The binding of leukotriene B4 (LTB4) to macrophages from the head kidney of the rainbow trout Oncorhynchus mykiss was measured. Binding of [3H]LTB4 achieved a steady state after approximately 30 min of incubation and was 30 % reversible in the presence of a minimum of 1000-fold excess of LTB4. Scatchard analysis of the kinetics of LTB4 binding over a range of [3H]LTB4 concentrations indicated the existence of only a single class of receptor with a dissociation constant, KD, of 0.14 nmol l−1 and a maximum receptor density, Bmax, of approximately 17 800 sites per macrophage. The LTB4 receptor antagonist LY223982 was ineffective in inhibiting the binding of [3H]LTB4 to trout macrophages, although another receptor antagonist, LTB4-dimethylamide, displaced a maximum of 25 % of the total binding. LTB5 was equally effective as LTB4 at displacing [3H]LTB4, while other eicosanoids tested were without significant effect. It is suggested that the putative receptors for LTB4 on trout macrophages are similar to the high-affinity receptors for this compound reported to occur on mammalian granulocytes, although any structural similarities of the binding sites await further investigation.

Leukotriene B4 [5(S),12(R)-8,10-trans-6,14-cis-eicosatetraenoic acid, LTB4] is a key pro-inflammatory compound in mammals promoting leucocyte chemotaxis, aggregation, superoxide anion generation and degranulation (Samuelsson et al. 1987). Production of LTB4 has been found in several cell types, including granulocytes, lymphocytes, mononuclear phagocytes and keratinocytes in which the required 5-lipoxygenase and LTA4 hydrolase activities are found (Samuelsson et al. 1987; Ford-Hutchinson et al. 1994). LTB4 interacts with a number of target cells via a single type of receptor, termed B-LT (Metters, 1995). This can exist in two states, at least in some cell types. First, high-affinity receptors which are coupled to guanine-nucleotide-binding G-proteins and are involved in chemotactic and chemokinetic responses in granulocytes (Sherman et al. 1988; Mong, 1991). The second state is represented by the low-affinity forms which are uncoupled from G-proteins and are associated with degranulation and secretion events in human granulocytes (e.g. Kreisle et al. 1985; Mong, 1991). Both forms of the receptor are interconvertible; for example, the receptor can be converted from high to low affinity by treatment with GTP analogues (Sherman et al. 1988; Slipertz et al. 1993). The B-LT protein has been identified as having an Mr of either 60 kDa (Goldman et al. 1991) or 70–80 kDa (Harvey et al. 1992), although further structural elucidation is still awaited.

Significantly less is known about LTB4 generation and its biological activities in non-mammalian vertebrates. In amphibians, LTB4 and LTB5 (the latter derived from eicosapentaenoic acid) cause contraction of bullfrog (Rana catesbeiana) lung (Andazola et al. 1992) but their potential pro-inflammatory ability is apparently untested. The generation and biological activities of leukotrienes in fish, in particular the rainbow trout Oncorhynchus mykiss, are well studied. LTB4 biosynthesis occurs both in macrophages and in platelet equivalent cells, termed thrombocytes, in rainbow trout (Pettitt et al. 1991; Lloyd-Evans et al. 1994). As eicosapentaenoate (20:5, n-3) is a common fatty acid in trout phospholipids, LTB5 is also generated in similar amounts to LTB4 by these cells. Unspecified cell types in the brain, skin, ovary, heart and alimentary canal also have LTB4/5 biosynthetic capacity (Knight et al. 1995). Functional studies have demonstrated that LTB4 is a chemotactic agent for trout leucocytes (Sharp et al. 1992), causes transient increases in intracellular [Ca2+] in macrophages (Knight et al. 1993) but does not affect enzyme secretion from trout macrophages (Knight, 1995; Rowley, 1996) or the uptake of foreign test particles by these cells (Knight et al. 1993). Experimentally induced inflammatory exudates in the peritoneal cavity of rainbow trout exhibit enhanced levels of LTB4 and other eicosanoids, including prostaglandin E2 and lipoxin A4 (Rowley, 1996). Despite this knowledge of the biosynthesis and functioning of LTB4 in trout, nothing is known about its binding to target cells and the specificity of this reaction. Therefore, the present study characterises the binding of LTB4 to rainbow trout macrophages. This work is the first report of eicosanoid binding sites on non-mammalian leucocytes.

Chemicals

[3H]LTB4 (specific activity 193 Ci mmol−1; 7.14 TBq mmol−1) was obtained from Amersham Life Sciences (Little Chalfont, UK), while LTB4, LTB4-dimethylamide, LTB5, LTC4, lipoxin A4 (LXA4), 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 12(S)-hydroxyeicosapentaenoic acid [12(S)-HEPE] were obtained from Cascade Biochem Ltd (Reading, UK). LY223982, an LTB4 receptor antagonist, was kindly provided by Lilly Research Laboratories (Indianapolis, USA). All other reagents were obtained from the Sigma Chemical Co. Ltd (Poole, UK), unless otherwise stated, and were the highest grade available.

Fish

Adult triploid rainbow trout, Oncorhynchus mykiss (Walbaum), approximate mass 200–250 g, were obtained from Pontlliw Trout Farm, (South Wales, UK), maintained in outdoor freshwater concrete tanks at 10–15 °C and fed ad libitum on Mainstream expanded diet (B.P. Nutrition Ltd, Cheshire, UK) prior to use.

Macrophage isolation and maintenance

Trout were killed by immersion in a lethal dose of ethyl p-aminobenzoate (0.1 g l−1), exsanguinated and the head kidneys removed into Ca2+/Mg2+-free Hanks balanced salt solution (HBSS). Each kidney was disrupted through a fine plastic mesh and the resulting cell suspension centrifuged (1500 g for 5 min at 4 °C) to remove soluble material and cell debris. The cell pellet was resuspended, layered onto a preformed 55 % Percoll gradient prepared as described previously (Pettitt et al. 1991) and centrifuged at 3000 g for 30 min at 4 °C. The layer containing macrophages and other contaminating leucocytes (mainly lymphocytes) was removed and the cells washed by centrifugation in Ca2+/Mg2+-free HBSS. The cells obtained from approximately six fish were then pooled and approximately 2×107 adherent cells were placed in 9 cm tissue-culture-grade Petri dishes (Nunc, Paisley, UK) containing 5 ml of HBSS and left to attach for 30–60 min. The dishes were then gently agitated to resuspend any non-adherent cells, which were discarded. The remaining adherent cells were incubated in 5 ml of RPMI (Gibco, Paisley, UK) containing 5 % heterologous fish serum overnight at 18 °C. After incubating the cells for approximately 15 min at 18 °C with Ca2+/Mg2+-free HBSS containing 5 mmol l−1 EDTA, the remaining cells were resuspended by gentle aspiration, washed once with Ca2+/Mg2+-free HBSS and counted using a haemocytometer. Typically in excess of 90 % of the recovered cells re-adhered to glass and were judged to be macrophages on the basis of their morphology.

Binding time-course experiments

Macrophages (2×107 ml−1) in Ca2+/Mg2+-containing HBSS ([Ca2+] 1.3 mmol l−1; [Mg2+] 0.4 mmol l−1) were incubated at less than 4 °C in a salt ice-water bath in parallel with either 0.03 nmol l−1 [3H]LTB4 alone or 0.03 nmol l−1 [3H]LTB4 together with a 104-fold excess of LTB4 to establish the levels of total and non-specific binding respectively. At designated time intervals (1, 5, 10, 30 and 60 min), samples (0.5 ml) were removed from both incubations and layered onto 350 μl of silicone oil (relative density 1.03) in Eppendorf tubes which were centrifuged (10 000 g for 1 min) to pellet the cells, isolating them from the unbound label. The upper aqueous phase was removed with a small amount of the silicone oil and the tube and oil interface were washed with methanol (750 μl) which was drawn off with the remainder of the oil. The pellet was then disrupted with 30 % ethanol, 20 % trichloroacetic acid in Ca2+/Mg2+-free HBSS (200 μl) and the tube and contents mixed with 5 ml of Pico Fluor 40 (Packard Instruments B.V., Groningen, The Netherlands). The radioactivity associated with the pellet was determined over a 5 min counting period using a 1217 Rackbeta liquid scintillation counter (LKB, Pharmacia, Milton Keynes, UK).

Determination of the quantity of cold homoligand required to displace specific binding

Macrophages (2×107 ml−1) in Ca2+/Mg2+-containing HBSS were incubated with 0.03 nmol l−1 [3H]LTB4 and a range of concentrations between 0.3 nmol l−1 and 0.3 μmol l−1 of cold homoligand (LTB4) for 30 min at less than 4 °C. Samples (0.5 ml) of the incubation mixture were then layered onto cushions of silicone oil and treated as described above.

Scatchard analysis of LTB4 binding to macrophages

Macrophages (2×107 ml−1) in Ca2+/Mg2+-containing HBSS were incubated with a range of concentrations of [3H]LTB4 (0.05, 0.1, 0.3, 0.6, 0.9 and 1.2 nmol l−1) both in the presence and in the absence of a 1000-fold excess of cold LTB4 for 30 min at less than 4 °C. Samples (0.5 ml) of the cell suspension were then removed and layered onto a silicone oil cushion and centrifuged to separate the cells from the unbound label. The upper aqueous phase was carefully removed, mixed with scintillation fluid and the quantity of label present was determined. The tube was then washed, the cell pellet disrupted and the quantity of label present was determined.

Determination of the specificity of the LTB4 binding sites

Macrophages (2×107 ml−1) in Ca2+/Mg2+-containing HBSS were incubated with a variety of related eicosanoids [LTB4, LTB5, LXA4, LTC4, 12(S)-HETE, 12(S)-HEPE] or the LTB4 receptor antagonists LY223982 and LTB4-dimethylamide, at three different concentrations (3×10−8, 3×10−9 or 3×10−10 mol l−1) together with 0.03 nmol l−1 [3H]LTB4 for 30 min at less than 4 °C. The results were expressed as a percentage of the label bound to cells incubated with radioactive ligand in the absence of a competing compound.

The non-specific association of the radioactive label with trout macrophages with time was established by incubating the cells with both [3H]LTB4 and a 104-fold excess of cold LTB4. Such conditions have been calculated to prevent 99.9 % of receptor occupancy by the radiolabel (Hulme and Birdsall, 1992), thereby identifying the quantity of radioactivity that had partitioned into the membrane or other hydrophobic areas, had become entrapped in the cell pellet or had become associated with the microcentrifuge tube. Throughout the incubation, the levels of non-specific binding did not exceed 8.2±1.0 % (mean ± S.D., N=6) of the total binding. The level of specific binding was calculated by subtracting the non-specific binding from the total amount of label associated with the cell pellet.

Both the total and specific binding of [3H]LTB4 achieved a steady state at approximately 30 min (Fig. 1). A 30 min incubation time was therefore used subsequently to ensure that a suitable concentration of unlabelled (cold) homoligand had been used to determine non-specific binding. At least a 1000-fold excess of cold LTB4 was required to compete with the specifically bound radioactive ligand to display a constant level of non-specific binding. Furthermore, once at equilibrium, this binding was partially reversible with approximately 30 % reduction in binding following the addition of a 1000-fold excess of cold LTB4 after 20 min of incubation. As shown in Fig. 2A, as the concentration of [3H]LTB4 increased, the specific binding approached saturation. When replotted as a Scatchard plot (Fig. 2B), linear regression analysis indicated that there is a single class of receptor sites with a KD of 0.14±0.003 nmol l−1 and a Bmax of 0.295±0.006 pmol per 107 macrophages or 17 800±3600 binding sites per cell (mean values ± S.E.M., N=3). The linear nature of the Scatchard plots did not suggest the existence of cooperativity, and the Hill plot, which yielded a coefficient of 1.03, confirmed this (Fig. 2C).

Fig. 1.

Time course of [3H]LTB4 binding to trout macrophages determined at less than 4 °C. Macrophages (2×107) were incubated with 0.03 nmol l−1 [3H]LTB4 in the absence (○) or presence (▪) of a 104-fold excess of cold homoligand. Specific binding (•) was calculated by subtracting the non-specific bound label from the total radiolabel. Results shown are means ± S.E.M of three separate experiments using different batches of cells performed with duplicate determinations at each time interval.

Fig. 1.

Time course of [3H]LTB4 binding to trout macrophages determined at less than 4 °C. Macrophages (2×107) were incubated with 0.03 nmol l−1 [3H]LTB4 in the absence (○) or presence (▪) of a 104-fold excess of cold homoligand. Specific binding (•) was calculated by subtracting the non-specific bound label from the total radiolabel. Results shown are means ± S.E.M of three separate experiments using different batches of cells performed with duplicate determinations at each time interval.

Fig. 2.

(A) Saturation analysis of [3H]LTB4 binding to rainbow trout macrophages. Values are means ± S.E.M of three separate experiments each performed in duplicate. (B) Scatchard analysis of [3H]LTB4 binding to rainbow trout macrophages. The lines represent linear regression analyses of data from three separate experiments where r=−0.887 (▪), −0.953 (▴) and −0.585 (▾). The linear nature of these plots suggests a single binding site with a mean KD of 0.14 nmol l−1 and Bmax of approximately 17 800 binding sites per cell. (C) Hill analysis of [3H]LTB4 binding to rainbow trout macrophages. The lines represent linear regression analyses of three experiments where r=0.994 (▪), 0.979 (▴) and 1.00 (▾). The mean Hill coefficient was determined to be 1.03.

Fig. 2.

(A) Saturation analysis of [3H]LTB4 binding to rainbow trout macrophages. Values are means ± S.E.M of three separate experiments each performed in duplicate. (B) Scatchard analysis of [3H]LTB4 binding to rainbow trout macrophages. The lines represent linear regression analyses of data from three separate experiments where r=−0.887 (▪), −0.953 (▴) and −0.585 (▾). The linear nature of these plots suggests a single binding site with a mean KD of 0.14 nmol l−1 and Bmax of approximately 17 800 binding sites per cell. (C) Hill analysis of [3H]LTB4 binding to rainbow trout macrophages. The lines represent linear regression analyses of three experiments where r=0.994 (▪), 0.979 (▴) and 1.00 (▾). The mean Hill coefficient was determined to be 1.03.

Fig. 3 demonstrates the ability of several other lipoxygenase products previously shown to be generated by trout macrophages [LXA4, 12-(S)HETE, 12-(S)HEPE, LTB5, LTC4; Pettitt et al. 1991; Knight et al. 1995] to antagonise the binding of [3H]LTB4 to macrophages. It was found that LTB5 interacted with the binding site in a similar manner to LTB4, with an estimated IC50 value of 8×10−10 mol l−1. The other predominant trout macrophage-derived lipoxygenase products had no significant effect upon the ability of the putative receptor to bind radiolabelled LTB4 at the concentrations tested. The LTB4 receptor antagonist LY223982 had no significant displacement effect on [3H]LTB4 binding to macrophages over the concentration range used, although LTB4-dimethylamide, a partial LTB4 receptor antagonist in mammals (Falcone and Aharony, 1990), did displace over 25 % of the total binding (IC50>1×10−8 mol l−1).

Fig. 3.

(A) Competition of [3H]LTB4 binding with structurally related compounds: LTB4 (▪), LTB5 (•), 12-(S)HETE (▫) and LXA4 (▴), and (B) with the known LTB4 mammalian receptor antagonists LY223982 (▪) and LTB4-dimethylamide (•). The results are expressed as a percentage of total binding in the absence of any competing compound and represent the mean ± S.E.M. of 3–4 separate experiments. For the sake of clarity, the results for two other compounds, 12-(S)HEPE and LTC4, are not shown, but they were not significantly different from those shown for LXA4 and 12-(S)HETE.

Fig. 3.

(A) Competition of [3H]LTB4 binding with structurally related compounds: LTB4 (▪), LTB5 (•), 12-(S)HETE (▫) and LXA4 (▴), and (B) with the known LTB4 mammalian receptor antagonists LY223982 (▪) and LTB4-dimethylamide (•). The results are expressed as a percentage of total binding in the absence of any competing compound and represent the mean ± S.E.M. of 3–4 separate experiments. For the sake of clarity, the results for two other compounds, 12-(S)HEPE and LTC4, are not shown, but they were not significantly different from those shown for LXA4 and 12-(S)HETE.

The results of the present work demonstrate that LTB4 binding to rainbow trout macrophages is structurally specific, apparently saturable, but only approximately 30 % reversible after 20 min in the presence of a 1000-fold excess of unlabelled homoligand. Whilst under ideal conditions binding would be fully reversible to non-specific binding levels, other authors have also reported similar difficulties with human granulocytes with levels of displacement of approximately 70 % (Goldman and Goetzl, 1982). This non-displacement may be due to internalisation of the label similar to that described by Lin et al. (1984) and is usually prevented in studies with mammalian cells by lowering the temperature sufficiently to prevent possible internalisation and metabolism of the label. The fish used in these experiments were maintained at temperatures between 10 and 15 °C and the experiments were performed at approximately 2 °C. Consequently, the temperature difference between the two conditions may not have been sufficient to prevent internalisation of the label especially as fish leucocytes show a high degree of homeoviscous adaptation, allowing them to maintain cellular processes, such as endocytosis, at low environmental temperatures (Bowden et al. 1996). The label may also have been internalised by a transport system similar to that described for LXA4 (Simchowitz et al. 1994), although the only similar system identified for LTB4 in humans results in the release of this compound from cells (Lam et al. 1990) and no import systems have been described.

Analysis of the kinetics of LTB4 binding over the [3H]LTB4 concentration range used indicated that only a single class of receptor was present with a KD of 0.14 nmol l−1 and a Bmax of 17 800 binding sites per macrophage. This does not appear to resemble the high-and low-affinity B-LT states found in some mammalian neutrophils and eosinophils (e.g. Goldman and Goetzl, 1984; Goldman et al. 1986; Maghni et al. 1991; Sehmi et al. 1992), although Kreisle et al. (1985) were able to identify only one high-affinity LTB4 receptor type on rat neutrophils.

The putative rainbow trout LTB4 receptors most closely resemble the mammalian high-affinity receptors, which transduce the chemotactic response to LTB4 (Sehmi et al. 1992). Trout head kidney leucocytes display both a chemotactic response to LTB4 (Sharp et al. 1992) and an increase in the intracellular concentration of Ca2+ (EC50=1.2 nmol l−1) in the presence of this eicosanoid (Knight et al. 1993). It is also interesting that Knight (1995) was unable to demonstrate an increase in degranulation of these cells in response to LTB4 at concentrations above 10−5 mol l−1, which is a cellular response attributed to the mammalian low-affinity receptors (Goldman and Goetzl, 1984). Whilst it is tempting to speculate that only one receptor state exists, and that receptor occupancy may result in a rise in intracellular [Ca2+] and induce a chemotactic response, further work demonstrating the antagonistic effects of certain compounds on both these responses and on receptor binding is required.

In the present study, it was not possible to demonstrate the inhibition of [3H]LTB4 binding by LY223982, although LTB4-dimethylamide was a partial antagonist and may demonstrate further inhibition at higher concentrations. The ineffectiveness of a further mammalian LTB4 receptor antagonist, LY255283, was also reported by Andazola et al. (1992) whilst characterising the bullfrog lung LTB4 receptor and together these results suggest that the piscine and anuran leukotriene receptors are both different from their mammalian counterparts in terms of their structure. Further insight into the potential dissimilarity between eicosanoid receptors in fish and mammals is reflected in the finding that the human cDNA probe for the LXA4 receptor on human granulocytes (Fiore et al. 1994) did not hybridize with trout macrophage mRNA (S. Fiore, C. N. Serhan, L. A. Bowden and A. F. Rowley, unpublished observations), suggesting that sequence homology between these two ‘receptors’ in fish and humans may be limited.

Finally, the structural specificity of this binding site elucidated by the competition studies is also similar to that for bullfrog lung (Andazola et al. 1992). Both display a higher affinity for the eicosapentaenoic-acid-derived lipoxygenase product LTB5 than for the equivalent mammalian receptor (Falcone and Aharony, 1990) and neither interacts with peptido-leukotrienes. This former observation is not unexpected since membranes of trout macrophages contain a high proportion of eicosapentaenoic acid and consequently produce similar amounts of LTB5 and LTB4 (Pettitt et al. 1989, 1991). Furthermore, both 4-and 5-series leukotrienes have similar biological potency in fish (Secombes et al. 1994), unlike the situation in mammals where LTB5 is a weak chemotactic factor in comparison with LTB4 (e.g. Lee et al. 1984). The similarity in receptor occupancy by LTB4 and LTB5 reported in the present study with trout is therefore not surprising and may reflect an evolutionary adaptation to the situation where both 4-and 5-series lipoxygenase products naturally exist in these animals.

We wish to thank Dr R. Newton, University of Wales Swansea, for helpful discussion during these studies. This work was supported by the Agricultural and Food Research Council (grant AG58/510 to A.F.R.), NIH (grants GM38765 and PO1-DK50305 to C.N.S.) and a studentship to L.A.B from the Natural Environment Research Council. We also thank Lilly Research Laboratories for the generous provision of LY223982.

Andazola
,
J. J.
,
Underwood
,
J. A.
,
Chiono
,
M.
,
Torres
,
O. A.
and
Herman
,
C. A.
(
1992
).
Leukotriene B4and leukotriene B5 have binding sites on lung membranes and cause contraction on bullfrog lung
.
J. Pharmac. exp. Ther.
263
,
1111
1117
.
Bowden
,
L. A.
,
Restall
,
C. J.
and
Rowley
,
A. F.
(
1996
).
The influence of environmental temperature on membrane fluidity, fatty acid composition and lipoxygenase product generation in head kidney leucocytes of the rainbow trout, Oncorhynchus mykiss
.
Comp. Biochem. Physiol. (in press)
.
Falcone
,
R. C.
and
Aharony
,
D.
(
1990
).
Modulation of ligand binding to leukotriene B4 receptors on guinea pig lung membranes by sulfhydryl modifying reagents
.
J. Pharmac. exp. Ther
.
255
,
565
571
.
Fiore
,
S.
,
Maddox
,
J. F.
,
Perez
,
H. D.
and
Serhan
,
C. N.
(
1994
).
Identification of human cDNA encoding a functional high affinity lipoxin A4 receptor
.
J. exp. Med.
180
,
253
260
.
Ford-Hutchinson
,
A. W.
,
Gresser
,
M.
and
Young
,
R. N.
(
1994
).
5-Lipoxygenase
.
A. Rev. Biochem
.
63
,
383
417
.
Goldman
,
D. W.
,
Enkel
,
H.
,
Gifford
,
L. A.
,
Chenoweth
,
D. E.
and
Rosenbaum
,
J. T.
(
1986
).
Lipopolysaccharide modulates receptors for leukotriene B4, C5a and formyl-methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes
.
J. Immunol.
137
,
1971
1976
.
Goldman
,
D. W.
,
Gifford
,
L. A.
,
Young
,
R. N.
,
Marotti
,
T.
,
Cheung
,
K. L.
and
Goetzl
,
E. J.
(
1991
).
Affinity labelling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4
.
J. Immunol
.
146
,
2671
2677
.
Goldman
,
D. W.
and
Goetzl
,
E. J.
(
1982
).
Specific binding of leukotriene B4to receptors on human polymorphonuclear leukocytes
.
J. Immunol.
129
,
1600
1604
.
Goldman
,
D. W.
and
Goetzl
,
E. J.
(
1984
).
Heterogeneity of human polymorphonuclear leukocyte receptors for leukotriene B
.
J. exp. Med.
153
,
482
487
.
Harvey
,
J. P.
,
Koo
,
C. H.
,
Boggs
,
J. M.
,
Young
,
R. N.
and
Goetzl
,
E. J.
(
1992
).
Mouse monoclonal antibody to a latent epitope of leucocyte receptors for leukotriene B4
.
Immunology
76
,
122
128
.
Hulme
,
E. C.
and
Birdsall
,
N. J. M.
(
1992
).
Strategy and tactics in receptor-binding studies
. In
Receptor Ligand Interactions
(ed.
E. C.
Hulme
), pp.
63
176
.
Oxford
:
Oxford University Press
.
Knight
,
J.
(
1995
).
Distribution and function of lipoxins and other eicosanoids in the rainbow trout, Oncorhynchus mykiss. PhD thesis, University of Wales
.
Knight
,
J.
,
Holland
,
J. W.
,
Bowden
,
L. A.
,
Halliday
,
K.
and
Rowley
,
A. F.
(
1995
).
Eicosanoid generating capacities of different tissues from the rainbow trout, Oncorhynchus mykiss
.
Lipids
30
,
451
458
.
Knight
,
J.
,
Lloyd-Evans
,
P.
,
Rowley
,
A. F.
and
Barrow
,
S. E.
(
1993
).
Effects of lipoxins and other eicosanoids on phagocytosis and intracellular calcium mobilisation in rainbow trout (Oncorhynchus mykiss) leukocytes
.
J. Leukocyte Biol
.
54
,
518
522
.
Kreisle
,
R. A.
,
Parker
,
C. W.
,
Griffin
,
G. L.
,
Senior
,
R. M.
and
Stenson
,
W. F.
(
1985
).
Studies of leukotriene B4-specific binding and function in rat polymorphonuclear leukocytes: absence of a chemotactic response
.
J. Immunol
.
134
,
3356
3363
.
Lam
,
B. K.
,
Gagnon
,
K.
,
Austen
,
F.
and
Soberman
,
R. J.
(
1990
).
The mechanisms of leukotriene B4export from human polymorphonuclear leukocytes
.
J. biol. Chem
.
265
,
13438
13441
.
Lee
,
T. H.
,
Mencia-Heurta
,
J.-M.
,
Shih
,
C.
,
Corey
,
E. J.
,
Lewis
,
R. A.
and
Austen
,
K. F.
(
1984
).
Characterisation of biologic properties of 5,12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene B5and the double lipoxygenase product
.
J. biol. Chem
.
259
,
2383
2389
.
Lin
,
A. H.
,
Ruggsed
,
P. L.
and
Gorman
,
R. R.
(
1984
).
Leukotriene B4binding to human neutrophils
.
Prostaglandins
28
,
837
842
.
Lloyd-Evans
,
P.
,
Barrow
,
S. E.
,
Hill
,
D. J.
,
Bowden
,
L. A.
,
Rainger
,
G. E.
and
Rowley
,
A. F.
(
1994
).
Eicosanoid generation and effects on the aggregation of thrombocytes from the rainbow trout, Oncorhynchus mykiss
.
Biochim. biophys. Acta
1215
,
291
299
.
Maghni
,
K.
,
De Brum-Fernandes
,
A. J.
,
Foldes-Filep
,
E.
,
Gaudry
,
M.
,
Borgeat
,
P.
and
Sirois
,
P.
(
1991
).
Leukotriene B4 receptors on guinea pig alveolar eosinophils
.
J. Pharmac. exp. Ther.
258
,
784
789
.
Metters
,
K. M.
(
1995
).
Leukotriene receptors
.
J. Lipid Mediators Cell Signalling
12
,
413
427
.
Mong
,
S.
(
1991
).
Receptors and receptor antagonists for mammalian 5-lipoxygenase products
. In
Lipoxygenases and Their Products
(ed.
S. T.
Crooke
and
A.
Wong
), pp.
185
206
.
London
:
Academic Press
.
Pettitt
,
T. R.
,
Rowley
,
A. F.
and
Barrow
,
S. E.
(
1989
).
Synthesis of leukotriene B and other conjugated triene lipoxygenase products by blood cells of the rainbow trout, Salmo gairdneri
.
Biochim. biophys. Acta
1003
,
1
8
.
Pettitt
,
T. R.
,
Rowley
,
A. F.
,
Barrow
,
S. E.
,
Mallet
,
A. I.
and
Secombes
,
C. J.
(
1991
).
Synthesis of lipoxins and other lipoxygenase products by macrophages from the rainbow trout, Oncorhynchus mykiss
.
J. biol. Chem.
266
,
8720
8726
.
Rowley
,
A. F.
(
1996
).
The evolution of inflammatory mediators
.
Mediators Inflammation
5
,
3
13
.
Samuelsson
,
B.
,
Dahlen
,
S.-E.
,
Lindgren
,
J. Å.
,
Rouzer
,
C. A.
and
Serhan
,
C. N.
(
1987
).
Leukotrienes and lipoxins: Structures, biosynthesis and biological effects
.
Science
237
,
1171
1176
.
Secombes
,
C. J.
,
Clements
,
K.
,
Ashton
,
I.
and
Rowley
,
A. F.
(
1994
).
The effect of eicosanoids on rainbow trout, Oncorhynchus mykiss, leucocyte proliferation
.
Vet. Immunol. Immunopathol.
42
,
367
378
.
Sehmi
,
R.
,
Rossi
,
A. G.
,
Kay
,
A. B.
and
Cromwell
,
O.
(
1992
).
Identification of receptors for leukotriene B4 expressed in guineapig peritoneal eosinophils
.
Immunology
77
,
129
135
.
Sharp
,
J. E.
,
Pettitt
,
T. R.
,
Rowley
,
A. F.
and
Secombes
,
C. J.
(
1992
).
Lipoxininduced migration of fish leukocytes
.
J. Leukocyte Biol
.
51
,
140
145
.
Sherman
,
J. W.
,
Goetzl
,
E. J.
and
Koo
,
C. H.
(
1988
).
Selective modulation by guanine nucleotides of the high affinity subset of plasma membrane receptors for leukotriene B4on human polymorphonuclear leukocytes
.
J. Immunol.
140
,
3900
3904
.
Simchowitz
,
L.
,
Fiore
,
S.
and
Serhan
,
C. N.
(
1994
).
Carriermediated transport of lipoxin A4 in human neutrophils
.
Am. J. Physiol
.
267
,
C1525
C1534
.
Slipertz
,
D. M.
,
Scoggan
,
K. A.
,
Nicholson
,
D. W.
and
Metters
,
K. M.
(
1993
).
Photoaffinity labelling and radiation inactivation of the leukotriene B4receptor in human myeloid cells
.
Eur. J. Pharmac.
244
,
161
173
.