- Email: [email protected]
Nitric oxide production by nurse shark (Ginglymostoma cirratum) and clearnose skate (Raja eglanteria) peripheral blood leucocytes
Fish & Shellfish Immunology 20 (2006) 40e46 www.elsevier.com/locate/fsi
Nitric oxide production by nurse shark (Ginglymostoma cirratum) and clearnose skate (Raja eglanteria) peripheral blood leucocytes Cathy J. Walsh a,*, Jason D. Toranto a,1, C. Taylor Gilliland a,2, David R. Noyes a,3, Ashby B. Bodine b, Carl A. Luer a a
Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA b Clemson University, Clemson, SC 29634, USA Received 5 January 2005; accepted 21 March 2005 Available online 31 May 2005
Abstract Reactive nitrogen intermediates, such as nitric oxide (NO), are important immunomodulators in vertebrate immune systems, but have yet to be identified as mediators of host defence in any member of class Chondrichthyes, the cartilaginous fishes. In the present study, production of NO by nurse shark (Ginglymostoma cirratum) peripheral blood leucocytes (PBL) stimulated with bacterial cell wall lipopolysaccharide (LPS) was investigated. PBL were cultured for 24 to 96 h following stimulation with LPS at concentrations ranging from 0 to 25 mg mlÿ1, in both serum-supplemented and serum-free culture conditions. Production of NO was measured indirectly using the Griess reaction, with maximal NO production occurring after 72 h using 10% FBS and 10 mg LPS mlÿ1. Application of these culture conditions to PBL from another cartilaginous fish (clearnose skate, Raja eglanteria) resulted in a similar NO response. Addition of a specific inhibitor of inducible nitric oxide synthase (iNOS), L-N6-(1-iminoethyl)lysine (L-NIL), resulted in a significant decrease in the production of NO by PBL from both species. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Nitric oxide; Nitric oxide synthase; Nurse shark; Ginglymostoma cirratum; Clearnose skate; Raja eglanteria; Elasmobranch; Peripheral blood leucocytes
* Corresponding author. Tel.: C1 941 388 4441; fax: C1 941 388 4312. E-mail address: [email protected] (C.J. Walsh). 1 Current address: University of Alabama at Birmingham, Birmingham, AL 35294, USA. 2 Current address: University of Florida, Gainesville, FL 32612, USA. 3 Current address: H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. 1050-4648/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2005.03.011
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
41
1. Introduction Immune cells from vertebrate animals produce a variety of molecules involved in innate mechanisms of host defence. Among the most potent are the reactive oxygen and nitrogen intermediates. The ability of vertebrate immune systems to combat bacterial or parasitic infections depends on the release of such molecules, including superoxide (Oÿ 2 ), peroxide, singlet O2 and nitric oxide (NO) [1]. Nitric oxide has many functions, including intercellular signaling, vasodilation, and defence against microbes and tumors [1e3]. Nitric oxide is produced in a variety of cell types by a group of enzymes referred to as nitric oxide synthases (NOS). Of the three known isoforms of NOS, only the inducible form, iNOS, is transcriptionally upregulated through bacterial or cytokine challenge [3]. Nitric oxide production has been demonstrated in several species of bony fish (class Osteichthyes) including goldfish, Carassius auratus [4], catfish, Clarias gariepinus [5], gilthead seabream, Sparus aurata L. [6], rainbow trout, Oncorhynchus mykiss [7,8], turbot, Scophthalmus maximus [9], and carp, Cyprinus carpio [10]. Nitric oxide production has also been demonstrated in three species of invertebrates including Forbe’s sea star, Asteria forbesi [11], a freshwater snail, Viviparus ater [12], and the Anopheles mosquito, Anopheles stephensi [13]. While immune function in cartilaginous fishes (class Chondrichthyes) is not completely characterised, it has been documented that components necessary for both an adaptive [14e16] and an innate immune system are present. Mechanisms of innate immunity demonstrated for elasmobranchs include phagocytosis [17,18], nonspecific cytotoxicity [19e22], and complement activation [23]. Nitric oxide production, however, has yet to be demonstrated for any species of elasmobranch fish. This paper describes the production of NO by LPS-stimulated PBL from two representative elasmobranch species. In addition, culture conditions, including serum source, LPS concentration, and time in culture that result in optimal NO production are reported. Finally, inhibition of NO production by the iNOS-specific inhibitor, L-N6-(1iminoethyl)lysine (L-NIL) is demonstrated for PBL from both species. The data presented provide the first evidence for the participation of reactive nitrogen intermediates in the immune system at the phylogenetic level of elasmobranch fishes.
2. Materials and methods 2.1. Experimental animals Nurse shark (Ginglymostoma cirratum) pups were collected in the Florida Keys and transported live to Mote Marine Laboratory, Sarasota, FL. Adult clearnose skates (Raja eglanteria) were collected in near shore waters off Anna Maria Island, FL. Animals were maintained in captivity under conditions previously described [24]. Body masses for animals bled for the studies described ranged from 3.6 to 5.0 kg for four immature nurse sharks and from 1.2 to 1.8 kg for three mature clearnose skates. 2.2. Collection of blood and isolation of PBL Whole blood was obtained via caudal venipuncture [25]. Animals were bled three times at approximately two-week intervals, with whole blood volumes of 5 ml drawn from each shark and 3 ml from each skate at each sampling. To monitor recovery of blood cell volumes, a small portion of each sample was drawn into heparin-coated microcapillary tubes for hematocrit determination. In all cases, hematocrit values fell within normal ranges for each species [25]. The remaining whole blood was immediately mixed with elasmobranch-modified ACD anticoagulant solution (E-ACD) at a ratio of 0.175 ml E-ACD per 1.0 ml
42
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
whole blood. E-ACD was prepared by supplementing ACD solution (40 mM citric acid, 70 mM sodium citrate, 140 mM dextrose) with sodium chloride to approximate the osmotic environment of elasmobranchs (final osmolarity between 900 and 1000 mOsm). PBL were isolated from whole blood through repetitive slow speed centrifugation, 10e20 min at 50 ! g for each spin. Centrifugation continued until erythrocytes formed a loose pellet and PBL remained as a partially concentrated suspension above the red cell layer. PBL were carefully aspirated and resuspended in elasmobranch-modified PBS (E-PBS; 450 mM NaCl, 10 mM NaH2PO4, pH 7.4). Cells were centrifuged at 200 ! g for 7 min, then resuspended in elasmobranchmodified RPMI (E-RPMI) culture medium (RPMI; Sigma Chemical Co., St. Louis, MO, modified by adding 360 mM urea and 115 mM NaCl to approximate the normal elasmobranch cellular environment). Before placing into culture, cells were enumerated using a haemocytometer and viability was assessed by trypan blue exclusion [26] using 0.2% trypan blue prepared in E-PBS. 2.3. Cell culture conditions PBL were cultured at 25 C in a 5% CO2 environment for up to 96 h using E-RPMI [15,27] as the culture medium. In addition, penicillin (50 units mlÿ1), streptomycin (50 mg mlÿ1), neomycin (0.1 mg mlÿ1), and amphotericin B (0.25 mg mlÿ1) were added to control bacterial and fungal growth. Sodium bicarbonate (23.8 mM) was added and the final pH of the medium was adjusted to 7.2e7.4. All reagents were sterilized using a 0.2-mm filter prior to use. To establish culture conditions resulting in optimal NO production, time in culture ranged from 0 to 96 h. Cells were cultured under both serum-free conditions as well as in the presence of 10% FBS (foetal bovine serum, HyClone). 2.4. Production of NO by nurse shark and clearnose skate PBL To analyse the effect of serum on production of nitric oxide by shark PBL, nurse shark PBL were cultured in serum-free medium, or in medium supplemented with 10% FBS or heterologous nurse shark serum (NSS). Serum was heat-inactivated at 56 C for 30 min before use. For this set of experiments, cells were cultured for 72 h at 25 C and 5% CO2 and stimulated with 0, 2.5, 5, 10, or 25 mg LPS mlÿ1 (lipopolysaccharide from Salmonella typhosa, Sigma Chemical Co., St. Louis, MO). To determine the time period during which maximal NO production occurs, nurse shark PBL were cultured at a final concentration of 2.5 ! 106 cells mlÿ1 and NO measured after 24, 48, 72, and 96 h of culture. Cells were stimulated with 5 mg LPS mlÿ1 and 10% FBS was included in the cell culture medium. Production of nitric oxide by clearnose skate PBL in culture was analysed using cells at a concentration of 2.5 ! 106 cells mlÿ1 in the presence of 10% heat-inactivated FBS. PBL were cultured for 72 h at 25 C and 5% CO2, and stimulated with 0, 2.5, 5, 10, and 20 mg LPS mlÿ1. 2.5. Measurement of NO activity NO production was determined indirectly by measurement of nitrite released into the culture medium using a colourimetric assay based on the Griess reaction [28]. Assays were conducted in 96-well microtiter plates with 200 ml cells per well and triplicate wells for each treatment. After incubation, supernatants were assayed for nitrite using a procedure adapted from McCarthy et al. [29]. Briefly, 100 ml aliquots of cell culture supernatants were removed from individual wells and transferred to a separate 96-well microtitre plate. One hundred microlitres of Griess reagent (1% sulfanilamide; 0.1% N-naphthyl-ethylenediamine; 2.5% H3PO4) was added to each sample well and plates were incubated at 25 C for 10 min. Absorbance at 540 nm was determined using a microplate reader (BioRad, Model 550). The molar concentration of nitrite in samples was determined from standard curves generated using known concentrations of sodium nitrite.
43
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
2.6. Effect of the iNOS-specific inhibitor, L-NIL, on NO production To evaluate the presence of iNOS in nurse shark and clearnose skate PBL, an iNOS-specific inhibitor, 6 -(1-iminoethyl)lysine (L-NIL), was included in cell cultures at final concentrations of 0, 10, and 30 mM. For these experiments, PBL were cultured at 2.5 ! 106 cells mlÿ1 and stimulated with 5 mg LPS mlÿ1. Cells were cultured for 72 h at 25 C and 5% CO2.
L-N
2.7. Statistical analyses Statistical analyses were performed using the computer program SAS, version 8.0, for Windows (SAS Institute Inc., Cary, NC). Data were compared using analysis of variance (ANOVA) and General Linear Model (GLM) analyses. When significant differences were detected, single degree of freedom contrast or response curve analyses was performed. Data points and error bars on the figures represent mean G standard error of the mean (SEM) responses for all animals tested in each experiment.
3. Results 3.1. Nitric oxide production by elasmobranch PBL Production of nitric oxide by nurse shark PBL cultured with three different conditions of serum supplementation at five different LPS concentrations is presented in Fig. 1. Overall, cultures including FBS and NSS resulted in significantly greater NO production than cultures without serum (P ! 0.001), although no significant difference was observed between serum types. With serum included in the culture, NO production increased linearly (P ! 0.05) with increasing LPS up to concentrations of 10 mg mlÿ1, after No Serum
14
FBS
µM NO produced
12
NSS
10 8 6 4 2 0
0
5
10
LPS (µg
15
20
25
ml-1)
Fig. 1. Production of nitric oxide by LPS-stimulated nurse shark PBL cultured under serum-free conditions or supplemented with foetal bovine serum (FBS) or nurse shark serum (NSS). Four nurse sharks were sampled three times each. Cells were cultured at 2.5 ! 106 cells mlÿ1 for 72 h at 25 C, 5% CO2 with 0, 2.5, 5, 10, or 25 mg LPS mlÿ1. Error bars represent standard error of the mean (SEM).
44
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
which production of NO tended to plateau (slight quadratic curvature, P Z 0.11). In addition to examining the effects of serum supplementation on NO production, studies to investigate the effects of time in culture on NO production by nurse shark PBL were performed. As shown in Fig. 2, NO production increased linearly (P ! 0.001) from 24 to 72 h following LPS stimulation, and decreased slightly after 96 h of culture. The ability of skate PBL to produce NO in response to LPS stimulation was also investigated. In response to LPS concentrations ranging from 0 to 20 mg mlÿ1, nitric oxide production by FBSsupplemented clearnose skate PBL was shown to increase exponentially (Fig. 3). 3.2. Inhibition of NO production by L-NIL Effects of the iNOS-specific inhibitor, L-NIL, on NO production by shark and skate PBL are presented in Figs. 2 and 3. No significant interactions between LPS and L-NIL were observed with either nurse shark or clearnose skate PBL. With both nurse shark and clearnose skate PBL (Figs. 2 and 3), increasing L-NIL concentration (10 and 30 mM) resulted in significantly (P ! 0.001) decreased NO production.
4. Discussion Data are presented demonstrating nitric oxide production by both nurse shark and clearnose skate immune cells in response to LPS stimulation, providing the first evidence of the participation of reactive nitrogen intermediates in the immune system at the phylogenetic level of elasmobranchs. Elasmobranch PBL responded to LPS stimulation by producing measurable amounts of NO. The maximal amount of NO production occurred after 72 h in culture, which is consistent with the time required to elicit maximal amount of NO production in mammals [29], teleost [4e10], and invertebrates [11,12]. The highest amounts of NO produced by elasmobranch PBL in response to LPS stimulation ranged from approximately 8 to 10 mM. These values are comparable to maximal NO production by mammalian species, which has been reported to range from 5 to 20 mM [29]. 0 µM L-NIL
9
10 µM L-NIL
µM NO produced
8
30 µM L-NIL
7 6 5 4 3 2 1 0
20
40
60
80
100
Time (h) Fig. 2. Production of nitric oxide by LPS-stimulated nurse shark PBL as a function of time. Four nurse sharks were sampled twice each. Nurse shark PBL were cultured at a concentration of 2.5 ! 106 cells mlÿ1 with 10% FBS. Nurse shark PBL were stimulated with 5 mg LPS mlÿ1 for 96 h in the presence of 0, 10 or 30 mM of the iNOS-specific inhibitor, L-NIL. Error bars represent SEM.
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
45
14
µM NO produced
12 10 8
0 µM L-NIL 10 µM L-NIL
6
30 µM L-NIL
4 2 0
0
5
10
15
20
LPS (µg ml-1) Fig. 3. Production of nitric oxide by LPS-stimulated clearnose skate PBL. Four skates were sampled one time each. Skate PBL were cultured at 2.5 ! 106 cells mlÿ1 for 72 h in the presence of 10% FBS. Skate PBL were stimulated with 0e20 mg LPS mlÿ1 in the presence of 0, 10 or 30 mM of the iNOS-specific inhibitor, L-NIL. Error bars represent SEM.
The inclusion of serum in the cell culture media significantly increased NO production by nurse shark PBL compared to serum-free cultures, although an overall difference was not observed when comparing commercially available FBS to heterologous nurse shark serum. For routine use, however, commercially available FBS is typically a very consistent and uniform product while heterologous NSS is potentially more variable. Production of NO by elasmobranch immune cells in response to LPS stimulation was similar to that seen in mammals, with NO produced in the 5e20 mM range after 72 h of culture. L-NIL, a selective inhibitor of inducible nitric oxide synthase (iNOS) [30], significantly reduced NO production by both nurse shark and clearnose skate PBL stimulated with LPS, suggesting that an iNOS is responsible for NO production by stimulated elasmobranch immune cells. The existence of iNOS within white blood cells of any species strongly indicates that NO is an antipathogenic agent in the immune system of that species [2]. Our findings suggest that elasmobranch PBL utilise NO as a chemical mediator in host defence and likely contain a phylogenetic equivalent to inducible nitric oxide synthase found in higher vertebrate immune cells.
Acknowledgements The authors gratefully acknowledge the use of facilities at Mote Marine Laboratory (Sarasota, FL). The authors would like to thank Dr. Jonathon Stamler at Duke University Medical Center for generously providing the iNOS inhibitor, L-NIL, used in this study. One of the authors (CAL) received partial support through grants from the Vernal W. and Florence H. Bates Foundation and the Disney Wildlife Conservation Fund. Student participation was made possible by a stipend to JDT from the Marjorie G. and Louis S. Gilbert Scholarship and by a grant to CTG from the Keating Family Foundation. Statistical analyses were performed with the assistance of Dr. Joe Toler at Clemson University.
46
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
References [1] [2] [3] [4]
[5] [6] [7] [8]
[9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
[19] [20] [21] [22] [23] [24] [25]
[26] [27] [28] [29] [30]
Nussler A, Billiar T. Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol 1993;54:171e85. Nathan CF, Hibbs JB. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr Opin Immunol 1991;3:65e70. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992;6:3051e64. Neumann NF, Fagan D, Belosevic M. Macrophage activating factor(s) secreted by mitogen stimulated goldfish kidney leukocytes synergize with bacterial lipopolysaccharide to induce nitric oxide production in teleost macrophages. Dev Comp Immunol 1995;19:473e82. Yin Z, Lam TJ, Sin YM. Cytokine-mediated antimicrobial immune response of catfish, Clarias gariepinus, as a defense against Aeromonas hydrophila. Fish Shellfish Immunol 1997;7:93e104. Mulero V, Meseguer J. Functional characterisation of a macrophage-activating factor produced by leucocytes of gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol 1998;8:143e56. Laing KJ, Hardie LJ, Aartsen W, Grabowski PS, Secombes CJ. Expression of an inducible nitric oxide synthase gene in rainbow trout Oncorhynchus mykiss. Dev Comp Immunol 1999;23:71e85. Campos-Perez JJ, Ward M, Grabowski PS, Ellis AE, Secombes CJ. The gills are an important site of iNOS expression in rainbow trout Oncorhynchus mykiss after challenge with the gram-positive pathogen Renibacterium salmoninarum. Immunology 2000;99:153e61. Tafalla C, Novoa B. Requirements for nitric oxide production by turbot (Scophthalmus maximus) head kidney macrophages. Dev Comp Immunol 2000;24:621e31. Saeij JP, Stet RJ, Groeneveld A, Verburg van Kemenade L, Muiswinkel W, Wiegertjes GF. Molecular and functional characterization of a fish inducible-type nitric oxide synthase. Immunogenetics 2000;51:339e46. Beck G, Ellis T, Zhang H, Lin W, Beauregard K, Habicht GS, et al. Nitric oxide production by coelomocytes of Asterias forbesi. Dev Comp Immunol 2001;25:1e10. Franchini A, Fontanili P, Ottaviani E. Invertebrate immunocytes: relationship between phagocytosis and nitric oxide production. Comp Biochem Physiol 1995;110B:403e7. Luckhart S, Rosenberg R. Gene structure and polymorphism of an invertebrate nitric oxide synthase gene. Gene 1999;232:25e34. Luer CA, Walsh CJ, Bodine AB, Wyffels JT, Scott TR. The elasmobranch thymus: anatomical, histological, and preliminary functional characterization. J Exp Zool 1995;273:342e54. Rast JP, Anderson MK, Strong SJ, Luer C, Litman RT, Litman GW. a, b, g, and d T cell antigen receptor genes arose early in vertebrate phylogeny. Immunity 1997;6:1e11. Flajnik MF, Rumfelt LL. The immune system of cartilaginous fish. Curr Top Microbiol Immunol 2000;248:249e70. Hyder SL, Cayer ML, Pettey CL. Cell types in peripheral blood of the nurse shark: an approach to structure and function. Tissue Cell 1983;15:437e55. Walsh CJ, Luer CA. Comparative phagocytic and pinocytic activities of leucocytes from peripheral blood and lymphomyeloid tissues of the nurse shark (Ginglymostoma cirratum Bonaterre) and the clearnose skate (Raja eglanteria Bosc). Fish Shellfish Immunol 1998;8:197e215. Pettey CL, McKinney EC. Mitogen-induced cytotoxicity in the nurse shark. Dev Comp Immunol 1981;5:53e64. Pettey CL, McKinney EC. Induction of cell-mediated cytotoxicity by shark 19S IgM. Cell Immunol 1988;111:28e38. Pettey CL, McKinney EC. Temperature and cellular regulation in the shark. Eur J Immunol 1983;13:133e8. McKinney EC, Haynes L, Droese AL. Macrophage-like effector of spontaneous cytotoxicity from the shark. Dev Comp Immunol 1986;10:497e508. Nonaka M, Smith SL. Complement system of bony and cartilaginous fish. Fish Shellfish Immunol 2000;10:215e27. Luer CA. Elasmobranchs (sharks, skates, and rays) as animal models for biomedical research. In: Woodhead AD, Vivirito K, editors. Nonmammalian animal models for biomedical research. Boca Raton, FL: CRC Press; 1989. p. 121e47. Walsh CJ, Luer CA. Elasmobranch hematology: identification of cell types and practical applications. In: Smith M, Warmolts D, Thoney D, Hueter R, editors. Elasmobranch husbandry manual: captive care of sharks, rays, and their relatives. Columbus, OH: Ohio Biological Survey, Inc.; 2004. p. 307e23. Baur H, Kasperek S, Pfaff E. Criteria of viability of isolated liver cells. Hoppe Seylers Z Physiol Chem 1975;356:827e38. Walsh CJ, Luer CA, Wyffels JT, Bodine AB. Dexamethasone-induced apoptosis in immune cells from peripheral circulation and lymphomyeloid tissues of juvenile clearnose skates, Raja eglanteria. Dev Comp Immunol 2002;26:629e39. Green LC, Wagner DDA, Glogowski J, Skepper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite and (15N)nitrate in biological fluids. Anal Biochem 1982;124:131e8. McCarthy JE, Redmond PH, Duggan SM, Watson RWG, Condron CM, O’Donnell JR, et al. Characterization of the defects in murine peritoneal macrophage function in the early postsplenectomy period. J Immunol 1995;155:387e96. Bryk R, Wolff DJ. Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine. Biochemistry 1988;37:4844e52.
Nitric oxide production by nurse shark (Ginglymostoma cirratum) and clearnose skate (Raja eglanteria) peripheral blood leucocytes Cathy J. Walsh a,*, Jason D. Toranto a,1, C. Taylor Gilliland a,2, David R. Noyes a,3, Ashby B. Bodine b, Carl A. Luer a a
Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA b Clemson University, Clemson, SC 29634, USA Received 5 January 2005; accepted 21 March 2005 Available online 31 May 2005
Abstract Reactive nitrogen intermediates, such as nitric oxide (NO), are important immunomodulators in vertebrate immune systems, but have yet to be identified as mediators of host defence in any member of class Chondrichthyes, the cartilaginous fishes. In the present study, production of NO by nurse shark (Ginglymostoma cirratum) peripheral blood leucocytes (PBL) stimulated with bacterial cell wall lipopolysaccharide (LPS) was investigated. PBL were cultured for 24 to 96 h following stimulation with LPS at concentrations ranging from 0 to 25 mg mlÿ1, in both serum-supplemented and serum-free culture conditions. Production of NO was measured indirectly using the Griess reaction, with maximal NO production occurring after 72 h using 10% FBS and 10 mg LPS mlÿ1. Application of these culture conditions to PBL from another cartilaginous fish (clearnose skate, Raja eglanteria) resulted in a similar NO response. Addition of a specific inhibitor of inducible nitric oxide synthase (iNOS), L-N6-(1-iminoethyl)lysine (L-NIL), resulted in a significant decrease in the production of NO by PBL from both species. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Nitric oxide; Nitric oxide synthase; Nurse shark; Ginglymostoma cirratum; Clearnose skate; Raja eglanteria; Elasmobranch; Peripheral blood leucocytes
* Corresponding author. Tel.: C1 941 388 4441; fax: C1 941 388 4312. E-mail address: [email protected] (C.J. Walsh). 1 Current address: University of Alabama at Birmingham, Birmingham, AL 35294, USA. 2 Current address: University of Florida, Gainesville, FL 32612, USA. 3 Current address: H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. 1050-4648/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2005.03.011
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
41
1. Introduction Immune cells from vertebrate animals produce a variety of molecules involved in innate mechanisms of host defence. Among the most potent are the reactive oxygen and nitrogen intermediates. The ability of vertebrate immune systems to combat bacterial or parasitic infections depends on the release of such molecules, including superoxide (Oÿ 2 ), peroxide, singlet O2 and nitric oxide (NO) [1]. Nitric oxide has many functions, including intercellular signaling, vasodilation, and defence against microbes and tumors [1e3]. Nitric oxide is produced in a variety of cell types by a group of enzymes referred to as nitric oxide synthases (NOS). Of the three known isoforms of NOS, only the inducible form, iNOS, is transcriptionally upregulated through bacterial or cytokine challenge [3]. Nitric oxide production has been demonstrated in several species of bony fish (class Osteichthyes) including goldfish, Carassius auratus [4], catfish, Clarias gariepinus [5], gilthead seabream, Sparus aurata L. [6], rainbow trout, Oncorhynchus mykiss [7,8], turbot, Scophthalmus maximus [9], and carp, Cyprinus carpio [10]. Nitric oxide production has also been demonstrated in three species of invertebrates including Forbe’s sea star, Asteria forbesi [11], a freshwater snail, Viviparus ater [12], and the Anopheles mosquito, Anopheles stephensi [13]. While immune function in cartilaginous fishes (class Chondrichthyes) is not completely characterised, it has been documented that components necessary for both an adaptive [14e16] and an innate immune system are present. Mechanisms of innate immunity demonstrated for elasmobranchs include phagocytosis [17,18], nonspecific cytotoxicity [19e22], and complement activation [23]. Nitric oxide production, however, has yet to be demonstrated for any species of elasmobranch fish. This paper describes the production of NO by LPS-stimulated PBL from two representative elasmobranch species. In addition, culture conditions, including serum source, LPS concentration, and time in culture that result in optimal NO production are reported. Finally, inhibition of NO production by the iNOS-specific inhibitor, L-N6-(1iminoethyl)lysine (L-NIL) is demonstrated for PBL from both species. The data presented provide the first evidence for the participation of reactive nitrogen intermediates in the immune system at the phylogenetic level of elasmobranch fishes.
2. Materials and methods 2.1. Experimental animals Nurse shark (Ginglymostoma cirratum) pups were collected in the Florida Keys and transported live to Mote Marine Laboratory, Sarasota, FL. Adult clearnose skates (Raja eglanteria) were collected in near shore waters off Anna Maria Island, FL. Animals were maintained in captivity under conditions previously described [24]. Body masses for animals bled for the studies described ranged from 3.6 to 5.0 kg for four immature nurse sharks and from 1.2 to 1.8 kg for three mature clearnose skates. 2.2. Collection of blood and isolation of PBL Whole blood was obtained via caudal venipuncture [25]. Animals were bled three times at approximately two-week intervals, with whole blood volumes of 5 ml drawn from each shark and 3 ml from each skate at each sampling. To monitor recovery of blood cell volumes, a small portion of each sample was drawn into heparin-coated microcapillary tubes for hematocrit determination. In all cases, hematocrit values fell within normal ranges for each species [25]. The remaining whole blood was immediately mixed with elasmobranch-modified ACD anticoagulant solution (E-ACD) at a ratio of 0.175 ml E-ACD per 1.0 ml
42
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
whole blood. E-ACD was prepared by supplementing ACD solution (40 mM citric acid, 70 mM sodium citrate, 140 mM dextrose) with sodium chloride to approximate the osmotic environment of elasmobranchs (final osmolarity between 900 and 1000 mOsm). PBL were isolated from whole blood through repetitive slow speed centrifugation, 10e20 min at 50 ! g for each spin. Centrifugation continued until erythrocytes formed a loose pellet and PBL remained as a partially concentrated suspension above the red cell layer. PBL were carefully aspirated and resuspended in elasmobranch-modified PBS (E-PBS; 450 mM NaCl, 10 mM NaH2PO4, pH 7.4). Cells were centrifuged at 200 ! g for 7 min, then resuspended in elasmobranchmodified RPMI (E-RPMI) culture medium (RPMI; Sigma Chemical Co., St. Louis, MO, modified by adding 360 mM urea and 115 mM NaCl to approximate the normal elasmobranch cellular environment). Before placing into culture, cells were enumerated using a haemocytometer and viability was assessed by trypan blue exclusion [26] using 0.2% trypan blue prepared in E-PBS. 2.3. Cell culture conditions PBL were cultured at 25 C in a 5% CO2 environment for up to 96 h using E-RPMI [15,27] as the culture medium. In addition, penicillin (50 units mlÿ1), streptomycin (50 mg mlÿ1), neomycin (0.1 mg mlÿ1), and amphotericin B (0.25 mg mlÿ1) were added to control bacterial and fungal growth. Sodium bicarbonate (23.8 mM) was added and the final pH of the medium was adjusted to 7.2e7.4. All reagents were sterilized using a 0.2-mm filter prior to use. To establish culture conditions resulting in optimal NO production, time in culture ranged from 0 to 96 h. Cells were cultured under both serum-free conditions as well as in the presence of 10% FBS (foetal bovine serum, HyClone). 2.4. Production of NO by nurse shark and clearnose skate PBL To analyse the effect of serum on production of nitric oxide by shark PBL, nurse shark PBL were cultured in serum-free medium, or in medium supplemented with 10% FBS or heterologous nurse shark serum (NSS). Serum was heat-inactivated at 56 C for 30 min before use. For this set of experiments, cells were cultured for 72 h at 25 C and 5% CO2 and stimulated with 0, 2.5, 5, 10, or 25 mg LPS mlÿ1 (lipopolysaccharide from Salmonella typhosa, Sigma Chemical Co., St. Louis, MO). To determine the time period during which maximal NO production occurs, nurse shark PBL were cultured at a final concentration of 2.5 ! 106 cells mlÿ1 and NO measured after 24, 48, 72, and 96 h of culture. Cells were stimulated with 5 mg LPS mlÿ1 and 10% FBS was included in the cell culture medium. Production of nitric oxide by clearnose skate PBL in culture was analysed using cells at a concentration of 2.5 ! 106 cells mlÿ1 in the presence of 10% heat-inactivated FBS. PBL were cultured for 72 h at 25 C and 5% CO2, and stimulated with 0, 2.5, 5, 10, and 20 mg LPS mlÿ1. 2.5. Measurement of NO activity NO production was determined indirectly by measurement of nitrite released into the culture medium using a colourimetric assay based on the Griess reaction [28]. Assays were conducted in 96-well microtiter plates with 200 ml cells per well and triplicate wells for each treatment. After incubation, supernatants were assayed for nitrite using a procedure adapted from McCarthy et al. [29]. Briefly, 100 ml aliquots of cell culture supernatants were removed from individual wells and transferred to a separate 96-well microtitre plate. One hundred microlitres of Griess reagent (1% sulfanilamide; 0.1% N-naphthyl-ethylenediamine; 2.5% H3PO4) was added to each sample well and plates were incubated at 25 C for 10 min. Absorbance at 540 nm was determined using a microplate reader (BioRad, Model 550). The molar concentration of nitrite in samples was determined from standard curves generated using known concentrations of sodium nitrite.
43
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
2.6. Effect of the iNOS-specific inhibitor, L-NIL, on NO production To evaluate the presence of iNOS in nurse shark and clearnose skate PBL, an iNOS-specific inhibitor, 6 -(1-iminoethyl)lysine (L-NIL), was included in cell cultures at final concentrations of 0, 10, and 30 mM. For these experiments, PBL were cultured at 2.5 ! 106 cells mlÿ1 and stimulated with 5 mg LPS mlÿ1. Cells were cultured for 72 h at 25 C and 5% CO2.
L-N
2.7. Statistical analyses Statistical analyses were performed using the computer program SAS, version 8.0, for Windows (SAS Institute Inc., Cary, NC). Data were compared using analysis of variance (ANOVA) and General Linear Model (GLM) analyses. When significant differences were detected, single degree of freedom contrast or response curve analyses was performed. Data points and error bars on the figures represent mean G standard error of the mean (SEM) responses for all animals tested in each experiment.
3. Results 3.1. Nitric oxide production by elasmobranch PBL Production of nitric oxide by nurse shark PBL cultured with three different conditions of serum supplementation at five different LPS concentrations is presented in Fig. 1. Overall, cultures including FBS and NSS resulted in significantly greater NO production than cultures without serum (P ! 0.001), although no significant difference was observed between serum types. With serum included in the culture, NO production increased linearly (P ! 0.05) with increasing LPS up to concentrations of 10 mg mlÿ1, after No Serum
14
FBS
µM NO produced
12
NSS
10 8 6 4 2 0
0
5
10
LPS (µg
15
20
25
ml-1)
Fig. 1. Production of nitric oxide by LPS-stimulated nurse shark PBL cultured under serum-free conditions or supplemented with foetal bovine serum (FBS) or nurse shark serum (NSS). Four nurse sharks were sampled three times each. Cells were cultured at 2.5 ! 106 cells mlÿ1 for 72 h at 25 C, 5% CO2 with 0, 2.5, 5, 10, or 25 mg LPS mlÿ1. Error bars represent standard error of the mean (SEM).
44
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
which production of NO tended to plateau (slight quadratic curvature, P Z 0.11). In addition to examining the effects of serum supplementation on NO production, studies to investigate the effects of time in culture on NO production by nurse shark PBL were performed. As shown in Fig. 2, NO production increased linearly (P ! 0.001) from 24 to 72 h following LPS stimulation, and decreased slightly after 96 h of culture. The ability of skate PBL to produce NO in response to LPS stimulation was also investigated. In response to LPS concentrations ranging from 0 to 20 mg mlÿ1, nitric oxide production by FBSsupplemented clearnose skate PBL was shown to increase exponentially (Fig. 3). 3.2. Inhibition of NO production by L-NIL Effects of the iNOS-specific inhibitor, L-NIL, on NO production by shark and skate PBL are presented in Figs. 2 and 3. No significant interactions between LPS and L-NIL were observed with either nurse shark or clearnose skate PBL. With both nurse shark and clearnose skate PBL (Figs. 2 and 3), increasing L-NIL concentration (10 and 30 mM) resulted in significantly (P ! 0.001) decreased NO production.
4. Discussion Data are presented demonstrating nitric oxide production by both nurse shark and clearnose skate immune cells in response to LPS stimulation, providing the first evidence of the participation of reactive nitrogen intermediates in the immune system at the phylogenetic level of elasmobranchs. Elasmobranch PBL responded to LPS stimulation by producing measurable amounts of NO. The maximal amount of NO production occurred after 72 h in culture, which is consistent with the time required to elicit maximal amount of NO production in mammals [29], teleost [4e10], and invertebrates [11,12]. The highest amounts of NO produced by elasmobranch PBL in response to LPS stimulation ranged from approximately 8 to 10 mM. These values are comparable to maximal NO production by mammalian species, which has been reported to range from 5 to 20 mM [29]. 0 µM L-NIL
9
10 µM L-NIL
µM NO produced
8
30 µM L-NIL
7 6 5 4 3 2 1 0
20
40
60
80
100
Time (h) Fig. 2. Production of nitric oxide by LPS-stimulated nurse shark PBL as a function of time. Four nurse sharks were sampled twice each. Nurse shark PBL were cultured at a concentration of 2.5 ! 106 cells mlÿ1 with 10% FBS. Nurse shark PBL were stimulated with 5 mg LPS mlÿ1 for 96 h in the presence of 0, 10 or 30 mM of the iNOS-specific inhibitor, L-NIL. Error bars represent SEM.
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
45
14
µM NO produced
12 10 8
0 µM L-NIL 10 µM L-NIL
6
30 µM L-NIL
4 2 0
0
5
10
15
20
LPS (µg ml-1) Fig. 3. Production of nitric oxide by LPS-stimulated clearnose skate PBL. Four skates were sampled one time each. Skate PBL were cultured at 2.5 ! 106 cells mlÿ1 for 72 h in the presence of 10% FBS. Skate PBL were stimulated with 0e20 mg LPS mlÿ1 in the presence of 0, 10 or 30 mM of the iNOS-specific inhibitor, L-NIL. Error bars represent SEM.
The inclusion of serum in the cell culture media significantly increased NO production by nurse shark PBL compared to serum-free cultures, although an overall difference was not observed when comparing commercially available FBS to heterologous nurse shark serum. For routine use, however, commercially available FBS is typically a very consistent and uniform product while heterologous NSS is potentially more variable. Production of NO by elasmobranch immune cells in response to LPS stimulation was similar to that seen in mammals, with NO produced in the 5e20 mM range after 72 h of culture. L-NIL, a selective inhibitor of inducible nitric oxide synthase (iNOS) [30], significantly reduced NO production by both nurse shark and clearnose skate PBL stimulated with LPS, suggesting that an iNOS is responsible for NO production by stimulated elasmobranch immune cells. The existence of iNOS within white blood cells of any species strongly indicates that NO is an antipathogenic agent in the immune system of that species [2]. Our findings suggest that elasmobranch PBL utilise NO as a chemical mediator in host defence and likely contain a phylogenetic equivalent to inducible nitric oxide synthase found in higher vertebrate immune cells.
Acknowledgements The authors gratefully acknowledge the use of facilities at Mote Marine Laboratory (Sarasota, FL). The authors would like to thank Dr. Jonathon Stamler at Duke University Medical Center for generously providing the iNOS inhibitor, L-NIL, used in this study. One of the authors (CAL) received partial support through grants from the Vernal W. and Florence H. Bates Foundation and the Disney Wildlife Conservation Fund. Student participation was made possible by a stipend to JDT from the Marjorie G. and Louis S. Gilbert Scholarship and by a grant to CTG from the Keating Family Foundation. Statistical analyses were performed with the assistance of Dr. Joe Toler at Clemson University.
46
C.J. Walsh et al. / Fish & Shellfish Immunology 20 (2006) 40e46
References [1] [2] [3] [4]
[5] [6] [7] [8]
[9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
[19] [20] [21] [22] [23] [24] [25]
[26] [27] [28] [29] [30]
Nussler A, Billiar T. Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol 1993;54:171e85. Nathan CF, Hibbs JB. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr Opin Immunol 1991;3:65e70. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992;6:3051e64. Neumann NF, Fagan D, Belosevic M. Macrophage activating factor(s) secreted by mitogen stimulated goldfish kidney leukocytes synergize with bacterial lipopolysaccharide to induce nitric oxide production in teleost macrophages. Dev Comp Immunol 1995;19:473e82. Yin Z, Lam TJ, Sin YM. Cytokine-mediated antimicrobial immune response of catfish, Clarias gariepinus, as a defense against Aeromonas hydrophila. Fish Shellfish Immunol 1997;7:93e104. Mulero V, Meseguer J. Functional characterisation of a macrophage-activating factor produced by leucocytes of gilthead seabream (Sparus aurata L.). Fish Shellfish Immunol 1998;8:143e56. Laing KJ, Hardie LJ, Aartsen W, Grabowski PS, Secombes CJ. Expression of an inducible nitric oxide synthase gene in rainbow trout Oncorhynchus mykiss. Dev Comp Immunol 1999;23:71e85. Campos-Perez JJ, Ward M, Grabowski PS, Ellis AE, Secombes CJ. The gills are an important site of iNOS expression in rainbow trout Oncorhynchus mykiss after challenge with the gram-positive pathogen Renibacterium salmoninarum. Immunology 2000;99:153e61. Tafalla C, Novoa B. Requirements for nitric oxide production by turbot (Scophthalmus maximus) head kidney macrophages. Dev Comp Immunol 2000;24:621e31. Saeij JP, Stet RJ, Groeneveld A, Verburg van Kemenade L, Muiswinkel W, Wiegertjes GF. Molecular and functional characterization of a fish inducible-type nitric oxide synthase. Immunogenetics 2000;51:339e46. Beck G, Ellis T, Zhang H, Lin W, Beauregard K, Habicht GS, et al. Nitric oxide production by coelomocytes of Asterias forbesi. Dev Comp Immunol 2001;25:1e10. Franchini A, Fontanili P, Ottaviani E. Invertebrate immunocytes: relationship between phagocytosis and nitric oxide production. Comp Biochem Physiol 1995;110B:403e7. Luckhart S, Rosenberg R. Gene structure and polymorphism of an invertebrate nitric oxide synthase gene. Gene 1999;232:25e34. Luer CA, Walsh CJ, Bodine AB, Wyffels JT, Scott TR. The elasmobranch thymus: anatomical, histological, and preliminary functional characterization. J Exp Zool 1995;273:342e54. Rast JP, Anderson MK, Strong SJ, Luer C, Litman RT, Litman GW. a, b, g, and d T cell antigen receptor genes arose early in vertebrate phylogeny. Immunity 1997;6:1e11. Flajnik MF, Rumfelt LL. The immune system of cartilaginous fish. Curr Top Microbiol Immunol 2000;248:249e70. Hyder SL, Cayer ML, Pettey CL. Cell types in peripheral blood of the nurse shark: an approach to structure and function. Tissue Cell 1983;15:437e55. Walsh CJ, Luer CA. Comparative phagocytic and pinocytic activities of leucocytes from peripheral blood and lymphomyeloid tissues of the nurse shark (Ginglymostoma cirratum Bonaterre) and the clearnose skate (Raja eglanteria Bosc). Fish Shellfish Immunol 1998;8:197e215. Pettey CL, McKinney EC. Mitogen-induced cytotoxicity in the nurse shark. Dev Comp Immunol 1981;5:53e64. Pettey CL, McKinney EC. Induction of cell-mediated cytotoxicity by shark 19S IgM. Cell Immunol 1988;111:28e38. Pettey CL, McKinney EC. Temperature and cellular regulation in the shark. Eur J Immunol 1983;13:133e8. McKinney EC, Haynes L, Droese AL. Macrophage-like effector of spontaneous cytotoxicity from the shark. Dev Comp Immunol 1986;10:497e508. Nonaka M, Smith SL. Complement system of bony and cartilaginous fish. Fish Shellfish Immunol 2000;10:215e27. Luer CA. Elasmobranchs (sharks, skates, and rays) as animal models for biomedical research. In: Woodhead AD, Vivirito K, editors. Nonmammalian animal models for biomedical research. Boca Raton, FL: CRC Press; 1989. p. 121e47. Walsh CJ, Luer CA. Elasmobranch hematology: identification of cell types and practical applications. In: Smith M, Warmolts D, Thoney D, Hueter R, editors. Elasmobranch husbandry manual: captive care of sharks, rays, and their relatives. Columbus, OH: Ohio Biological Survey, Inc.; 2004. p. 307e23. Baur H, Kasperek S, Pfaff E. Criteria of viability of isolated liver cells. Hoppe Seylers Z Physiol Chem 1975;356:827e38. Walsh CJ, Luer CA, Wyffels JT, Bodine AB. Dexamethasone-induced apoptosis in immune cells from peripheral circulation and lymphomyeloid tissues of juvenile clearnose skates, Raja eglanteria. Dev Comp Immunol 2002;26:629e39. Green LC, Wagner DDA, Glogowski J, Skepper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite and (15N)nitrate in biological fluids. Anal Biochem 1982;124:131e8. McCarthy JE, Redmond PH, Duggan SM, Watson RWG, Condron CM, O’Donnell JR, et al. Characterization of the defects in murine peritoneal macrophage function in the early postsplenectomy period. J Immunol 1995;155:387e96. Bryk R, Wolff DJ. Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine. Biochemistry 1988;37:4844e52.