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Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin
Comparative Biochemistry and Physiology Part C 123 (1999) 53 – 59
Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin Christopher A. Ottinger 1, Stewart C. Johnson *, K. Vanya Ewart, Laura L. Brown, Neil W. Ross Institute for Marine Biosciences, National Research Council Canada, 1411 Oxford Street, Halifax, No6a Scotia, B3H 3Z1, Canada Received 1 September 1998; received in revised form 11 January 1999; accepted 27 January 1999
Abstract We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on Aeromonas salmonicida viability and the anti-A. salmonicida activity of Atlantic salmon macrophages. In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml − 1 to virulent A. salmonicida failed to significantly reduce (P\ 0.05) cell viability. However, binding of the lectin to A. salmonicida did result in significant (P50.05) dose-dependent increases in phagocytosis, and bactericidal activity. Significant increases (P 50.05) were also observed in phagocyte respiratory burst activity within the lectin concentration range of 4.0 – 20.0 ng ml − 1 but the stimulation was not dose dependent at these lectin concentrations. At the lowest lectin concentration tested (0.32 ng ml − 1), a significant decrease (P50.05) in respiratory burst was observed. The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A. salmonicida. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Aeromonas salmonicida; Atlantic salmon; Furunculosis; Innate immunity; Lectin; Macrophage; Mannose binding; Salmo salar
1. Introduction Lectins are sugar-binding proteins that may be intracellular, extracellular or membrane bound. In animals, extracellular and soluble lectins recognize specific carbohydrate arrays on pathogen surfaces and enable the host to identify pathogens as non-self. Once bound to pathogens, lectins can stimulate increased uptake by phagocytosis and complement-mediated cell lysis. In mammals, lectins such as human mannose-binding protein (MBP) are known to play an important role in
* Corresponding author. Tel.: +1-902-426-2630; fax: + 1-902-4269413. E-mail address: [email protected] (S.C. Johnson) 1 Current address: National Fish Health Research Laboratory, Kearneysville, WV 25430, USA.
innate immunity and disease avoidance [15,24,25,31,47]. Lectins have been isolated from serum, plasma, surface mucus, egg surfaces and egg components from a wide variety of fish species [18,19]. Generally, little is known about the biological functions of these lectins in fish, although they are thought to play a role in protection against bacterial pathogens and in the prevention of polyspermy. A number of MBPs have been isolated from salmon serum using mannose–agarose beads [4]. One of these was a calcium-dependent lectin, which exists as a disulfide-linked multimer of a polypeptide having a relative molecular mass (Mr) of 17,000. The concentration of this lectin in serum was estimated to be approximately 5 mg ml − 1. This lectin was demonstrated to bind to the surfaces of the fish bacterial pathogens Vibrio anguillarum and Aeromonas salmonicida [4].
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To investigate the functional properties of this lectin we used A. salmonicida, the causative agent of furunculosis, as it is an economically important pathogen of Atlantic salmon and other salmonids. This bacterium has a mannose-containing cell wall [2], secretes a mannose-rich extracellular polysaccharide [2,11] and produces a capsule in which mannose is a principal component [11]. Virulence factors of A. salmonicida, including some associated with the surface A-layer, suppress immune function as well as protect the bacteria from intracellular killing [3,8,10,12,27,30,33,35,49]. In this study, we evaluated the ability of this mannose-binding lectin to inhibit the growth of wild-type (virulent) A. salmonicida and to enhance Atlantic salmon macrophage anti-A. salmonicida activity in vitro. We also investigated dose-dependent enhancement of phagocytosis, bactericidal and respiratory burst activity.
2. Materials and methods
2.1. Fish Post-smolt Atlantic salmon (38 – 122 g) were maintained in 200-l tanks supplied with flow-through sea water at 10–12°C and a photoperiod that matched local seasonal changes. Prior to removal of the head kidney, fish were fully anesthetized using tricaine methanesulfonate (TMS; Syndel Laboratories, Vancouver, BC, Canada) and bled from the caudal artery.
2.2. Lectin Purified lectin was obtained using the methods described elsewhere [4]. Briefly, lectin was purified from the serum of Atlantic salmon using a two-step chromatography procedure. Mannose-binding compounds were absorbed from serum using mannose-coated beads. Following elution, the semi-purified solution was passed through a Sepharose 4B column to remove any proteins that did not specifically bind to the mannose beads. The concentration of the purified lectin was determined by using a modified colloidal protein–gold quantification system (ISS Protein Gold; Integrated Separation Systems, MA).
2.3. Bacteria Virulent Aeromonas salmonicida (strain 80204) [33] was used in all experiments. This strain possesses the A-layer, lipopolysaccharide (LPS) and the macrophage cytotoxic factor [33]. The presence of the A-layer and LPS was confirmed by sodium dodecyl sulfate –polyacrylamide electrophoresis (SDS – PAGE) [32] using whole-cell lysates of both heat-killed and live cells (see
below). For all applications, bacteria were grown to log phase in tryptic soy broth at 15°C. Bacteria were washed three times in Hank’s balanced salt solution (HBSS) at 4°C, re-suspended in cold HBSS, and cell number estimated by measuring absorbance at 600 nm. The relationship between absorbance and cell number was determined previously by plate counts. In experiments involving dead bacteria, cells were heat-killed by incubation at 70°C for 30 min, allowed to cool and then washed and cell number estimated as described above.
2.4. Macrophage isolation Macrophages were isolated from the anterior kidney following a modification of the methods described elsewhere [45]. Anterior kidneys were removed aseptically, placed in 5 ml of L-15 media supplemented with 2% fetal bovine serum (FBS), 100 units ml − 1 penicillin/ streptomycin (P/S) and 10 units ml − 1 heparin (L-15/ 2%) and stored on ice until processing. Kidney tissue was dissociated by repeated passage through a 3-ml syringe. Tissue fragments were allowed to settle for 10 min and the cells in suspension were removed. The cell suspensions were pelleted (500× g for 10 min at 4°C), washed twice in 10 ml L-15/2%, then layered on 34/51% Percoll discontinuous gradients and centrifuged at 400× g for 20 min at 4°C. The macrophage enriched fraction from the 34/51% interface was collected, pelleted by centrifugation at 500× g for 10 min at 4°C, washed with 10 ml of L-15/2%, and then re-suspended in 5 ml of L-15 medium supplemented with 0.1% FBS and 100 units ml − 1 P/S (L-15/0.1%). The number of viable cells was determined by trypan blue exclusion and the cell density adjusted to 1× 107 cells ml − 1. Cells were plated at 100 ml per well in 96-well or at 200 ml per well in 24-well tissue culture plates. When 24-well plates were used, 800 ml media (L-15/0.1%) and a 12-mm circular cover glass were added to the wells prior to the addition of the cells. Plates were incubated for 2 h at 17°C in a humidified incubator after which the media containing the non-adherent cells was removed and replaced with either 100 ml or 1 ml of L-15/5% FBS/100 units ml − 1 P/S (L-15/5%). Plates were then incubated for an additional 24 h at 17°C in a humidified incubator.
2.5. Lectin opsonization effects on A. salmonicida 6iability A. salmonicida was suspended at 1× 109 viable colony-forming units (cfu) ml − 1 in HBSS containing lectin concentrations ranging from 0 to 20 ng ml − 1 and incubated with gentle mixing for 2 h at room temperature. Both the opsonized and non-opsonized control bacteria were pelleted, washed three times with cold
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HBSS and re-suspended in HBSS at a concentration of 1× 109 bacteria ml − 1. The bacteria were then serially diluted in peptone saline and 3×25 ml drops of each dilution were plated on duplicate tryptic soy agar plates. After 48 h incubation at 15°C colonies were counted for the 10 − 5 dilution. The viability of opsonized bacteria was compared to the non-opsonized using analysis of variance (ANOVA) of the pooled data from the duplicate plates.
2.6. Lectin opsonization of phagocytosis in macrophages Macrophage isolates from ten salmon were screened for lectin-opsonized phagocytosis of A. salmonicida [37]. Fifty ml per well (50 ml bacteria solution at A600 = 1 in a total volume of 1050 ml per well =5.0 ×107 cfu per well) of either lectin-opsonized or non-opsonized heatkilled A. salmonicida were added to 24-well plates containing macrophages (2× 106 cells per well) adhered to cover glasses. The plates were incubated for 3 h at 17°C. Following incubation, the macrophages on the cover glasses were fixed with 2% formaldehyde, permeabilized by 30 s exposure to 250 ml of 1 mg ml − 1 Zwittergent (Sigma Chemicals) and washed twice with L-15 media. The macrophages were then incubated with 250 ml per well of rabbit anti-A. salmonicida antibodies (Microtek International) diluted 1:50 in L-15 for 15 min at room temperature, washed with L-15, then incubated with 250 ml per well fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG (Sigma) diluted 1:100. Following incubation with the secondary antibody the macrophages were washed twice in L-15, counterstained with 250 ml per well 0.01% bis-benzimide (Hoechst 33258; Polysciences Inc., Warrington, PA) in L-15 and then washed twice again. Cells were observed using epifluorescence microscopy. Macrophages were identified by the lateral position and kidney bean shape of their stained nuclei. The occurrence of phagocytosis was determined by the presence of a minimum of five FITC-labeled bacteria in the macrophages. Visualization of non-phagocytosed A. salmonicida was rare. FITC fluorescence was not observed in non-permeabilized macrophages or in permeabilized cells not exposed to the bacteria. To determine if there was a significant dose response the data were Logit transformed and a regression analysis was performed.
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lectin as described above. Then 10 ml per well of an A600 = 0.01 solution of either lectin-opsonized or nonopsonized bacteria (1×105 cfu per well) were added to each well containing 1× 106 macrophages per well. Controls consisted of either lectin-opsonized or non-opsonized bacteria added to wells that contained only media. Plates were incubated for 3 h at 17°C in a humidified incubator. Following lysis of the macrophages with 0.2% Tween-20 in tissue culture grade water, the bacteria were resuspended and serially diluted (10-fold) in peptone saline. Then 25-ml drops of each dilution were plated in triplicate on tryptic soy agar plates and the plates were incubated for 24 h at 15°C. The number of cfu for each dilution was represented by the triplicate mean. Dose dependency was assessed by regression analysis.
2.8. Lectin-induced macrophage respiratory burst acti6ity Macrophage isolates from 31 salmon were screened for respiratory burst activity in 96-well plates using the nitroblue tetrazolium (NBT) method [36]. Prior to treatment cells (1×106 cells per well) were washed with HBSS and then 100 ml of NBT in HBSS (NBT/HBSS) were added to each well. Triplicate or quadruplicate (depending on the number of available cells per fish) wells then received either 10 ml of opsonized heat-killed A. salmonicida in NBT/HBSS (1× 105 cfu per well), 10 ml of non-opsonized A. salmonicida (1× 105 cfu per well), in NBT/HBSS or 10 ml NBT/HBSS (negative control). Plates were incubated for 3 h at 17°C prior to fixation and solubilization of the NBT, and then reading at 650 nm in a multiscan spectrophotometer. As a control experiment, respiratory burst activity was measured in isolated macrophages exposed to the same concentrations of lectin alone, in the absence of bacteria. Purified lectin in NBT/HBSS was added to triplicate wells that contained cells harvested and treated as above. Respiratory burst activity was measured as above. Values for each group represented the mean of the triplicate or quadruplicate wells per fish. Data were pooled and the effect of the bound lectin was evaluated by regression analysis and single-tailed t-tests that compared doses to each other and to the negative control. Cells from two fish that showed no NBT reduction were excluded from the analysis.
2.7. Bactericidal acti6ity 3. Results Macrophages isolated from ten salmon were used to assess lectin enhancement of macrophage killing of A. salmonicida [12]. In our assay, the 96-well plates were not shaken or centrifuged prior to incubation with the bacteria. Viable A. salmonicida were opsonized with
A 2-h incubation of viable A. salmonicida with 0, 0.8, 4.0 or 20.0 ng ml − 1 of purified lectin in the absence of other factors did not significantly (one-way ANOVA, P\0.05) reduce the viability of the bacteria (Fig. 1).
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Incubation of heat-killed A. salmonicida with 0, 0.16, 0.8, 4.0 or 20.0 ng ml − 1 of purified lectin resulted in significant (regression analysis, P B 0.001) dose-dependent opsonization of the bacteria for phagocytosis by Atlantic salmon macrophages (Fig. 2). At lectin concentrations as low as 0.8 ng ml − 1 there was statistically significant enhancement (t-test, P 5 0.05) of phagocytic activity when compared to the non-opsonized bacteria control. Incubation of viable A. salmonicida with 0, 0.32, 1.6 or 20.0 ng ml − 1 of purified lectin resulted in a significant (regression analysis, P 5 0.05) dose-dependent lectin enhancement of bactericidal activity by Atlantic salmon macrophages (Fig. 3). A. salmonicida incubated in the absence of macrophages but otherwise treated Fig. 3. Effects of mannose-binding lectin on macrophage antiAeromonas salmonicida bactericidal activity. Viable A. salmonicida were incubated for 2 h at room temperature with various concentration of mannose-binding lectin after which they were incubated for 3 h at 17°C with (white bars) or without (black bars) Atlantic salmon macrophages. Following incubation the macrophages were lysed, the bacteria resuspended and serially diluted in peptone saline and plated on tryptic soy agar. Values are mean+S.E. cfu. Results significantly different (P 50.05) from control are indicated by an asterisk (*). N =10 fish.
Fig. 1. Viability of the bacterium Aeromonas salmonicida incubated for 2 h at room temperature with varying concentrations of mannosebinding lectin in the absence of other factors. Values are mean +S.E. cfu.
Fig. 2. Mannose-binding lectin enhanced phagocytosis of Aeromonas salmonicida. Heat-killed A. salmonicida were incubated for 2 h at room temperature with varying concentrations of the mannose-binding lectin. The lectin-opsonized and non-opsonized bacteria were then incubated for 3 h at 17°C with Atlantic salmon head kidney macrophages after which they were fixed, permeabilized, stained with FITC, and counterstained with bis-benzimide. Values are the mean percentage +S.E. of macrophages that contained a minimum of five FITC-labeled bacteria. Results significantly different (P5 0.05) from control are indicated by an asterisk (*). N= 10 fish.
the same as those with macrophages showed no significant (t-tests, P\0.05) lectin-associated reduction in bacterial growth. Incubation of heat-killed A. salmonicida with 4.0 and 20.0 ng ml − 1 purified lectin resulted in significant increases (t-test, P5 0.05) in respiratory burst activity relative to the activity observed in macrophages incubated alone or with non-opsonized A. salmonicida as measured in NBT assays (Fig. 4). There was a significant decrease (t-tests, P5 0.05) in respiratory burst activity in macrophages incubated with heat-killed A. salmonicida which had been incubated with 0.32 ng ml − 1 of lectin relative to all other treatment and control groups. Respiratory burst activity in macrophages incubated with non-opsonized A. salmonicida was not significantly different (t-test, P\ 0.05) from macrophages incubated in the absence of the bacteria. Also, in the absence of bacteria, there was no significant difference in respiratory burst activity in macrophages exposed to the various lectin concentrations when compared to control macrophages; those exposed to no lectin (data not shown).
4. Discussion The lectin used in our study has been shown to bind to the salmon pathogens Vibrio anguillarum and A. salmonicida [4]. In this study, incubation of viable A. salmonicida with nanogram amounts of purified lectin in the absence of macrophages had no effect on in vitro
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growth. Lectins isolated from the eggs of Pacific salmon have previously been shown to inhibit growth of various bacterial pathogens except A. salmonicida when used at higher concentrations. Inhibition of growth of various fish pathogens after incubation with a purified D-galactose-L-rhamnose-specific lectin isolated from the ova of the chinook salmon (Oncorhynchus tshawytscha) has been reported [48]. Incubation with 150 mg ml − 1 chinook ova lectin for 1 h resulted in a 99% reduction of growth of V. anguillarum. A 1-h incubation with 0.5 mg ml − 1 chinook ova lectin reduced the growth of Yersinia ruckeri, Aeromonas hydrophila and Edwardsiella tarda by 76, 77 and 36% respectively. At the highest concentration tested, the chinook ova lectin had no effect on A. salmonicida or Renibacterium salmoninarum growth. A galactose-binding lectin has been purified from coho salmon eggs that bound to A. salmonicida cells but not to the other pathogens A. hydrophila, V. anguillarum, Vibrio ordalii, or R. salmoninarum [51]. This lectin showed no antibacterial activity against A. salmonicida [52]. The concentration of the lectin described in the present study has been estimated to be approximately 5 mg ml − 1 serum [4]. This is considerably higher than the maximum concentrations used in our study and it is possible that at higher concentrations this lectin may show antimicrobial activity. It is also possible that A. salmonicida is generally resistant to the antibacterial activities of lectins and that the lectin we studied might show some antimicrobial activity against other bacterial species.
Fig. 4. Mannose-binding lectin-induced respiratory burst activity of Atlantic salmon head kidney macrophages. Heat-killed A. salmonicida were incubated for 2 h at room temperature with various concentration of mannose-binding lectin. The opsonized bacteria were then incubated with macrophages for 3 h at 17°C prior to fixation and solubilization of the NBT. Values are mean +S.E. OD650. Results significantly different (P5 0.05) from control are indicated by an asterisk (*) ** indicates significant difference in the macrophages exposed to bacteria opsonized with 0.32 ng ml − 1 lectin compared to control macrophages and other treatment groups. N= 31 fish.
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In our study, opsonization of viable and heat-killed A. salmonicida with a Ca2 + -binding type mannosebinding lectin purified from the serum of Atlantic salmon resulted in dose-dependent increases in phagocytosis, bactericidal activity and a significant increase in respiratory burst activity by Atlantic salmon macrophages. Previous reports have described opsonization of microbial pathogens and parasites with antibody or complement resulting in enhanced fish leukocyte adherence [34], phagocytosis [1,2,13,16,20, 22,28,29,38,41,42,46] and killing [50]. Although a wide variety of lectins have been isolated from fish, this is the first report of opsonization by a purified lectin and its effects on fish immunity: bactericidal activity, phagocytosis and enhancement of respiratory burst activity. In mammals the effects of lectins on these processes have been well documented [14,21,39]. The maximum lectin concentration of 20.0 ng ml − 1 that was used in this study is well below the estimated 5 mg ml − 1 concentration of this lectin in the serum [4]. However, the physiologically relevant concentration of this lectin in tissues or mucus (sites at which it is likely to be an important contributor to innate immunity) may be in the ng/ml range. Given the number of variables in this experimental system, it seemed prudent to use the heat-killed A. salmonicida for the phagocytosis and respiratory burst assays to eliminate the possibility of cytotoxicity associated with this virulent strain [33]. It is possible that binding of this lectin to the surface of virulent A. salmonicida may reduce cytotoxicity, thereby enhancing levels of phagocytosis and macrophage bactericidal activity. Respiratory burst activity in macrophages is an important mechanism by which A. salmonicida are killed following phagocytosis [44] although there is evidence that the bacterium can resist damage by reactive oxygen species [9]. When A. salmonicida was incubated with increasing amounts of lectin there was a significant increase in bactericidal activity of the macrophages. Assuming that increased quantities of lectin relate to an increased number of A. salmonicida with lectin on their surface, our results suggest that the lectin-labeled bacteria are interacting directly with the macrophages. Interactions between lectin and the macrophage may be occurring via receptors on the macrophage surface. The occurrence of such receptors has been postulated for phagocytic cells in mammals [15,43]. There was not a linear relationship between lectin concentration and respiratory burst activity, although a significant increase in respiratory burst activity was seen at 4 and 20 ng ml − 1 when compared to controls. This suggests that other mechanisms may also be involved in the stimulation of oxygen radical formation by macrophages. It is possible that macrophages interacting with lectin-bound A. salmonicida are induced to
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produced cytokines, such as macrophage activating factor (MAF). These cytokines may then stimulate other macrophages that have not yet encountered lectin-labeled bacteria. The ability of MAF from stimulated macrophage cultures to induce respiratory burst activity in otherwise unstimulated cells has been reported for rainbow trout [17]. Although the ability of isolated Atlantic salmon macrophages to produce MAF has yet to be demonstrated, leukocytes from the anterior kidney of this species have been shown to produce MAF when stimulated with intact A. salmonicida [26] or with outer membrane proteins obtained from A. salmonicida [7]. In this model, a minimum lectin dose would be required for activation of the macrophage population since cytokines would provide an amplification of its effect. It is not clear why a significantly reduced respiratory burst activity in the 0.32 ng ml − 1 treatment was found when compared to the controls. As our results were obtained in a culture system, it could be argued that the media or cell secretory components could have contributed to the observed activities. We minimized this possibility by using heat-inactivated fetal bovine serum in our media so that neither immunoglobulin nor active complement components were provided. The remaining media components are not known to enhance respiratory burst, phagocytosis or bactericidal activity. Enhancement could have also resulted from antibody or complement components produced by the leukocytes in the cultures. Our leukocyte cultures were enriched for macrophages, but they were not pure and thus antibodies capable of binding to the A. salmonicida could have been present. Although fish macrophages have not been shown to produce complement components, macrophage-derived complement has been shown to opsonize pathogens occurring outside the vascular system in mammals [5,6]. However, if our results were due to factors present in the media or produced in the culture then we would expect the observed enhancements to have been independent of lectin concentration, which was not the case. A cortical lectin from the oocytes of the loach, Rutilus rutilus, has been shown to stimulate mitogenic activity and the release of soluble factors (IL-2, TXB2, PGE2) from human lymphocyte cultures [23]. The human MBP is known to function as an opsonin independent of complement and immunoglobulins, as well as to mediate complement deposition resulting in killing of organisms normally resistant to complement-mediated lysis in the absence of MBP [40]. In this study, we provide the first evidence of lectin-enhanced macrophage activity in a fish. This mannose-binding lectin may substantially contribute to innate immunity of Atlantic salmon both through its direct effects on macrophages, as well as its effects on other cells and interactions with other molecules of the innate immune system similar to those described in the above studies.
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[36] Rook GAW, Steele J, Umar S, Dockrell HM. A simple method for the solubilisation of reduced NBT, and its use as a colormetric assay for activation of human macrophages by g-interferon. J Immunol Methods 1985;82:161 – 7. [37] Rose AS, Levine RP. Complement-mediated opsonisation and phagocytosis of Renibacterium salmoninarum. Fish Shellfish Immunol 1992;2:223 – 40. [38] Sakai DK. Opsonization by fish antibody and complement in the immune phagocytosis by peritoneal exudate cells isolated from salmonid fishes. J Fish Dis 1984;7:29 – 38. [39] Sastry K, Esekowitz RA. Collectins: pattern recognition molecules involved in first line host defense. Curr Opin Immunol 1993;5:59 – 66. [40] Schweinle JE, Ezekowitz RAB, Tenner AJ, Kuhlman M, Joiner KA. Human mannose-binding protein activates the alternative complement pathway and enhances serum bactericidal activity on a mannose-rich isolate of Salmonella. J Clin Invest 1989;84:1821 – 9. [41] Scott AL, Rogers WQA, Klesius PH. Chemiluminescence by peripheral blood phagocytes from channel catfish: function of opsonin and temperature. Dev Comp Immunol 1985;9:241–50. [42] Secombes CJ. Immunological activation of rainbow trout macrophages induced in vitro by sperm autoantibodies and factors derived from testis sensitised leucocytes. Vet Immunol Immunopathol 1986;12:193 – 201. [43] Sharon N. Bacterial lectins, cell – cell recognition and infectious disease. FEBS Lett 1987;217:145 – 57. [44] Sharp GJE, Secombes CJ. The role of reactive oxygen species in the killing of the bacterial fish pathogen Aeromonas salmonicida by rainbow trout macrophages. Fish Shellfish Immunol 1993;3:119 – 29. [45] Sharp GJE, Pike AW, Secombes CJ. Leucocyte migration in rainbow trout (Oncorhynchus mykiss [Walbaum]): optimization of migration condition and responses to host an pathogen (Diphyllobothrium dendriticum [Nitzsch]) derived chemoattractants. Dev Comp Immunol 1991;15:295 – 305. [46] Stave JW, Roberson BS, Hetrick FM. Factors affecting the chemiluminescent response of fish phagocytes. J Fish Biol 1984;25:197 – 206. [47] Turner MW. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol Today 1996;17:532–40. [48] Voss EW Jr, Fryer JL, Banowetz GM. Isolation, purification, and partial characterization of a lectin from chinook salmon ova. Arch Biochem Biophys 1978;186:25 – 34. [49] Weeks-Perkins BA, Ellis AE. Chemotactic responses of Atlantic salmon (Salmo salar) macrophages to virulent and attenuated strains of Aeromonas salmonicida. Fish Shellfish Immunol 1995;5:313 – 23. [50] Whyte SK, Chappell LH, Secombes CJ. Cytotoxic reactions of rainbow trout, Salmo gairdneri Richardson, macrophages for larvae of the eye fluke Diplostomum spathaceum (Digenea). J Fish Biol 1989;35:333 – 45. [51] Yousif AN, Albright LJ, Evelyn TPT. Purification and characterization of a galactose-specific lectin from the eggs of coho salmon Oncorhynchus kisutch, and its interaction with bacterial fish pathogens. Dis Aquat Org 1994;20:127 – 36. [52] Yousif AN, Albright LJ, Evelyn TPT. Interaction of coho salmon Oncorhynchus kisutch egg lectin with the fish pathogen Aeromonas salmonicida. Dis Aquat Org 1995;21:193 – 9.
Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin Christopher A. Ottinger 1, Stewart C. Johnson *, K. Vanya Ewart, Laura L. Brown, Neil W. Ross Institute for Marine Biosciences, National Research Council Canada, 1411 Oxford Street, Halifax, No6a Scotia, B3H 3Z1, Canada Received 1 September 1998; received in revised form 11 January 1999; accepted 27 January 1999
Abstract We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on Aeromonas salmonicida viability and the anti-A. salmonicida activity of Atlantic salmon macrophages. In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml − 1 to virulent A. salmonicida failed to significantly reduce (P\ 0.05) cell viability. However, binding of the lectin to A. salmonicida did result in significant (P50.05) dose-dependent increases in phagocytosis, and bactericidal activity. Significant increases (P 50.05) were also observed in phagocyte respiratory burst activity within the lectin concentration range of 4.0 – 20.0 ng ml − 1 but the stimulation was not dose dependent at these lectin concentrations. At the lowest lectin concentration tested (0.32 ng ml − 1), a significant decrease (P50.05) in respiratory burst was observed. The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A. salmonicida. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Aeromonas salmonicida; Atlantic salmon; Furunculosis; Innate immunity; Lectin; Macrophage; Mannose binding; Salmo salar
1. Introduction Lectins are sugar-binding proteins that may be intracellular, extracellular or membrane bound. In animals, extracellular and soluble lectins recognize specific carbohydrate arrays on pathogen surfaces and enable the host to identify pathogens as non-self. Once bound to pathogens, lectins can stimulate increased uptake by phagocytosis and complement-mediated cell lysis. In mammals, lectins such as human mannose-binding protein (MBP) are known to play an important role in
* Corresponding author. Tel.: +1-902-426-2630; fax: + 1-902-4269413. E-mail address: [email protected] (S.C. Johnson) 1 Current address: National Fish Health Research Laboratory, Kearneysville, WV 25430, USA.
innate immunity and disease avoidance [15,24,25,31,47]. Lectins have been isolated from serum, plasma, surface mucus, egg surfaces and egg components from a wide variety of fish species [18,19]. Generally, little is known about the biological functions of these lectins in fish, although they are thought to play a role in protection against bacterial pathogens and in the prevention of polyspermy. A number of MBPs have been isolated from salmon serum using mannose–agarose beads [4]. One of these was a calcium-dependent lectin, which exists as a disulfide-linked multimer of a polypeptide having a relative molecular mass (Mr) of 17,000. The concentration of this lectin in serum was estimated to be approximately 5 mg ml − 1. This lectin was demonstrated to bind to the surfaces of the fish bacterial pathogens Vibrio anguillarum and Aeromonas salmonicida [4].
0742-8413/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 2 - 8 4 1 3 ( 9 9 ) 0 0 0 0 9 - 2
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To investigate the functional properties of this lectin we used A. salmonicida, the causative agent of furunculosis, as it is an economically important pathogen of Atlantic salmon and other salmonids. This bacterium has a mannose-containing cell wall [2], secretes a mannose-rich extracellular polysaccharide [2,11] and produces a capsule in which mannose is a principal component [11]. Virulence factors of A. salmonicida, including some associated with the surface A-layer, suppress immune function as well as protect the bacteria from intracellular killing [3,8,10,12,27,30,33,35,49]. In this study, we evaluated the ability of this mannose-binding lectin to inhibit the growth of wild-type (virulent) A. salmonicida and to enhance Atlantic salmon macrophage anti-A. salmonicida activity in vitro. We also investigated dose-dependent enhancement of phagocytosis, bactericidal and respiratory burst activity.
2. Materials and methods
2.1. Fish Post-smolt Atlantic salmon (38 – 122 g) were maintained in 200-l tanks supplied with flow-through sea water at 10–12°C and a photoperiod that matched local seasonal changes. Prior to removal of the head kidney, fish were fully anesthetized using tricaine methanesulfonate (TMS; Syndel Laboratories, Vancouver, BC, Canada) and bled from the caudal artery.
2.2. Lectin Purified lectin was obtained using the methods described elsewhere [4]. Briefly, lectin was purified from the serum of Atlantic salmon using a two-step chromatography procedure. Mannose-binding compounds were absorbed from serum using mannose-coated beads. Following elution, the semi-purified solution was passed through a Sepharose 4B column to remove any proteins that did not specifically bind to the mannose beads. The concentration of the purified lectin was determined by using a modified colloidal protein–gold quantification system (ISS Protein Gold; Integrated Separation Systems, MA).
2.3. Bacteria Virulent Aeromonas salmonicida (strain 80204) [33] was used in all experiments. This strain possesses the A-layer, lipopolysaccharide (LPS) and the macrophage cytotoxic factor [33]. The presence of the A-layer and LPS was confirmed by sodium dodecyl sulfate –polyacrylamide electrophoresis (SDS – PAGE) [32] using whole-cell lysates of both heat-killed and live cells (see
below). For all applications, bacteria were grown to log phase in tryptic soy broth at 15°C. Bacteria were washed three times in Hank’s balanced salt solution (HBSS) at 4°C, re-suspended in cold HBSS, and cell number estimated by measuring absorbance at 600 nm. The relationship between absorbance and cell number was determined previously by plate counts. In experiments involving dead bacteria, cells were heat-killed by incubation at 70°C for 30 min, allowed to cool and then washed and cell number estimated as described above.
2.4. Macrophage isolation Macrophages were isolated from the anterior kidney following a modification of the methods described elsewhere [45]. Anterior kidneys were removed aseptically, placed in 5 ml of L-15 media supplemented with 2% fetal bovine serum (FBS), 100 units ml − 1 penicillin/ streptomycin (P/S) and 10 units ml − 1 heparin (L-15/ 2%) and stored on ice until processing. Kidney tissue was dissociated by repeated passage through a 3-ml syringe. Tissue fragments were allowed to settle for 10 min and the cells in suspension were removed. The cell suspensions were pelleted (500× g for 10 min at 4°C), washed twice in 10 ml L-15/2%, then layered on 34/51% Percoll discontinuous gradients and centrifuged at 400× g for 20 min at 4°C. The macrophage enriched fraction from the 34/51% interface was collected, pelleted by centrifugation at 500× g for 10 min at 4°C, washed with 10 ml of L-15/2%, and then re-suspended in 5 ml of L-15 medium supplemented with 0.1% FBS and 100 units ml − 1 P/S (L-15/0.1%). The number of viable cells was determined by trypan blue exclusion and the cell density adjusted to 1× 107 cells ml − 1. Cells were plated at 100 ml per well in 96-well or at 200 ml per well in 24-well tissue culture plates. When 24-well plates were used, 800 ml media (L-15/0.1%) and a 12-mm circular cover glass were added to the wells prior to the addition of the cells. Plates were incubated for 2 h at 17°C in a humidified incubator after which the media containing the non-adherent cells was removed and replaced with either 100 ml or 1 ml of L-15/5% FBS/100 units ml − 1 P/S (L-15/5%). Plates were then incubated for an additional 24 h at 17°C in a humidified incubator.
2.5. Lectin opsonization effects on A. salmonicida 6iability A. salmonicida was suspended at 1× 109 viable colony-forming units (cfu) ml − 1 in HBSS containing lectin concentrations ranging from 0 to 20 ng ml − 1 and incubated with gentle mixing for 2 h at room temperature. Both the opsonized and non-opsonized control bacteria were pelleted, washed three times with cold
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HBSS and re-suspended in HBSS at a concentration of 1× 109 bacteria ml − 1. The bacteria were then serially diluted in peptone saline and 3×25 ml drops of each dilution were plated on duplicate tryptic soy agar plates. After 48 h incubation at 15°C colonies were counted for the 10 − 5 dilution. The viability of opsonized bacteria was compared to the non-opsonized using analysis of variance (ANOVA) of the pooled data from the duplicate plates.
2.6. Lectin opsonization of phagocytosis in macrophages Macrophage isolates from ten salmon were screened for lectin-opsonized phagocytosis of A. salmonicida [37]. Fifty ml per well (50 ml bacteria solution at A600 = 1 in a total volume of 1050 ml per well =5.0 ×107 cfu per well) of either lectin-opsonized or non-opsonized heatkilled A. salmonicida were added to 24-well plates containing macrophages (2× 106 cells per well) adhered to cover glasses. The plates were incubated for 3 h at 17°C. Following incubation, the macrophages on the cover glasses were fixed with 2% formaldehyde, permeabilized by 30 s exposure to 250 ml of 1 mg ml − 1 Zwittergent (Sigma Chemicals) and washed twice with L-15 media. The macrophages were then incubated with 250 ml per well of rabbit anti-A. salmonicida antibodies (Microtek International) diluted 1:50 in L-15 for 15 min at room temperature, washed with L-15, then incubated with 250 ml per well fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit IgG (Sigma) diluted 1:100. Following incubation with the secondary antibody the macrophages were washed twice in L-15, counterstained with 250 ml per well 0.01% bis-benzimide (Hoechst 33258; Polysciences Inc., Warrington, PA) in L-15 and then washed twice again. Cells were observed using epifluorescence microscopy. Macrophages were identified by the lateral position and kidney bean shape of their stained nuclei. The occurrence of phagocytosis was determined by the presence of a minimum of five FITC-labeled bacteria in the macrophages. Visualization of non-phagocytosed A. salmonicida was rare. FITC fluorescence was not observed in non-permeabilized macrophages or in permeabilized cells not exposed to the bacteria. To determine if there was a significant dose response the data were Logit transformed and a regression analysis was performed.
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lectin as described above. Then 10 ml per well of an A600 = 0.01 solution of either lectin-opsonized or nonopsonized bacteria (1×105 cfu per well) were added to each well containing 1× 106 macrophages per well. Controls consisted of either lectin-opsonized or non-opsonized bacteria added to wells that contained only media. Plates were incubated for 3 h at 17°C in a humidified incubator. Following lysis of the macrophages with 0.2% Tween-20 in tissue culture grade water, the bacteria were resuspended and serially diluted (10-fold) in peptone saline. Then 25-ml drops of each dilution were plated in triplicate on tryptic soy agar plates and the plates were incubated for 24 h at 15°C. The number of cfu for each dilution was represented by the triplicate mean. Dose dependency was assessed by regression analysis.
2.8. Lectin-induced macrophage respiratory burst acti6ity Macrophage isolates from 31 salmon were screened for respiratory burst activity in 96-well plates using the nitroblue tetrazolium (NBT) method [36]. Prior to treatment cells (1×106 cells per well) were washed with HBSS and then 100 ml of NBT in HBSS (NBT/HBSS) were added to each well. Triplicate or quadruplicate (depending on the number of available cells per fish) wells then received either 10 ml of opsonized heat-killed A. salmonicida in NBT/HBSS (1× 105 cfu per well), 10 ml of non-opsonized A. salmonicida (1× 105 cfu per well), in NBT/HBSS or 10 ml NBT/HBSS (negative control). Plates were incubated for 3 h at 17°C prior to fixation and solubilization of the NBT, and then reading at 650 nm in a multiscan spectrophotometer. As a control experiment, respiratory burst activity was measured in isolated macrophages exposed to the same concentrations of lectin alone, in the absence of bacteria. Purified lectin in NBT/HBSS was added to triplicate wells that contained cells harvested and treated as above. Respiratory burst activity was measured as above. Values for each group represented the mean of the triplicate or quadruplicate wells per fish. Data were pooled and the effect of the bound lectin was evaluated by regression analysis and single-tailed t-tests that compared doses to each other and to the negative control. Cells from two fish that showed no NBT reduction were excluded from the analysis.
2.7. Bactericidal acti6ity 3. Results Macrophages isolated from ten salmon were used to assess lectin enhancement of macrophage killing of A. salmonicida [12]. In our assay, the 96-well plates were not shaken or centrifuged prior to incubation with the bacteria. Viable A. salmonicida were opsonized with
A 2-h incubation of viable A. salmonicida with 0, 0.8, 4.0 or 20.0 ng ml − 1 of purified lectin in the absence of other factors did not significantly (one-way ANOVA, P\0.05) reduce the viability of the bacteria (Fig. 1).
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Incubation of heat-killed A. salmonicida with 0, 0.16, 0.8, 4.0 or 20.0 ng ml − 1 of purified lectin resulted in significant (regression analysis, P B 0.001) dose-dependent opsonization of the bacteria for phagocytosis by Atlantic salmon macrophages (Fig. 2). At lectin concentrations as low as 0.8 ng ml − 1 there was statistically significant enhancement (t-test, P 5 0.05) of phagocytic activity when compared to the non-opsonized bacteria control. Incubation of viable A. salmonicida with 0, 0.32, 1.6 or 20.0 ng ml − 1 of purified lectin resulted in a significant (regression analysis, P 5 0.05) dose-dependent lectin enhancement of bactericidal activity by Atlantic salmon macrophages (Fig. 3). A. salmonicida incubated in the absence of macrophages but otherwise treated Fig. 3. Effects of mannose-binding lectin on macrophage antiAeromonas salmonicida bactericidal activity. Viable A. salmonicida were incubated for 2 h at room temperature with various concentration of mannose-binding lectin after which they were incubated for 3 h at 17°C with (white bars) or without (black bars) Atlantic salmon macrophages. Following incubation the macrophages were lysed, the bacteria resuspended and serially diluted in peptone saline and plated on tryptic soy agar. Values are mean+S.E. cfu. Results significantly different (P 50.05) from control are indicated by an asterisk (*). N =10 fish.
Fig. 1. Viability of the bacterium Aeromonas salmonicida incubated for 2 h at room temperature with varying concentrations of mannosebinding lectin in the absence of other factors. Values are mean +S.E. cfu.
Fig. 2. Mannose-binding lectin enhanced phagocytosis of Aeromonas salmonicida. Heat-killed A. salmonicida were incubated for 2 h at room temperature with varying concentrations of the mannose-binding lectin. The lectin-opsonized and non-opsonized bacteria were then incubated for 3 h at 17°C with Atlantic salmon head kidney macrophages after which they were fixed, permeabilized, stained with FITC, and counterstained with bis-benzimide. Values are the mean percentage +S.E. of macrophages that contained a minimum of five FITC-labeled bacteria. Results significantly different (P5 0.05) from control are indicated by an asterisk (*). N= 10 fish.
the same as those with macrophages showed no significant (t-tests, P\0.05) lectin-associated reduction in bacterial growth. Incubation of heat-killed A. salmonicida with 4.0 and 20.0 ng ml − 1 purified lectin resulted in significant increases (t-test, P5 0.05) in respiratory burst activity relative to the activity observed in macrophages incubated alone or with non-opsonized A. salmonicida as measured in NBT assays (Fig. 4). There was a significant decrease (t-tests, P5 0.05) in respiratory burst activity in macrophages incubated with heat-killed A. salmonicida which had been incubated with 0.32 ng ml − 1 of lectin relative to all other treatment and control groups. Respiratory burst activity in macrophages incubated with non-opsonized A. salmonicida was not significantly different (t-test, P\ 0.05) from macrophages incubated in the absence of the bacteria. Also, in the absence of bacteria, there was no significant difference in respiratory burst activity in macrophages exposed to the various lectin concentrations when compared to control macrophages; those exposed to no lectin (data not shown).
4. Discussion The lectin used in our study has been shown to bind to the salmon pathogens Vibrio anguillarum and A. salmonicida [4]. In this study, incubation of viable A. salmonicida with nanogram amounts of purified lectin in the absence of macrophages had no effect on in vitro
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growth. Lectins isolated from the eggs of Pacific salmon have previously been shown to inhibit growth of various bacterial pathogens except A. salmonicida when used at higher concentrations. Inhibition of growth of various fish pathogens after incubation with a purified D-galactose-L-rhamnose-specific lectin isolated from the ova of the chinook salmon (Oncorhynchus tshawytscha) has been reported [48]. Incubation with 150 mg ml − 1 chinook ova lectin for 1 h resulted in a 99% reduction of growth of V. anguillarum. A 1-h incubation with 0.5 mg ml − 1 chinook ova lectin reduced the growth of Yersinia ruckeri, Aeromonas hydrophila and Edwardsiella tarda by 76, 77 and 36% respectively. At the highest concentration tested, the chinook ova lectin had no effect on A. salmonicida or Renibacterium salmoninarum growth. A galactose-binding lectin has been purified from coho salmon eggs that bound to A. salmonicida cells but not to the other pathogens A. hydrophila, V. anguillarum, Vibrio ordalii, or R. salmoninarum [51]. This lectin showed no antibacterial activity against A. salmonicida [52]. The concentration of the lectin described in the present study has been estimated to be approximately 5 mg ml − 1 serum [4]. This is considerably higher than the maximum concentrations used in our study and it is possible that at higher concentrations this lectin may show antimicrobial activity. It is also possible that A. salmonicida is generally resistant to the antibacterial activities of lectins and that the lectin we studied might show some antimicrobial activity against other bacterial species.
Fig. 4. Mannose-binding lectin-induced respiratory burst activity of Atlantic salmon head kidney macrophages. Heat-killed A. salmonicida were incubated for 2 h at room temperature with various concentration of mannose-binding lectin. The opsonized bacteria were then incubated with macrophages for 3 h at 17°C prior to fixation and solubilization of the NBT. Values are mean +S.E. OD650. Results significantly different (P5 0.05) from control are indicated by an asterisk (*) ** indicates significant difference in the macrophages exposed to bacteria opsonized with 0.32 ng ml − 1 lectin compared to control macrophages and other treatment groups. N= 31 fish.
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In our study, opsonization of viable and heat-killed A. salmonicida with a Ca2 + -binding type mannosebinding lectin purified from the serum of Atlantic salmon resulted in dose-dependent increases in phagocytosis, bactericidal activity and a significant increase in respiratory burst activity by Atlantic salmon macrophages. Previous reports have described opsonization of microbial pathogens and parasites with antibody or complement resulting in enhanced fish leukocyte adherence [34], phagocytosis [1,2,13,16,20, 22,28,29,38,41,42,46] and killing [50]. Although a wide variety of lectins have been isolated from fish, this is the first report of opsonization by a purified lectin and its effects on fish immunity: bactericidal activity, phagocytosis and enhancement of respiratory burst activity. In mammals the effects of lectins on these processes have been well documented [14,21,39]. The maximum lectin concentration of 20.0 ng ml − 1 that was used in this study is well below the estimated 5 mg ml − 1 concentration of this lectin in the serum [4]. However, the physiologically relevant concentration of this lectin in tissues or mucus (sites at which it is likely to be an important contributor to innate immunity) may be in the ng/ml range. Given the number of variables in this experimental system, it seemed prudent to use the heat-killed A. salmonicida for the phagocytosis and respiratory burst assays to eliminate the possibility of cytotoxicity associated with this virulent strain [33]. It is possible that binding of this lectin to the surface of virulent A. salmonicida may reduce cytotoxicity, thereby enhancing levels of phagocytosis and macrophage bactericidal activity. Respiratory burst activity in macrophages is an important mechanism by which A. salmonicida are killed following phagocytosis [44] although there is evidence that the bacterium can resist damage by reactive oxygen species [9]. When A. salmonicida was incubated with increasing amounts of lectin there was a significant increase in bactericidal activity of the macrophages. Assuming that increased quantities of lectin relate to an increased number of A. salmonicida with lectin on their surface, our results suggest that the lectin-labeled bacteria are interacting directly with the macrophages. Interactions between lectin and the macrophage may be occurring via receptors on the macrophage surface. The occurrence of such receptors has been postulated for phagocytic cells in mammals [15,43]. There was not a linear relationship between lectin concentration and respiratory burst activity, although a significant increase in respiratory burst activity was seen at 4 and 20 ng ml − 1 when compared to controls. This suggests that other mechanisms may also be involved in the stimulation of oxygen radical formation by macrophages. It is possible that macrophages interacting with lectin-bound A. salmonicida are induced to
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produced cytokines, such as macrophage activating factor (MAF). These cytokines may then stimulate other macrophages that have not yet encountered lectin-labeled bacteria. The ability of MAF from stimulated macrophage cultures to induce respiratory burst activity in otherwise unstimulated cells has been reported for rainbow trout [17]. Although the ability of isolated Atlantic salmon macrophages to produce MAF has yet to be demonstrated, leukocytes from the anterior kidney of this species have been shown to produce MAF when stimulated with intact A. salmonicida [26] or with outer membrane proteins obtained from A. salmonicida [7]. In this model, a minimum lectin dose would be required for activation of the macrophage population since cytokines would provide an amplification of its effect. It is not clear why a significantly reduced respiratory burst activity in the 0.32 ng ml − 1 treatment was found when compared to the controls. As our results were obtained in a culture system, it could be argued that the media or cell secretory components could have contributed to the observed activities. We minimized this possibility by using heat-inactivated fetal bovine serum in our media so that neither immunoglobulin nor active complement components were provided. The remaining media components are not known to enhance respiratory burst, phagocytosis or bactericidal activity. Enhancement could have also resulted from antibody or complement components produced by the leukocytes in the cultures. Our leukocyte cultures were enriched for macrophages, but they were not pure and thus antibodies capable of binding to the A. salmonicida could have been present. Although fish macrophages have not been shown to produce complement components, macrophage-derived complement has been shown to opsonize pathogens occurring outside the vascular system in mammals [5,6]. However, if our results were due to factors present in the media or produced in the culture then we would expect the observed enhancements to have been independent of lectin concentration, which was not the case. A cortical lectin from the oocytes of the loach, Rutilus rutilus, has been shown to stimulate mitogenic activity and the release of soluble factors (IL-2, TXB2, PGE2) from human lymphocyte cultures [23]. The human MBP is known to function as an opsonin independent of complement and immunoglobulins, as well as to mediate complement deposition resulting in killing of organisms normally resistant to complement-mediated lysis in the absence of MBP [40]. In this study, we provide the first evidence of lectin-enhanced macrophage activity in a fish. This mannose-binding lectin may substantially contribute to innate immunity of Atlantic salmon both through its direct effects on macrophages, as well as its effects on other cells and interactions with other molecules of the innate immune system similar to those described in the above studies.
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