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

I. Jang,

Department

Laura

ofZoology,

J. Hardie,

University

and Christopher

ofAberdeen,

Tillydrone

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. Words: bow trout

Key



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

macrophage supernatants

J. Secombes Avenue,

Aberdeen

United

Kingdom

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. Jang’s 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

AND

MATERIALS

METHODS

Fish Rainbow trout from College kept at 14*C were

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

acclimatized

use.

Isolation

of head

kidney

Phagocyte-ennched scribed

head

previously

[20].

macrophages kidney

Briefly,

cell head

suspensions kidney

were leukocyte

obtained

as de-

suspensions

pre-

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 18’C), 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%.

Production Three phages; Sigma),

of macrophage

and

supernatants

from

mitogen-stimulated

trout

macro(LPS,

British head

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 18’C before being washed five times with Li5 medium warmed to 18’C 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 4’C 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.

Detection

As can be macrophages

seen with

burst

in Figure

1,

incubation

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

of head kidney impact on their variance demonafter both 24 and

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-

of MAF activity

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

RESULTS

respiratory

supernatants

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,

Biotechnology)

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 Student’s t-test.

Journal

of Leukocyte

Biology

Volume

57, June

1995

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

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).

of

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h

12

0

0

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0 0.

0 U) 0

0

E C

Prior exposure mAb significantly

E 0

of

U.0

0

(1)0

LL

LLCJ)

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