Chitooligosaccharides Stimulate Atlantic Salmon, Salmo salar L., Head Kidney Leukocytes to Enhanced Superoxide Anion Production In Vitro

Comp. Biochem. Physiol. Vol. 118B, No. 1, pp. 105–115, 1997 Copyright  1997 Elsevier Science Inc. All rights reserved.

ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00021-7

Chitooligosaccharides Stimulate Atlantic Salmon, Salmo salar L., Head Kidney Leukocytes to Enhanced Superoxide Anion Production In Vitro James Hoffman,1,2 Audny Johansen,1 Kari Steiro,1 Asbjørn Gildberg,1 Even Stenberg,1 and Jarl Bøgwald1,2 1

2

Norwegian Institute of Fisheries and Aquaculture, N-9002 Tromsø, Norway; and The Norwegian College of Fishery Science, University of Tromsø, Breivika, N-9037 Tromsø, Norway ABSTRACT. Chitosans and chitooligosaccharides stimulated Atlantic salmon, Salmo salar L., head kidney leukocytes in vitro to produce elevated levels of superoxide anion. Both soluble and insoluble chitooligosaccharides were stimulatory 2 and 7 days after addition. Protein-chitooligosaccharide conjugates were also stimulatory in vitro both at 2 and 7 days after addition. Deacetylation seemed to be of little importance for the stimulatory capacity. High concentrations of the 80% deacetylated chitosan/chitooligosaccharides were toxic to the leukocytes as judged by reduced reduction of nitroblue tetrazolium and morphology. comp biochem physiol 118B;1: 105–115, 1997.  1997 Elsevier Science Inc. KEY WORDS. Atlantic salmon leukocytes, chitosan, chitooligosaccharides, stimulation, superoxide anion, nitroblue tetrazolium reduction

INTRODUCTION The respiratory burst is probably one of the most important bactericidal mechanisms of phagocytes (3,9). There are several reports on the respiratory burst activity of fish phagocytes (2,4,5,10,25). The respiratory burst activity was shown to correlate with increased killing activity of fish pathogenic bacteria (6,11,22). Recently, several potent immunomodulators have been studied, examples are small molecular weight peptides (2,12), lipopolysaccharide (LPS) (4,20), βD-glucans (18), peptidoglycan, and muramyl dipeptide (MDP) (13). Chitin is a polysaccharide that is widely distributed in nature. It is composed of β-1,4 N-acetyl-Dglucosamine subunits. On treatment of chitin with base, the acetamido groups are hydrolysed to give free amino groups. In mammals, deacetylated chitin derivatives have been reported to stimulate nonspecific resistance (in mice) against Escherichia coli infection (14) and suppression of the growth of Meth-A tumours in syngeneic Balb/c mice (14), adjuvant activity in mice and guinea pigs (15), and stimulation of cytokine production in mice (16). Also, the deacetylated chitin derivative showed protective activity against Sendai virus infection in mice (8). Stimulation of cultured human monocytes by chitosan to induce production of TNF-α has Address reprint requests to: Jarl Bøgwald, The Norwegian College of Fishery Science, University of Tromsø, Breivika, N-9037 Tromsø, Norway. Tel. 147-7-764-6069; Fax 147-7-764-6020; E-mail: [email protected]. Received 9 May 1996; revised 19 February 1997; accepted 26 February 1997.

been reported (17). To our knowledge there are no reports on the direct stimulatory effect of chitin/chitosan derivatives on cells of the nonspecific defence system in Atlantic salmon. The aim of the present investigation was to study the effect of various chitosans and chitooligosaccharides on Atlantic salmon leukocytes in vitro. To our knowledge, this is the first time it has been shown that both soluble and insoluble chitooligosaccharides are able to stimulate cultured Atlantic salmon head kidney leukocytes to produce enhanced amounts of the superoxide anion.

MATERIALS AND METHODS Preparation of Chitooligosaccharides Chitooligosaccharides were prepared from chitosan by sodium nitrite degradation. Chitosan, 10 and 80% deacetylated (100 mg, chitosan-10/80) was solubilized in 0.5 M acetic acid (20 ml). Sodium nitrite (6.69, 3.34, 1.67 mg) was added to produce chitooligosaccharides with chainlengths of 6, 12, and 24 monosaccharide units, respectively, and the solution was incubated at room temperature for 2 hr with agitation. The pH was adjusted to 8.0 with ammonia. The mixture was freeze dried, solubilized in a small volume of 50 mM ammonium acetate, pH 4.5 and gel chromatographed on a P-2 column. The products were denoted DP6, DP12, DP24, and NAcDP6, NAcDP12, NAcDP24 after degradation of 80% and 10% deacetylated chitosans, respectively.

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FIG. 1. Superoxide anion pro-

duction in Atlantic salmon leukocytes after in vitro stimulation for 2 and 7 days with high-deacetylated chitooligosaccharides (DP6, DP12, DP24) and chitosan (80% deacetylated, CHIT80). The results are expressed as OD 620 3 10 5 cells 21 and data represent three readings for each concentration 1 SD of two fish (*P , 0.05).

Preparation of Chitooligosaccharide–Human Serum Albumin Conjugates Chitooligosaccharides, NAcDP6, NAcDP12, and NAcDP24 (16.9 mg) were solubilized in 0.2 M phosphate buffer, pH 6.5. Human serum albumin (HSA, 6.8 mg) and sodiumcyanoborohydride (NaBH 3CN) were added. The mixtures were incubated with agitation at 37°C for 24 hr, dialyzed against 50 mM Tris-buffer, pH 8.6, centrifuged to remove some insoluble substance, and chromatographed on a DEAE-Sepharose ion exchange column (1 3 10 cm) at pH 8.6. The product was eluted with a 0–0.5 M NaCl gradient in 50 mM Tris-buffer, pH 8.6, desalted on a P-2 column and freeze-dried. Isolation of Head Kidney Leukocytes The head kidney was collected and pushed through a 100 µm nylon cell strainer (Falcon, Becton Dickinson, NJ, USA) in Leibovitz L-15 medium (osmolarity 360 mOsm kg 21 ) with glucose (0.33 g l 21 ), HEPES (pH 7.3, 3.57 g l 21 ) and caffeine (15 mM) as anticoagulant. The cell suspension was placed on top of a 54% Percoll solution (Pharmacia, Uppsala, Sweden) and centrifuged at 500 3 g for 15 min to isolate the leukocyte fraction. The cells were resuspended in L-15 medium and plated in flat-bottomed 96-well tissue culture plates (Nunc, Roskilde, Denmark), at 5 3 10 5 cells per well in 200 µl medium, and incubated overnight at 12°C in air. Nonadherent cells were removed by washing the cultures twice in medium. The cells were cultured in L-15 medium (200 µl per well) supplemented with 5% FCS, penicillin (100 IU ml 21 ), streptomycin (100 µg ml 21 ) and gentamycin (50 µg ml 21 ).

In Vitro Stimulation of Leukocytes One day after seeding (immediately after washing), the cells were stimulated by chitosans and the low-molecular weight chitooligosaccharides in various concentrations. Freezedried substances were dissolved in L-15 medium supplemented with 5% FCS. The soluble chitooligosaccharides were solubilized to final concentrations of 50 and 250 µg ml 21 and filtered (0.2 µm). Insoluble chitooligosaccharides and chitosans were suspended to 25 and 100 µg ml 21 and subjected to ultrasonic disintegration to obtain a homogenous suspension. Superoxide Anion Production Respiratory burst activity of leukocytes was quantified by measuring the superoxide anion production using the procedure of reduction of nitroblue tetrazolium (NBT) to formazan (3). The culture medium was removed, and the NBT solution (1 mg ml 21 medium, 100 µl well 21 ) was added supplemented with phorbol myristate acetate (PMA, 0.2 µg ml 21 ) as the trigger (21). After a reaction period of 30 min at 12°C, the medium was removed and the cultures washed twice in phosphate buffered saline (PBS, osmolarity 360 mOsm kg 21 ) before fixation in 70% ethanol for 3 min. The formazan was dissolved in 120 µl/2 M potassium hydroxide (KOH) and 140 µl dimethylsulfoxide (DMSO). After extensive solubilization, the optical density of the solutions was measured at 620 nm. Parallel cultures were used for determination of cell numbers by adding 1% Tween 20 and 0.05% crystal violet in 0.1 M citric acid to solubilize the cell membranes and release intact nuclei, which were counted in a Bu¨rker haematocytometer. The results are ex-

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FIG. 2. Superoxide anion production in Atlantic salmon leukocytes after in vitro stimulation for 2 and 7 days with low-deacetylated chitooligosaccharides (NAcDP6, NAcDP12, NAcDP24) and chitosan (10% deacetylated, CHIT10). The results are expressed as OD 620 3 10 5 cells 21 and data represent three readings for each concentration 1 SD of two fish (*P , 0.05).

FIG. 3. Superoxide anion production in Atlantic salmon leukocytes after in vitro stimulation for 2 and 7 days with low-deacetylated chitooligosaccharides conjugated to human serum albumin. The results are expressed as OD 620 3 10 5 cells 21 and data represent three readings for each concentration 1 SD of two fish (*P , 0.05).

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FIG. 4. Pictures of Atlantic salmon leukocytes after incubation for 2 days with stimulants. (A) Unstimulated control. (B) LPS

(5 mg ml 21 ). (C) Chitooligosaccharide DP6 (50 mg ml 21 ). (D) Chitooligosaccharide DP6 (250 mg ml 21 ). (E) Chitosan-80 (25 mg ml 21 ). (F) Chitosan-10 (25 mg ml 21 ).

pressed as means of OD 620 per 10 5 cells 1 SD of at least three replicate wells. Morphology of Cells The morphology of living (unfixed) cells was observed in the inverted microscope during the cultivation period (7 days). Pictures were taken with a Kodak Gold 100 ASA colour film.

Viability Tests The viability of the cells in culture was checked by the trypan blue exclusion test by incubation in 0.25% Trypan Blue in PBS for 15–30 sec. In addition, the cytoplasmic enzyme, lactate dehydrogenase (LDH, EC 1.1.1.27), was measured by use of a kit from Sigma Chemical Co. (St Louis, MO) In short, pyruvic acid is converted to lactic acid by LDH. An intensely coloured

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FIG. 4. (continued)

phenylhydrazone formed from residual pyruvic acid is measured colorimetrically and is inversely proportional to LDH activity. The decrease in absorbance at 464 nm is a measure of the enzyme activity.

significant when P , 0.05. The vertical bars in Figs 1–3 represent standard deviations (SD).

Statistical Analysis

RESULTS Stimulation of Leukocytes by High-Deacetylated Chitooligosaccharides

Student’s t-test was used to determine the statistical significance of the results. Differences were considered statistically

Both soluble and insoluble high-deacetylated (80%) lowmolecular weight chitooligomers were stimulatory as mea-

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FIG. 4. (continued)

sured by production of high amounts of superoxide anion. Figure 1 shows the reduction of NBT 2 and 7 days after stimulation with oligosaccharides with DP6, DP12, and DP24, respectively. As may be seen from Fig. 1, there was a strong enhancement of the superoxide anion production in leukocytes stimulated with 50 µg ml 21 of soluble oligosaccharides and with 25 µg ml 21 of insoluble oligosaccharides. Stimulation of cells with as high doses as 250 µg ml 21 of

soluble and 100 µg ml 21 of insoluble saccharides resulted in reduced reduction of NBT (Fig. 1). Seven days after stimulation, the cells still produced enhanced levels of superoxide compared with the controls (Fig. 1), although the activity was somewhat lower. Stimulation of cells with the unhydrolyzed high-molecular weight chitosan-80 was low compared with low-molecular weight chitooligosaccharides (Fig. 1) at 2 days. Seven

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days after the addition of both 25 and 100 µg ml 21 chitosan80, the leukocytes produced high levels of superoxide anion (Fig. 1). LPS in 5 µg ml 21 was included as a positive control. Stimulation of Leukocytes by Low-Deacetylated Chitooligosaccharides Two days after addition, the NAcDP6 oligosaccharide stimulated the cells to increased production of superoxide, both at 25 and 100 µg ml 21 comparable to LPS at 5 µg ml 21 (Fig. 2). The stimulatory effect was higher at day 7 with regard to the oligosaccharide NAcDP6 and LPS. Two days after addition, NAcDP12 and NAcDP24 did not stimulate leukocytes to produce increased levels of superoxide (Fig. 2). Some stimulation was, however, observed with NAcDP12 and NAcDP24 in doses of 25 µg ml 21 7 days after addition (Fig. 2). Stimulation of the leukocytes with the high-molecular weight chitosan-10 was low after 2 days and high after 7 days (Fig. 2). Stimulation of Leukocytes by Deacetylated Chitooligosaccharides Conjugated to Human Serum Albumin The stimulatory capacity of these conjugates are shown in Fig. 3. On day 2, NAcDP12-HSA and NAcDP24-HSA were the most stimulatory (Fig. 3), and on day 7 there was even an increased production of superoxide anion, compared with 2 days as determined by increased reduction of NBT (Fig. 3). Morphology of Cells The morphology of the leukocytes stimulated with low-molecular weight chitooligosaccharides DP6 50 µg ml 21 and 250 µg ml 21 for 2 days (Fig. 4 c and d) showed comparable spreading with unstimulated cells (Fig. 4 a) and LPS 5 µg ml 21 (Fig. 4 b). Cells stimulated by the unhydrolyzed chitosans for 2 days were more spread on the plastic surface compared with the controls. Seven days after stimulation, the cells that were stimulated with the lowest concentration (50 µg ml 21 with regard to soluble substances and 25 µg ml 21 with regard to insoluble substances) had an even more highly spread appearance compared with the cells at 2 days (Fig. 5). However, the highest concentration was toxic to the cells as evaluated by morphology, Trypan Blue exclusion test, and the amount of the cytoplasmic enzyme lactate dehydrogenase, when cultured for an extended time (7 days) in the presence of high concentrations of the oligosaccharides (Fig. 5 d). Also, at day 7 after stimulation the unhydrolyzed chitosan-stimulated cells showed more spreading than the controls (Fig. 5 e and f), but not as highly spreading as the cells stimulated by low concentrations of the chi-

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tooligosaccharides (Fig. 5 c). Cells stimulated for 7 days with LPS showed a more spread appearance than the controls (Fig. 5 a and b) but not as highly spread as cells stimulated by 50 µg of the soluble chitooligosaccharide DP6 (Fig. 5c). Viability Tests We found a correlation between the two viability tests, the Trypan Blue exclusion test and the test for measuring the amount of lactate dehydrogenase in the cells. Cultures stimulated for 7 days with DP6 (250 µg ml 21 ) showed reduced content of LDH (c. 50%, compared to nonstimulated controls). About 50% of the cells in these cultures also showed staining by the Trypan Blue exclusion test. Cultures stimulated with DP6 (50 µg ml 21 ), chitosan-10 (25 µg ml 21 ) and chitosan-80 (25 µg ml 21 ) showed a viability of 85–90%. Nonstimulated control cultures and the LPS (5 µg ml 21 ) stimulated cultures showed a viability of 95%. DISCUSSION The present work shows that chitosans and chitooligosaccharides can stimulate Atlantic salmon head kidney leukocytes in vitro to produce elevated levels of superoxide anion. The immunological activity of chitin/chitosan and chitooligosaccharides in mammals is well documented (7,14,24). Recently, Sakai et al. (19) reported immunostimulating effects of chitin in rainbow trout (Oncorhynchus mykiss). They found increased resistance against infection with Vibrio anguillarum after intraperitoneal injection of 100 mg chitin kg 21 body weight. The effects of chitin seemed to be mediated by nonspecific activation mechanisms. Leukocytes isolated from the chitin-stimulated fish showed increased phagocytic activity and increased chemiluminescence production. Our results support these observations, since cultured leukocytes from Atlantic salmon head kidney had enhanced capacities to reduce NBT after in vitro stimulation. Oral administration of chitosan to rainbow trout was found to cause elevated oxidative radical release, myeloperoxidase activity, and phagocytosis in polymorphonuclear leukocytes from blood (23). Also, the immunostimulated fish seemed to be more resistent to infection with Aeromonas salmonicida. The same authors found protection in brook trout (Salvelinus fontinalis) against A. salmonicida after injection or immersion in chitosan (1). The duration of protection was, however, very short. Our results show that low-molecular weight soluble and insoluble chitooligosaccharides stimulate Atlantic salmon leukocytes in vitro. These results correspond to results obtained by Suzuki et al. (24) on the stimulatory capacity of polymorphonuclear cells from mice by N-acetylchitooligosaccharides with a chain length of four to seven monosaccharide units. As may be seen from Fig. 1, chitooligosaccharides derived from the highly deacetylated chitosan (80%) were most stimulatory in low con-

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FIG. 5. Pictures of Atlantic salmon leukocytes after incubation for 7 days with stimulants: (A) Unstimulated control, (B) LPS

(5 mg/ml 21 ), (C) Chitooligosaccharide DP6 (50 mg/ml 21 ), (D) Chitooligosaccharide DP6 (250 mg/ml 21 ), (E) Chitosan-80 (25 mg/ml 21 ), (F) Chitosan-10 (25 mg/ml 21 ).

centrations and at 2 days of stimulation. At 7 days of stimulation the reductive NBT-capacity of the cells was reduced. This result may be as a consequence of decreased viability (50%) of cells due to toxicity of positively charged molecules at neutral pH, although it does not seem to be a correlation of viability and reduced NBT capacity in cells stimu-

lated with low concentrations (50 µg ml 21 DP6). Cells stimulated by nonhydrolyzed chitosan (chitosan-80) for 7 days showed increased capacity to reduce NBT, compared with at 2 days. The viability of these cells was 85–90% of unstimulated controls. One explanation why this substance did not seem to be that toxic to the cells may be the physical

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FIG. 5. (continued)

nature of chitosan compared with the chitooligosaccharides. Unhydrolyzed chitosan formed flakes or big aggregates that would be difficult to be ingested by phagocytic cells, and the toxicity of the substances may be stronger intracellularly than extracellularly. The low-deacetylated chitooligosaccharides (10%, NAcDP’s) or the proteinchitooligosaccharide complexes (HSA-NAcDP’s) did not show toxic effects to any appreciable extent (viability 85– 90%). In these situations the reduction of NBT was strong-

est 7 days after stimulation (Fig. 2 and Fig. 3). The use of a protein component with specificity (e.g., antibodies) would secure that the stimulatory chitooligosaccharides would localize to desirable positions, e.g., in live animals. A possible role for chitosan/chitooligosaccharides as adjuvants in vaccines will be investigated. High amounts of the polysaccharide/oligosaccharides may be produced from the shells of crustaceans to a relatively low cost. This fact makes chitosan especially suitable as feed additives for do-

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FIG. 5. (continued)

mestic animals and aquacultured fish. In addition, immersion of juveniles in chitosan/chitooligosaccharide suspensions/solutions may cause protection against disease when the fish are too small to receive a vaccine by injection or in situations where the specific immune system is not fully developed. Financial support from The French-Norwegian Foundation for Scientific and Technical Research and Development (FNS) and The Norwegian Research Council grant no. 103080/130 is acknowledged.

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