Elevation of rainbow trout Oncorhynchus mykiss respiratory burst activity with macrophage-derived

Seon I. Jang, Laura J. Hardie, and Christopher

Tillydrone

macrophage supernatants

J. Secombes
Avenue, Aberdeen United Kingdom

Department

ofZoology,

University

ofAberdeen,

Abstract: A variety of supernatants were prepared by stimulating rainbow trout Oncorhynchus mykiss head kidney macrophages with lipopolysaccharide (LPS), tumor necrosis factor a (TNF-ct), or a leucocyte-derived macrophage-activating factor (1-MAF), individually and in combination. If generated using a 12-h stimulation period, such supernatants were found to elevate significantly the respiratory burst activity of target macrophages; that is, they contained a macrophage-derived MAF (m-MAF), but supernatants generated using a shorter incubation penod showed no significant activity. Combinations of these treatments were particularly effective in generating m-MAF--containing supernatants. The elevation of respiratory burst activity by supernatants generated using combined treatments could be partially inhibited by prior treatment of the target macrophages with anti-TNF-cz receptor 1 (TNFR1) monoclonal antibodies (mAbs). Similarly, treatment of macrophages with combinations of 1-MAF and m-MAF generated supernatants with potent m-MAF activity and this activity was partially inhibited by prior treatment of the target cells with anti-TNFR1 mAb. In addition, the presence of anti-transforming growth factor 13i (TGF-1) serum while generating these latter supernatants resulted in significantly increased m-MAF activity. Such data suggest that fish leukocytes secrete a variety of potent macrophage-activating (TNF-a) and -deactivating (TGF-f3) factors. J. Leukoc. Biol. 57: 943-947; 1995.
Key

Words: bow trout

macrophages TNF-ct

MAF

respiratory

burst

rain-

INTRODUCTION It is well established that fish macrophages can be activated in vivo and in vitro for enhanced bactericidal activity [i, 2]. Although many factors are known to induce this phenomenon, the mechanism(s) of in vivo activation has still to be elucidated. On the other hand, it is clear that in vitro this can be brought about by one or more molecules, termed macrophage-activating factors (MAFs), released from head kidney and peripheral blood leukocytes [3]. Several lines of evidence suggest that fish T cells release factors with MAF activity. MAF is released following stimulation of leucocytes with a T cell mitogen (concanavalin A) [3], and its release in response to specific antigen can be enhanced by prior immunization [4], the hallmark of lymphocyte responses. MAF release following Con A stimulation is temperature sensitive [5], as are other knOwn activities of fish T cells [6], and removal of surface immunoglobulin-negative cells by panning prevents MAY release [7]. In mammals, the predominant

MAF present in supernatants from mitogen-stimulated leukocytes is interferon-y (IFN-y) [8]. Similarly, the MAF activity in supernatants from mitogen-stimulated trout leukocytes cofractionates with IFN activity, and both activities share similar temperature and pH sensitivities, suggesting that a cytokine akin to IFN-y is released from fish T cells [9]. Cytokines from other leucocyte types can also activate mammalian macrophages, including macrophage products themselves [iO]. In particular, tumor necrosis factor a (TNFa) is a potent autocrine signal [1 1], able to elevate macrophage respiratory burst [12] and microbicidal activity [i3], and to synergise with IFN-y for the induction of microbicidal [14, 15] and tumoricidal activity [16, i7]. Recently, it has been shown that rainbow trout lymphocytes, macrophages, and neutrophils can respond to recombinant human TNF-cz [i8, 19] and that these responses can be inhibited by prior treatment of the macrophages with monoclonal antibodies (mAbs) to the 55-kDa TNF-a receptor (TNFR1) [19]. Such findings strongly suggest that this TNFR1 has been conserved within the vertebrates. In addition, responsiveness to MAF activity in trout leukocyte supernatants can be partially inhibited by treatment of test macrophages with the anti-TNFRi mAb [19]. This implies that either TNF-a was present in such supernatants as a component of the MAF activity or that it was released from MAF-stimulated macrophages. To investigate these observations further, the present study was undertaken to confirm whether trout macrophages can be triggered to release factors with MAF activity, following incubation with a variety of stimulatory signals. In addition, the ability of anti-TNFR1 mAb to inhibit the activities of such supernatants was studied to elucidate, indirectly, whether fish macrophage-derived MAY (m-MAF) activity involves a TNF-a-like molecule.

Abbreviations: Con A, concanavalin A; FCS, fetal calf serum; HBSS, Hanks balanced salt solution; IFN-y, interferon-i 1-MAF, leukocyte-derived MAF; LPS, lipopolysaccharide; mAb, monoclonal antibody; MAF, macrophage-activating factor; m-MAF, macrophage-derived MAF; PMA, phorbol myristate acetate; TCF-f31 transforming growth factor i; TNF-a, tumor necrosis factor a; TNFR1, TNF.a receptor. Reprint requests: Christopher J. Secombes, Department of Zoology, University ofAberdeen, Tillydrone Avenue, Aberdeen, AB9 2TN, United Kingdom. Seon I. Jangs present address: Department of Aquaculture, National Fisheries University of Pusan, Republic of Korea. Received May 2, 1994; acceptedJanuary 25, 1995.

Journal

of Leukocyte

Biology

Volume

57, June

1995

943

MATERIALS

AND

METHODS

Fish
Rainbow trout from College kept at 14*C
were acclimatized

Oncorhynchus mykiss, weighing 300-500 g, were obtained Mill Trout Farm, Almond Bank, Perthshire. They were and fed twice daily on a commercial pelleted diet. Fish to the aquarium system for at least 2 weeks prior to

use.

Isolation
scribed

of head
previously

kidney
head
[20].

macrophages
kidney cell
head

Phagocyte-ennched

suspensions
kidney leukocyte

were

obtained
suspensions

as depre-

Briefly,

pared in Leibovitz medium (L15, Gibco) were separated on a 34%/51% Percoll density gradient, and the phagocyte-enriched fraction at the interface was collected. These cells were washed twice in L15, adjusted to I x io viable cells/ml L15 plus 0.1% fetal calf serum (FCS, Gibco), and either 100 p1 was added to wells of a 96-well tissue culture plate (Nunc) or I ml was added to wells of a 24-well plate (Nunc), in triplicate per treatment. Following an adherence period (3 h at 18C), the cells were washed and incubated in L15 medium plus 5% FCS overnight at 18C. Macrophages were washed vigorously with L15 medium three times prior to use, leaving approximately 20% of the cell number in the wells and giving a macrophage purity of 90-95%.

with the target macrophages for 1 h, at 6 and 0.45 pg/mI, respectively, before addition of the macrophage supernatants. These mAbs have their activity ablated in the presence of soluble TNFR1 (personal observation) and are able to neutralize the responsiveness of trout macrophages to human TNF-a at the above concentrations [19]. Other mAbs used alongside 5R2 and 5R16 have no effect on trout macrophage respiratory burst activity [19]. Macrophage respiratory burst activity was assessed via the reduction of ferricytochrome c by released superoxide anion (02), following stimulation of the cells with phorbol myristate acetate (PMA, Sigma) [20]. Macrophages were washed twice in phenol red-free Hanks balanced salt solution (HBSS, Gibco) before 100 tl of 2 mg/ml fen-icytochrome c plus 1 tg/ml PMA in phenol red-free HBSS was added to triplicate wells per treatment. Macrophages incubated with PMA and superoxide dismutase (Sigma), at 300 IU/ml, were used as the blank. Optical density values were taken at 550 nm after 30 mm, on a multiscan spectrophotometer (MDC), and converted to nmole 02 according to Pick [22]. Data were analyzed by one-way analysis of variance and Students t-test.

RESULTS
As can be macrophages

seen
with

in Figure

1,

incubation

Production
Three phages; Sigma),

of macrophage

supernatants
macro(LPS,

respiratory

burst

types of treatment were used to stimulate head kidney Escherichia coli (serotype 01 1 1 :B4) lipopolysaccharide recombinant human tumor necrosis factor a (TNF-a,
and supernatants from mitogen-stimulated trout

British
head

Biotechnology)

kidney .leukocytes. The trout leukocyte supernatants could increase macrophage respiratocy burst activity and thus were deemed to contain leukocyte-derived MAF (1-MAF) activity [3]. The concentrations of LPS that were stimulatory for trout macrophages were first determined on macrophage monolayers in 96-well plates from five fish. After washing the cells three times with L15 medium, 100 il of medium containing 0.1-100 jig LPS/ml L15 plus 5% FCS was added to triplicate wells per concentration, and their respiratory burst activity was analyzed (as described below) following a 24- or 48-h incubation at 18C. Subsequently, LPS was used only at a concentration of 50 pg/ml to stimulate macrophages. Concentrations of TNF-a and 1-MAY supernatants deemed to have optimal stimulatory effects on trout macrophages have been determined previously [19] and were 25 IU/ml and a dilution of 1:4, respectively. In experiments to investigate whether trout macrophages could release factors with MAY activity, macrophages in 24-well plates, from four to five fish, were stimulated with MAF, LPS, and ThF-a alone, or in various combinations (at the above concentrations), in triplicate per treatment. Macrophages incubated with medium were used to generate control supernatants. The macrophages were then cultured for 3, 6, and 12 h at 18C before being washed five times with Li5 medium warmed to 18C and cultured for a further 24 h in the absence of exogenous stimuli. Finally, the supernatants from these macrophages were collected after centrifugation of the culture medium at 400g for 30 mm at 4C and stored at -70C prior to testing for m-MAF activity. In the same manner, generated m-MAF supernatants deemed to be the most stimulatory for target macrophages (see below) were diluted 1:4 and used in combination with l-MAF supernatants to stimulate macrophages for 12 h (i.e., to give a potential mixture of fish lymphocyte and macrophage cytokines). The macrophages were then washed and supernatants harvested 24 h later as described above. Finally, in one set of such experiments a chicken antiporcine transforming growth factor I3 (TGF-31) gamma globulin preparation (British Biotechnology) at 2 tg/ml was added together with the above supernatants to generate m-MAF, because TGF4I1 is a potent macrophage-deactivating factor that could be present in the 1-MAF supernatants. Indeed, studies have shown that fish macrophages can respond to natural bovine TGF-1 and that this responsiveness is inhibited by preincubation of the TCF431 with the above antibody [21]. The anti-TCF-1 gamma globulin preparation had no effect on macrophage respiratory burst activity in the absence of TGF-31.

strated a significant 48 h of culture in the presence of LPS, with maximal increases seen using 50 tg/ml for 24 h. At both incubation times higher concentrations of LPS (100 jig/ml) gave significantly lower (P < .05) respiratory burst activity relative to levels seen using 50 j.tg/ml, although the nanomoles 02 produced were still significantly (P < .05) above background levels in the absence of LPS. Incubation of target macrophages for 24 h with I :4 diluted macrophage supernatants from control cultures had a small but significant (P < .05) impact on respiratory burst activity compared with target macrophages incubated with medium alone (Figs. 2-5). Incubation of target macrophages for 24 h with 1:4 diluted supernatants from macrophages stimulated for 3 and 6 h with LPS, TNF-a and 1-MAF alone and in various combinations had no significant impact on respiratory burst activity relative to control supernatants (Fig. 2). However, incubation with 1:4 diluted supernatants prepared by stimulating macro-

LPS had a significant activity. Analysis of dose effect (P < .01)

of head kidney impact on their variance demonafter both 24 and

Detection

of MAF activity LPS (tg/ml)

Fig. 1. Respiratory burst activity of rainbow trout head kidney phages incubated with varying concentrations of E. coli LPS for 48 h prior to stimulation with PMA for 30 mm. Data are means five fish. macro24 and + SE for

m-MAF activity was assessed by incubating test supernatants with target macrophages in a 96-well plate, prior to determination of their respiratory burst activity. Supernatants were diluted from 1:2 to 1:32 in L15 medium plus 5% FCS and 100-tl aliquots added to washed macrophages (as for LPS above) in triplicate for 24 h at 18C. In some experiments, two anti-TNFRI mAbs, 5R2 and 5R16 (Celltech), were preincubated

944

Journal

of Leukocyte

Biology

Volume

57, June

1995

03h
6h

of

12

a
0

0
0.

0
U) 0

E
C

the different treatments had the greatest impact on respiratory burst activity relative to control supernatants, as evidenced by higher respiratory burst activity. In addition, with the exception of l-MAF versus 1-MAF + LPS, all paired comparisons between single and combined treatments were significantly (P < .05) different. Furthermore, dilution of the supernatants revealed that those from macrophages given combined treatments retained their ability to elevate significantly respiratory burst activity of target macrophages beyond that of supernatants from macrophages given a single treatment (Fig. 3). However, the use of all three treatments to stimulate macrophages did not generate supernatants with more m-MAF activity compared with those from macrophages given two treatments (Figs. 2 and 3).
Prior exposure mAb significantly of target macrophages with anti-TNFR1

E
0

U.0

0 C

(1)0 cC _J.

LL

LLCJ)

LLSC1)

from macrophages treatments to elevate ity (Fig. 4). Anti-TNFR1

reduced stimulated
macrophage

the ability of supernatants for 12 h with the combined

respiratory burst activ-

exposure

did

not

result despite activity

in signifi-

<

<
+

<a.

<,o.
+

g
0. U)

cant

reductions
given to the

in

activity
single low or

using
treatments, insignificant

supernatants

from
a similar of such

macrophages trend, due

supernatants.

Supernatants
Fig. 2. Respiratory burst activity of rainbow trout head kidney (target) macrophages incubated for 24 h with medium alone or 1:4 diluted macrophage supernatants prior to stimulation with PMA for 30 mm. The macrophage supernatants were prepared by incubating head kidney macrophages for 3, 6, or 12 h with medium (control supernatant) or various combinations of 1:4 diluted 1-MAF, 25 lU/mI TNF-a, and 50 pg/mI LPS. Following this incubation period the macrophages were washed and the supernatants harvested 24 h later. The data are means + SE for four fish. *J < #{149}#{216}5, **P < #{216}, and *** < .001 compared with the respiratory burst activity of macrophages incubated with control supernatants.

from

macrophages

stimulated

with

com-

binations of trout 1-MAFand m-MAF-containing supernatants also possessed m-MAF activity. Thus, they were able to increase significantly the production of 02 by target macrophages above that of macrophages incubated with control supernatants (Fig. 5). Indeed, these supernatants were more active than supernatants from macrophages stimulated with 1-MAF alone used in the same experiment (P < .05), and as above only the supernatants generated using the combined supernatants were significantly affected by prior treatment of the target macrophages with anti-TNFR1 mAb. The addition of

phages

for 12 h did significantly respiratory burst activity of target eral, supernatants from macrophages

increase

(P < .05) the macrophages. In gengiven combinations

anti-TGF-1
also had

serum
a significant

when
impact

generating
(P
<

these
.05) on

supernatants
their ability to

elevate

macrophage

respiratory

burst

activity

(Fig. 5). The

g Medium alone
U Control
I-MAF 8 supernatant (1:4)

LPS(50tg/mI)
+

U l-MAF

TNF-a
LPS

i;

I-MAF
I-MAF

+ +

TNF-cz

LPS

0 0 0 0 0

0 0.4 Fig. 3. Respiratory burst activity of rainbow trout head kidney (target) macrophages incubated for 24 h with medium alone or varying dilutions ofmacrophage supernatants prior to stimulation with PMA for 30 mm. The macrophage supernatants were prepared by incubating head kidney macrophages for 12 h with medium (control supernatant) or various combinations of 1:4 diluted 1-MAF, 25 lU/mI TNF-a, and 50 ig/ml LPS. Following this incubation period the macrophages were washed and the supernatants harvested 24 h later. The data are means + SE for five fish. * < 05, ** < and *** < .001 compared with the respiratory burst activity of macrophages incubated with control supernatants.

0
U)

0 0

E
C

0 1:8
Dilution

fang

et al.

Elevation

of

trout

macrophage

respiratory

burst

activity

945

0 Cells alone

D U
0

Cells Cells

+ +

5R16 5R2

m-MAF supernatants was at least as high as those seen with 1-MAF, which result in increased bactericidal activity against the fish pathogen Aeromonas salmonicida [3]. The activity in the macrophage supernatants generated with combined treatments was significantly inhibited by treatment of the target macrophages with an anti-TNFR1 mAb. Thus, part of their MAF activity is potentially due
to released

0 C)

TNF-ct.

It

has

been

shown

previously [18,

0.

0
U)

E
C

trout leukocytes can respond to human TNF-a and that the anti-TNFR1 mAb used in the present could inhibit these responses [19]. Furthermore, of anti-TNFR1 mAb has no effect on the increased ratory burst activity induced directly by Con A (personal observation), ruling out the possibility

that 19]

study addition respior LPS that cell

surface binding of any antibody ity of these cells. Such studies

TNFR plies
E0
CU U.Q

could decrease the activsuggest that at least one

leukocytes, which itself im-

is conserved on that a TNF-ct-like

trout

g
LA.
0
_j .0

LL.u <
-

a-C) LL +

<

_
<a.

LL.t?CI)

trout
warranted.

leukocytes,

molecule may be released from although more definitive proof is clearly

0. U)

-I-.

Fig. 4. Respiratocy burst activity of rainbow trout head kidney (target) macrophages incubated for 24 h with medium alone or 1:4 diluted macrophage supernatants prior to stimulation with PMA for 30 mm. The macrophage supernatants were prepared by incubating head kidney macrophages for 12 h with medium (control supernatant) or various combinations of 1:4 diluted 1-MAY, 25 lU/mi TNF-a, and 50 tg/ml LPS.
Following this incubation period the macrophages were washed and the

supernatants harvested 24 h later. In some cases the target macrophages were preincubated with anti-TNFR1 mAb for I h at 6 tg/ml(5R2) or 0.45 ig/ml (5R16) before addition ofthe macrophage supernatants. The data are means + SE for four fIsh. *J < 05 and ** < .01 compared with the respiratory burst activity of macrophages incubated with the respective supernatants without anti-TNFR1 treatment.

That human TNF-cZ was used to generate some of these supernatants in the present study is also cause for caution, because it is not possible to exclude entirely the potential carryover of some human TNF-cz in the macrophage supernatants. However, supernatants generated using TNF-cz alone were clearly not very stimulatory, nor inhibitable by anti-TNFR1 mAb, and some combinations of treatments lacking TNF-a (e.g., 1MAF-containing supernatants plus LPS) were as stimulatory as those generated with TNF-cx. TNF-cx is well known to synergize with other cytokines [23, 24], and with respect to effects on macrophages it can 10

0 Cells alone
DCells
+ +

I Cells
presence of anti-TGF-1 serum produced with significantly more m-MAF activity (P respective supernatants generated without serum [e.g., 1-MAF + m-MAF (b) in Fig. 5].
<

5R16 5R2

supernatants .05) than the

i

7.5

5.

DISCUSSION
2.5 It seems

remarkably easy to induce rainbow trout macrophages to release factors that have an autocrine effect on their respiratory burst activity. Although it is true that other cells were present in the cultures in addition to the macrophages, the small percentage of contaminating leukocytes maximally represents 2 x io lymphocytes per well, and this has been shown previously to be insufficient

E0
CU

u-0
<C

+ <Li.

+ U. <LI

+ <LI-

Q
0.
(I)

<

<
.J.

<

to generate

bioassay-detectable

1-MAF

supernatants

[7].
Fig. 5. Respiratory
macrophages macrophage macrophage macrophages 1-MAF plus

Thus, macrophages do appear to be the source of these factors. A range of treatments were used to stimulate the release of these factors, including bacterial products (LPS), a recombinant cytokine (TNF-a) known to cross-react with trout leukocytes [18, 19], and a trout leukocyte supernatant known to activate trout macrophages [3, 21]. Although these treatments were effective when used individually, the most active supernatants were generated using these treatments in pairs. However, combining all three treatments did not elevate the m-MAF activity of the generated supernatants further, although this could have been due to a limit on the maximum response possible by the fixed number of target cells present per well. The increase in respiratory burst activity induced by the latter

kidney (target) or I :4 diluted for 30 mm. The head kidney or 1:4 diluted 1:4. The m-MAF supernatants were prepared by stimulating head kidney macrophages for 12 h with 1:4 diluted l-MAF plus 25 IU/ml TNF.a (a), a plus 50 tg/ml LPS (b), or b plus 2 jig/ml rabbit anti-TGF-31 serum (c) In all cases, following the 12-h incubation period the macrophages were washed and the supernatants harvested 24 h later. In some cases the target macrophages were preincubated with anti-TNFR1 mAb for 1 h at 6 tg/ml(5R2) or 0.45 tg/ml (5R16) before addition of the macrophage supernatants. The data are means + SE for five fish. * < 05 and ** < .01 compared with the respiratory burst activity of target macrophages incubated with the respective supernatants without anti-TNFRI treatment.

burst activity of rainbow trout head incubated for 24 h with medium alone supernatants prior to stimulation with PMA supernatants were prepared by incubating for 12 h with medium (control supernatant) various m-MAF supernatants (a, b, c) diluted

946

Journal

of

Leukocyte

Biology

Volume

57,

June

1995

synergize with IFN-y to increase microbicidal [14, 15] and tumoricidal activity [16, 17]. Human TNF-a has also been shown to synergize with trout 1-MAY-containing supernatants to enhance macrophage respiratory burst activity [18]. Because these leukocyte supernatants contain IFN activity that coelutes with MAF activity, it has been suggested previously that their MAF activity may be mediated via an IFN-y-like molecule [9] and hence the synergy with human TNF-a. Thus, in the present study the ability of these two types of trout supernatant, potentially containing trout molecules functionally equivalent to TNF-cx and IFN-y, to stimulate macrophages when used in combination was also investigated. Supernatants generated in this manner certainly had a far larger stimulatory effect on target macrophages than supernatants generated by stimulation with 1-MAF alone, and this effect was significantly inhibited by treatment with anti-TNFR1 mAb as with the supernatants generated using combined treatments discussed earlier. Whether these supernatants were acting synergistically in the present study is not known, because the macrophage supernatants were not tested alone. Finally, the presence of anti-TGF-fi serum during the generation of macrophage supernatants significantly increased their MAF activity. It has been shown previously that leukocyte MAF supernatants often have inhibitory effects on macrophage activity when used at suboptimal concentrations [7, 9], possibly due to the presence of suppressive factors. More recendy, it has been shown that trout macrophages can respond to natural bovine TGF-1 [21]. Coincubation of macrophages with leukocyte MAF supernatants plus TGF-1 resulted in a significant decrease in macrophage respiratory burst activity, and incubation of activated macrophages with TGF-31 deactivated them. In mammals and birds the mature TGF431 peptide is 99-100% identical [25] but has not been isolated from other vertebrate groups. Similarly, the mature TGF-2 peptide is 95% identical across amphibians, birds, and mammals. Even TGF-f5, to date a TGFunique to amphibians, is 75% identical to TGF-1. So TGF43s are very conserved and consequently polyclonal anti-TGF-31 sera show considerable cross-species reactivity. In the present study, the addition of anti-TGF-1 serum eliminated a small but significant suppressive effect of the supernatants on macrophage activity, and experiments are ongoing to characterize trout TGF-.

4.

5.

6.

7.

8. 9. 10.

11.

12.

Marsden, M.J., Cox, D., Secombes, C.J. (1994) Antigen-induced release of macrophage activating factor from rainbow trout Oncorhynchus mykiss leucocytes. Vet. Immunol. Immunopathol. 42, 199-208. Hardie, L.J., fletcher, T.C., Secombes, C.J. (1994) Effect of temperature on macrophage activation and the production of macrophage activating factor by rainbow trout (Oncorhynchus mykiss) leucocytes. Dev. Comp. Immunol. 18, 57-66. Clem, L.W., Miller, N.W., Bly, J.E. (1991) Evolution of lymphocyte subpopulations, their interactions, and temperature sensitivities. In Phylogenesis oflmmune Functions (C. Warr and N. Cohen, eds) CRC Press, Boca Raton, FL, 191-213. Craham, S., Secombes, C.J. (1990) Cellular requirements for lymphokine secretion by rainbow trout Salmo gairdneri leucocytes. Dcv. Comp. ImmunoL 14, 59-68. Trinchieri, C., Perussia, B. (1985) Immune interferon: a pleiotropic lymphokine with multiple effects. Immunol. Today 6, 131-136. Craham, S., Secombes, C.J. (1990) Do fish lymphocytes secrete interferon-y.J. Fish BioL 36, 563-573. Adams, D.O., Hamilton, T.A. (1992) Molecular basis of macrophage activation: Diversity and its origins. In The Mac.rciphage (C.E. Lewis andJ.O.D. McCee, eds) IRL Press, New York, 75-114. Manogue, K.R., van Denter, S.J.H., Cerami, A. (1991) Tumour necrosis factor alpha or cachectin. In The Cytokine Handbook (A. Thomson, ed) Academic Press, London, 241-256. Sheehan, K.C.F., Schreiber, R.D. (1992) The synergy and antagonism ofinterferon-yand TNF. In TumorNecrosis Faaors: The Molecules and TheirEmergingRole in Medicine(B. Beutler, ed) Raven Press, New

York, 145-178.
13. Theodos, C.M., Povinelli, L, Molma, R., Sherry, B., Titus, R.C. (1991) Role of tumor necrosis factor in macrophage leishmanicidal activity in vitro and resistance to cutaneous leishmaniasis in vivo. Inftct. Immun. 59, 2839-2842. Liew, F.Y., Millott, S. (1990) Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishmania major through the induction ofnitric oxide.J. ImmunoL 145, 4306-4310. Bogdan, C., Moll, H., Solbach, W., Rollinghoff, M. (1990) Tumor necrosis factor-alpha in combination with interferon-gamma, but not with interleukin 4 activates murine macrophages for elimination of Leishmania major amastigotes. Eur.J. Immunol. 20, 1131-1135. Chen, L., Suzuki, Y., Wheelock, E.F. (1987) Interferon-y synergizes with tumor necrosis factor and with interleukin 1 and requires the presence of both monokines to induce antitumor cytotoxic activity in macrophages.j ImmunoL 139, 4096-4101. Sibley, L.D., Adams, LB., Fukutomi, Y., Krahenbuhl, J.L. (1991) Tumor necrosis factor-a triggers antitoxoplasmal activity of IFN-y primed macrophages.j ImmunoL 147, 2340-2345. Hardie, L.J., Chappell, L.H., Secombes, C.J. (1994) Human tumor necrosis factor a influences rainbow trout Oncorhynchus mykiss lencocyte responses. Vet. ImmunoL ImmunopathoL 40, 73-84. Jang, 5.1., Mulero, V., Hardie, L.J., Secombes, C.J. (1995) Inhibition ofrainbow trout phagocyte responsiveness to human tumor necrosis factor a(hTNFa) with monoclonal antibodies to the hTNFa 55 kDa receptor. Fish Shellfish ImmunoL5, 61-69. Secombes, C.J. (1990) Isolation ofsalmonid macrophages and analysis of their killing activity. In Techniques in Fish Immunology, VoL 1 (J.S. Stolen, T.C. fletcher, D.P. Anderson, B.S. Robertson, and W.B. van Muiswinkel, eds) SOS Publications, Fair Haven, NJ, 137-154. Jang, 5.1., Hardie, L.J., Secombes, C.J. (1994) The effects of transforming growth factor on rainbow trout Oncorhynchus mykiss macrophage respiratory burst activity. Dcv. Comp. ImmunoL 18, 315-323. Pick, E. (1986) Microassays for superoxide and hydrogen peroxide production and nitroblue tetrazolium reduction using an enzyme immunoassay microplate reader. Methods EnzymoL 132, 407-421. Vilcek, J., Le, J. (1994) Immunology of cytokines: an introduction. In The Cytokine Handbook 2nd edition (A. Thomson, ed) Academic Press, London, 1-19. Munoz-Fernandez, M.A., Fernandez, M.A., Fresno, M. (1992) Synergism between tumor necrosis factor.a and interferon-y on macrophage activation for the killing of intracellular Ttypanosoma cruzi through a nitric oxide-dependent mechanism. Eur.J. ImmunoL 22, 301-307. Roberts, A.B., Sporn, M.B. (1990) The transforming growth factorfls. In Peptide Growth Factors and Their Receptors Vol. I (M.B. Sporn and A.B. Roberts, eds) Springer-Verlag, Berlin, 419-472.

14.

15.

16.

17.

18.

19.

20.

ACKNOWLEDGMENTS
This for work the was supported by a grant from AFRC (no. Many thanks to Celltech Ltd, Slough, Berks, generous gift of the anti-TNFR1 mAb.

21.

AG1/551).

22.

23.

REFERENCES
24. 1. 2. 3. Secombes, protective Secombes, Immune C.J., Fletcher, T.C. (1992) The role of phagocytes in the mechanisms offish. Annu. Rev. Fish Dis. 2, 53-71. C.J. (1994) Macrophage activation in fish. Modulators Fish Responses 1, 49-57. Craham, S., Secombes, C.J. (1988) The production ofa macrophage activating factor from rainbow trout Salmo gairdneri leucocytes. Immunology 65, 293-297.

25.

Jang

et al.

Elevation

of trout

macrophage

respiratory

burst

activity

947