Stimulation of respiratory burst and phagocytic activity in Atlantic salmon (Salmo salar L.) macrophages by lipopolysaccharide

Fish & Shellfish Immunology (1995) 5, 475-491

Stimulation of respiratory burst and phagocytic activity in Atlantic salmon (Salmo salar L.) macrophages by lipopolysaccharide STEIN TORE SOLEM*, JORUNN B. JORGENSEN AND BORRE ROBERTSEN

Department of Marine Biochemistry, Norwegian College of Fishery Science, University of Tromso, N-9037 Tromso, Norway (Received 2 August 1994, accepted in revised form 18 April 1995) The present paper describes the effect of lipopolysaccharides (LPS) from Aeromonas salmonicida and other Gram-negative bacteria on the respiratory burst, phagocytosis and bactericidal activity of head kidney macrophages from Atlantic salmon (Salmo salar L.) in vitro. Macrophages were first cultured in the presence of various concentrations of LPS from A. salmonicida for 1, 2 and 5 days and then tested for respiratory burst activity (reduction of nitroblue tetrazolium) after exposure to phorbol myristate acetate (PMA). The most marked increase in respiratory burst activity of LPS-treated macrophages was observed after 5 days of incubation with 1, 10 and 100/~g LPS ml '. The increase appeared to be dose-dependent with a maximal response at 10/~g m l - ' . At this LPS-concentration and incubation time the respiratory burst activity was 3-9 times larger in the treated macrophages than in the control macrophages. LPS from three other Gram-negative bacterial salmon pathogens and two non-fish pathogens also enhanced the respiratory burst activity of salmon macrophages. Macrophages incubated with 10 and 50/~g LPS ml-1 also showed a significant increase in PMA-stimulated H20 2production after 5 days of incubation. LPS also stimulated the phagocytic activity of Atlantic salmon macrophages against opsonized and nonopsonized glucan particles, and glutaraldehyde-fixed sheep red blood cells. LPS-treated macrophages showed an increased ability to kill an avirulent A-layer lacking strain of A. salmonicida, but not a virulent A-layer positive strain. :C) 1995 Academic Press Limited Key words:

LPS, macrophages, fish, Atlantic salmon, respiratory burst, superoxide, hydrogen peroxide, phagocytosis, bactericidal activity.

I. I n t r o d u c t i o n L i p o p o l y s a c c h a r i d e (LPS), the principal cell wall c o m p o n e n t of G r a m - n e g a t i v e b a c t e r i a is k n o w n to be a m o n g the most p o t e n t m o d u l a t o r s of m a m m a l i a n i m m u n e f u n c t i o n s (Burrell, 1990). LPS is k n o w n as e n d o t o x i n due to its ability to elicit h a r m f u l effects in m a m m a l s s u c h as lethal shock, h y p o t e n s i o n a n d fever (Bone, 1991). On the o t h e r hand, LPS m a y also e n h a n c e the overall *Author to whom correspondence should be addressed. 475 1050-4648/95[070475+17 $12.00/0

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resistance to bacterial and viral infections, and cancer in mammals (Nowotny, 1983). LPS interacts with a multitude of humoral factors and cells in the body (Burrell, 1990). Many of the immunomodulatory and inflammatory effects of LPS can be ascribed to its ability to modulate macrophage functions. LPS stimulates mammalian macrophages to synthesise and secrete important mediators such as prostaglandins (Kunkel et al., 1986) and cytokines like tumour necrosis factor-a (TNF-a) (Sayers et al., 1987). TNF-a is in part responsible for the toxic effects of LPS in mammals (Mathison et al., 1988), but also contributes to its positive effects on defence against microbial infections and cancer (Old, 1985). LPS has also been shown to up-regulate antimicrobial functions in mammalian macrophages by stimulation of respiratory burst (Pabst & Johnston, 1980; Yagawa et al., 1984) and phagocytosis activity (Cooper et al., 1984). In conjunction with interferon-y (IFN-),), a potent macrophage activating factor (MAF) secreted mainly by activated T-lymphocytes and natural killer cells (Arai et al., 1990), LPS can stimulate macrophages to a fully activated state in which they can kill several pathogenic bacteria and even tumour cells (Adams & Hamilton, 1987). LPS consists of three parts; the O-antigen chain, the core oligosaccharide and lipid A. It is well established that lipid A, the structurally most conserved part of LPS molecules, is most important for the biological activity of LPS including its ability to activate mononuclear phagocytes (Raetz, 1990). So far little is known about the immunomodulatory effects of LPS in fish. It has been shown that LPS induces an acute-phase hypoferremic response in rainbow trout (Congelton & Wagner, 1991), stimulates polyclonal proliferation of salmonid lymphocytes (Warr & Simon, 1983), and elicits the production of IL-1 like compounds in channel catfish macrophages (Clem et al., 1985). The overall effect of LPS in fish and mammals differs, since the toxic effects of LPS seen in mammals is not observed in several fish species (Berczi et al., 1966; Wedemeyer et al., 1969; Harbell et al., 1979). As an example, doses equivalent to a dose 10 times the mouse LD50 failed to produce mortality or significant changes in haematocrit of coho salmon (Oncorhynchus kisutch) (Harbell et al., 1979). Studies of how LPS influences fish macrophages might explain why these molecules have such different toxicity in lower and higher vertebrates. The present paper describes the effect of LPS from A. salmonicida and other Gram-negative bacteria, on respiratory burst activity, phagocytosis activity, and bactericidal activity of head kidney macrophages in vitro.

II. M a t e r i a l s a n d M e t h o d s CHEMICALS

A stock solution of I mg phorbol myristate acetate (PMA; Sigma, St Louis, MO, U.S.A.) m1-1 dimethylsulfoxide (DMSO; Fluka Chemie AG, Buchs, Switzerland) was prepared and stored in small aliquots at - 20°C. Zymosan A (Sigma) was suspended in 0"9% NaC1 and boiled in a waterbath for 30 min, washed twice in phosphate buffered saline (PBS), and opsonized by incubation with 40% salmon serum (4 mg m l - 1) for 1 h at 14°C and gently shaken. The

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opsonized zymosan was then washed twice, resuspended in L-15 medium (Gibco, Renfrewshire, Scotland), pH 7.4, 380 mOsm, and sonicated at 60 W for 1 min, using a Bandelin Sonoplus HD60. Finally the concerLtration was diluted to 0'5 mg m l - l . This solution was prepared just prior to use. Phenol red sodium salt (Sigma) was prepared as a stock solution of 10 mg m l - 1 sterile distilled water, and stored at 4°C. Horseradish peroxidase (HRP; Sigma, type II, salt free powder, 175 purpurogallin units mg-1) was suspended in phenol red free Hanks balanced salt solution (HBSS; Gibco) at 380 mOsm, pH 7.4 to a concentration of 10 mg m l - 1 and kept frozen at -80°C until use. Hydrogen peroxide was obtained from Merck, Germany. Nitroblue tetrazolium (NBT; Sigma) was suspended in L-15 maintenance medium (see below) and kept frozen at - 20°C until use. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) was suspended in sterile distilled water at a concentration of 5 mg MTT m l - 1 and stored at -20°C. Superoxide dismutase (SOD) and trifluoperazine (TPF) were obtained from Sigma. M-Glucan, a microparticulate yeast glucan from cell walls of Saccharomyces cerevisiae, was obtained from KS Biotec-Mackzymal, Tromso, Norway. Glucan particles were opsonized by incubation (10s particles ml-1) with 40% salmon serum at 14°C for 1 h. The particles were then washed, resuspended, and sonicated as described for zymosan. BACTERIA

Aeromonas salmonicida strain #3175/88, an A-layer positive, virulent strain, was obtained from Vikan Veterinary Fish Research Station, Namsos, Norway. A. salmonicida strain MT004 (A-layer negative and relatively non-virulent) and strain MT 423 (A-layer positive and virulent) were kindly given by Dr C. J. Secombes, Department of Zoology, University of Aberdeen, Aberdeen, U.K. The presence of A-protein was routinely checked by growing the bacterium at 17°C for 24-48 h on tryptic soy agar (Difco) containing 0-1 mg Coomassie Brilliant Blue R-250 (Bio-Rad) ml - 1. The strains of A. salmonicida were stored in culture medium containing 20% glycerol at -80°C until use. When needed the A. salmonicida strains were grown in tryptic soy broth (TSB, Difco) for 24-48 hours at 17°C, then subcultured in TSB for a further 24-48 h to obtain log-phase cultures. LIPOPOLYSACCHARIDE(LPS) LPS from Escherichia coli 0111: B4, Salmonella typhimurium, and diphosphoryl lipid A from Salmonella minnesota Re-595 were obtained from Sigma. LPS from Vibrio anguillarum AL 112 (serotype 01), AL 104 (serotype 02), and V. salmonicida AL 1133 was a gift from Dr J. B~gwald, Fiskeriforskning, Troms~, Norway. LPS from A. salmonicida (#3175/88) was prepared by the phenol-water extraction method described by Westphal & J a n n (1965) and lyophilized. The different LPS-types were suspended in sterile PBS (pH 7.4, 380 mOsm) to a concentration of 1 mg m l - 1 and stored in small aliquots at -20°C. Prior to use, the suspensions were sonicated in a Bandelin Sonorex RT255 water bath sonicator for 1 min.

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FISH

Atlantic salmon (Salmo salar L.) weighing 1-2 kg were used for isolation of head kidney (HK) macrophages. The fish were obtained from the Centre of Aquaculture Research, Tromse, where they were held in aquaculture pens. MACROPHAGE ISOLATION, CULTIVATION AND STIMULATION

HK macrophages were isolated according to the method described by Braun-Nesje et al. (1981) with modifications of the Percoll densities. Briefly, the cell suspension was layered onto a 37%/51% Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient and centrifuged at 400 g for 30 rain. The macrophage-enriched suspension at the interface was collected, adjusted to 1-5 x 107 cells ml - i L-15 medium (pH 7.4 and 380 mOsm) containing 0.1% fetal calf serum (FCS; Hyc]one, Lund, Sweden), 100 units penicillin (Gibco) ml - i and 0-I mg streptomycin (Gibco) ml - i, and i00/~] was added per well in flat bottomed 96-wells microtitre plates (Nunc, Roskilde, Denmark). The macrophages were allowed to adhere for 3 h at 14°C before non-adherent cells were washed off. The remaining monolayers were cultured with L-15 medium supplemented with 5% FCS plus I00 units penicillin ml - i and 0"i mg streptomycin m] -I (maintenance medium). The cells were maintained 0-2 days before stimulation. Any residual non-adherent cells were removed by washing the cells just prior to use. The macrophages were cultured in the presence of various concentrations of bacterial LPS or M-Glucan suspended in maintenance medium. At various time intervals the macrophages were assayed for respiratory burst activity, hydrogen peroxide production, phagocytosis activity and bactericidal activity as described below. The mean cell number per well for each microtitre plate was determined by counting nuclei released after removal of the medium and addition of 100/~I lysis buffer containing 0'1 M citric acid (Merck), I% Tween 20 (Sigma) and 0-05% crystal violet (Secombes, 1990). MEASUREMENT OF RESPIRATORY BURST ACTIVITY

Respiratory burst activity of macrophages was quantified using the reduction of NBT to formazan as a measure of superoxide anion production as described by Secombes (1990). The cells were triggered with I00/~] of a solution containing PMA (200 ng m]-1) or opsonized zymosan (500/Ig m l - ' ) in maintenance medium containing NBT (I mg m] - i) and incubated for 20 min at 14°C. The cells were then fixed in 70% methanol, air dried and the formazan dissolved by the addition of 120/II KOH (Merck) and 140/II DMSO. Optical density (O.D.) was measured at 620 nm with KOH/DMSO as a blank using a THERMOmaxmu]tiscan spectrophotometer (Molecular Devices Corporation). The results are expressed as O.D.62o per 105 cells. INHIBITORSTUDIES Two inhibitors of superoxide production were tested, superoxide dismutase (SOD) which dismutates 0 2- to H202 and trifluoperazine (TFP), an inhibitor

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of NADPH-oxidase (Cross, 1990). H K macrophages were preincubated for 10 min with SOD (300 units m l - 1 ) or TPF (25pM) in maintenance medium prior to the NBT-assay. MEASUREMENT OF HYDROGEN PEROXIDE

P r o d u ctio n of H202 by macrophages was quantified as described by Secombes (1990). Briefly, macrophage monolayers were incubated with 10 and 50~g A. salmonicida LPS m l - 1 for 3, 5, and 7 days. The macrophages were then washed twice in PBS (380 mOsm, pH 7"4), and the secretion of H20 2 was determined in 100~1 phenol red free HBSS at 380 mOsm, supplemented with 0'56 mM phenol red and 0-1 mg HRP m l - 1, finally adjusted to pH 7-0. To this solution, PMA was added to a final c o n c e n t r a t i o n of 200 ng ml - 1. After 40 min the reaction was stopped by the addition of 10gl of 1 N NaOH. The amount of H20 e was determined spectrophotometrically at O.D. 620 nm from a standard curve and the results were expressed as nmol H~O 2 per 105 cells. MEASUREMENT OF PHAGOCYTOSIS ACTIVITY

Phagocytosis activity of head kidney macrophages was measured using glutaraldehyde-fixed sheep red blood cells (GSRBC, Sigma) as target particles. A colorimetric method adapted from Gebran et al. (1992) was used, and will be published separately. The method is based on quant i t at i on of red blood cells by measurement of the specific oxidation of 2,7-diaminofluorene (DAF) to fluorene blue by the pseudoperoxidase activity of haemoglobin. Head kidney macrophages were isolated and cultured as described above. Groups of eight macrophage-wells were either left u n t r e a t e d for 5 days or A. salmonicida LPS was added to a final c o n c e n t r a t i o n of 10/~g m l - 1 on day 5, 4, 3 and 2 days before the phagocytosis assay. On day 5 half of the macrophages in each t r e a t m e n t (4 wells) were fixed with 0-5% (v/v) paraformaldehyde in PBS at 14°C for 60 min prior to addition of GSRBC. Wells with fixed macrophages were used as a measurement of attached, but not ingested GSRBC. All wells were washed once with medium and then 100 gl GSRBC (1'8 × l0 T cells m l - 1 ) in m a i n t e n a n c e medium was added. Phagocytosis was allowed to proceed for 15 min at 14°C whereafter the wells were washed three times with medium to remove non-ingested and non-attached GSRBC. Medium was next replaced with 1001d 0.2 M Tris-HC1 in 6 M urea at pH 7.4 and left for 5 min at room t e m p e r a t u r e before addition of 100 itl of DAF substrate solution (Gebran et al., 1992). The plates were incubated for 5 min at room t em perat ure and the O.D. was determined at 620 nm in the T H E R M O m a x multiscan spectrophotometer. Results are given as O.D.62o values. The values of control wells (fixed macrophages) were subtracted from the values of wells with intact macrophages within each t r e a t m e n t group. Macrophage cell numbers in each well was estimated to be 5 × 104 by counting nuclei. Phagocytosis of opsonized and normal glucan particles was studied using macrophages seeded in 24-well culture plates supplied with 13-mm diameter T h e r m a n o x coverslips (Nunc) by the procedure described by Engstad & Robertsen (1993). Macrophages were cultured for 3, 4, and 5 days with medium containing 10~g A . salmonicida LPS ml -1; or medium alone (control). The cells were washed twice in PBS and I × 10s glucan particles in L-15 containing

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0"1% Medicult ~ Synthetic Serum Replacement (SSR2) (Medi-Cult A/S, Copenhagen, Denmark) were added to each well. The culture plates were then incubated for 15 min at 14°C. Next, the macrophages were washed five times in PBS to remove u n a t t a c h e d particles and fixed with 1 ml per well cold 2.5% glutaraldehyde in PBS for a minimum of 2 h. After fixation the coverslips were washed three times with distilled H20, stained with Giemsa, and mounted on micro slides with histokit (Chemie Teknik, Germany). For each t r e a t m e n t three coverslips were used and 200 macrophages per coverslip were examined by light microscopy. The mean number of particles per macrophage (phagocytic index) was determined. THE BACTERICIDAL ASSAY

The macrophage bactericidal assay using MTT was conducted as previously described (J~rgensen & Robertsen, 1994). Two strains of A. salmonicida were used, the relatively non-virulent strain MT004, an A-layer lacking strain, and the virulent strain MT423, an A-layer possessing strain. Log phase of bacteria were adjusted to a starting c o n c e n t r a t i o n of l0 s cells m l - 1 and diluted to the c o n c e n t r a t i o n used in the assay in m ai nt enance medium without antibiotics. For the avirulent strain a ratio of 10 bacteria per cell was used and for the virulent strain bacteria:cell ratio of 1:1 was used. The number of bacteria added to the macrophages was based on counted nuclei as described above. The bacterial dilutions used were added to quadruplicate wells containing macrophages. The plates were incubated at 14°C for 3 h. The remaining bacterial dilutions in m a i nt e na nc e medium without antibiotics were coincubated with the plates and used as controls. The macrophages were lysed by the addition of 20% (v/v) Tween 20 in distilled water. A standard curve of bactericidal activity was prepared from the different dilutions of bacteria t h a t had been added to the macrophages. Bacteria were diluted in ma i nt e na nc e medium without antibiotics to correspond to 0, 30, 60 and 90% reduction in cell number. The different dilutions were added to wells with lysed macrophages and the plates incubated at 17°C for 8 or 15 h, MT004 and MT423 respectively. The tetrazolium salt MTT was added to all wells and the plates were incubated for 30 min at room temperature. The formazanbacteria precipitate was solubilised using isopropanol (Merck) and O.D. at 550 nm determined. By linear regression analysis the O.D. corresponding to 0 and 90% killing for each c o n c e n t r a t i o n of bacteria added to the macrophages was established. The percentage of bacteria killed by the macrophages was calculated by the formula: 1 --

O'D'sample -- O'D'90% killing

×

90%

O'D'0% killing--O'D'90% killing

Controls included were macrophages incubated with no bacteria before lysing with Tween 20. STATISTICAL METHOD

The two tailed Student's t-test was used to determine the statistical probability.

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III. R e s u l t s PRIMING OF RESPIRATORY BURST ACTIVITY OF MACROPHAGES BY INCUBATION WITH A. S A L M O N I C I D A LPS

Reduction of NBT by macrophages triggered with PMA or opsonized zymosan has frequently been used to measure respiratory burst activity in such cells from both mammals and fish (Rook et al., 1985; Secombes, 1990). In the present study we used the NBT-method to measure burst activity in LPS-stimulated Atlantic salmon macrophages. LPS did not trigger the respiratory burst of the macrophages directly at the concentrations used in these experiments (data not shown). The effect of incubation time and concentration of LPS on the priming of respiratory burst activity of salmon macrophages is illustrated in Fig. 1. In these experiments macrophages from three fish were cultured in the presence of various concentrations of A. salmonicida LPS for 1, 2 and 5 days and then tested for respiratory burst activity after exposure to PMA. Yeast fl-glucan was included as a positive control since in previous studies it was found to be a potent primer of respiratory burst activity in salmon macrophages (Jorgensen & Robertsen, 1994). Only a minor increase in respiratory burst activity was observed after 1 day of incubation of macrophages with 10 and 100pg LPS m l - 1. A marked increase in respiratory burst activity of LPS-treated macrophages from all three individual fish was observed after 5 days of incubation. At this time point a significant increase was observed for one individual at 0-1/~g LPS m l - 1 and for all three individuals at 1, 10 and 100/~g LPS m l - 1 The increase appeared to be dose-dependent with a maximal response at 10/lg ml-1. At this LPS-concentration and time the respiratory burst activity was 3.9 times larger in the treated macrophages than in the control macrophages. The increase in respiratory burst activity obtained by treatment of macrophages with 1 to 100/~g LPS ml - 1 was similar to the burst activity obtained by treatment with 1/~g glucan m l - 1 Priming of respiratory burst activity of salmon macrophages with LPS from A. salmonicida could also be observed using opsonized zymosan, a microparticulate stimulus, to trigger the burst [Fig. 2(b)]. As shown in Fig. 2, NBT-reduction by control and LPS-treated macrophages exposed to PMA or opsonized zymosan was significantly inhibited by SOD and TFP which confirm that the assay is specific for superoxide production. EFFECT OF A. S A L M O N I C I D A LPS ON THE CAPACITY OF MACROPHAGES TO PRODUCE H202

As shown in Fig. 3, macrophages incubated with 10 and 50pg LPS m1-1 showed a significant (P<0-01) increase in PMA-stimulated H202-production after 5 days of incubation but not after 3 or 7 days. PRIMINGOF RESPIRATORYBURSTACTMTYOF MACROPHAGESBYINCUBATIONWITHLPS FROMVARIOUSPATHOGENICANDNON-PATHOGENICBACTERIA TO test whether LPS with different O-antigen chains had a different effect on the priming of respiratory burst activity of Atlantic salmon macrophages, LPS

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Concentration (pg/ml) of LPS or Glucan (t) Fig. 1. The effect of incubation time and concentration of LPS from A. salmonicida on the respiratory burst activity of HK macrophages. Macrophages were incubated in medium (control) or in medium containing various concentrations of LPS or l # g ml-1 glucan for 1 (a), 2 (b), and 5 days (c). PMA-induced 0 2 - production was measured by the NBT-method and expressed as O.D.62o per 108 macrophages. Results are means of quadruplicate readings from three fish. Vertical bars are standard deviation (S.D.) of the mean. *P<0.05, **P<0.01 compared to PMA-stimulated control macrophages.

( 1 0 # g m1-1) from v a r i o u s p a t h o g e n i c a n d n o n - p a t h o g e n i c G r a m - n e g a t i v e b a c t e r i a were tested in the b u r s t a s s a y with m a c r o p h a g e s from t h r e e fish. As s h o w n in Fig. 4 the m a c r o p h a g e s r e s p o n d e d to all the v a r i o u s LPS-types.

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Fig. 2. T h e effect of e x o g e n o u s s u p e r o x i d e d i s m u t a s e (300 u n i t ml ') (D) and trifiuop e r a z i n e (25pM) ([1) on t h e r e d u c t i o n of N B T by L P S - s t i m u l a t e d or u n s t i m u l a t e d H K m a c r o p h a g e s . M a c r o p h a g e s from one fish w e r e i n c u b a t e d in m e d i u m w i t h or w i t h o u t 1 0 p g A . s a l m o n i c i d a L P S m l - ' for 5 days p r i o r to assay. T h e r e s p i r a t o r y b u r s t was i n d u c e d by P M A (200 ng ml 1) (a) or o p s o n i z e d z y m o s a n (0.5 mg ml - 1) (b). R e d u c t i o n of N B T was m e a s u r e d and e x p r e s s e d as O.D.62o per 105 m a c r o p h a g e s . R e s u l t s a r e m e a n s i S D . of six r e a d i n g s . *P<0"05, **P<0"01 c o m p a r e d to P M A or o p s o n i z e d z y m o s a n - i n d u c e d N B T - r e d u c t i o n by m a c r o p h a g e s u n t r e a t e d w i t h inhibitors (•).

PRIMING OF RESPIRATORY BURST ACTIVITY OF MACROPHAGES BY INCUBATION WITH DIPHOSPHORYL LIPID A

To study the effect of lipid A on the priming of respiratory burst activity of salmon macrophages, various concentrations of diphosphoryl lipid A were tested in the burst assay with macrophages from one fish. As illustrated in Fig. 5 the macrophages incubated for 5 days with 10pg diphosphoryl lipid A m l - 1 showed a significant increase in respiratory burst activity, equivalent to that of 10pg A. salmonicida LPS m l - 1 STIMULATION OF PHAGOCYTOSIS BY A. SALMONICIDA LPS

The uptake of glutaraldehyde fixed sheep red blood cells (GSRBC) by HK macrophages was measured following treatment with 10pg A. salmonicida

S.T. SOLEM ET AL.

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Fig. 3. The effect of LPS from A. salmonicida on the c a p a c i t y of m a c r o p h a g e s to produce H202. Macrophages were i n c u b a t e d in medium alone (control) ( I ) or medium containing 10 ([~) or 50#g LPS m l - ' ([]) for 3, 5, and 7 days. The formation of H202 was determined by t r i p l i c a t e r e a d i n g s per fish, and expressed as nmol per 10 ~ macrophages after s t i m u l a t i o n with PMA. Results are means + S.D of nmol H202 p r o d u c e d by m a c r o p h a g e s from t h r e e fish. *P<0-05, **P<0.01 c o m p a r e d to P M A - s t i m u l a t e d control macrophages. (@) Control m a c r o p h a g e s w i t h o u t PMA. 0.8

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L P S m l - 1 f o r 0, 2, 3, 4 a n d 5 d a y s . A s s h o w n i n F i g . 6, t h e u p t a k e o f GSRBC was enhanced by LPS-treatment at all incubation times. Maximal phagocytosis activity appeared to occur after 4 days of incubation

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Fig. 6. Stimulation of phagocytosis by A. salmonicida LPS. Macrophages from one fish were incubated in medium alone for 5 days (control) or in medium containing LPS (10~g m l - ' ) for 2, 3, 4, and 5 days before the uptake of GSRBC was measured. Phagocytic activities are expressed as O.D.62o per 10'~ macrophages and are means of quadruplicate readings + S.D. *P<0"05, **P<0"01 compared to control macrophages. a l t h o u g h only the d a t a from 2 and 4 days of i n c u b a t i o n were significantly different. The q u a n t i t a t i v e effect of L P S on p h a g o c y t o s i s a c t i v i t y was studied by the e n g u l f m e n t of n o n o p s o n i z e d (NG) and opsonized g l u c a n particles (OG) by H K m a c r o p h a g e s t r e a t e d with 1 0 ~ g A. salmonicida L P S m l - 1 for 3, 4, a n d 5 days. The p h a g o c y t i c index of the m a c r o p h a g e s was significantly (P<0"01) e n h a n c e d after 3, 4, a n d 5 days of i n c u b a t i o n with LPS. The e n h a n c e d a c t i v i t y was d e t e c t e d for b o t h NG a n d OG (Fig. 7).

486

S.T. SOLEM E T AL. 10

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Fig. 7. The effect of LPS on the phagocytic index (mean particles per macrophage).

Macrophages were incubated in medium alone (control) ( - 0 - ) , or in medium with LPS (10/~g ml-1) from A. salmonicida (-@-). The phagocytosis of nonopsonized (a) and opsonized (b) glucan particles was determined by counting 200 macrophages. Results are means ± S,D of triplicate countings and expressed as phagocytic index. THE ABILITYOF LPS-STIMULATEDMACROPHAGESTO KILLA. SALMONICIDA M a c r o p h a g e s were i n c u b a t e d with 10gg A. salmonicida L P S m l - 1 for 4 days and t h e n tested for ability to kill an a v i r u l e n t and a v i r u l e n t s t r a i n of A. salmonicida. As s h o w n in Fig. 8 a significant i n c r e a s e d killing of the a v i r u l e n t s t r a i n of A. salmonicida was o b s e r v e d by m a c r o p h a g e s from two of t h r e e individuals. LPS did not, however, e n h a n c e the b a c t e r i c i d a l a c t i v i t y of the m a c r o p h a g e s a g a i n s t the v i r u l e n t s t r a i n (data not shown).

IV. D i s c u s s i o n The p r e s e n t w o r k d e m o n s t r a t e s t h a t L P S from A. salmonicida and o t h e r G r a m - n e g a t i v e b a c t e r i a h a v e the ability to i n c r e a s e the r e s p i r a t o r y b u r s t a c t i v i t y of A t l a n t i c salmon m a c r o p h a g e s . The largest i n c r e a s e was o b s e r v e d after 5 days of i n c u b a t i o n w i t h 10 ~g LPS m l - 1. H i g h e r c o n c e n t r a t i o n s did not give a f u r t h e r i n c r e a s e in r e s p i r a t o r y b u r s t a c t i v i t y and v i r t u a l l y no effect was d e t e c t e d w i t h an L P S - c o n c e n t r a t i o n of 0-1 g g ml - 1. The v a l i d i t y of using the NBT-assay to m e a s u r e s u p e r o x i d e p r o d u c t i o n in m a c r o p h a g e s exposed to L P S

STIMULATION OF RESPIRATORY BURST 100

487

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Fig. 8. The ability of LPS-primed macrophages to kill an avirulent strain (MT004) of A. salmonicida. Macrophages from three different fishes were incubated with medium alone (control) (m) or with medium containing 10~lg A. salmonicida LPS ml- ' (D) for 4 days and then tested for bactericidal activity against A. salmonicida MT 004. Results are means of quadruplicate readings expressed as percent bacteria killed + S.D. *P<0'05, **P<0"01 compared to the bactericidal activity of control macrophages.

was confirmed by showing t hat SOD and the respiratory burst inhibitor T F P markedly reduced the NBT-reduction of macrophages following exposure to PMA or zymosan. A f ur t her confirmation was obtained by the observation t hat macrophages t r eat ed with LPS for 5 days also produced increased amounts of H202 after exposure to PMA. A . s a l m o n i c i d a LPS and yeast fl-glucan showed similar enhancements of respiratory burst activity. The dose response curves are different, however, because glucan showed optimal priming at about l g g m l - 1 and no effect at 10~g m l - ' (Jorgensen & Robertsen, 1994). LPS had a maximal effect at 10~g m l - ' and the effect did not decline dramatically at higher concentrations. It is well known t hat LPS has the ability to enhance the respiratory burst activity of mammalian m ononucl ear phagocytes, but the mechanism of the priming is not fully understood (Pabst & Johnston, 1980; Yagawa et al., 1984). The enzyme system responsible for production of superoxide is a NADPH oxidase (Morel et al., 1991) and this enzyme complex appears to be present in salmonid macrophages as well (Secombes et al., 1992). NADPH oxidase is a multicomponent electron t r ans por t chain requiring for its activity the participation of a membrane-bound b-type cytochrome and several cytosolic oxidase proteins (Morel et al., 1991). The cytochrome b is a heterodimer of a 91 kDa glycoprotein and a 22 kDa polypeptide. Mouse peritoneal macrophages t hat were activated by injection of LPS showed an enhanced respiratory burst activity which was associated with a higher Vmax and a lower K m for the superoxide producing NADPH oxidase compared to normal cells (Sasade et al., 1983). The increase of respiratory burst activity of LPS-prime.d Atlantic salmon macrophages might be caused by modification of the kinetic parameters of the NADPH oxidase. Augmentation of respiratory burst activity of h u man monocyte-derived macrophages with IFN-), was shown to be associated with an induction of cytochrome b heavy chain gene expression whereas

488

S.T. SOLEM ET AL.

expression of the light chain was unaffected (Newburger et al., 1988). This was confirmed by Cassatella et al. (1990) who in addition showed that both LPS and IFN-7 induce enhancement of mRNA levels for the 91 kDa cytochrome b chain of human neutrophils. LPS may have similar effects on gene expression of NADPH oxidase in fish macrophages. There was apparently no clear difference between the ability of LPS from pathogenic and non-pathogenic bacteria to stimulate respiratory burst activity. However, LPS from V. a n g u i l l a r u m serotype O1 gave a smaller increase in respiratory burst activity than the others. Also diphosphoryl lipid A stimulated respiratory burst activity to approximately the same level as LPS from A. salmonicida. These data indicate that the most conserved portion of the LPS-molecule, possibly lipid A, is responsible for the priming effect of the LPS-types. Lipid A is also known to be the most important part of the molecule for the modulatory effects of LPS on mammalian macrophages (Raetz, 1990). The O antigen chain which is highly variable between species and the core oligosaccharide which shows some variability have also been shown to influence the activity of LPS on mouse macrophages (Chen et al., 1992). Thus, these parts may have some influence on the activity of the LPS from serotype O1 of V. anguillarum. The present work shows several interesting differences between the LPSresponse of mammalian and fish macrophages. Enhanced respiratory burst can be detected in mouse macrophages as early as 30 min after addition of LPS and reaches a maximum after 24 h (Pabst & Johnston, 1980). Mouse macrophages also respond to much smaller LPS-concentrations than salmon macrophages. Optimal priming in mouse macrophages was obtained with LPS-concentrations of 0-01-1~g m1-1, while 10~g m1-1 was less effective, presumably because it had a toxic effect on the cells (Pabst & Johnston, 1980). The difference in sensitivity of mammalian and fish macrophages to LPS may partially explain the differences in the toxicity of LPS to higher and lower vertebrates. Fish macrophages possibly have receptors with less affinity for LPS than the mammalian macrophages. LPS/lipid A appear to exert its effect on mammalian macrophages either by binding to CD 14 after complexing with LPS-binding protein (Wright et al., 1990), an acute phase serum protein, or by direct binding to the 80 kDa, later shown to be 73 kDa, LPS-receptor (Morrison et al., 1992). Lower vertebrates like frog and chicken appear to lack the serum-independent 73 kDa LPS-receptor typical for mammals (Roeder et al., 1989). On the basis of these results it was suggested t h a t the presence of specific LPS-binding proteins on mononuclear cells may be important for endotoxin susceptibility of animals. Similar to mouse macrophages (Cooper et al., 1984), LPS also stimulated the phagocytic activity of Atlantic salmon macrophages. This effect on phagocytosis was evident both with respect to GSRBC, and opsonized and nonopsonized glucan particles. Phagocytosis of GSRBC was enhanced already after 2 days of incubation of macrophages with LPS, and showed a maximum after 4 days of incubation. Phagocytosis of NG and OG was largest 3 days after incubation and then declined. The difference in the influence of incubation time on the stimulation of phagocytic activity against GSRBC and glucan particles may be apparent only because of the difference in techniques used to

STIMULATION OF RESPIRATORYBURST

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study phagocytosis. We did not detect differences in the ability of LPSstimulated macrophages to phagocytize NG compared to OG. These observations indicate t h a t LPS has a general effect on the phagocytic activity of salmon macrophages rather than an effect on the expression of specific receptors such as complement- and glucan-receptors. As for respiratory burst activity, stimulation of phagocytic activity of salmon macrophages occurred much later after addition of LPS and at much higher concentrations t h a n for mouse macrophages (Cooper et al., 1984). The in vitro priming of salmon macrophages with LPS to higher phagocytic activity and greater capacity to produce 0 2- and H202 represents at least some steps in the process of macrophage activation. LPS-stimulated macrophages would therefore be expected to have an increased capacity to kill bacteria because both superoxide and hydrogen peroxide are known to be toxic to a range of bacteria including a number of virulent and avirulent strains of A. salmonicida (Sharp & Secombes, 1993). This could thus explain why in vitro stimulation of salmon macrophages with LPS resulted in a significantly enhanced ability to kill the avirulent strain of A . salmonicida. However, the virulent strain of A. salmonicida was not killed by LPSstimulated macrophages. This was also observed for glucan-stimulated macrophages (Jorgensen & Robertsen, 1994) and suggests t h a t killing of virulent A. salmonicida by salmon macrophages requires a higher state of activation of these macrophages involving additional defence mechanisms. Graham & Secombes (1988) have shown t h a t while non-activated rainbow trout macrophages possess the ability to kill avirulent MT004 A. salmonicida they are unable to kill the virulent strain 184. However, following activation with MAF the macrophages acquire the ability to augment their killing capacity to kill virulent 184 A. salmonicida. In vivo LPS would be expected to interact with B and T cells as well as macrophages (Burrell, 1990). A combination of direct effects of LPS on macrophages and indirect effects via lymphokines such as MAF might raise the macrophages to a higher state of activation than that obtained in the present in vitro experiment. Such combined effects of LPS and IFN-y is well known in mammalian systems both with respect to killing of microbial pathogens and lysis of tumours (Adams & Hamilton, 1987). References Adams, D. O. & Hamilton, T. A. (1987). Molecular transductional mechanisms by which IFN-7 and other signals regulate macrophage development. Immunological Reviews 97, 5-27. Arai, K.-I., Lee, F., Miyajima, A., Miyatake, S., Arai, N. & Yokota, T. (1990). Cytokines: coordinators of immune and inflammatory responses. Annual Review of Biochemistry 59, 783-836. Berczi, I., Bertok, L. & Bereznai, T. (1966). Comparative studies of the toxicity of Escherichia coli lipopolysaccharide endotoxin in various animal species. Canadian Journal of Microbiology 12, 1070-1071. Bone, R. C. (1991). The pathogenesis of sepsis. Annals of Internal Medicine 115, 457-469. Braun-Nesje, R., Bertheussen, K., Kaplan, G. & Seljelid, R. (1981). Salmonid macrophages: separation, in vitro culture and characterization. Journal of Fish Diseases 4, 141-151.

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Burrell, R. (1990). Immunomodulation by bacterial endotoxin. Critical Reviews in Microbiology 17, 189-208. Cassastella, M. A., Bazzoni, F., Flyan, R. M., Dusi, S., Trinchieri, G. & Rossi, F. (1990). Molecular basis of interferon-~ and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Journal of Biological Biochemistry 265, 20241-20246. Chen, T., Lei, M. & Morrison, D. C. (1992). Lipopolysaccharide receptors and signal transduction pathways in mononuclear phagocytes. Current Topics in Microbiology and Immunology 181, 169-188. Clem, L. W., Sizemore, R. C. & Elsaesser, C. F. (1985). Monocytes as accessory cells in fish immune responses. Development and Comparative Immunology 9, 803-809. Congleton, J. L. & Wagner, E. J. (1991). Acute phase hypoferremic response to lipopolysaccharide in rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology 93A, 195-200. Cooper, P. H., Mayer, P. & Baggiolini, M. (1984). Stimulation of phagocytosis in bone marrow derived mouse macrophages by bacterial lipopolysaccharide: correlation with biochemical and functional parameters. Journal of Immunology 133, 913-922. Cross, A. R. (1990). Inhibitors of the leukocyte superoxide generating oxidase: Mechanism of action and methods for their elucidation. Free Radical Biology and Medicine 8, 71-93. Engstad, R. E. & Robertsen, B. (1993). Recognition of yeast cell wall glucan by Atlantic salmon (Salmo salar L.) macrophages. Developmental and Comparative Immunology 17, 319-330. Gebran, S. J., Romano, E. L., Pons, H. A., Cariani, L. & Sonayo, A. N. (1992). A modified colorimetric method for the measurement of phagocytosis and antibodydependent cell cytotoxicity using 2,7-diaminofluorene. Journal of Immunological Methods 15, 255-260. Graham, S. & Secombes, C. J. (1988). The production of a macrophage-activating factor from rainbow trout, Salmo gairdneri, leucocytes. Immunology 11, 389-396. Harbell, S. C., Hodkins, H. O. & Schiewe, M. H. (1979). Studies on the pathogenesis of vibriosis in coho salmon Onchorhynchus kisutch (Walbaum). Journal of Fish Diseases 2, 391-404. J~rgensen, J. B. & Robertsen, B. (1995). Yeast fl-glucan stimulates respiratory burst activity of Atlantic salmon macrophages (Salmo salar L.). Developmental and Comparative Immunology, 19, 43-57. Kunkel, S. L., Chensue, S. W. & Phan, S. H. (1986). Prostaglandins as endogenous mediators of interleukin 1 production. Journal of Immunology 136, 186-192. Mathison, J. C., Wolfson, E. & Ulevitch, R. C. (1988). Participation of tumor necrosis factor in the mediation of Gram-negative bacterial lipopolysaccharide-induced injury in rabbits. Journal of Clinical Investigation 81, 1925-1937. Morel, F., Doussiere, J. & Vignais, P. V. (1991). The superoxide-generating oxidase of phagocytic cells. Physiological, molecular and pathological aspects. European Journal of Biochemistry 201, 523-546. Morrison, D. C., Lei, M., Chen, T., Flebbe, L. M., Halling, J. & Field, S. (1992). Identification and characterization of mammalian cell membrane receptors for LPS-endotoxin. In: Microbial Infections (H. E. A. Friedman, ed.) pp. 23-29. New York: Plenum Press. Newburger, P. E., Ezekowitz, A. B., Whitney, C., Wright, J. & Orkin, S. H. (1988). Induction of phagocyte cytochrome b heavy chain gene expression by interferon ~,. Proceedings of the National Academy of Science U.S.A. 85, 5125-5219. Nowotny, A. (1983). Beneficial Effects of Endotoxins. New York, London: Plenum Press. Old, L. J. (1985). Tumor necrosis factor (TNF). Science 230, 630-632. Pabst, M. J. & Johnston, J. R. B. (1980). Increased production of superoxide anion by macrophages exposed in vitro to muramyl dipeptide and lipopolysaccharide. Journal of Experimental Medicine 151, 101-114.

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Raetz, C. H. R. (1990). Biochemistry of endotoxins. Annual Reviews in Biochemistry 59, 129-170. Roeder, D. J., Lei, M.-G. & Morrison, D. C. (1989). Endotox~c-lipopolysaccharidespecific binding proteins on lymphoid cells of various animal species: Association with endotoxin susceptibility. Infection and Immunity 57, 1054-1058. Rook, G. A. W., Steele, J., Umar, S. & Dockrell, H. M. (1985). A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by y-interferon. Journal of Immunological Methods 82, 161-167. Sasada, M., Pabst, M. J. & Johnston, R. B. (1983). Activation of mouse peritoneal macrophages by lipopolysaccharide alters the kinetic parameters of the superoxide-producing NADPH-oxidase. Journal of Biological Chemistry 258, 9631-9635. Sayers, T. J., Macher, I., Chung, J. & Kugler, E. (1987). The production of tumor necrosis factor by mouse bone marrow-derived macrophages in response to bacterial lipopolysaccharide and a chemically synthesized monosaccharide precursor. Journal of Immunology 138, 2935-2940. Secombes, C. J. (1990). Isolation of salmonid macrophages and analysis of their killing activity. In: Techniques in Fish Immunology (J. S. Stolen, T. C. Fletcher, D. P. Anderson, B. S. Robertson & W. B. van Muiswinkel, eds) pp. 139-154. NJ 07704-3303, U.S.A.: SOS Publications. Secombes, C. J., Cross, A. R., Sharp, G. J. E. & Garcia, R. (1992). NADPH oxidase-like activity in rainbow trout Oncorhyncus mykiss (Walbaum) macrophages. Developmental and Comparative Immunology 16, 405-414. Sharp, G. J. E. & Secombes, C. J. (1993). The role of reactive oxygen species in the killing of the bacterial fish pathogen Aeromonas salmonicida by rainbow trout macrophages. Fish & Shellfish Immunology 3, 119-129. Warr, G. W. & Simon, R. C. (1983). The mitogenic response potential of lymphocytes from the rainbow trout (Salmo gairdneri) re-examined. Developmental and Comparative Immunology 7, 379-384. Wedemeyer, G., Ross, A. J. & Smith, L. (1969). Some metabolic effects of bacterial endotoxins in salmonid fishes. Journal Fisheries Research Board of Canada 26, 115-119. Westphal, O. & Jann, K. (1965). Bacterial lipopolysaccharides. Extraction with phenolwater and further applications of the procedure. Methods in Carbohydrate Chemistry 5, 83-91. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J. & Mathison, J. C. (1990). CD14, a receptor for complexes of lipopolysaccharides (LPS) and LPS binding protein. Science 249, 1431-1433. Yagawa, K., Kaku, M., Ichinose, Y., Nagao, S., Tanaka, A. & Tomoda, A. (1984). Biphasic effects of muramyl dipeptide or lipopolysaccharide on superoxide anion-generating activities of macrophages. Infection and Immunity 45, 82-85.