradykinin has been shown to induce pain in a dose related manner in the dog knee joint (15), and it was observed that 0.05 ~g bradykinin, a subliminal dose, produced no pain. In preliminary experiments, LTB (i ~g) when injected intrasynovially before bradykinin did not produce 4 significant hyperalgesia in response to a subthreshold dose of bradykinin (0.05 ~g). Under identical conditions, PGE 2 (i ug) was effective in inducing hyperalgesia (14). These data support the importance of PGEp in pai~ production during MSU inflammation and imply that LTB 4 does nor play a critical and contributory role in this model
582
OCTOBER 1986 VOL. 32 NO. 4
PROSTAGLANDINS
LTB4(Iug)
120
100
Q.
80
Q
6o
~ ao
~ i
0
0.5
I
1
Q
Q
i
i
i
i
1.5
2
2.5
3
Time after intrasynovial injection (hr) Figure 3.
Time course of left paw pressure after the injection of LTB 4, PGE2, combination of LTB 4 and PGE 2 and MSU into the dog's synovial space (n = 3-5 dogs/point).
100 90 q~ E
80
,=
70 x
o=
60 50
30 20
10 0
Figure 4.
PGE2 (101~g)
LTB4 (Ipg)
PGE2 + LTB4 (10Mg) (iMg)
NA URATE(Smg)
Leukocyte concentrations at 3 hrs after the injection of LTB4, PGE2, combination of LTB 4 and PGE2, and urateo Number of dogs used are indicated in the parenthesis.
O C T O B E R 1986 V O L . 32 NO. 4
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PROSTAGLANDINS
of inflammation. It should be noted, however, that LTB. has been shown to produce slight to moderate hyperalgesia in normal an~ yeast injected rat paws (16, 17). The concentration of LTC. at 3 hr in the urate-induced inflammatory 4 exudate dropped slightly compared to preinjection values (1.8 versus 1.4 ng/ml). However, this concentration of LTC A at 3 hr post urate is probably sufficient to produce with the higfily elevated PGEp a synergistic increase in capillary permeability as demonstrated by Williams (18,19) in rabbit skin. In conclusion, urate-induced synovitis in the dog produced augmented levels of PGEp in synovlal exudate but there is no evidence for the presence of LTB. or its metabolites. Furthermore, LTB. injected alone into the dog's ~nee Joint was not inflammatory. Together, these observations argue against a role for LTB 4 in MSU-induced synovitls in the dog. ACKNOWLEDGMENTS The authors wish to thank Laura Marlnari for determining eicosanoid concentrations, Paul Freimuth for the graphic arts and Bernadette Reczek for preparing the manuscript. REFERENCES
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Ford-Hutchinson, A.W., Bray, M.A., Dolg, M.V., Shipley, M.E. and M.J.H. Smith. Leukotriene B: A potent chemokinetlc and aggregating substance released from polymorphonuclear leucocytes. Nature (London) 286: 264-265, 1980.
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Van Arman, C.G. and R.P. Carlson. The two distinct phases of inflammatory response in the dog's knee joint. In, Bradykinin and Related Kinins: Cardiovascular Biochemical and Neural Actions (Ed. F. Sicuteri, M. Rocha e Silva and Nathan Back), Plenum Press, New York, 1970, pp. 525-533.
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Rackham, A. and A.W. Ford-Hutchinson. Inflammation and pain sensitivity: Effect of leukotrienes D., B. and prostaglandin E in the rat paw. Prostaglandins 25: 193-2~3, 9983.
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Levine, J.D., Lau, W. and G. Kwiat. Leukotriene B 4 produces hyperalgesia that is dependent on polymerphonuclear leukocytes. Science 225: 743-745, 1984.
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Williams, T.J. Interactions between prostaglandlns, leukotrienes and other mediators of inflammation. Br. Med. Bull. 39: 239-242, 1983.
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Williams, T.J. Prostaglandins E2, prostaglandin Ip and the vascular changes of inflammation. Br. J. Pharmacol. 65: 517-524, 1979.
Editor:
h. R a i s z
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A c c e p t e d :9-4-86
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