Eicosanoids and their role in immune modulation in fish—a brief overview

Fish & Shellfish Immunology (1995) 5, 549-567

E i c o s a n o i d s a n d t h e i r role in i m m u n e m o d u l a t i o n in fish--a brief overview ANDREW F. ROWLEY*, JOHN KNIGHT, PAUL LLOYD-EVANS,JASON W. HOLLANDAND PHILIP J. VICKERS'~

School of Biological Sciences, University of Wales, Swansea, Singleton Park, Swansea, SA2 8PP, U.K., and tMerck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Quebec, Canada Eicosanoids have been demonstrated to play a central role in immune regulation in mammals brought about by their direct effects on cells such as macrophages and lymphocytes or by their indirect effects via cytokines. Studies have shown that fish mononuclear phagocytes, granulocytes and thrombocytes synthesize and release both cyclooxygenase- and lipoxygenasederived products such as prostaglandin E._,, leukotriene B~I and lipoxin A 4. Whether lymphocytes have the ability to generate leukotrienes and lipoxins is still unclear but they do appear to have 12-1ipoxygenase activity that leads to the generation of 12-hydroxy fatty acid derivatives. As in mammals, leukotriene and lipoxin biosynthesis requires the presence of a 5-1ipoxygenase activating protein-like molecule that is sensitive to the action of the specific inhibitor, MK-886. The prostaglandin-generating ability of trout macrophages can be altered by incubation with lipopolysaccharide suggesting the possible presence of an inducible cyclooxygenase activity. Prostaglandins have been found to suppress the mitogen-induced proliferation of trout leucocytes and the generation of humoral antibody and plasma cells both in vivo and in vitro. The lipoxygenase products, leukotriene B,I and lipoxin A 4, have more variable effects ranging from inhibition to stimulation depending on the assay system employed. Overall, there is clear evidence that eicosanoids play a role in immune regulation in fish in a similar way to that reported in mammals. i: 1995 Academic Press Limited Key

words:

Eicosanoid, lipoxygenase, cyclooxygenase, 5-1ipoxygenase activating protein, prostaglandins, leukotrienes, lipoxins, leucocytes, macrophages, lymphocytes, immune regulation, fish, rainbow trout.

I. I n t r o d u c t i o n

One of the h a l l m a r k s of the m a m m a l i a n i m m u n e system is the exquisite a r r a y of r e g u l a t o r y m e c h a n i s m s t h a t i n t e g r a t e the cellular activities o p e r a t i n g in this h o m e o s t a t i c response to foreign i n v a d e r s or altered 'self'. The i n t e r p l a y b e t w e e n cytokines, e i c o s a n o i d s a n d v a r i o u s l e u c o c y t e types well exemplifies the c o m p l e x i t y of this system. N o t only can cytokines, s u c h as interleukin-1 (IL-1), i n d u c e p r o s t a g l a n d i n E,_, (PGE2; a type of eicosanoid) g e n e r a t i o n from *To whom all correspondence should be addressed. ¶IPresent address: Department of Molecular Sciences, Pfizer Central Research, Sandwich, Kent, CT139NJ. 549 1050 4648/95/080549+19 $12.00/0

c 1995 Academic Press Limited

550

A.F. ROWLEYET AL.

human monocytes (e.g. Browning & Ribolini, 1987) but this eicosanoid can also cause changes in the levels of IL-1 and tumour necrosis factor-~ production in monocytes (Knudsen et al., 1986; Hart et al., 1989) and IL-2 and IL-3 generation in lymphocytes (e.g. Minakuchi et al., 1990; Daculsi et al., 1993). Furthermore, down regulation of IL-2 receptors on mononuclear phagocytes by recombinant granulocyte-macrophage colony-stimulating factor is also caused by the induction of PGE synthesis in these cells (Hancock et al., 1988). As exemplified by these, and other reports, PGE 2 has been found to have many indirect and direct effects on lymphocytes and mononuclear phagocytes. Thus, this molecule is considered to be a key immune modulator because of its effects on lymphocytes and macrophages (Goodwin & Ceuppens, 1983; Phipps et al., 1991). Significantly less is known about the potential immunomodulatory effects of leukotrienes, such as LTBj, although this compound augments the expression of IL-2 receptor-fl expression on CD56 ÷ and CD8 ÷ lymphocytes (Stafikov~ et al., 1992) and immunoglobulin synthesis by Staphylococcus aureus-stimulated B cells is augmented by the presence of LTB 4 (Yamaoka et al., 1989). Finally, IL-4 specifically induces 15-1ipoxygenase (an enzyme involved in eicosanoid synthesis) mRNA in human monocytes, while interferon ;' (IFN-;,) inhibits this reaction (Conrad et al., 1992). Thus, eicosanoids can regulate the synthesis and expression of receptors for various ILs while in turn these cytokines can affect the biosynthesis of eicosanoids by a variety of mechanisms hence providing a feedback loop. The piscine immune system has been shown to contain many of the factors and cell types characteristic of its mammalian counterpart. In particular, concomitant with the evolution of the first vertebrates was probably the appearance of 'true' lymphocytes and the ability to synthesize immunoglobulin in response to immunogenic challenge (see review by Manning, 1994, for further details). Until recently, little was known about the potential role of eicosanoids in immune modulation in fish, therefore this review is designed to provide a timely overview of recent findings and incorporates a number of unpublished observations from our laboratories. II. W h a t Are E i c o s a n o i d s a n d H o w A r e T h e y F o r m e d ? Eicosanoids are oxygenated derivatives of polyunsaturated fatty acids formed by the metabolism of membrane phospholipids by the action of phospholipases. The principal substrate is arachidonic acid (AA; 20:4, n-6) with a 20-carbon backbone, but both eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3) are also important substrates in fish due to their propensity in membrane phospholipids in these organisms (see review by Henderson & Sargent, 1985 for further details). There are two main pathways involved in eicosanoid generation. The first is catalysed by lipoxygenases to yield a range of monohydroxy fatty acids (e.g. 5(S)-hydroxy-eicosatetraenoic acid; 5-HETE derived from AA) while di- and tri-hydroxy fatty acids, such as leukotrienes (LT) and lipoxins (LX), are also formed via epoxy intermediates (Fig. 1). Central in the synthesis of leukotrienes is the highly unstable epoxide, LTA 4 (5(S),6(S)-trans-5,6-oxido7,9-trans-ll,14-cis-eicosatetraenoic acid); formed from 5(S)-hydroperoxy-6trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE). This epoxide is rapidly

FISH IMMUNE MODULATION

551

12-HETE

12-HPETE 12-LOl -] 15-LO

15-HPETE - - - ~ 15-HETE

5"LO1 LXA4 ~.~xide

hydrolases

5-HPETE . . . . . . . -~ 5-HETE 5-LO~

5(6)Epoxytetraene ~ LTA4 ~ LTB4 12-LO ~ LTAhydrolase , LXB4

LTC4

6-trans-LTB 4, 6_trans_12.epi.LTB4,

L~D4

5(S),6(R)-DiHETE

LTE4 Fig. 1. Main biosynthetic routes for the generation of lipoxygenase (LO) products. Solid lines represent enzymatic steps while dotted lines represent non-enzymatic reactions. For simplicity, not all products generated are shown. AA=arachidonic acid.

hydrolysed enzymatically to LTB 4 (5(S), 12(R)-dihydroxy-6,14-cis-8,10-transeicosatetraenoic acid) or non-enzymatically to several dihydroxyeicosat e t r a e n o a t e s (e.g. 6-trans-LTB,, 6-trans-12-epi-LTB,t). Leukotriene A,, can also be converted to cysteinyl leukotrienes, LTC 4, LTD 4 and LTE,~. The lipoxins, LXA, (5(S),6(R),15(S)-trihydroxy-7,9,13-trans,ll-cis-eicosatetraenoic acid) and LXB 4 (5(S),14(R),15(S)-trihydroxy-6,10,12-trans,8-cis-eicosatetraenoic acid), have also been shown to be formed from LTA 4 in mammals by the activities of 12-1ipoxygenase and epoxide hydrolases (Serhan, 1994). Additional biosynthetic routes for lipoxin generation are also present in both mammals (Serhan, 1994) and fish (Rowley et al., 1994). Mammalian 5-1ipoxygenase (5-LO) has been extensively characterised in the last few years as a result of molecular studies in which the genomic sequences have been studied (see review of Ford Hutchinson et al. (1994) for more details). Human 5-LO is a 78 kDa protein localised in the nuclear membrane of leucocytes (Woods et al., 1993; Brock et al., 1994). Unlike other lipoxygenases, it has a requirement for Ca .'+ and a novel protein termed 5-1ipoxygenase activating protein (FLAP). FLAP is an 18kDa protein located in the nuclear membrane and, by the use of specific inhibitors such as MK-886 (L-663,536; 3-[l-(chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]2,2-dimethylpropanoic acid), it has been shown to be essential for leukotriene biosynthesis in intact cells (Gillard et al., 1989; Dixon et al., 1990). The exact mechanism of FLAP activity is unclear but it appears to function

552

A.F. ROWLEYET AL.

by facilitating the transfer of AA (or presumably EPA) to 5-LO thereby allowing the enzymatic reactions to occur in an optimal manner (Mancini et al., 1993; Ford-Hutchinson et al., 1994). As yet, there are no reports of similar requirements for FLAP-like proteins for the activity of either 12- or 15-LO. The second main pathway of eicosanoid biosynthesis is often referred to as the cyclooxygenase (COX) cascade and leads to the generation of prostaglandins (PG), prostacyclin (PGI) and thromboxanes (TX). The enzyme t h a t plays a central rate-limiting role in the generation of these three types of eicosanoids is COX, also termed prostaglandin H synthase (PGHS). The constitutive form of this enzyme (COX-1/PGHS-1; EC 1.14.99.1) is a 72 kDa molecule widely distributed in many cells and tissues while a second form more recently identified, christened inducible COX (COX-2, PGHS-2), has been demonstrated in macrophages (Masferrer et al., 1992; Lee et al., 1992; Riese et al., 1994), fibroblasts (Hulkower et al., 1994), mast cells (Murakami et al., 1994), mesangial cells (Kester et al., 1994), preovulatory follicles (Sirois, 1994) and unspecified cells in the human amnion (Teixeira et al., 1994) following stimulation. This latter isoform is induced in macrophages and fibroblasts by various proinflammatory stimuli including lipopolysaccharide, IL-lfl, phorbol esters, tumour necrosis factor-a (TNF-a) and IFN-~,, and in preovulatory follicles by human chorionic gonadotrophin (Sirois, 1994). The COX activity of PGHS yields PGG2 from AA, while its peroxidase activity reduces PGG9 to the 15-hydroxy analogue, termed PGH 2 (Smith, 1989). Both PGG 2 and PGHe are unstable and this latter compound acts as a substrate for the synthesis of PGE 2, an important immune regulatory molecule (Phipps et al., 1990) as well as TXA 2, PGI 2 (prostacyclin) and a multitude of other PGs (Fig. 2). The bioactive PGs are rapidly converted to inactive metabolites. For example, TXA 2 is a potent platelet aggregatory compound while TXB 2 is inactive (Hamberg et al., 1975). Similarly, PGEe injected intravascularly has a half-life of < l m i n and is metabolised by a dehydrogenase found in the lung and other tissues (GranstrSm & Kumlin, 1987) ultimately to be excreted as a biologically inactive form in the urine. Lipoxygenase products such as 5-HETE, LTC 4, LTB 4, LXA 4 and LXB:I are also subject to further metabolism. For example, LTB~ undergoes ~)-oxidation to yield 20-OH-LTB 4 and subsequently to 20-COOH-LTB4 (Hansson et al., 1981; Powell, 1984) while LXA4 is rapidly metabolised (>60% within 30s) to 15-oxo-LXA4, 3,14-dihydro-15-oxo-LXA4 and 13,14dihydro-LXAt by human monocytes by a dehydrogenase-type mechanism (Serhan et al., 1993; Serhan, 1994). Similarly, human neutrophils metabolise 5-HETE to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE; Powell et al., 1992). Lipoxin B 4 is subject to •-oxidation in the liver by a cytochrome P-450 dependent mechanism to yield 20-OH-LXB4 and subsequently 20-COOH-LXB 4 (Mizukami et al., 1994). The bioactive potential of the breakdown products of LXA 4 and LXB 4 are unreported, although 20-OHLTB 4 and 20-COOH-LTB4 are less biologically active than LTB 4 (Camp et al., 1982), while in contrast 5-oxo-ETE is 100 fold more active at inducing Ca 2÷ mobilisation in neutrophils than the parent molecule, 5-HETE (Powell et al., 1993).

FISH IMMUNE MODULATION

553

AA

PGG2 >~ O

PGHz

PGI2 I

T 6-keto-PGFla

PGD2

PGE2

PGF2rL

TXA2 I

• TXB2

Fig. 2. Main biosynthetic routes for the generation of prostanoids. COX= cyclooxygenase, TX = thromboxane, PG = prostaglandin, AA = arachidonic acid.

III. E i c o s a n o i d Generation by Fish L e u c o c y t e s and T h r o m b o c y t e s The cellular sources of eicosanoids for immune regul at ory activity are likely to be those cells at the site of inflammation and immunological reactions, especially as eicosanoids have short half-lives in vivo and are hence unlikely to be transported from organ to organ. Therefore, fixed cells in lymphoid organs such as macrophages, reticular cells, dendritic cells and endothelial cells are prime candidates for the provision of eicosanoids but free leucocytes must also contribute to the total pool of these compounds generated. Previous studies using a range of cartilaginous and bony fish have examined the eicosanoid-generating potential of leucocytes. In the cartilaginous dogfish, Scyliorhinus canicula, unf r act i onat ed leucocytes have been found to generate a range of COX and LO-derived metabolites including PGE z, TXB z, 5-HETE, 15-HETE and non-enzymatic hydrolysis products of LTA 4 (e.g. 6-trans-LTB4, 6-trans-12-epi-LTB4) but no LTB 4 or LTB~, suggesting a lack of LTA hydrolase activity (Rowley et al., 1987; Pet t i t t & Rowley, 1991; see Fig. 1). The profile of eicosanoid generation in some of the other species examined has been shown to be markedly different. For example, rainbow t r o u t (Oncorhynchus mykiss) u n f r a c t i o n a t e d peripheral blood leucocytes and head kidney macrophages generate significant amounts of lipoxins (LXA 4, LXA 5, 11-transLXA 4 and 11-trans-LXA~) as well as 12-HETE, 12-hydroxyeicosapentaenoic acid (12-HEPE; derived from EPA), LTB 4, LTB 5 and PGE z (Pettitt et al., 1989a,b; P ett i t t et al., 1991; Rowley et al., 1994; Table 1). A similar profile of products is also released by thrombocytes (platelet equivalent cells) in O. mykiss following challenge with calcium ionophore, A23187 but the overall levels are c. 20-fold less t han from the leucocytes (Lloyd-Evans et al., 1994; Table 1). Some, but not all, of the leucocytes from other fish species also have the capacity to synthesise lipoxins. Carp (Cyprinus carpio) head kidney macrophages generate large amounts of both LXA 4 and LXB 4, although tilapia

eO 0

ri~ ~

,i~

-H -H -H

,-...~ ¢.-, o-a ¢Xl

~D -H -H -H

.-3

-H "~

,B

C'x1

~2o ~,11~

0

-H -H -H ¢0

,i~

,,.~ ~ oO eO ,i~

E~

-H -H -H L ~ L~- CO

~b~o

~.~ ~ . : m

0D

o

to

-H -H -H b.- b -

oco

eQ

-~ G~bo -H -H -H

© II Z

m

©

X

-H -H -H eQ cO o o

II

~

~-~

c,i

m~e II

O~

~

R~ -H

. ~©

~.~

b o

FISH I M M U N E M O D U L A T I O N

555

(Oreochromis niloticus), rudd (Scardinius erythrophthalmus) and catfish (Ictalurus punctatus) macrophages only produce LTs and mono-HETEs (Rowley, 1991). Neutrophils from the peripheral blood of the plaice, Pleuronectes platessa, appear to have a similar profile of lipoxygenase products as mononuclear phagocytes of other species (Tocher & Sargent, 1987) suggesting that monocytes/macrophages, granulocytes and thrombocytes all have significant eicosanoid-generating capacity in fish. DO FISH I,YMPHOCYTES GENERATE LIPOXYGENASE PRODUCTS?

Whether mammalian lymphocytes have the capacity to generate lipoxygenase products proved to be a controversial subject for nearly twenty years. Although several groups of researchers claimed the apparent generation of LTBL, from purified populations of B and/or T lymphocytes (e.g. Goetzl, 1981) the contamination of these cells by other leucocytes, such as granulocytes and monocytes, led to the suggestion that this product was formed as a result of the presence of the contaminating cells and not the lymphocytes (Goldyne, 1988). Use of lymphoblastic B- and T-cell lines and molecular probes to localise LO and FLAP activity have enabled the debate to be settled proving that 5-LO-derived products, such as LTB.j and 5-HETE, can be generated from lysates of B- but not T-lymphocytes or intact B-lymphocytes co-incubated with ionophore and glutathione depleting agents (Claesson et al., 1992; Jacobsson et al., 1992). Surprisingly, although various lymphoblastic T cell lines were found not to express 5-1ipoxygenase activity they did observe FLAP mRNA in these cells (Jacobsson et al., 1992). To our knowledge, no lymphoblastic cell lines such as those used by Jacobsson et al. (1992) are available from fish and the paucity of molecular probes tested with fish leucocytes has made it difficult to investigate whether fish lymphocytes can generate any LO-derived eicosanoids. One approach taken by Knight & Rowley (unpublished observations) has been to incubate mixtures of density gradient isolated adherent leucocytes (mainly macrophages) with non-adherent leucocytes (mainly lymphocytes) produced by several stages of removal of contaminating cells by adherence to plastic. Mixtures of these two populations with different ratios of adherent: non-adherent cells were subsequently challenged with calcium ionophore and LO products separated and quantified by reverse phase high performance liquid chromatography (RP-HPLC). The initial results suggest that no significant amounts of LTBI or LXA~ (indicative of 5-LO activity) are synthesised by the non-adherent cells but that the 12-LO products, 12-HETE and 12-HEPE, are produced (Fig. 3). Hence, it appears that 12-LO activity is present in fish lymphocytes which would be expected as it has been shown that this enzyme is widely distributed in many fish tissues (Knight et al., 1995). These initial studies do not rule out the possibility that 5-LO activity is present in fish lymphocytes as this enzyme may behave like its cryptic counterpart in mammalian lymphocytes therefore making it difficult to demonstrate its presence. Further studies are required to obtain a definitive answer to the question of the possession of 5-LO in fish lymphocytes.

556

A.F. ROWLEY ET AL. 250

300

12-HETE

250

200

12-HEPE

200

150

150 100

100

50 %

50 [

0

200 150 100 50 0

I

I

I

60

80

.• 20

40

40

0

200

LTB4

20

100

I

I

I

I

20

40

60

80

LXA4

100

//~=

150 100 5O

60

80 100 0 20 40 % Adherent contamination

60

80

100

Fig. 3. Generation of LXA, LTB, 12-HETE and 12-HEPE by calcium ionophore, A23187, challenged mixtures of non-adherent (mainly lymphocytes) and adherent (monocytes and neutrophils). Total leucocytes were recovered fi'om Percoll density gradient centrifugation and aliquots (5 ml containing c. 1 × 107 cells) were incubated for 15 min at 18° C in plastic tissue culture grade 25 cm :~ flasks to remove adherent cells. This process was repeated and the non-adherent cells (mainly lymphocytes) removed, counted and purity assessed microscopically. Non-adherent cells were resuspended in 1 2 ml of Ca/Mg-containing Hank's balanced salt solution (HBSS) and incubated with varying percentages of adherent cells to give a total cell number of 1 x 107 cells. These cells were challenged with 5p~t calcium ionophore, A23187, for 20 min at 18° C, the supernatants extracted and lipoxygenase products separated and quantified as described previously (Pettitt et al., 1991; Rowley et al., 1994).

IV.

FLAP

in Fish

Leucocytes

As described in Section II of this review, the presence of F L A P (5-1ipoxygenase a c t i v a t i n g protein) has been s h o w n to be r e q u i r e d for leukotriene b i o s y n t h e s i s in i n t a c t leucocytes. To i n v e s t i g a t e if F L A P , or its p h y l o g e n e t i c p r e c u r s o r , is p r e s e n t in fish leucocytes, we p r e - i n c u b a t e d m a c r o p h a g e s or u n f r a c t i o n a t e d p e r i p h e r a l blood l e u c o c y t e s from the r a i n b o w t r o u t O. m y k i s s with the specific F L A P inhibitor, MK-886 a n d s u b s e q u e n t l y c h a l l e n g e d these cells with c a l c i u m i o n o p h o r e to elicit e i c o s a n o i d g e n e r a t i o n . We f o u n d t h a t MK-886 has a c o n c e n t r a t i o n d e p e n d e n t i n h i b i t o r y effect on the g e n e r a t i o n of LTB4 a n d LXA 4, b o t h 5-1ipoxygenase products, while the a p p e a r a n c e of 12-1ipoxygenase products, 12-HETE and 12-HEPE was unaffected by the presence of this i n h i b i t o r (Figs 4 & 5). These p r e l i m i n a r y i n v e s t i g a t i o n s s t r o n g l y s u g g e s t e d the p r e s e n c e of a F L A P - l i k e molecule in these cells and to f u r t h e r s u b s t a n t i a t e this we s e p a r a t e d m a c r o p h a g e and p e r i p h e r a l blood cell lysates by p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s and blotted

FISHIMMUNEMODULATION

557

100

(.a) 50

3~

e-

o

i00

0 (

250

0

250

500

750

1000

50

0 500 750 MK 886 (ng m1-1}

1000

Fig. 4. Effect of MK-866, a specific 5-1ipoxygenase activating protein inhibitor, on the generation of LXA, (O), LTB, (m) and 12-HETE ('~) by head kidney macrophages (a) or unfractionated peripheral blood leucocytes (b) of the rainbow trout, O. mykiss. Cells were preincubated with MK-886 (1-1000 ng ml-~) for 5 rain at 18°C and subsequently incubated with 5#M calcium ionophore A23187 for 20 min at 18° C. Supernatants were extracted and lipoxygenase products separated, identified and quantified as described previously (Pettitt et al., 1991). Values are means i SD, N=3-4.

t h e s e o n t o n i t r o c e l l u l o s e . T h e s e W e s t e r n blots were p r o b e d w i t h a n t i b o d i e s raised a g a i n s t c e r t a i n h i g h l y c o n s e r v e d regions of h u m a n F L A P (Miller et al., 1990). B o t h cell p r e p a r a t i o n s s h o w e d a distinct i m m u n o r e a c t i v e p r o d u c t w i t h a m o l e c u l a r m a s s of 18 k D a (Fig. 6) w i t h the s a m e Rf v a l u e as h u m a n F L A P s t a n d a r d , f u r t h e r s t r e n g t h e n i n g the a s s e r t i o n t h a t F L A P h a s a long e v o l u t i o n a r y h i s t o r y d a t i n g b a c k to the e v o l u t i o n of s a l m o n i d fish. Clearly, it would be p a r t i c u l a r l y i n t e r e s t i n g to a s c e r t a i n if m o r e p r i m i t i v e fish s u c h as l a m p r e y s a n d hagfishes also h a v e this F L A P - l i k e molecule. Finally, m e n t i o n should be m a d e of the r e c e n t finding of M a r t i n e z et al. (1994) who

558

A.F. ROWLEY

ET AL.

Control 0.03

L X A ~ LXAI PGB 2

0.1

12-HEPE

12HETE~

ll'trans'LXA4"~k~ ~k ~i

--0.007

I,l,l,i,l,l,I 0

I 5

, , 10

0.03

ll-trans-LXA.I LXA~ \

15

i I

PGB2 ~

-0.007

~ 20

,IsL,l,l,Ijl,l,l,l~[ 25

30

MK-886 0.1

LXA.I

<

-0.007 0

I,l,l,[,I,t,l,[,I,I,b,I, 5 10 R e t e n t i o n t i m e (min)

,I,I 15

-0.007

I,l,l~l,l,l~L,t,i,L~l 20 25 30 R e t e n t i o n t i m e (min)

Fig. 5. Reverse phase high performance liquid chromatography (RP-HPLC) chromato-

grams showing the effect of MK-886 (100 ng ml- 1) on the generation of various lipoxygenase products. Note the lack of effect of MK-886 on the synthesis of the 12 lipoxygenase products, 12-HETE and 12-HEPE. See Figure 4 legend for experimental details. described the specific inhibitory effect of MK-886 on leukotriene generation by bullfrog brain implying that a FLAP-like protein also exists in these animals.

V. Modulation of Eicosanoid Generation in Fish by Inflammatory Stimuli The exciting finding t h a t at least several mammalian cell types can express a second form of COX activity (COX-2/PGHS-2) following stimulation has important ramifications for both our understanding of the role of PGs in inflammatory disorders such as rheumatoid arthritis and in immune regulation phenomena. Stimulation of mammalian macrophages by certain cytokines and microbial products (e.g. IL-I+LPS) therefore results in the elevation in the ability of these cells to generate PGs and probably other COX-derived products. More modest stimulation of the generation of LO products may also occur following the application of some types of stimuli that cause the differentiation of cells (e.g. Steinhilber et al., 1993). To determine if the generation of LO- and/or COX-derived products by fish macrophages can be stimulated by proinftammatory agents, trout head kidney

FISH IMMUNE MODULATION

559

-68

-18 -14 Fig. 6. Immunoblot analysis of FLAP from trout leucocytes. 100,000 × g membrane

preparations were made from lysates of peripheral blood leucocytes (PB1 and PB2) or head kidney macrophages (HK1 and HK2) and aliquots (40/1g/lane) separated by electrophoresis and analyzed by immunoblotting as described previously (Reid et al., 1990). The polyclonal antipeptide antiserum used in these studies, termed 709, interacts with amino acid residues 1-39 of FLAP and has been shown to recognize this molecule in 10 species of mammals (Vickers et al., 1992). The migration positions of molecular weight standards are indicated• macrophages were placed in culture in the presence of LPS and the ability of these cells to generate PGE determined by enzyme immunoassay. Macrophages incubated for 12 or 24 h with E. coli LPS showed no significant alteration in their PGE generating capacity compared with those cells incubated without LPS. After 48 h incubation, however, there was a statistically significant increase in the amount of PGE immunoreactive material released by these cells compared with the controls (Fig. 7). Whether this is caused by an increase in the expression of constitutive COX or the appearance of inducible COX (i.e. COX-2) is at present u n k n o w n but the subject of further study. Previous attempts to modify the generation of LO-derived products by trout macrophages stimulated by phorbol 12-myristate 13-acetate failed to yield any increase in LTB 4 and LXA4 biosynthesis (Secombes & Rowley, unpublished observations). VI. The Role o f E i c o s a n o i d s in I m m u n e R e g u l a t i o n in F i s h The first report suggesting t h a t eicosanoids might influence the immune system of fish was t h a t of L a u d a n et al. (1986). They observed t h a t the

560

A . F . ROWLEY E T A L .

%

O

~

m

48

24

Incubation time (h)

Fig. 7. Effect of lipopolysaccharide on the PGE-generating ability of trout macrophages. Head kidney macrophages were incubated with 501tg ml ' E. coli 0111:B4 lipopolysaccharide for periods of >48 h at 15° C. Subsequently, some cells were challenged with 5gM calcium ionophore, A23187, for 20min at 15°C to cause eicosanoid generation and release. The levels of PGE-immunoreactive material were determined in cell-free supernatants using a commercially available enzyme immunoassay method for PGE 2. Values shown are means ± S.D., N=3-6. *P<0"05 compared with control. [], - Ionophore - LPS; m, - Ionophore+LPS; ~], +Ionophore - LPS; and [], +Ionophore+LPS.

i m m u n o s u p p r e s s i v e a c t i v i t y of a m i c r o s p o r i d a n p a r a s i t e , Glugea stephani, in the w i n t e r flounder, Pseudopleuronectes americanus, c o u l d be r e v e r s e d if a n i m a l s w e r e also i n j e c t e d on a r e g u l a r basis d u r i n g the e x p e r i m e n t w i t h the COX inhibitor, i n d o m e t h a c i n . T h e s e i n t e r e s t i n g e x p e r i m e n t s w e r e i n t e r p r e t e d s u c h t h a t P G s w e r e t h o u g h t to be i n v o l v e d in the i m m u n o s u p p r e s s i v e a c t i v i t y of the p a r a s i t e . A s i m i l a r a p p r o a c h was t a k e n by R a i n g e r et al. (1992) in w h i c h r a i n b o w t r o u t w e r e i m m u n i s e d w i t h Aeromonas salmonicida in t h e p r e s e n c e or a b s e n c e of e i t h e r i n d o m e t h a c i n or the LO i n h i b i t o r , n o r d i h y d r o g u a i a r e t i c acid (NDGA) a n d the r e s u l t a n t t i t r e s of specific a n t i b o d y d e t e r m i n e d . U n l i k e t h e p r e v i o u s s t u d y by L a u d a n et al. (1986), h o w e v e r , t h e y f o u n d t h a t i n d o m e t h a c i n h a d a v a r i a b l e r e s p o n s e on a n t i b o d y g e n e r a t i o n f r o m i n h i b i t i o n to s t i m u l a t i o n (Table 2). I n h i b i t i o n of LO p r o d u c t g e n e r a t i o n by N D G A c a u s e d a dosed e p e n d e n t i n h i b i t o r y effect on a n t i b o d y s y n t h e s i s a g a i n s t A. salmonicida. A w e a k n e s s of the a p p r o a c h t a k e n in b o t h of t h e s e r e p o r t s is the use of i n h i b i t o r s of e i c o s a n o i d g e n e r a t i o n in vivo w h e r e it is i m p o s s i b l e to m o n i t o r the specificity of the a c t i o n of t h e s e drugs. A m o r e s u i t a b l e e x p e r i m e n t a l design is to e i t h e r c a r r y o u t s u c h e x p e r i m e n t s in in vitro m o d e l s y s t e m s or a l t e r n a t i v e l y

c~ tin

0

<

0

0

0

0

0 o9 c~

0

~

o:

0

~1

0

:n c~ 0

0

0

o

0 .,~

0 ..~

o c~

0 l:l ?:dO c~ 0 I[

o~

~

~ o

.~

~

I

c~

o,i

c~

:> CO 0

~.~o

~ o~

Z H Z

562

A. F. ROWLEY E T AL.

100 90 80 7O 60 % 50 40 30 2O 10 0

0

200

Conc. 16,16 DiMe PGE 2 (Bg kg -1)

Fig. 8. Effect of 16,16-dimethyl PGEe on the number of plaque forming cells (PFC) derived from spleens of rainbow trout vaccinated 15 days previously with sheep erythrocytes (1-25 x l0 s cells). Fish were injected every two days intraperitoneally with either saline or 200/lg kg ~ body weight 16,16-dimethyl PGEz. For further details see Knight & Rowley (1995). Mean values 4- S.D., N=3.

to a d m i n i s t e r eicosanoids to fish r a t h e r t h a n i n h i b i t o r s of t h e i r g e n e r a t i o n . This l a t t e r a p p r o a c h has the problem of e i c o s a n o i d stability as discussed previously in Section II, but stable a n a l o g u e s are available, such as 16,16dimethyl P G E 2, t h a t mimics the biologic a c t i v i t y of PGE,. T h e r e f o r e , a series of e x p e r i m e n t s were p e r f o r m e d to d e t e r m i n e if a d m i n i s t r a t i o n of prostaglandins causes any c h a n g e in the dynamics of a n t i b o d y f o r m a t i o n in the r a i n b o w trout, O. mykiss to e i t h e r A. salmonicida or sheep e r y t h r o c y t e s (SRBC) ( K n i g h t & Rowley, 1995). A clear i m m u n o s u p p r e s s i v e a c t i o n of 16, 16-dimethyl PGE2 on the g e n e r a t i o n of plaque forming cells (PFC) to SRBC and specific a n t i b o d y to A. salmonicida was observed (Fig. 8; Table 2). Similarly, c u l t u r e of t r o u t splenic l e u c o c y t e s with SRBC in the p r e s e n c e of the prostaglandins, PGE,, PGE:~ or 16,16-dimethyl PGE.,, caused a dose d e p e n d e n t i n h i b i t i o n of PFC, while the LO-products, LXA a and LTB~ (10 7_10- lo M) were w i t h o u t clear effect ( K n i g h t & Rowley, 1995). These results c o n t r a s t to e x p e r i m e n t s e x a m i n i n g the effect of PGs, LTB~ and LXA 4 on the p r o l i f e r a t i v e response of t r o u t l e u c o c y t e s from the h e a d k i d n e y to p h y t o h a e m a g g l u t i n i n - P where LTB 4 and LTBs significantly s t i m u l a t e d this response in a dosed e p e n d e n t m a n n e r (Secombes et al., 1994). F u r t h e r m o r e , the effect of lipoxins on l e u c o c y t e p r o l i f e r a t i o n a l t h o u g h smaller in m a g n i t u d e t h a n t h a t of LTB4/LTB.~ was still i n h i b i t o r y at b e t w e e n 1 0 - a and 1 0 - 7 M. P r o s t a g l a n d i n s E 2 and E:3, however, were p o t e n t i n h i b i t o r s of the mitogenic effect of p h y t o h a e m a g g l u t i n i n - P (Table 2).

FISH IMMUNE MODULATION

563

VII. Concluding R e m a r k s R e c e n t e x p e r i m e n t s as d e s c r i b e d in this r e v i e w h a v e r e v e a l e d t h a t eicosanoids c a n m o d u l a t e s o m e a s p e c t s of the i m m u n e r e s p o n s e in fish. H o w e v e r , t h e r e is still a p a u c i t y of i n f o r m a t i o n a v a i l a b l e on h o w e i c o s a n o i d s a c t d u r i n g these e v e n t s in fish. It is likely t h a t some of the i m m u n e m o d u l a t o r y effects of e i c o s a n o i d s o p e r a t e via c y t o k i n e e x p r e s s i o n in these animals. To date, no c y t o k i n e s h a v e b e e n fully c h a r a c t e r i s e d in fish (Secombes, 1994) a n d this s i t u a t i o n will h a m p e r f u r t h e r i n v e s t i g a t i o n s a t t e m p t i n g to p r o b e how e i c o s a n o i d s a l t e r the i m m u n e and i n f l a m m a t o r y r e a c t i o n s of fish. We wish to thank Merck Frosst for the kind provision of MK-886. The unpublished studies of the authors quoted in this review were supported by the Science and Engineering Research Council (grant no. GR/G05179) and the Agricultural and Food Research Council (grant no AG58/510). JK and JWH were supported by research studentships from the Science and Engineering Research Council and the Natural Environment Research Council respectively.

References Abramovitz, M., Wong, E., Cox, M. E., Richardson, C. D., Li, C. & Vickers, P. J. (1993). 5-Lipoxygenase-activating protein stimulates the utilization of arachidonic acid by 5-1ipoxygenase. European Journal of Biochemistry 215, 105 111. Brock, T. G., Paine, R. III, Peters-Golden, M. (1994). Localization of 5-1ipoxygenase to the nucleus of unstimulated rat basophilic leukemic cells. Journal of Biological Chemistry 269, 22059 22066. Browning, J. L. & Ribolini, A. (1987). Interferon blocks interleukin 1-induced prostaglandin release from human peripheral monocytes. Journal of Immunology 138, 2857-2863. Camp, R. D., Wollard, P. M., Mallet, A. I., Fincham, N. J., Ford-Hutchinson, A. W. & Bray, M. A. (1982). Neutrophil aggregating and chemokinetic properties of 5,12,20-trihydroxy-6,8,10,14-eicosatetraenoic acid. Prostaglandins 23, 631-64]. Claessom H.-E., Odlander, B. & Jakobsson. P.-J. (1992). Leukotriene B~ in the immune system. International Journal of hnmunopharmacology 14, 441 449. Conrad, D. J., Kfihn, H.. Mulkins. M., Highland, E. & Sigal, E. (1992). Specific inflammatory cytokines regulate the expression of human monocyte 15-1ipoxygenase. Proceedings of the National Academy of Sciences, U.S.A. 89, 217-222. Daculsi, R., Vaillier, D. & Gualde, N. (1993). Regulation by PGE., of IL-2, IL-3 and IFN production by cortico-resistant thymocytes. Immunology Letters 38, 229-235. Dixon, R. A. F., Diehl, R. E., Opas, E., Rands, E., Vickers. P. J., Evans, J. F., Gillard, J. W. & Miller, D. K. (1990). Requirement of a 5-1ipoxygenase-activating protein for leukotriene synthesis. Nature (London) 343, 282-284. Ford-Hutchinson, A. W., Gresser, M. & Young, R. N. (1994). 5-Lipoxygenase. Annual Review of Biochemistry 63, 383-417. Gillard, J., Ford-Hutchinson, A. W.. Chan, C., Charleson, S., Denis, D., Foster, A., Fortin, R., Leger, S.. McFarlane, C. S., Morton, H., Piechuta, H., Riendeau, D., Rouzer, C. A., Rokach, J., Young, R., MacIntyre, D. E., Peterson, L., Bach, T., Eiermann, G., Hopple, S., Humes, J., Hupe, L., Luell, S., Metzger, J., Meurer, R., Miller, D. K., Opas, E. & Pacholok, S. (1989). L-663,536 (MK886) (3-[1-(4chlorobenzyl)-3-t-butyl-thio-5-isopylindol-2-yl]-2,2-dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor. Canadian Journal of Physiology and Pharmacology 67, 456-464.

564

A.F. ROWLEY ET AL.

Goetzl, E. (1981). Selective feed-back inhibition of the 5-1ipoxygenation of arachidonic acid in human T lymphocytes. Biochemical and Biophysics Research Communications 101, 344-350. Goldyne, M. (1988). Lymphocytes and arachidonic acid metabolism. In Arachidonate Metabolism in Immunologic Systems (L. Levine, ed.) pp. 140-152. Basel: Karger. Goodwin, J. S. & Ceuppens, J. (1983). Regulation of the immune response by prostaglandins. Journal of Clinical Immunology 3, 295-315. GranstrSm, E. & Kumlin, M. (1987). Metabolism of prostaglandins and lipoxygenase products: relevance for eicosanoid assay. In Prostaglandins and Related Substances: A Practical Approach (C. Beneddetto, R. G. McDonald-Gibson, S. Nigam & T. F. Slater, eds). pp 5-27. Oxford: IRL Press. Hamberg, M., Svensson, J. & Samuelsson, B. (1975). Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proceedings National Academy of Sciences, U.S.A. 72, 2994-2998. Hancock, W. W., Pleau, M. E. & Kobzik, L. (1988). Recombinant granulocytemacrophage colony-stimulating factor down-regulates expression of IL-2 receptor on human mononuclear phagocytes by induction of prostaglandin E. Journal of Immunolog~y 140, 3021-3025. Hansson, G., Lindgren, J.X~., Dahl6n, S.-E., Hedquist, P. & Samuelsson, B. (1981). Identification and biological activity of novel U-oxidized metabolites of leukotriene B4 from human leukocytes. Federation of European Biochemical Societies Letters 130, 107-112. Hart, P. H., Whitty, G. A., Piccoli, D. S. & Hamilton, J. A. (1989). Control by IFN- 7 and PGE.~ of TNFa and IL-1 production by human monocytes. Immunology 66, 376-383. Henderson, R. J. & Sargent, J. R. (1985). Fatty acid metabolism in fish. In Nutrition and Feeding in Fish (C. B. Cowey, A. M. Mackie & J. G. Bell, eds.) pp. 349-364, London: Academic Press. Hulkower, K. I., Wertheimer, S. J., Levin, W., Coffey, J. W., Anderson, C. M., Chen, T., DeWitt, D. L., Crowl, R. M., Hope, W. C. & Morgan, D. W. (1994). Interleukin -lfl induces cytosolic phospholipase A2 and prostaglandin H synthase in rheumatoid synovial fibroblasts. Arthritis and Rheumatism 37, 653-661. Jakobsson, P.-J., Steinhilber, D., Odlander, B., R~dmark, O., Claesson, H.-E. & Samuelsson, B. (1992). On the expression and regulation of 5-1ipoxygenase in human lymphocytes. Proceedings of the National Academy of Sciences, U.S.A. 89, 3521-3525. Kester, M., Coroneos, E., Thomas, P. J. & Dunn, M. J. (1994). Endothelin stimulates prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis through a tyrosine kinase-signaling pathway in rat mesangial cells. Journal of Biological Chemistry 269, 22574-22580. Knight, J. (1994). Distribution and functions of lipoxins and other eicosanoids in the rainbow trout, Oncorhynchus mykiss. PhD Dissertation, University of Wales. Knight, J. & Rowley, A. F. (1995). Immunoregulatory activities of eicosanoids in the rainbow trout, Oncorhynchus mykiss. Immunology 85, 389-393. Knight, J., Lloyd-Evans, P., Rowley, A. F. & Barrow, S. E. (1993). Effect of lipoxins and other eicosanoids on phagocytosis and intracellular calcium mobilisation in rainbow trout (Oncorhynchus mykiss) leukocytes. Journal of Leukocyte Biology 54, 518-522. Knight, J., Holland, J. W., Bowden, L. A., Halliday, K. & Rowley, A. F. (1995). Eicosanoid generating capacities of different tissues from the rainbow trout, Oncorhynchus mykiss. Lipids 30, 451-458. Knudsen, P. J., Dinarello, C. A. & Strom. T. B. (1986). Prostaglandins posttranscriptionally inhibit monocyte expression of interleukin 1 activity by increasing intracellular cyclic adenosine monophosphate. Journal of Immunology 137, 3189-3194. Laudan, R., Stolen, J. & Call, A. (1986). Immunoglobulin levels of the winter flounder (Pseudopleuronectes americanus) and the summer flounder (Paralichthys

FISH IMMUNE MODULATION

565

dentatus) injected with the microsporidan parasite Glugea stephani. Developmental & Comparative Immunology 10, 331-340. Lee, S. H., Soyoola, E., Chanmugam, P., Hart, S., Sun, W., Zhong, H., Liou, S., Simmons, D. & Hwang, D. (1992). Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. Journal of Biological Chemistry 267, 25934-25938. Lloyd-Evans, P. L., Barrow, S. E., Hill, D. J., Bowden, L. A., Rainger, G. E., Knight, J. & Rowley, A. F. (1994). Eicosanoid generation and effects on the aggregation of thrombocytes from the rainbow trout, Oncorhynchus mykiss. Biochimica et Biophysica Acta 1215, 291-299. Mancini, J. A., Abramovitz, M., Cox, M. A., Wong, E., Charleson, S., Perrier, H., Wang, Z., Prasit, P. & Vickers, P. (1993). 5-Lipoxygenase-activating protein is an arachidonate binding protein. Federation of European Biochemical Societies Letters 318, 277-281. Manning, M. J. (1994). Fishes. In Immunology a Comparative Approach (R. J. Turner, ed.) pp. 69-100. Chichester: J. Wiley & Sons. Martinez, J. M., Chapunoff, D., Romero, M. A. & Herman, C. A. (1994). Eicosanoid synthesis by warm- and cold-acclimated American bullfrog (Rana catesbiana) brain. Journal of Experimental Zoology 269, 298-307. Masferrer, J. L., Seibert, K., Zweifel, B. & Needleman, P. (1992). Endogenous glucocorticoids relgulate an inducible cyclooxygenase enzyme: Proceedings of the National Academy of Sciences, U.S.A. 89, 3917-3921. Miller, D. K., Gillard, J. W., Vickers, P. J., Sadowski, C., Leveille, J. A., Mancini, P., Charleson, P., Dixon, R. A. F., Ford-Hutchinson, A. W., Fortin, R., Gauthier, J. Y., Rodkey, J., Rosen, R., Rouzer, C. A., Sigal, I. S., Strader, C. D. & Evans, J. F. (1990). Identification and isolation of a membrane protein necessary for leukotriene biosynthesis. Nature (London) 343, 278-281. Minakuchi, R., Wacholtz, M. C., Davis, L. S. & Lipsky, P. E. (1990). Delineation of the mechanism of inhibition of human T cell activation by PGE.,. Journal of Immunology 145, 2616-2625. Mizukami, Y., Sumimoto, H., Isobe, R., Minakami, S. & Takeshige, K. (1994). t)-Oxidation of lipoxin B., by rat liver. Identification of an ~-carboxy metabolite of lipoxin B~. European Journal of Biochemistry 224, 959-965. Murakami, M., Matsumoto, R., Austen, K. F. & Arm, J. P. (1994). Prostaglandin endoperoxide synthase-1 and -2 couple to different transmembrane stimuli to generate prostaglandin D,, in mouse bone marrow-derived mast cells. Journal of Biological Chemistry 269, 22269-22275. Pettitt, T. R. & Rowley, A. F. (1991). Fatty acid composition and lipoxygenase metabolism in blood cells of the lesser spotted dogfish, Scyliorhinus canicula. Comparative Biochemistry and Physiology 99B, 647-652. Pettitt, T. R., Rowley, A. F. & Secombes, C. J. (1989a). Lipoxins are major lipoxygenase products of rainbow trout macrophages. Federation of European Biochemical Societies Letters 259, 168-170. Pettitt, T. R., Rowley, A. F. & Barrow, S. E. (1989b). Synthesis of leukotriene B and other conjugated triene lipoxygenase products by blood cells of the rainbow trout, Sahno gairdneri. Biochimica et Biophysica Acta 1603, 1-9. Pettitt, T. R., Rowley, A. F., Barrow, S. E., Mallet, A. I. & Secombes, C. J. (1991). Synthesis of lipoxins and other lipoxygenase products by macrophages from the rainbow trout, Oncorhynchus mykiss. Journal of Biological Chemistry 266, 8720-8726. Phipps, R. P., Stein, S. H. & Roper, R. L. (1991). A new view of prostaglandin E regulation of the immune response. Immunology Today 12, 349-352. Powell, W. S. (1984). Properties of leukotriene B4 20-hydroxylase from polymorphonuclear leukocytes. Journal of Biological Chemistry 259, 3082-3089. Powell, W. S., Gravelle, F. & Gravel, S. (1992). Metabolism of 5(S)-hydroxy6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific

566

A.F. ROWLEY ET AL.

dehydrogenase in human polymorphonuclear leukocytes. Journal of Biological Chemistry 267, 19233-19241. Powell, W. S., Gravel, S., MacLeod, R. J., Mills, E. & Hashefi, M. (1993). Stimulation of human neutrophils by 5-oxo-6,8,11,14-eicosatetraenoic acid by a mechanism independent of the leukotriene B~ receptor. Journal of Biological Chemistry 268, 928~9286. Rainger, G. E., Rowley, A. F. & Pettitt, T. R. (1992). Effect of inhibitors of eicosanoid biosynthesis on the immune reactivity of the rainbow trout, Oncorhynchus mykiss. Fish & Shellfish Immunology 2, 143-154. Reddy, S. T. & Herschman, H. R. (1994). Ligand-induced prostaglandin synthesis requires expression of TIS10/PGS-2 prostaglandin synthase gene in murine macrophages and fibroblasts. Journal of Biological Chemistry 269, 15473-15480. Reid, G. K., Kargman, S., Vickers, P. J., Mancini, J. A., Leveille, C., Ethier, D., Miller, D., Gillard, J. W., Dixon, R. A. & Evans, J. F. (1990). Correlation between expression of 5-1ipoxygenase activating protein, 5-1ipoxygenase and cellular leukotriene synthesis. Journal of Biological Chemistry 265, 19818-19823. Reise, J., Hoff, T., Nordhoff, A., DeWitt, D. L., Resch, K. & Kaever, V. (1994). Transient expression of prostaglandin endoperoxide synthase-2 during mouse macrophage activation. Journal of Leukocyte Biology 55, 476-482. Rowley, A. F. (1991). Lipoxin formation in fish leucocytes. Biochimica et Biophysica Acta 1084, 303-306. Rowley, A. F., Barrow, S. E. & Hunt, T. C. (1987). Preliminary studies on eicosanoid production by fish leucocytes using GC-mass spectrometry. Journal of Fish Biology 31A, 107-111. Rowley, A. F., Lloyd-Evans, P. L., Barrow, S. E. & Serhan, C. N. (1994). Lipoxin biosynthesis by trout macrophages involves the formation of epoxide intermediates. Biochemistry 33, 856-863. Secombes, C. J. (1994). The phylogeny of cytokines. In The Cytokine Handbook, 2nd. edn. (A. Thomson, ed.) pp. 567-594. London: Academic Press Inc. Secombes, C. J., Clements, K., Ashton, I. & Rowley, A. F. (1994). The effect of eicosanoids on rainbow trout, Oncorhynchus mykiss, leucocyte proliferation. Veterinary Immunology & Immunopathology 42, 367 378. Serhan, C. N. (1994). Lipoxin biosynthesis and its impact in inflammatory and vascular events. Biochimica et Biophysica Acta 1212, 1-25. Serhan, C. N., Fiore, S., Brezinski, D. A. & Lynch, S. (1993). Lipoxin A,, metabolism by differentiated HL-60 cells and human monocytes: conversion to novel 15-oxo and dihydro products. Biochemistry 32, 6313-6319. Sirois, J. (1994). Induction of prostaglandin endoperoxide synthase-2 by human chorionic gonadotrophin in bovine preovulatory follicles in vivo. Endocrinology 135, 841-848. Smith, W. L. (1989). The eicosanoids and their biochemical mechanisms of action. Biochemical Journal 259, 315-324. Stafikov~, J., Gagnon, N. & Rola-Pleszczynski, M. (1992). Leukotriene B4 augments interleukin-2 receptor-beta (IL-2Rfl) expression and IL-2Rfl-mediated cytotoxic response in human peripheral blood lymphocytes. Immunology 76, 258 263. Steinhilber, D., Hoshiko, S., Grunewald, J., Radmark, O. & Samuelsson, B. (1993). Serum factors regulate 5-1ipoxygenase activity in maturing HL-60 cells. Biochimica et Biophysica Acta 1178, 1-8. Teixeira, F. J., Zakar, T., Hirst, J. J., Guo, F., Sadowsky, D. W., Machin, G., Demianczuk, N., Resch, B. & Olson, D. M. (1994). Prostaglandin endoperoxide-H synthase (PGHS) activity and immunoreactive PGHS-1 and PGHS-2 levels in human amnion throughout gestation, at term, and during labor. Journal of Clinical Endocrinology and Metabolism 78, 1396-1402. Tocher, D. R. & Sargent, J. R. (1987). The effect of calcium ionophore A23187 on the metabolism of arachidonic and eicosapentaenoic acids in neutrophils from a marine teleost rich in (n-3) polyunsaturated fatty acids. Comparative Biochemistry and Physiology 87B, 733-739.

FISH IMMUNE MODULATION

567

Vickers, P. J., O'Neill, G. P., Mancini, J. A., Charleson, S. & Abramovitz, M. (1992). Cross-species comparison of 5-1ipoxygenase-activating protein. Molecular Pharmacology 42, 1014-1019. Woods, J., Evans, J. F., Ethier, D., Scott, S., Vickers, P. J., Hearn, L., Heibein, J. A., Charleson, S. & Singer, I. I. (1993). 5-Lipoxygenase and 5-1ipoxygenase activating protein are localized in the nuclear envelope of activated human leukocytes. Journal of Experimental Medicine 178, 1935 1946. Yamaoka, K. A., Cla~sson, H.-E. & Ros6n, A. (1989). Leukotriene B, enhances activation, proliferation and differentiation of human B lymphocytes. Journal of Immunology 143, 1996-2000.