Claesson, Hans-Erik

Feinmark, Steven J.

Lindgren, Jan Åke

Malmsten, Curt

Samuelsson, Bengt

Elsevier - Publisher Connector 

Stimulation of human leukocyte degranulation by leukotriene B4 and its ω-oxidized metabolites 

Volume 136, number 1 FEBS LETTERS December 198 1 
STIMULATION OF HUMAN LEUKOCYTE DEGRANULATION BY LEUKOTRIENE B4 AND 
ITS o-OXIDIZED METABOLITES 
Steven J. FEINMARK, Jan Ake LINDGREN, Hans-Erik CLAESSON, Curt MALMSTEN and 
Bengt SAMUELSSON 
Department of Physiological Chemistry, Karolinska Znstitutet, 104 01 Stockholm, Sweden 
Received 2 November 1981 
1. Introduction 2. Materials and methods 
Leukotriene (LT)B4 has been indicated to play an 
important role in inflammatory reactions uch as 
leukocyte chemotaxis [ 1,2] and adhesion to post- 
capillary venules [3]. In [4-61 LTB., was suggested 
to be a weak stimulator of polymorphonuclear leuko- 
cyte (PMNL) degranulation. However, it is now clear 
that previous purification procedures for biosyntheti- 
tally prepared LTB4 were inadequate. The newly 
discovered LTB4 isomer, SQ,12Q-DHETE, co-chro- 
matographs with LTB4 in the reverse phase high- 
pressure liquid chromatography (HPLC) systems 
commonly used [7]. As a consequence, arlier studies 
probably tested preparations which contained a mix- 
ture of both isomers with the non-leukotriene product 
predominating. Therefore, we have investigated the 
role of LTB4, SQ,12(S)-DHETE and their o-oxida- 
tion products (fig.1) [8] in leukocyte degranulation 
in vitro. In addition, the activity of the non-enzymati- 
tally formed LTB4 isomers was investigated. 
Cytochalasin B, phenolphthalein glucuronic acid, 
and dried Micrococcus lysodeikticus were purchased 
from Sigma (St Louis MO). Dextran T-500 was 
obtained from Pharmacia Fine Chemicals (Uppsala) 
and fMLP was a product of UCB SA (Brussels). 
2.1. Cell preparation and incubations 
This study shows that LTB4-stimulated degranula- 
tion at lOa M; the o-oxidation products of LTB4 
were less potent while 5@),12(S)-DHETE had no 
activity even at 2 X low6 M. Furthermore, the latter 
compound inhibited enzyme release induced by LTB4. 
Purified polymorphonuclear leukocytes were pre- 
pared from leukocyte concentrates of human periph- 
eral blood (Karolinska Hospital, Stockholm) by 
dextran sedimentation followed by hypotonic lysis of 
residual erythrocytes [9]. The cells were diluted to 
5 X 1 O6 cells/ml in Dulbecco’s phosphate-buffered 
saline @H 7.4). Aliquots (l-2 ml) of the cell suspen- 
sion were preincubated for 5 min with cytochalasin B
(5 Erg/ml in DMSO 1 pg/ml) in plastic vessels at 37°C. 
Test substances were added as free acids in either 
ethanol, methanol or DMSO. At the levels used, these 
solvents had no effect on spontaneous release. Jncu- 
bations were terminated after 5 mm by transfer to an 
ice bath followed by rapid centrifugation. The cell-free 
supernatants were subsequently assayed for the 
presence of lysozyme, /3-glucuronidase nd lactic acid 
dehydrogenase (LDH). 
Abbreviations: S(,S),lZQ-DHETE, 5@),12(S),dihydroxy- 
6-trans,8cis,lO-trans,l4ciseicosatetraenoic acid; fMLP, 
IV-formyl-methionyl-leucyl-phenylalanine; LTB,, 50, 
12(R)dihydroxydcis,8,10-trans,l4ciseicosatetraenoic acid; 
20-OH-LTB,, S(S),1 2(R),20-trihydroxydcis,8,1 O-trans, 
14cis-eicosatetraenoic acid; 20COOH-LTB,, 50,12(R)- 
dihy&oxydcis,8,1O-trans,l4ciseicosatetraen-l,20dioic 
acid;SQ,12(S),2O=THETE, 5(.9),12(~,20-trihydroxyd-trans, 
8cis,lO-trans,l4-cis-eicosatetraenoic acid 
2.2. Enzyme assays 
fl-Glucuronidase was measured by a modification 
of the procedure in [lo]. Supematant (0.2 ml) was 
added to 2 ml acetate buffer (0.1 M, pH 4.6) followed 
by 0.1 ml phenolphthalein glucuronic acid (0.01 M, 
pH 7.0) as substrate. The reaction was terminated 
after 18 h at 37’C by the addition of 5 ml glycine 
buffer (0.2 M in 0.2 M NaCl, pH 10.4). The absorb- 
Published by ElsevierfNorth-Holland Biomedicul Press 
00145793/81/0000-0000/$02.75 0 1981 Federation of European Biochemical Societies 141 
Volume 136, number 1 FEBS LETTERS December 1981 
czzc” 
Arachidonic acid 
i \ &CO”” _ &H 
LTAL 5S-H(P)ETE H(0):~cooH 
12 S-H (P)ETE 
I 
COOH 
LTB4 
6-tram-LfBL and 
12-epi-6-tram-LTB& 5S,12S-DHETE 
I 
H, OH 
COOH 
OH- ’ / _ LOOH *OH 
H OH 
20 -OH- LTB, 20 -COOH - LT Bb 5S, 12S, 20-THETE 
Fig.1. Products of arachidonic acid metabolism by human polymorphonuclear leukocytes: (--+) proposed conversions. 
ante of each sample was determined at 540 nm vs a 
glycine buffer blank. 
Lysozyme was determined by a turbidimetric 
assay [ 1 l] based on the ability of this enzyme to lyse 
suspensions ofMicrococcus lysodeikticus. Supernatant 
(0.2 ml) was added to 0.8 ml M. lysodeikticus suspen- 
sion (0.03 mg/ml in 0.1 M phosphate buffer, pH 7.0) 
and the fall in absorbance was monitored at 450 nm 
in a Cary 219 recording spectrophotometer. 
LDH was monitored according to [ 121 and never 
significantly exceeded control levels. 
The obtained enzyme release was corrected for 
spontaneous release and then calculated as a percen- 
tage of the total cellular enzyme content. This was 
determined by assaying the 10 000 X g supernatant 
of sonicated cell suspensions. 
2.3. Leukotriene preparation 
LTB,, 5@),12(S)-DHETE [7], 6-trans-LTB4, 
1 2-epi-6-trans-LTB4 [ 131 and 20-OH-LTB4 [8] were 
142 
prepared as reported. 20-COOH-LTB4 was prepared 
by a further purification of the major polar fraction 
in [8]. This fraction was subjected to reverse-phase 
HPLC using methanol/water/acetic acid (50:50:0.01, 
by vol.) followed by rechromatography eluting with 
methanol/water/acetic acid (45:55:0.01, by vol.). 
The purity of this compound was verified on straight- 
phase HPLC [8] after conversion to the dimethyl 
ester. 
3. Results 
A series of arachidonic acid metabolites was tested 
for the ability to induce degranulation in cytochalasin 
B-treated human PMNL in vitro. The compounds 
studied were LTB4, 20-OH-LTB4, 20-COOH-LTB4, 
6-frans-LTB4, 12epi-6-trans-LTB4, 5@),12(S)-DHETE 
and 5@),12@‘),20-THETE. LTB4 and its metabolites, 
20-OH-LTB4 and 20-COOH-LTB4, induced the release 
of both lysozyme and /3-glucuronidase (fig.2). 6-trans- 
Volume 136, number 1 FEBS LETTERS December 198 1 
10 9 8 7 6 
-LOG CONC. ( M I -LOG CONC. (M 1 
Fig.2. The release of granular enzymes from human polymorphonuclear leukocytes stimulated with varying concentrations of 
LTB, (o), 20-OH-LTB, (o), 20COOH-LTB, (=), S(S),lZ(S)-DHETE (X) and fhILP (0). Error bars denote standard errors of the 
mean of triplicate observations: (A) lysozyme; (B) fl-glucuronidase. The curves representing fMLP-induced release are presented 
for comparison to other studies in which this agent is frequently used. Although only a few points are plotted, the shape of the 
curve is supported by data in [ 14,151. 
LTB4, 12-epi-6-trans-LTB4, 5(S),12(S)-DHETE and 
5(S),12(S),20-THETE were all unable to induce 
enzyme release. LTB4 was found to yield a half-maxi- 
mal release of lysozyme at 3.5 X lOa M (mean of 
4 expt). 20-OH-LTB4 and 20-COOH-LTB4 were able 
to stimulate enzyme release equal to half the LTB4 
maximum only at levels lo- loo-times higher (on a 
molar basis). At 5 X lo-’ M, the LTB4-induced lyso- 
zyme release was 17.2%. When added at the same 
molar concentration, 20-OH-LTB4 and 20COOH- 
LTB4 caused 5.3% and 2.6% release, respectively. 
The activity of LTB4 was compared to that of the 
chemotactic peptide, fMLP. Maximal enzyme release 
induced by LTB4 was 35-50% of the enzyme released 
by fMLP. 
5(5),12(S)-DHETE has been found to make up a 
large (60-75%) fraction of the ‘LTBa-peak’ isolated 
from reverse-phase HPLC [7]. Therefore, the possibil- 
ity that the two isomers interacted to alter the activity 
of LTB4 alone was tested. 5@),12(S)-DHETE, at 
10m6 M, inhibited half-maximal LTB4 stimulation of 
lysozyme release by 42% (p < 0.001). Furthermore, 
this concentration of 5(,9,12(S)-DHETE had no effect 
on the release of lysozyme induced by fMLP. 
4. Discussion 
Several groups have investigated the enzyme- 
releasing effects of LTB4 on peripheral leukocytes. 
LTB4 was found to induce degranulation of cyto- 
chalasin B-treated human peripheral neutrophils but 
this occurred at a half-maximal dose of >l 0m6 M [4,6]. 
Earlier biosynthetic LTB4 preparations obtained from 
human leukocyte suspensions contained a non-leuko- 
triene isomer, 5@),12(S)-DHETE [7]. Although the 
ratio of these two conjugated trienes is variable, 
5@),12(S)-DHETE can make up considerably >50% 
of the ultraviolet light-absorbing material eluting in 
the ‘LTB,-peak’ from reverse-phase HPLC. These 
143 
Volume 136, number 1 FEBS LETTERS December 198 1 
isomers can now be separated using straight-phase 
HPLC [7]. It was therefore of interest o re-evaluate 
the biological activity of LTB4, S(S),12(S)DHETE 
and the mixture of the 2 compounds. 
LTB4 was found to be a potent inducer of enzyme 
release in human polymorphonuclear leukocytes. 
When added at as low as 10-a M, LTB4 caused the 
release of significant amounts of lysozyme. The 
w-oxidized metabolites, 20-OH-LTB4 and 20COOH- 
LTB4 were much less potent, indicating that o-oxida- 
tion may be a first step of biological inactivation of 
LTB4. 
The maximal amount of enzyme released by LTB4 
was always lower than the amount released from 
fMLP-stimulated cells. Thus, LTB4 is roughly 50% as 
efficacious as the chemotactic peptide (fig.2). This 
difference does not seem to be due to a selective 
discharge of one granule-type since markers for both 
azurophil (lysozyme) and specific gramdes @-glucu- 
ronidase) yielded similar esults. Instead, this observa- 
tion suggests hat different receptor populations may 
be involved. 
5($),12(S)-DHETE did not stimulate nzyme 
release. 6-trans-LTB4, 12-epi-6~trans-LTB4 andS(S), 
12(S),20-THETE were also inactive in this respect. 
This shows that LTB4-induced egranulation is a 
highly stereospecific effect. Similar observations have 
been made for LTB4-induced chemotaxis [2]. HOW- 
ever, at high relative concentrations, 5(S),12@)- 
DHETE can interfere with LTB4 activity. This com- 
petition, presumably at a cell-surface receptor, results 
in an observed inhibition of LTB4-induced enzyme 
release. Previous tudies, therefore, probably under- 
estimated the potency of LTB4 in two important ways: 
(i) The concentration of ultraviolet light-absorbing 
material included large amounts of S(S),12(S) 
DHETE, which was biologically inactive; 
(ii) This compound acted as a competitive antagonist 
of the LTB4-induced enzyme releasing activity. 
This report indicates that LTB4 is sufficiently potent 
to play an important role in leukocyte degranulation 
in vivo. Further studies to clarify this role are in 
progress. 
Acknowledgements 
We thank MS Inger Tollman-Hansson a d MS 
Margareta Hovgard for their excellent echnical assis- 
tance. This study was supported by grants from 
Riksforeningen mot Reumatism, Konung Gustaf V:s 
808rsfond, M. Bergvalls Stiftelse, the World Health 
Organization, the Swedish Medical Research Council 
(proj. 03X-2 17) and the North Atlantic Treaty Orga- 
nization .
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Stimulation of human leukocyte degranulation by leukotriene B4 and its ω-oxidized metabolites

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