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. 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

or 48-h incubation at 18C. RESULTS As can be macrophages seen with in Figure 1. although the nanomoles 02 produced were still significantly (P < .45 pg/mI. Incubation of target macrophages for 24 h with I :4 diluted macrophage supernatants from control cultures had a small but significant (P < . The trout leukocyte supernatants could increase macrophage respiratocy burst activity and thus were deemed to contain leukocyte-derived MAF (1-MAF) activity [3].e. weighing 300-500 g. before addition of the macrophage supernatants. Macrophages were washed twice in phenol red-free Hanks’ balanced salt solution (HBSS. with the target macrophages for 1 h. were used as the blank.05) above background levels in the absence of LPS. 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. to give a potential mixture of fish lymphocyte and macrophage cytokines). macro24 and + SE for m-MAF activity was assessed by incubating test supernatants with target macrophages in a 96-well plate. The macrophages were then cultured for 3. Sigma) [20]. Data are means five fish. June 1995 . Finally. Macrophage respiratory burst activity was assessed via the reduction of ferricytochrome c by released superoxide anion (02). Almond Bank. in triplicate per treatment. macrophages kidney cell head Phagocyte-ennched suspensions kidney leukocyte were obtained suspensions as depre- Briefly. or in various combinations (at the above concentrations). Finally. strated a significant 48 h of culture in the presence of LPS. 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. 6. 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].05) impact on respiratory burst activity compared with target macrophages incubated with medium alone (Figs. The anti-TCF-1 gamma globulin preparation had no effect on macrophage respiratory burst activity in the absence of TGF-31. Fish to the aquarium system for at least 2 weeks prior to use. Isolation scribed of head previously kidney head [20]. and converted to nmole 02 according to Pick [22]. In some experiments.1% fetal calf serum (FCS. pared in Leibovitz medium (L15. At both incubation times higher concentrations of LPS (100 jig/ml) gave significantly lower (P < . of macrophage supernatants macro(LPS. In the same manner. prior to determination of their respiratory burst activity. Data were analyzed by one-way analysis of variance and Student’s t-test. macrophages in 24-well plates. the cells were washed and incubated in L15 medium plus 5% FCS overnight at 18C. adjusted to I x io viable cells/ml L15 plus 0. Indeed. were preincubated 944 Journal of Leukocyte Biology Volume 57. 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. 1.tg/ml. 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.leukocytes. The macrophages were then washed and supernatants harvested 24 h later as described above. two anti-TNFRI mAbs. They were and fed twice daily on a commercial pelleted diet. 2). Respiratory burst activity of rainbow trout head kidney phages incubated with varying concentrations of E. and the phagocyte-enriched fraction at the interface was collected. These cells were washed twice in L15. Subsequently. respectively. leaving approximately 20% of the cell number in the wells and giving a macrophage purity of 90-95%. 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. Optical density values were taken at 550 nm after 30 mm. and supernatants from mitogen-stimulated trout British head Biotechnology) kidney . on a multiscan spectrophotometer (MDC). and ThF-a alone.. coli LPS for 48 h prior to stimulation with PMA for 30 mm. because TGF4I1 is a potent macrophage-deactivating factor that could be present in the 1-MAF supernatants. 5R2 and 5R16 (Celltech). were stimulated with MAF. 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. Macrophages incubated with PMA and superoxide dismutase (Sigma). at 300 IU/ml. Other mAbs used alongside 5R2 and 5R16 have no effect on trout macrophage respiratory burst activity [19].MATERIALS AND METHODS Fish Rainbow trout from College kept at 14*C were acclimatized Oncorhynchus mykiss.1-100 jig LPS/ml L15 plus 5% FCS was added to triplicate wells per concentration. TNF-a and 1-MAF alone and in various combinations had no significant impact on respiratory burst activity relative to control supernatants (Fig. with maximal increases seen using 50 tg/ml for 24 h. The concentrations of LPS that were stimulatory for trout macrophages were first determined on macrophage monolayers in 96-well plates from five fish. in triplicate per treatment. from four to five fish. were obtained Mill Trout Farm. Analysis of dose effect (P < . 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]. at 6 and 0. Sigma). 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. following stimulation of the cells with phorbol myristate acetate (PMA. incubation with 1:4 diluted supernatants prepared by stimulating macro- LPS had a significant activity. 100 il of medium containing 0. Macrophages were washed vigorously with L15 medium three times prior to use. LPS was used only at a concentration of 50 pg/ml to stimulate macrophages. Gibco) were separated on a 34%/51% Percoll density gradient. 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.01) of head kidney impact on their variance demonafter both 24 and Detection of MAF activity LPS (tg/ml) Fig.05) respiratory burst activity relative to levels seen using 50 j. and their respiratory burst activity was analyzed (as described below) following a 24. incubation Production Three phages. Perthshire. In experiments to investigate whether trout macrophages could release factors with MAY activity. After washing the cells three times with L15 medium. 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). However. 2-5). Incubation of target macrophages for 24 h with 1:4 diluted supernatants from macrophages stimulated for 3 and 6 h with LPS. Macrophages incubated with medium were used to generate control supernatants. Following an adherence period (3 h at 18’C). Gibco). respectively.

The data are means + SE for four fish. * < 05. Prior exposure mAb significantly of target macrophages with anti-TNFR1 E 0 U. and 50 ig/ml LPS. as evidenced by higher respiratory burst activity.05) on supernatants their ability to elevate macrophage respiratory burst activity (Fig. 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. 25 lU/mI TNF-a. Following this incubation period the macrophages were washed and the supernatants harvested 24 h later. *J < #{149}#{216}5. 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. these supernatants were more active than supernatants from macrophages stimulated with 1-MAF alone used in the same experiment (P < . Following this incubation period the macrophages were washed and the supernatants harvested 24 h later.05). 6.001 compared with the respiratory burst activity of macrophages incubated with control supernatants.05) the macrophages. 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.0 0 C (1)0 cC _J.001 compared with the respiratory burst activity of macrophages incubated with control supernatants. ‘C” 0 U) 0 0 E C 2 0 1:8 Dilution fang et al. The macrophage supernatants were prepared by incubating head kidney macrophages for 3. 5). or 12 h with medium (control supernatant) or various combinations of 1:4 diluted 1-MAF. 25 lU/mI TNF-a. U) cant reductions given to the in activity single low or using treatments. Supernatants Fig. and *** < . In addition. Thus.05) different. I-MAF I-MAF + + TNF-cz + LPS 0 0 0 0 0 6 0 0. **P < #{216}. all paired comparisons between single and combined treatments were significantly (P < . 5). Furthermore. <. supernatants from macrophages increase (P < . they were able to increase significantly the production of 02 by target macrophages above that of macrophages incubated with control supernatants (Fig.03h 6h of U 12 h 0 0 a 0 0 0. and 50 pg/mI LPS. The data are means + SE for five fish. with the exception of l-MAF versus 1-MAF + LPS. However. 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. 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.o. insignificant supernatants from a similar of such macrophages trend. 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. 0 U) 0 0 E C the different treatments had the greatest impact on respiratory burst activity relative to control supernatants. Elevation of trout macrophage respiratory burst activity 945 . + Q g 0. The addition of phages for 12 h did significantly respiratory burst activity of target eral. In gengiven combinations anti-TGF-1 also had serum a significant when impact generating (P < these . 3. Indeed.4 Fig. from macrophages stimulated with com- binations of trout 1-MAFand m-MAF-containing supernatants also possessed m-MAF activity. 2 and 3). due supernatants. 2. ** < and *** < . 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. 3). LL LLCJ) LLSC1) from macrophages treatments to elevate ity (Fig. 4). The g Medium alone U Control I-MAF 8 supernatant (1:4) J LPS(50tg/mI) + U l-MAF TNF-a LPS i.

nor inhibitable by anti-TNFR1 mAb.u < - a-C’) LL + < _ <a. Although it is true that other cells were present in the cultures in addition to the macrophages. The head kidney or 1:4 diluted 1:4. <LI + <LI- Q 0. It has been shown previously [18. c) diluted 946 Journal of Leukocyte Biology Volume 57.J.a (a). TNF-cx is well known to synergize with other cytokines [23. Respiratory macrophages macrophage macrophage macrophages 1-MAF plus Thus. A range of treatments were used to stimulate the release of these factors.01 compared with the respiratory burst activity of target macrophages incubated with the respective supernatants without anti-TNFRI treatment. b. Furthermore. 0. (I) < < . Fig. < to generate bioassay-detectable 1-MAF supernatants [7]. of anti-TNFR1 mAb has no effect on the increased ratory burst activity induced directly by Con A (personal observation). following the 12-h incubation period the macrophages were washed and the supernatants harvested 24 h later. + U.45 tg/ml (5R16) before addition of the macrophage supernatants. 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.5 5. and a trout leukocyte supernatant known to “activate” trout macrophages [3. macrophages do appear to be the source of these factors. DISCUSSION 2. In some cases the target macrophages were preincubated with anti-TNFR1 mAb for 1 h at 6 tg/ml(5R2) or 0. 0 _j . < 5R16 5R2 supernatants .5 It seems remarkably easy to induce rainbow trout macrophages to release factors that have an autocrine effect on their respiratory burst activity.g. * < 05 and ** < . 5]. In some cases the target macrophages were preincubated with anti-TNFR1 mAb for I h at 6 tg/ml(5R2) or 0. 25 lU/mi TNF-a. although this could have been due to a limit on the maximum response possible by the fixed number of target cells present per well.t?CI) trout warranted. 19]. Although these treatments were effective when used individually. ruling out the possibility that 19] study addition respior LPS that cell surface binding of any antibody ity of these cells. The activity in the macrophage supernatants generated with combined treatments was significantly inhibited by treatment of the target macrophages with an anti-TNFR1 mAb. combining all three treatments did not elevate the m-MAF activity of the generated supernatants further. supernatants generated using TNF-cz alone were clearly not very stimulatory. leukocytes. the small percentage of contaminating leukocytes maximally represents 2 x io lymphocytes per well. 5. *J < 05 and ** < . The data are means + SE for four fIsh. 1MAF-containing supernatants plus LPS) were as stimulatory as those generated with TNF-cx. molecule may be released from although more definitive proof is clearly o 0. 24].g. which itself im- is conserved on that a TNF-ct-like trout g LA.0 LL. 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.05) than the i 7. which result in increased bactericidal activity against the fish pathogen Aeromonas salmonicida [3]. and some combinations of treatments lacking TNF-a (e. because it is not possible to exclude entirely the potential carryover of some human TNF-cz in the macrophage supernatants. Such studies TNFR plies E0 CU U. “a” plus 50 tg/ml LPS (b). the most active supernatants were generated using these treatments in pairs. The data are means + SE for five fish. and 50 tg/ml LPS. The increase in respiratory burst activity induced by the latter kidney (target) or I :4 diluted for 30 mm. 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... U) -I-. June 1995 . LL. or “b’ plus 2 jig/ml rabbit anti-TGF-31 serum (c) In all cases. including bacterial products (LPS). 0 U) 0 0 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]. 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.45 ig/ml (5R16) before addition ofthe macrophage supernatants.0 Cells alone D U 0 Cells Cells + + 5R16 5R2 m-MAF supernatants was at least as high as those seen with 1-MAF. 21]. 4. and this has been shown previously to be insufficient E0 CU u-0 <C + <Li. part of their MAF activity is potentially due to released 0 0 C) TNF-ct. 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. However. + Fig. However. a recombinant cytokine (TNF-a) known to cross-react with trout leukocytes [18. Following this incubation period the macrophages were washed and the supernatants harvested 24 h later. Thus.Q could decrease the activsuggest that at least one leukocytes.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. 1-MAF + m-MAF (b) in Fig.

Le. 61-69. Fukutomi. T. protective Secombes. to date a TGFunique to amphibians. Adams.J. ed) Raven Press.j ImmunoL 139. Y. C.. to stimulate macrophages when used in combination was also investigated. Vet. ACKNOWLEDGMENTS This for work the was supported by a grant from AFRC (no. Lewis andJ. 73-84. Cerami. Coincubation of macrophages with leukocyte MAF supernatants plus TGF-1 resulted in a significant decrease in macrophage respiratory burst activity. Fair Haven. 1. FL. 5. In mammals and birds the mature TGF431 peptide is 99-100% identical [25] but has not been isolated from other vertebrate groups. 15.rciphage (C. it has been shown that trout macrophages can respond to natural bovine TGF-1 [21]. ImmunoL 14. 25. M.. Craham. 9]. eds) Springer-Verlag. Fish Dis. Sherry. Roberts. and temperature sensitivities. 199-208.J. LB.. In The Cytokine Handbook (A. ImmunoL 18. and this effect was significantly inhibited by treatment with anti-TNFR1 mAb as with the supernatants generated using combined treatments discussed earlier. 18. Eur.D. (1992) The synergy and antagonism ofinterferon-yand TNF. Jang. T. Y.. Millott. Manogue. L. Thus. Fish BioL 36. (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. Berks. ed) Academic Press. A. (1995) Inhibition ofrainbow trout phagocyte responsiveness to human tumor necrosis factor a(hTNFa) with monoclonal antibodies to the hTNFa 55 kDa receptor.B. generous gift of the anti-TNFR1 mAb. Fresno. 20. is 75% identical to TGF-1. their interactions.S.J. Anderson. ImmunoL ImmunopathoL 40.P. Stolen. eds) CRC Press. S. Annu. J. C. Bly. 301-307. Adams.. Hardie. Liew. London. McCee. Dev. 191-213. Hamilton. 131-136. Secombes.’D.. and mammals..J. Miller. 2340-2345. C. K. (1990) Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishmania major through the induction ofnitric oxide.W. Secombes.. Secombes.. Immunol.synergize with IFN-y to increase microbicidal [14. B.J. C. (1992) Molecular basis of macrophage activation: Diversity and its origins.. Theodos. Solbach.. AG1/551). 1131-1135. Human TNF-a has also been shown to synergize with trout 1-MAY-containing supernatants to enhance macrophage respiratory burst activity [18]. 42.J. Perussia. In Phylogenesis oflmmune Functions (C. in the present study the ability of these two types of trout supernatant. Wheelock. Boca Raton. 145-178. 4306-4310.. van Denter. Secombes. L. Comp. Secombes. Vilcek. NJ. 137-154.A.J. J. (1985) Immune interferon: a pleiotropic lymphokine with multiple effects.. 5. Fernandez. Sporn and A. (1990) Tumor necrosis factor-alpha in combination with interferon-gamma. T. K. the presence of anti-TGF-fi serum during the generation of macrophage supernatants significantly increased their MAF activity. Dcv. Hardie. C.. (1991) Evolution of lymphocyte subpopulations. H. Sibley. In TumorNecrosis Faaors: The Molecules and TheirEmergingRole in Medicine(B. Munoz-Fernandez.B. New York.J.O. Dcv. 6. C. L. Rollinghoff. Because these leukocyte supernatants contain IFN activity that coelutes with MAF activity. More recendy. birds. In The Mac. Secombes... Roberts.. Secombes.Y. C. fletcher.J. Cohen. Marsden. Moll. 2. London. Pick. E.J. (1991) Tumor necrosis factor-a triggers antitoxoplasmal activity of IFN-y primed macrophages.. R. Rev. Jang et al. ImmunoL 145. 5. 23. M. 13. A. 7. Immunol. Immun.J. 407-421.J. L. potentially containing trout molecules functionally equivalent to TNF-cx and IFN-y. possibly due to the presence of suppressive factors. 14. 1-19.C. C. In the present study. M. R. 563-573. W.J.. 9.W.J. S. (1990) The transforming growth factorfls. and W. S.. 2.1. Similarly.J. Fish Shellfish ImmunoL5. Chen. Secombes. Molma. Thomson.S.. 16. Secombes.F. Warr and N. Immunol. but not with interleukin 4 activates murine macrophages for elimination of Leishmania major amastigotes.D.O. Immunol. I (M. 315-323. M. the mature TGF-2 peptide is 95% identical across amphibians. Modulators Fish Responses 1. (1994) Effect of temperature on macrophage activation and the production of macrophage activating factor by rainbow trout (Oncorhynchus mykiss) leucocytes. R. fletcher. In The Cytokine Handbook 2nd edition (A. REFERENCES 24. (1990) Do fish lymphocytes secrete interferon-’y. Comp. Trinchieri..B.H. 18. T.F. van Muiswinkel. Sheehan. S. L. 20. B. Immunology 65. C. (1988) The production ofa macrophage activating factor from rainbow trout Salmo gairdneri leucocytes.. 15] and tumoricidal activity [16. the addition of anti-TGF-1 serum eliminated a small but significant suppressive effect of the supernatants on macrophage activity. 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(1991) Tumour necrosis factor alpha or cachectin.R. Immunopathol.L. Beutler.J.J. (1986) Microassays for superoxide and hydrogen peroxide production and nitroblue tetrazolium reduction using an enzyme immunoassay microplate reader. Clem. 4096-4101. So TGF43s are very conserved and consequently polyclonal anti-TGF-31 sera show considerable cross-species reactivity. F. (1990) Cellular requirements for lymphokine secretion by rainbow trout Salmo gairdneri leucocytes.1. Craham. Whether these supernatants were acting synergistically in the present study is not known. VoL 1 (J.. Hardie.. eds) IRL Press. (1992) The role of phagocytes in the mechanisms offish. Elevation of trout macrophage respiratory burst activity 947 .B. It has been shown previously that leukocyte MAF supernatants often have inhibitory effects on macrophage activity when used at suboptimal concentrations [7. Secombes. Sporn.B.. 19. Jang. Thomson. 2839-2842.. J. L. 10. Finally. Many thanks to Celltech Ltd. Immune C. (1994) Human tumor necrosis factor a influences rainbow trout Oncorhynchus mykiss lencocyte responses. 4. Today 6. 17]. 59-68.E. Supernatants generated in this manner certainly had a far larger stimulatory effect on target macrophages than supernatants generated by stimulation with 1-MAF alone. J.j ImmunoL 147.. (1991) Role of tumor necrosis factor in macrophage leishmanicidal activity in vitro and resistance to cutaneous leishmaniasis in vivo.J. 3. ImmunoL 22. 57-66. Mulero.J. In Peptide Growth Factors and Their Receptors Vol. Schreiber.. 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. 49-57. Craham. N.C. Slough. 53-71. eds) SOS Publications. and incubation of activated macrophages with TGF-31 deactivated them. 241-256. Chappell. Methods EnzymoL 132. 11.E. C. 59. Povinelli.M. Suzuki.

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