Leukotriene B4, polymorphonuclear leukocytes and inflammatory exudates in the rat

PROSTAGLANDINS Leukotriene B4, Folymorphonuclear Leukocytes and Inflamatory Exudates in the Rat A.W. Ford-Hutchinson,

G. Rrunet, P. Savard and S. Charleson

Merck Frosst Canada Inc. P.O. Box lflO5, Pointe Claire-Dorval, OuPbec, H9R 4P8, Canada Abstract: Leukocyte numbers and Leukotriene B4- (LTB4-) and LTC4immunoreactivity were measured in inflammatory exudates obtained from sponges impregnated with several irritants implanted subcutaneously in the rat. Sponges containing 1% uric acid, carrageenan or zymosan Increases in were implanted for 5h and compared to saline sponges. leukocyte numbers and LTB4-immunoreactivity were found in the presence of irritants, the highest concentrations being observed in the presence of zymosan. The presence of LTB4 was confirmed by liquid chromatographic (HPLC) analysis. A time course study was carried out with zymosan-impregnated sponges and the maximal rate of leukocyte infiltration was found to coincide with the maximal levels The LTC4-immunoreactivity was low and of LTB4-immunoreactivity. following analysis by HPLC was concluded to be unrelated to leukotrienes. The levels of LTB4-immunoreactivity, but not the numbers of leukocytes, were elevated compared to corresponding controls in sponges containing 0.01% ionophore A23187 (untreated rats) or in sponges containing zymosan (rats pretreated with ind methacin; 3 and 10 mg/_kg p.0.). Impregnation of sponges with 3 x 10'8M LTB4 but not 3 x Ill and 3 x IO- M LTR induced a significant leukocyte migration. It was concluded that L4 B4 can induce leukocyte migration into sponge exudates in the rat but that measurements of LTB4 in such exudates can not be correlated with the degree of leukocyte infiltration. Introduction: Leukotriene B4 (LTB4) [5S,12R-dihydroxy-6,14-cis-8,lfl-trans eicosatetraenoic acid] is a product of arachidonic acid metabolism which may be produced by polymorphonuclear leukocytes (PMM) (1,2). LTB is a potent chemokinetic and chemotactic agent for PMNs in vitro (3,$,5) and when injected -in vivo induces leukocyte accumulat=nT and vascular permeability changes through a leukocyte dependent mechanism (7,8). LTB4 has been detected in synovial fluid obtained from patients with rheumatoid arthritis, spondyloarthritis and gout (9,10), in skin chambers applied to patients with psoriasis (11) and in inflammatory exudates obtained following sponge implantation in the rat (12). LTB4 is one of a number of chemotactic factors that may be produced in inflammatory exudates and its relative importance as compared to other factors, such as the complement derived peptide C5a and platelet activating factor, has not been determined: In the present study we have monitored production of LTB4 and LTC4 in

JULY 1984VOL. 28 NO. 1

13

PROSTAGLANDINS

inflammatory exudates in the rat produced following the implantation of polyester sponges impregnated with various substances and have correlated the concentrations of leukotrienes with the changes in leukocyte numbers. Materials and fkthods Materials Materials used were as follows; C3H]-LTC4 (34-43 Ci/mmol), C3H] LTB4 (34 Ci/mmol), Biofluor (New England Nuclear Corporation); Goat anti-rabbit immunoglobulin (Bio-Rad), lyophylized normal rabbit serum (Miles Laboratories); polyester sponges (Woolco); Zymosan, Bovine serum albumin, Fraction V, N-formyl-methionyl-leucyl-phenylalanine, uric acid, phenylmethylsulfonyl fluoride (Sigma); ionophore A23187 (Calbiochem); 12-HETE and 9-hydroxy and 13-hydroxy octadecadienoic acid (racemic mixtures) were a gift from Dr. R. Camp at the Institute of Dermatology, London; leukotrienes and other lipoxygenase products were synthesized at Merck Frosst. Inflamatorv

Exudates

Polyester sponges (circular disks 12 x 7 rmn) soaked in either 0.9% saline or various substances dissolved or suspended in 0.9% (w/v) saline were implanted subcutaneously in male Sprague-Dawley rats (200-250 gm) as previously described (13). Animals were killed at various times (2-24 hr) after sponge implantation and the sponges carefully squeezed and the exudates collected into polypropylene tubes. An aliquot (100 ul) was removed for determination of leukocyte numbers using a haemocytometer and in some cases a differential count was also performed. The remainder of the exudate was immediately centrifuged at 800 g for 10 min to remove cells and debris. The supernatants were removed immediately and stored at -7O'C prior to radioin..,iunoassay for leukotrienes. Radioimnunoassay Leukotriene C and B4-like immunoreactivity was assayed by double-antibody rad. ioimmunoassay (RIA) essentially 3s previously3 described with the following modifications (14). [ HI-LTC4 or [ HlLTB4, immune rabbit sera and goat anti-rabbit immunoglobulin were diluted in buffered saline (0.135 M NaCl, 0.02 M sodium phosphate pH 7.2, 0.02% (w/v) sodium azide, 0.1 mM ghenylmethylsylfonyl fluoride and 1% (w/v) bovine serum albumin). [ HI-LTC, or [ HI-LTB4 and normal rabbit serum we e added to b ffer so that 50 ~1 of solution contained 10,000 dpm [5 HI-LTC4 or [Y HI-LTB4 lo.1 - 0.15 pmole) and Immune rabbit sera were diluted 3.5 ,,Jof normal rabbit serum. appropriately so that 50 ,,lof these solutions would contain final dilutions of 1:9600 of anti-LTC serum or 1:4800 of anti-LTB4 serum This 1. ilution of anti-serum precipitated in the reaction mixture. 50% of the radio-labelled ligand. 14

JULY

1984 VOL. 28 NO. 1

PROSTAGLANDINS

The double-antibody RIA was carried out as follows. Competing ligand (100 Pl of a 1:2 dilution of exudate for LTC determinations; 100 ~1 of a 1:lO or 1:20 dilution of exudate for LT! 4 determinations) was added to 1.5 ml polypropylene microfuge tubes containing 50 ~1 of diluted anti-LTC4 or anti-LTB4 serum. Usually assays were carried out in duplicate. In control tubes buffer was substituted for ligand and/or rabbit immune serum. Prepared radioactive solution (50 ~1) was added to each tube, the contents vortexed gently and incubated for 2 hr at 4°C. Goat anti-rabbit immunoglobulin (400 pl of a 1:4 dilution in buffer) was added and the mixture vortexed and maintained at 4°C for 18-24 hr. Tubes were centrifuged at 12,000 g for 3 min and 300 Pl aliquots of the supernatant transferred to 4 ml plastic scintillation vials. Following the addition of 3 ml of Biofluor, the radioactivity was determined in a liquid scintillation counter (Beckman LS 9000) equipped with RIA data reduction module. Extraction and olrumatography of Leukotrienes Within Inflamatory exudates Small amounts of exudate from 10 separate sponge implants (1-2 ml) were applied to Cl8 SepPak cartridges (Waters) previously treated with 20 ml of MeOH followed by 20 ml H20. The cartridge was then washed with 1D ml H 0 and the leukotrienes eluted with 1fIml MenH. Two hundred Pl of 5E (w/v) NaHCG3 was added to the methanol wash and the sample was taken to dryness under vacua. The residue was taken up in 200 ~1 of H20 followed by a wash of 51,~1 of H2n for column (Waters) and eluted application (150 ~1) to a Rondapak C with CH3CN: H2D: Acetic acid (35:65: !I8 .l) pH5 at a flow rate of 1 or 2 ml/min. Retention times of standard leukotrienes were determined before and after application of the sample. Fractions were collected at 1 or 2 min intervals (2 ml) into tubes containing 200 ~1 of 5% (w/v) NaHC03 and the contents lyophylised. Water (250 ~1) was added to each tube and 100 ~1 aliquots removed for radioimmunoassay. Statistics: Data were analyzed by the equal or unequal variance t-test. Results Specificity of Antisera Previous studies have shown that the LTC4 anti-sera used in the present study was highly specific for peptido-lipid leukotrienes and did not cross react with LTB4, prostaglandins and various other compounds (14). Very similar specificities were obtained in the present study (Table 1). Concentrations required for 50% inhibition for LTC4, LTD4, LTE4 and LTF4 were 0.25, 0.3, 5.0 and 1.0 pmoles respectively. The LTB4 antisera showed a high degree of selectivity for LTB4 (IC5 0.7 pmoles) and failed significantly to cross react with peptido- 9. ipid leukotrienes and a number of other mono- and dihydroxy-fatty acids.

JULY 1984 VOL. 28 NO. 1

15

3 c

%

6 .r

z!

$.

2

4

Amount required for inhibition of [Y HI-LTB4 (pmoles)

rabbit antiserum.

0.7 40.0 ,'iBtiHLTB > 10000.0 20 COOH-L 4 B4 21.5 6 trans.-LTB4 290.0 5S,12S-LTB4 660.0 5(S),12(S),6 trans LTB4 3300.0 (5S,6R)-LTC4 1250.0 LTD4 950.0 LTE4 > 3000.0 5-hydroxyceicosatetraenoic acid 1850.0 12-hydroxyceicosatetraenoic acid > 4600.0 9-hydroxyoctadecadienoic acid > 4600.0 13-hydroxyoctadecadienoic acid 8S,15S-dihydroxy-5,11-cis9,13-trans eicosatetraenoic acid > 3500.0 8R,15S-dihydroxy-5,11-cis9,13-trans eicosatetraenoic acid > 6100.0 8S,15S-dihydroxy-5,13-cis9,11-trans eicosatetraenoic acid > 3200.0 8S,15R-dihydroxy-5,13-cis9,11-trans eicosatetraenoic acid > 2900.0

Ligand

of the C3H] ligand to the appropriate Results are shown as inhibition of binding Each value represents the mean of 2-16 determinations.

0.25 0.38 18.0 2.4 1.0 0.3 0.7 5.0 14.5 1.0 3.75 > 500.0 380.0 > 3000.0 > 4600.0 > 4600.0

-

Amount required for !$%,i;:~;;;~t.e~;

(5S,6R)-LTC4 (5R,6R)-LTC4 (5S,6S)-LTC4 (5R,6S)-LTC4 LTC4-sulfone LTD4 LTD4 -sulfone LTE4 LTE4-sulfone LTF4 LTF4-sulfone LTB4 5-hydroxyceicosatetraenoic acid 12-hydroxyceicosatetraenoic acid 9-hydroxydodecanoic acid 13-hydroxydodecanoic acid

Ligand

Specificity of LTC4 AND LTB4 antisera

Table 1

az

5 >

2

PRWTAGLANDINS

Cell kcumulation

and Leukotriene Bntent

of Sponges

Previous studies have shown elevated leukocyte migration and prostaglandin levels in sponges impregnated with 1% (w/v) zymosan or carrageenan and implanted in rats for 5 h as compared to saline treated sponges implanted under the same conditions (15). Table 7 shows the leukocyte accumulation and LTR4- and LTC4-immunoreactivity in sponges treated either with saline or 1% (w/v) zymosan, uric acid or carrageenan and implanted for 5 h in the rat. Insertion of irritants in the sponge produced a significant increase in leukocyte migration; differential counts indicated that > 95% of the cells were PMNs. Zymosan-treated sponges in particular showed very large increases in both LTB4-immunoreactivity and leukocyte accumulation. Similar increases in LTB4-immunoreactivity were observed following implantation of sponges treated with 0.01% ionophore A23187 but unlike zymosan a reduction rather than an increase in leukocyte accumulation was observed. The apparent LTC4-immunoreactivity in contrast was much lower than the LTB4-immunoreactivity and did not reflect the changes in either LTB4 concentrations or the leukocyte numbers. The concentration of ionophore A23187 used does not induce the release of lactic dehydrogenase from caseinate-elicited rat PMNs in vitro. It is thus unlikely that the absence of leukocytes in the exudateis due to a direct toxic effect of the ionophore on the migrating cells. Because the highest LTR4-immunoreactivity was observed in the sponges containing zymosan a time course study was carried out using zymosan-treated sponges and the results are shown in figure I. Maximum LTB4-immunoreactivity was observed 8 h after sponge implantation which correlated with the maximal rate of leukocyte infiltration. After 12 h, LTB4-immunoreactivity had declined about 80% and remained at that level for up to 48 h. Leukocyte accumulation occured rapidly over the first 8 h, was maximal after 15 h and was substantially reduced after 24 and 48 h. Differential counts indicated the presence of PMt+s (> 95%) at all time intervals. The apparent LTC4-immunoreactivity was low over the period of maximal neutrophil infiltration (l-8 hr) and remained low up to 48 h.

In order to confirm the presence of leukotrienes within sponge exudates, pooled exudates obtained from zymosan (1% w/v)-treated sponges implanted for 5,8 and 24 h in the rat were analyzed by reverse phase high performance liquid chromatography (HPLC) followed by radioimmunoassay of all the column fractions. At all three time intervals > 90% of the LTB4-immunoreactivity was associated with a peak with the retention time of authentic LTB4. In contrast in sponge exudates obtained 5 and 24 h after implantation, all the LTC4immunoreactive material was associated with the initial solvent peak and none with authentic LTC4, LTD4 or LTE4. After 8 h (see Figure 2) part of the LTC -immunoreactive material was associated with the solvent peak and part had a retention time between that of authentic

JULY

1984 VOL. 28 NO. 1

2.0*

0.6*

21.0 +

2.4 ?

1% zymosan

0.4*

0.2*

0.05

- 52)

120.0 + 11.9*

123.9 f 13.4*

2.8 f

0.9 f

0.1 +

* p < O.nO5 compared to sponges with saline only.

Results are shown as means

SEM (n = 10

2.5*

13.5 +

1% carrageenan

e

1.4

7.2 +

1% uric acid

0.01% A23187

0.6

ng/mT

X10_6/ml

5.7 f

LTR4 immunoreactivity

Cell Count

Saline alone

to sponge

Stimuli added

0.1

5.0 f

4.1 +

1.7 +

0.6

0.7

0.1

1.4 ?r 0.4

0.8 +

ng/mT

LTC4 immunoreactivity

Contents and Cell Numbers in

fi-hour Sponge Exudates in the Rat

Leukotriene

Table 2

3

z

b

3

PROSTAGLANDINS

0

10

20

30

40

50

TIME (h)

Figure 1: Levels of leukocytes (U), LTB4-immunoreactivty (0) and apparent LTC4-immunoreactive (A) within sponge exudates obtained from rats at various times after implantation of polyester sponges soaked in 1% (w/v) zymosan. Results are shown as means with SFIM (n = 10-50). LTC4 and LTD4. In this particular experiment the amount of LTB immunoreactivity material present with retention times between 44 and 46 min was equal to 90% of the LTB -immunoreactivity present in the sample injected onto the column. 4 his indicates that loss of LTB4 during the extraction and chromatography procedures is minimal. These results confirmed the ~presence of LTB4 within the exudates but suggest that the apparent LTC4-immunoreactive material observed was probably unrelated to peptido-lipid leukotrienes. To study the effects of chemotactic factors within sponge exudates upon subsequent leukocyte migration, sponges treated with various chemotactic agents were implanted in the rat and leukocyte content and LTB4-immunoreactivity det_grmined (Takle 3). Sponge - 3 x 1D- M LTB4 showed high exudates containing initially 3 x 10 concentrations of LTB4-immunoreactivity 5 h later. The amounts observed were approximately l&20% of the initial concentration added to the sponge indicating that LTB4 is incompletely metabolized in this situation. In contrast to the high LTR4-immunoreactivity present in the exudates, little or no change in leukocyte accumulation was observed and only 3 x lo- M LTR4 produced an increase in leukocyte numbers. Experiments were also carried out with another chemotactic factor, N-for l-methionyl-leucylphenylalanine (F-met-leu-phe). At lo-"ry M a small increase in leukocyte numbers and "_g increase in LTR4-immunoreactivity was observed whereas at 10 M no significant increase in leukocyte counts was noted despite a small but significant increase in LTR4immunoreactivity.

JULY 1984 VOL. 28 NO. 1

19

4 8

12

18

24

28

RETENTION TIME (mid

20

32

38

40

44

48

1

Figure 2: Levels of LTB4- and LTC4-immunoreactivity in reverse phase HPLC fractions obtained following the chromatography of a pooled sample of inflammatory exudate obtained from rats implanted for 8 h with sponges containing 1X (w/v) zymnsan. Retention times of authentic leukotrienes are indicated. Results are expressed as ng of immunoreactive in each fraction and in addition the ultraviolet absorbtion (280 nm) of the effluent from the chromatogram is shown.

01 0

LTD.,

19.6’

AUTHENTIC

LTC4

10.3’

AUTHENTIC

L

t4

added

*P

** P G O.nnl

are shown as means

c 0.015,

Results

(lK%l)

F-met-leu-phe

agents sponge

0.R

k i.i**

7.2 +

in.3

n.f!

to sponges

containing

saline

only.

n.i

1 n, 7*

0.2 1 0.5

11.0**

2.1**

0.1

! 229.fi**

:

5.1

1777.5

70.3 t

’

9.4 1.1**

14.2 r

4.2 f rl.6

5.7 +

0.1 t

ng/ml

LTR4 immunoreactivity

n.6

Xln-6/ml

and

in the rat

miqration

exudates

on leukocyte

3

Cell Count

in q-hour

1. SEM (n = 10-36)

compared

R4 (3 x lfl-"Y)

Lcukotriene

(10s7M)

R4 (3 x lo-'M)

Leukotriene

F-met-leu-phe

B4 (3 x 10e7M)

alone

Leukotriene

Saline

to sponge

Stimuli

LLTR4 concentrations

Effect of chemotactic

Table

PROSTAGLANDINS Implantation of zymosan-treated sponges in the rat for 5 h may serve as a useful model for studying the effects of anti-inflammatory agents upon leukotriene production in vivo. Accordingly, rats were pretreated with either indomethacinT3 and 10 mg/kg, p.o.) 1 h prior to sponge implantation or dexamethasone (2 mg/kg, p.o.) 18 h and 1 h Indomethacin significantly inhibited prior to sponge implantation. leukocyte migration at the higher dose and at both doses significantly elevated the levels of LTB4-immunoreactivity (Table 4). Dexamethasone at the dose used, had no effect upon either Preliminary studies have, however, shown that higher parameter. amounts of dexamethasone, unrelated to therapeutic concentrations, (15 mg/kg, P.o., 18 and 1 h prior to sponge implantation) reduce both the levels of LTB4-immunoreactivity and influx of leukocytes. Discussion:

In the present study radioimmunoassays specific for either LTB4 or peptido-lipid leukotrienes were used to determine the levels of leukotrienes within inflammatory exudates formed following sponge implantation in the rat. In studies using sponges soaked either in saline or in various irritants increased leukocyte accumulation was associated with increased LTB4-immunoreactivity but not LTC4immunoreactivity. In particular, zymosan was shown to be an extremely potent inducer of both leukocyte accumulation and production of LTR4-immunoreactivity material, a property that may be associated with the ability of zymosan to activate the alternate pathway of complement. A time course study was carried out using zymosan-impregnated sponges and maximal LTR4-immunoreactivity correlated with the fastest rate of leukocyte infiltration. Similar results have been reported using sponges implanted with 0.5% (w/v) carrageenan (12), although with zymosan the LTR -immunoreactivity was approximately 18-fold greater. In previous stud*ies the presence of LTB4 in sponge exudates was confirmed by reverse phase HPLC (12). Similar results were obtained in the present study confirming the validity of the LTB4 radioimmunoassay. In contrast to the changes in LTB4-immunoreactivity observed at various times, the apparent LTC4-immunoreactivity were low and did not change greatly suggesting that these measurements may be due to some background immunoreactivity unrelated to leukotrienes. HPLC analyses confirmed this hypothesis and indicated that peptido-lipid leukotrienes are not present within these inflammatory exudates in significant quantities. Previous studies have suggested that the source of LTB4 within sponge exudates was the incoming PMNs (12). LTB4 is rapidly metabolized through w-oxidation by PMNs, rat PMNs having one sixth of the activity of human PMNs (16). Thus although considerable levels of LTB4-immunoreactivity were detected 5 h after implantation of sponges soaked in LTR4 (Table 3), the levels of LTR4-immunoreactivity in zymosan-treated sponges declined rapidly between 8 and 12 h after

22

JULY

1984 VOL. 28 NO. 1

w

t4

compared

+ SEM (n = ln-52)

lR, 1

4.4

control.

27.0 +

2.5*

i5.n +

1

2.5

22.5 +

1

2.0

2i.n f

X10W6/ml

Cell count

1

(h)

to sponge

implantation

Treatment

drug

13.4

126.4 + 8.5

217.1 f lo.R***

179.4 f l&l**

123.9 f

nglml

LTB4-immunoreactivity

exudates

migration

sponge

in the rat)

treated

on leukocyte

to vehicle

implantation

prior

* p = 0.05, ** p = 0.01, *** p = 0.005

drugs

4

in zymosan

Time of

(5-hour

LTB4 concentrations

as means

(? mg/kg)

Dexamethasone

are shown

(10 mg/kg)

Indomethacin

Results

(3mg/kg)

control

Indomethacin

Vehicle

Treatment

Drug

and

Effect of anti-inflammatory

Table

PROSTAGLANDINS implantation (fig. 1). In the former case few PMNs were present and hence rapid metabolism of LTB4 would not be expected. In the latter case the decline in the levels of LTBa-immunoreactivity corresponded to the peak neutrophil influx. This suggests that in the presence of large numbers of PMNs the levels of LTB4 will be dependent on the balance between the rate of synthesis and the rate of metabolism of LTB4. The decline in the levels of LTB4-immunoreactivity in the zymosan-treated sponges may reflect a decrease in the rate of biosynthesis of LTB4 and previous workers have suggested that this may be due to the production of an endogenous inhibitor of leukotriene biosynthesis (12). In this context leukotriene R4 production by activated rat peritoneal polymorphonuclear leukocytes can be inhibited by prostaglandin E2 (17). The present studies would tend to support such a conclusion, although LTR4 release from other sources may be possible because in the experiment with ionophore A23187, high LTB4-immunoreactivity was observed in the sponge exudate with no concomitant increase in leukocyte migration. What has not been determined previously is the significance, of LTR4 concentrations in relation to leukocyte migration within the sponge and the relative contribution of LTB4 as a chemotactic agent compared with other factors, such as the complement derived peptide C5a. A number of observations in the present work suggests that there is no correlation between LTB4 concentrations within the sponge exudate and subsequent leukocyte migration. First, following implantation of sponges containing ionophore A23187, equivalent concentrations of LTB4 to those seen with zymosan were observed and this was associated with a decrease rather than an increase in leukocyte counts. Secondly, in the experiments with zymosan-treated sponges, indomethacin pretreatment induced an increase in the concentration of LTB4 and at the higher dose a decrease in leukocyte numbers. Previous studies have shown that indomethacin inhibits leukocyte accumulation into sponge exudates through a mechanism unrelated to inhibition of prostaglandin biosynthesis (18). Other studies have also shown an elevation of LTB4 production in sponge exudates following indomethacin pretreatment (19). Suprisingly pretreatment of rats with 2 mg/kg dexamethasone, 1 and 18 hr prior to implantation of zymosan-impregnated sponges, had no significant effect on the production of LTB4 or the leukocyte infiltration. The third series of observations relating to the levels of LTR4 were the experiments reported in table 3 in which sponges containing either LTB4 or the synthetic chemotactic peptide, F-met-leu-phe, were implanted in rats. LTB w s tested at three doses and only at the intermediate dose (3 x 4 0 -8M) was a significant increase in leukocyte migration observed. The failure to induce a large degree of leukocyte infiltration could not be accounted for by rapid and complete metabolism of LTB4 because significant concentrations of LTB4 were still observed 5 h after implantation. Failure to induce leukocyte accumulation at the highest dose of LTB4 may be related to the ability of chemotactic factors to immobilize leukocytes at high

JULY

1984 VOL. 28 NO. 1

PROSTAGLANDINS

Results with another chemotactic factor, F-met-leuconcentrations. phe, were also obtained and, as with LTB4, an increqse in leukocyte numbers was observed at only one concentration (10' M). The present results show that impregnation of sponges with certain concentrations of LTB4 can induce subsequent leukocyte However, the discrepancies observed in the results accumulation. suggest that measurement of LTB4 concentrations within sponge Two explanations exudates can not predict the leukocyte migration. for this discrepancy are possible. Firstly, other chemotactic factors than LTB4 may be quantitatively more important within sponge exudates in the rat. In this context leukocyte migration into sponges in the rat has been shown to be nearly abolished by pretreatment with cobra venom factor, suggesting a major role for the complement derived chemotactic peptides (20). Secondly, the sponge exudate is an artificial system lacking any vasculature. It may be much more physiologically important to know the levels of chemotactic factors close to the vascular network where the critical steps in the initiation of leukocyte migration occur. The relative contribution of LTB4 as a chemotactic factor in inflammatory exudates will probably only be ascertained when specific leukotriene biosynthesis inhibitors or LTB4-antagonists with. a suitable biodistribution are available for testing in inflammatory models. Acknowledgements: We would like to acknowledge the help of Dr. E. Hayes in setting up the leukotriene radioimmunoassays.

References : 1.

Borgeat, P. and Samuelsson, 6. Arachidonic acid metabolism in polymorphonuclear leukocytes: effects of ionophore A23187. Proc. Natl. Acad. Sci., 76, 2148-2152, 1979.

2. . Borgeat, P. and Samuelsson, B.

acid by rabbit polymorphonuclear dihydroxyeicosatetraenoic acid. 2646, 1979.

Transformations of arachidonic leukocytes. Formation of novel J. Biol. Chem., 2fi4, 2643-

3.

Ford-Hutchinson, A.W., Bray, M.A., Doig, M.V., Shipley, M.E. and Smith, M.J.H. Leukotriene B: a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature, 286, 264-265, 1980.

4.

Smith, M.J.H., Ford-Hutchinson, A.W. and Bray, M.A. Leukotriene B: a potential mediator of inflammation. J. Pharm. Pharmacol., -' 32 517-518, 1980.

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1984 VOL. 28 NO. 1

25

PROSTAGLANDINS

5.

Goetzl, E.J. and Pickett, W.C. The human PMN leukocyte chemotactic activity of complex hydroxy-eicosatetraenoic acids (HETEs). J. Immunol . , 125, 1789-1791, 1980.

6.

Bray, M.A., Cunningham, F.M., Ford-Hutchinson, A.W. and Smith, M.J.H. Leukotriene B : an inflammatory mediator --in vivo. Br. J. Pharmac., -22 483-486, 1981.

7.

Bray, M.A., Cunningham, F.M., Ford-Hutchinson, A.W. and Smith, M.J.H. Leukotriene B4: a mediator of vascular permeability. Br. J. Pharmac., 72_, 483-486, 1981.

8.

Wedmore, C.V. and Williams, T.J. permeability by polymorphonuclear Nature, 284, 646-650, 1980.

9.

Klickstein, L.B., Shapleigh, C. and Goetzl, E.J.. Lipoxygenation of archidonic acid as a source of polymorphonuclear leukocyte chemotactic factors in synovial fluid and tissue in rheumatoid arthritis and spondyloarthritis. 1166-il7n, 19811. J. Clin. Invest., 2,

The control of vascular leukocytes in inflammation.

10.

Leukotriene R4: Rae, S.A., Davidson, E.M. and Smith, M.J.H. inflammatory mediator in gout. Lancet, ii, 1122-1123, 1982.

11.

Brain, S.n., Camp, R.D.R., Dowd, P.M., Rlack, A.K., Wollard, P.M., Mallet, A.I. and &eaves, M.W. Psoriasis and leukotriene B4* Lancet, ii, 762-763, 1982.

12.

Simmons, P.A., Salmon, J.A. and Moncada, S. The release of leukotriene B during experimental inflammation. Biochem. 1983. Pharmacol., -’3$ 1353-1359,

13.

Ford-Hutchinson, A.W., Walker, J.R. and Smith, M.J.H. Assessment of anti-inflammatory activity by sponge implantation techniques. J. Pharmac. Methods, 1, 3-7, 1978.

14.

Hayes, E.C., Lombardo, D.L., Girard, Y., Maycock, A.L., Rokach, J ., Rosenthal A.S., Young, R.N., Egan, R.W. and Zweerink, H.J. Measuring leukotrienes of slow-reacting substances of anaphylaxis: development of a specific radioimmunoassay. J. Immunol., 131, 429-433, 1983.

15.

Ford-Hutchinson, A.W., Walker, J.R., Connor, N.S. and Smith, Prostaglandins and leukocyte migration into inflammatory M.J.H. exudates. J. Pharm. Pharmacol., 28, 106-112, 1977.

16.

Powell, W.S. Properties of Leukotriene R4 20-hydroxylase from polymorphonuclear leukocytes. J. Riol. Chem., 259, 3082 - 3089, 1984.

26

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1984 VOL. 28 NO. 1

PROSTAGLANDINS

17.

Ham, E.A., Soderman, D.D., Zanetti, M.E., Dougherty, H.W., McCauley, E. and Kuehl, F.A. Inhibition by prostaglandins of leukotriene B release from activated neutrophils. Proc. Natl. Acad. Sci. U. 4 .A., 80, 4349-4353 (1983).

18.

Walker, J.R., Smith, M.J.H. and Ford-Hutchinson, A.W. Antiinflammatory drugs, prostaglandins and leukocyte migration. Agents Actions, 2, 602-606, 1976.

19.

Salmon, J.A., Simmons, P.M. and Moncada, S. The effects of BW755C and other anti-inflammatory drugs on eicosanoid concentrations and leukocyte accumulation in experimentallyinduced acute inflammation. J. Pharm. Pharmacol., 2, 803-813, 1983.

20.

Wiener, S., Lendvai, S., Rogers, B., Urivetzky, M. and Meilman, in vivo. Am. J. Pathol., 73, 807-816, E. Non-immune chemotaxis -1973.

Editor: L. Jackson Roberts, II Received: 3-14-84 Accepted: 5-31-84

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1984 VOL. 28 NO. 1

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