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Characterization of serum complement activity of saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles
Comparative Biochemistry and Physiology, Part A 143 (2006) 488 – 493 www.elsevier.com/locate/cbpa
Characterization of serum complement activity of saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles Mark Merchant a,⁎, Adam Britton b a
Department of Chemistry, McNeese State University, Box 90455, Lake Charles, LA, 70609, USA b Wildlife Management Incorporated, Darwin, Northern Territory, Australia Received 12 October 2005; received in revised form 3 January 2006; accepted 8 January 2006 Available online 17 February 2006
Abstract We employed a spectroscopic assay, based on the hemolysis of sheep red blood cells (SRBCs), to assess the innate immune function of saltwater and freshwater crocodiles in vitro. Incubation of serum from freshwater and saltwater crocodiles with SRBCs resulted in concentrationdependent increases in SRBC hemolysis. The hemolytic activity occurred rapidly, with detectable activity within 2 min and maximum activity at 20 min. These activities, in both crocodilian species, were heat sensitive, unaffected by 20 mM methylamine, and completely inhibited by low concentrations of EDTA, suggesting that the alternative serum complement cascade is responsible for the observed effects. The hemolytic activities of the sera were inhibited by other chelators of divalent metal ions, such as phosphate and citrate. The inhibition of SRBC hemolysis by EDTA could be completely restored by the addition of 10 mM Ca2+ or Mg2+, but not Ba2+, Cu2+ or Fe2+, indicating specificity for these metal ions. The serum complement activities of both crocodilians were temperature-dependent, with peak activities occurring at 25–30 °C and reduced activities below 25 °C and above 35 °C. © 2006 Elsevier Inc. All rights reserved. Keywords: Crocodilian; Immunology; Innate immunity; Humoral immunity
1. Introduction The serum complement system, an important portion of the innate immune system, is composed of nine proteins that can be activated to initiate the inflammatory response, recruit leukocytes to the site of infection, mediate opsinization of particulate foreign materials and to kill microorganisms directly by the assembly of a multiprotein membrane attack complex in the outer membrane of microbes (Muller-Eberhard, 1986; Dalmasso et al., 1989). Complement proteins are expressed and circulated as inactive precursor proteins that can be activated in a very precise and highly coordinated manner (Campbell et al., 1988). The complement cascade can be initiated by three distinct mechanisms: an antibody-dependent classical pathway, an antibody-independent alternative pathway, and a lectin pathway, that result in the modulation of immune function.
⁎ Corresponding author. Tel.: +1 337 475 5773; fax: +1 337 475 5950. E-mail address: [email protected] (M. Merchant). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.01.009
The serum complement system is an ancient mechanism of innate immunity that developed prior to the appearance of vertebrates, and is found in early invertebrate species, including deuterostomes. The development of the alternative pathway of complement activation probably predated the classical and lectin-mediated pathways, since the alternative pathway involves direct interaction of complement proteins with microbial membranes and does not require the presence of a specialized recognition molecule (Zhu et al., 2005). The alternative and the lectin pathways have been characterized in echinoderms (AlSharif et al., 1998; Smith et al., 2001) and tunicates (Smith et al., 1999; Nonaka, 2001), while the classical pathway seems to have appeared for the first time in jawed fishes. Both cartilaginous and teleost fish contain complement components that participate in the alternative, lectin, and classical pathways of complement activation. Serum complement proteins C3, C4, and C5 have been identified, cloned, and purified in teleost fish (Smith, 1998; Nonaka and Smith, 2000). Thus far, teleost fish are the only animals in which multiple forms of functionally active C3 proteins, that are the products of different genes, have been
M. Merchant, A. Britton / Comparative Biochemistry and Physiology, Part A 143 (2006) 488–493
found (Sunyer et al., 1996, 1997a,b, 1998; Kuroda et al., 2000; Nakao et al., 2000). The mode of activation of the classical pathway in this teleost fish is very similar to that of mammals (Boshra et al., 2004). The results from several recent studies in our laboratory have indicated that crocodilians exhibit serum complement activities (Merchant et al., 2005a,b). The results from this study strongly suggest a potent complement system exists in the serum of the saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles.
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4. Statistics and controls For each SRBC hemolysis assay, a complete lysis positive control was acquired by rapidly syringing a solution of 1% SRBCs through a 31 ga needle in the presence of 1% (v / v) Triton X-100. This action resulted in 100% lysis of the SRBCs, as inspected by phase contrast microscopy at 400× magnification. This control sample was used as a comparison for all other samples. Each sample was analyzed in triplicate so that valid statistical results could be obtained. All results represent the means ± standard deviations.
2. Materials and methods 5. Results 2.1. Chemicals
2.2. Treatment of animals Captive saltwater crocodiles (1.6–1.7 m) were housed in outdoor 15 × 15 m pens. They were fed chicken heads ad libitum 4 times per week. Captive freshwater crocodiles (1.0–1.5 m) were housed in a 10 × 8 m enclosure and fed chicken heads ad libitum 3 times per week. Wild saltwater crocodiles were captured from a boat at night with the aid of a spotlight. The crocodiles were captured from the South Alligator River, Kakadu National Park in the Northern Territory of Australia. The animals were captured using a harpoon with three 1.9-cm barbs. Blood was harvested from the spinal vein (Olson et al., 1977; Zippel et al., 2003) using an 18 ga needle and a 60 mL syringe. The blood was allowed to clot at room temperature for at least 4 h before centrifugation at 3000 ×g for 15 min. The serum was removed and pooled. 3. SRBC hemolysis assay One milliliter of crocodile serum was incubated with 1 mL of 2% unsensitized sheep red blood cells (SRBCs). The samples were incubated for 20 min, except during the kinetic study, and centrifuged at 2000 ×g for 2 min. The resulting supernatant was transferred to a plastic cuvette and the optical density was measured at 540 nm. For the kinetic hemolysis analysis, 30 mL of 25% crocodile serum was added to 30 mL of 2% SRBCs (v / v). Two milliliter aliquots were removed at various times. The samples were centrifuged at 2000 ×g and the resulting supernatant was removed and the optical density measured at 540 nm. For the kinetic study, 30 mL of crocodile serum was mixed with 30 mL of 2% SRBCs (v / v) and aliquots were removed and centrifuged (2000 ×g) at various time points. The temperature-dependent study was conducted by preincubating aliquots of SRBCs and crocodile serum at specific temperatures for 10 min. The samples and SRBCs were then mixed and incubated for 20 min at the respective temperatures.
The data in Fig. 1 show the concentration-dependent hemolysis of unsensitized SRBCs by serum from saltwater and freshwater crocodiles. Incubation of increasing concentrations of serum from both crocodilian species with SRBCs in vitro resulted in increased hemolytic activities. The calculated CH50 values were 473 and 451 mL for serum derived from the saltwater and freshwater crocodiles, respectively. The CH50 values derived from these data represent the concentrations of serum required to produce 50% of maximum hemolysis of 1% SRBCs in a 1 mL reaction volume. This value is used clinically to determine the relative complement activity of a serum sample. The data in Table 1 reveal the effects of heat, protease, EDTA, phosphate, citrate, and methylamine on the hemolysis of SRBCs by crocodile serum. Mild heat treatment of serum (56 °C, 30 min) completely obliterated the SRBC hemolysis by serum from both crocodile species. In addition, treatment of the sera samples with 10 units of a protease derived from Streptomyces griseus (Sigma Chemical Co, St. Louis, MO, USA) for 30 min at 37 °C also reduced the hemolysis to baseline levels. Treatment of 110
Crocodylus porosus Crocodylus johnstoni
100
SRBC Hemolysis (% maximum)
Fresh 10% sheep red blood cells (v / v) were purchased from IMVS Veterinary Services Biological Products, South Australia. EDTA, MgCl2, CaCl2, CuCl2, Fe SO4, BaCl2, and Triton X-100 were purchased from Sigma Chemical Company (St. Louis, MO).
90 80 70 60 50 40 30 20 10 0 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Serum titer (mL) Fig. 1. Concentration-dependent hemolysis of SRBCs by saltwater and freshwater crocodile serum. SRBCs were incubated with different concentrations of sera from freshwater and saltwater crocodiles for 20 min. The samples were centrifuged and the optical densities of the supernatants were measured at 450 nm. Each sample was analyzed in triplicate and the results represent the means ± standard deviations.
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Table 1 Effects of heat, protease and EDTA treatment on the hemolysis of SRBCs by serum from the saltwater and freshwater crocodiles Hemolysis (% maximum)
None Heat-treated (56 °C, 30 min) Protease (10 U, 30 min, 37 °C) EDTA (10 mM) Sodium phosphate (30 mM) Potassium citrate (30 mM) Methylamine (20 mM)
Freshwater crocodile
Saltwater crocodile
98 ± 2 4±1 2±1 2±0 7±3 5±1 95 ± 4
101 ± 4 6±3 3±2 2±1 7±2 9±4 98 ± 4
Undiluted crocodile serum was heat-treated, or treated with protease, EDTA, phosphate, citrate, or methylamine, and then incubated with 1% SRBCs for 30 min at room temperature. The samples were then centrifuged at 2000 ×g and the optical densities of the supernatants were measured at 540 nm. The results represent the means ± standard deviations for three independent determinations.
Hemolysis (% maximum)
sera with divalent metal chelators Na2 EDTA (10 mM, pH 8.0), Na2HPO4 (30 mM), or K3 citrate (30 mM) for just 1 min at ambient temperature resulted in substantial reductions in the ability of the sera to disrupt the integrity of the SRBCs. However, treatment of the serum samples with 30 mM Na2HSO4 or KCl did not affect the hemolysis of SRBCs by the crocodile sera (data not shown), eliminating the possibility that the observed inhibition of the SRBC hemolysis by the chelators was due to elevated ionic strength of the solution. The kinetic analyses of the hemolysis of SRBCs by serum from the saltwater and freshwater crocodiles are depicted in Fig. 2. The sera showed little activity at 5 min exposure to SRBCs. However, the saltwater and freshwater crocodile sera exhibited 56.3% and 65.6% of maximal activity, respectively, after 10 min. The hemolytic response was maximal (98.2%) for the saltwater crocodile and near maximal (88.2%) for the freshwater crocodile at 15 min. Fig. 3 displays the concentration-dependent effects of EDTA on the hemolysis of SRBCs by crocodile serum. SRBC hemolysis was not affected in the presence of 1 mM EDTA. However, the hemolytic activities for serum from both crocodile species
Hemolysis (% maximum)
Serum treatment
Crocodylus porosus Crocodylus johnstoni
100
80
60
40
20
0 0
1
2
3
4
5
6
7
8
9
10
[EDTA] (mM) Fig. 3. Concentration-dependent effects of EDTA on the hemolysis of SRBCs by sera from freshwater and saltwater crocodiles. SRBCs were incubated with sera from freshwater and saltwater crocodiles in the presence of varying amounts of EDTA for 20 min. The samples were centrifuged and the optical densities of the supernatants were measured at 450 nm. Each sample was analyzed in triplicate and the results represent the means ± standard deviations.
were reduced to 77.6% and 15.4% in the presence of 2 mM EDTA, respectively. The ability of the sera from both crocodiles to hemolyze SRBCs was completely inhibited by 3 mM EDTA. The inhibition of the hemolytic activities of sera from both crocodiles could be completely reversed by the addition of either 30 mM Ca2+ or Mg2+ (Table 2). However, 30 mM Ba2+, Cu2+, or Fe2+ did not affect the inhibition of innate immune activity by serum from either crocodile species. The data in Fig. 4 illustrate the temperature-dependent effects of the hemolysis of SRBCs by serum from the two crocodiles. Peak serum complement activity was observed at 25–30 °C in the freshwater crocodile and 20–35 °C in the saltwater crocodile. However, the complement activity was reduced at temperatures below 20 °C. The hemolysis of SRBCs by the freshwater crocodile serum was reduced to 47.3%, 13.5%, and 9.2% of maximum at temperatures of 15, 10, and
100 Table 2 Effects of different divalent metal ions on EDTA-inhibited hemolysis of SRBCs by crocodile serum
80 60
Crocodylus porosus Crocodylus johnstoni
40 20 0 0
10
20
30
40
50
60
Time (min) Fig. 2. Kinetic analyses of serum complement activities of sera from saltwater and freshwater crocodiles. SRBCs were incubated with an equal volume of 25% sera from freshwater and saltwater crocodiles for various amounts of time. The samples were centrifuged and the optical densities of the supernatants were measured at 450 nm. Each sample was analyzed in triplicate and the results represent the means ± standard deviations.
Serum treatment
Hemolysis (% maximum) Freshwater crocodile
Saltwater crocodile
None 5 mM EDTA 5 mM EDTA + 10 5 mM EDTA + 10 5 mM EDTA + 10 5 mM EDTA + 10 5 mM EDTA + 10
102 ± 4 3±1 97 ± 4 99 ± 3 2±2 3±2 5±3
98 ± 6 2±1 100 ± 2 97 ± 2 0±0 2±1 2±1
mM Mg2+ mM Ca2+ mM Cu2+ mM Ba2+ mM Fe2+
Crocodile serum samples were treated with 5 mM EDTA, while other samples were treated with EDTA plus 10 mM Ca2+, Mg2+, Ba2+, Cu2+, or Fe2+. The samples were incubated with 1% SRBCs for 30 min at ambient temperature and then centrifuged at 2000 ×g and the optical densities of the supernatants were measured at 540 nm. The results represent the means ± standard deviations for three independent determinations.
SRBC Hemolysis (% maximum)
M. Merchant, A. Britton / Comparative Biochemistry and Physiology, Part A 143 (2006) 488–493
Crocodylus porosus Crocodylus johnstoni
100 80 60 40 20 0
5
10
15
20
25
30
35
40
o
Temperature ( C) Fig. 4. Temperature-dependent hemolysis of SRBCs by sera from freshwater and saltwater crocodiles. SRBCs were incubated with sera from freshwater and saltwater crocodiles at different temperatures for 20 min. The samples were centrifuged and the optical densities of the supernatants were measured at 450 nm. Each sample was analyzed in triplicate and the results represent the means ± standard deviations.
5 °C, respectively. A parallel reduction of activity for serum from the saltwater crocodile was observed (Fig. 4). For the freshwater crocodile, activity was reduced slightly to 92.4% at 35 °C, and further reduced to 69.4% at 40 °C. The saltwater crocodile serum exhibited maximal activity (96.4%) at 35 °C, but was reduced to 74.9% at 40 °C. 6. Discussion Several studies have focused on the antimicrobial properties of serum from crocodilians. Shaharabany et al. (1999) showed that crude extracts from the tissues of the Nile crocodile (Crocodylus niloticus) exhibit antimicrobial properties. More recent studies in our laboratory showed that serum from the American alligator (Alligator mississippiensis) exhibits potent antibacterial (Merchant et al., 2003), amoebacidal (Merchant et al., 2004), and antiviral (Merchant et al., 2005c) properties. Further studies showed that the antimicrobial activities of alligator serum were due to the alternative serum complement pathway (Merchant et al., 2005a). Still more recent studies in our laboratory have demonstrated innate immunity in all 23 extant crocodilian species, and also showed that these activities follow distinct taxonomical crocodilian lineages (Merchant et al., 2006). The ability of serum to hemolyze SRBCs has been linked to serum complement activity in several reptilian species (Dias de Silva et al., 1984; Mansour et al., 1980). We have employed this assay to characterize the serum complement activity of the saltwater (C. porosus) and freshwater (C. johnstoni) crocodiles. The data displayed in Fig. 1 reveal that the lysis of SRBCs by serum from the saltwater and freshwater crocodiles occurs in a concentration-dependent manner. The incubation of serum derived from both saltwater and freshwater crocodiles with 1% SRBCs resulted in strong hemolytic activity, as determined by an increase in optical density at 540 nm due to the release of hemoglobin by the disrupted erythrocytes. The hemolytic activities are similar to each other, and also comparable to that of the American alligator (Merchant et al., 2005a).
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The results from mechanistic experiments displayed in Table 2 show that the ability of the sera samples' ability to disrupt SRBCs is severely limited by modest heat treatment (56 °C, 30 min). These conditions are classical complement inactivation conditions. Human complement contains heatlabile proteins that are thermally inactivated under these conditions. The fact that the crocodilian hemolytic activities were obliterated by mild heat treatment eliminates the possibility that these activities are due to the presence of heat-stable antimicrobial peptides. In addition, the inhibition of hemolytic activities by protease indicates that the molecules responsible for the SRBC hemolysis are proteinaceous in nature. The data in Table 2 also illustrate that the hemolytic activities are severely limited in the presence of 10 mM EDTA. It is long been known that human serum requires the presence of divalent metal ions, specifically Mg2+ and Ca2+, to hemolyze SRBCs in vitro (Levine et al., 1953a; Levine et al., 1953b). Other studies have shown that divalent metal ions are required for alternative pathway-mediated complement activity, but not for classical pathway activation (Vogt et al., 1977). Furthermore, the addition of methylamine had no effect on the hemolysis of SRBCs by sera from either crocodilian species (Table 2). It is known that primary amines inhibit activation of the classical complement cascade by interacting with the thioester in the C4 protein, which is not involved in the alternative pathway (Gorski and Howard, 1980). The fact that the hemolysis of unsensitized SRBCs by crocodile serum is heat sensitive, requires divalent metal ions, and is unaffected by methylamine strongly suggests that the alternative pathway of complement activation is responsible for the hemolytic action. In addition, these results suggest similarities in the molecular mechanisms of complement activation between these crocodilians and that of humans. The kinetic profiles of serum from the saltwater and freshwater crocodiles are very similar (Fig. 2). These data show that the hemolysis of SRBCs occurs very rapidly when exposed to the serum of both crocodilian species. The maximum activities are reached within 15–20 min, which is remarkably similar to the kinetics of complement-mediated antibacterial activity of human serum (Wright and Levine, 1981). In addition, these kinetic curves are extremely similar to that previously reported for the American alligator (Merchant et al., 2005a). The hemolytic activities of serum from both crocodile species require divalent metals ions for activity (Fig. 3, Tables 1 and 2). These SRBC hemolytic activities are completely inhibited by EDTA concentrations as low as 3 mM. The hemolytic activity can also be inhibited by other chelators of divalent metal ions, such as phosphate, and citrate. In addition, the inhibition of SRBC hemolysis by EDTA can be restored by the addition of excess (30 mM) Ca2+ or Mg2+. These data are in contrast to the need of human serum for both Ca2+ and Mg2+ (Levine et al., 1953a,b), but is consistent with the fact that alligator serum requires either Ca2+ or Mg2+ (Merchant et al., 2005a) for complement activity. However, the crocodile serum complement activation does not show a complete lack of specificity, as addition of 30 mM Ba2+, Cu2+, or Fe2+ did not restore the EDTA-inhibited hemolysis of SRBCs. These data
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indicate that the mechanisms of complement activities for diverse crocodilian species are similar. Since crocodiles are ectothermic vertebrates whose body temperatures exhibit marked season and diurnal variations, we investigated the effectiveness of the serum complement system of the saltwater and freshwater crocodiles at a broad spectrum of temperatures in vitro. Grigg et al. (1998) described the thermal regulation of free-ranging saltwater crocodiles (C. porosus) throughout the year and reported body temperatures ranging from 20 to 35 °C. These animals maintained body temperatures that were near or at the optimal temperatures for humoral immune activity. In another study, Seebacher and Grigg (1997) measured body temperature of wild freshwater crocodiles (C. johnstoni) and found that their body temperatures ranged from 16 to 36 °C. Our data (Fig. 4) show that the serum complement activity of the freshwater crocodile falls to 85% at 20 °C and is further reduced to 47.3% at 15 °C, indicating that the freshwater crocodiles may be somewhat immunocompromised during the winter months when their body temperatures fall below 20 °C. However, this potentially depressed innate immunity may not be biologically relevant as the animals are not as active, and microbial growth is not as abundant during the winter months. The data presented in this study strongly suggest that the freshwater and saltwater crocodiles exhibit alternative serum complement activities. However, this study does rule out the presence of a classical complement cascade in these crocodilians. It is interesting to note that previous studies have shown that the spectrum of antibacterial activity of serum from A. mississippiensis is substantially different from than that of both C. johnstoni and C. porosus (Merchant et al., 2005b). However, the present study reveals striking similarities in the serum complement mechanisms of these crocodilians. We propose that, although the mechanisms of complement activation may be similar, the proteins that act to stimulate the complement cascade are activated by different microorganisms. Alternatively, the proteins that form the membrane attack complex may show different specificities for different types of microbial membrane compositions. Acknowledgements The authors wish to thank Tommy Nichols, Service Ranger with the Northern Territory Parks and Wildlife Service, and Garry Lindner, Park Ranger at Kakadu National Park, for their help in the capture of wild saltwater crocodiles used in this study. We also wish to acknowledge Ms. Erin O'Brien for her help in the capture and bleeding of crocodiles. References Al-Sharif, W.Z., Sunyer, J.O., Lambris, J.D., Smith, L.C., 1998. Sea urchin coelomocytes specifically express a homologue of the complement component C3. J. Immunol. 160, 2983–2997. Boshra, H., Gelman, A.E., Sunyer, J.O., 2004. Structural and functional characterization of complement C4 and C1s-like molecules in teleost fish: insights into the evolution of classical and alternative pathways. J. Immunol. 173, 349–359.
Campbell, R.D., Law, S.K.A., Reid, K.B.M., Sim, R.B., 1988. Structure, organization, and regulation of the serum complement genes. Ann. Rev. Immunol. 6, 161. Dalmasso, A.P., Falk, R.J., Raij, L., 1989. The pathobiology of the terminal complement complexes. Complement Inflam. 6, 36–48. Dias de Silva, W., Calich, V.L., Kipnis, T.l.L., Rosen, F.S., Alper, C.A., 1984. Complement in the serum and venom of Brazilian snakes (Crotalidae). Acta Pathol. Microbiol. Immunol. Scand. Suppl. 284, 97–103. Gorski, J.P., Howard, J.B., 1980. Effect of methylamine on the structure and function of the fourth component of human complement, C4. J. Biol. Chem. 255, 10025–10028. Grigg, G.C., Seebacher, F., Beard, L., Morris, D., 1998. Thermal relations of large crocodiles, Crocodylus porosus, free-ranging in a naturalistic situation. Proc. R. Soc. Lond., B 265, 1793–1799. Kuroda, N., Naruse, K., Shima, A., Nonaka, M., Sasaki, M., 2000. Molecular cloning and linkage analysis of complement C3 and C4 genes of the Japanese medaka fish. Immunogenetics 51, 117–128. Levine, L., Cowan, K.M., Osler, A.G., Mayer, M.M., 1953a. The role of calcium and magnesium ions in complement fixation and immune hemolysis. I. Uptake of complement nitrogen by specific precipitates and its inhibition by ethylenediaminetetraacetate. J. Immunol. 71, 359–366. Levine, L., Osler, A.G., Mayer, M.M., 1953b. The role of calcium and magnesium ions in complement fixation and immune hemolysis. III. The respective roles of calcium and magnesium ions in immune hemolysis. J. Immunol. 71, 374–379. Mansour, M.H., El Ridi, R., Badir, N., 1980. Surface markers of lymphocytes in the snake, Spalerosophis diadema. I. Investigation of lymphocyte surface markers. Immunology 40, 605–611. Merchant, M., Roche', C., Elsey, R.M., Prudhomme, J., 2003. Antibacterial properties of serum from the American alligator (Alligator mississippiensis). Comp. Biochem. Physiol., B 136 (3), 505–513. Merchant, M., Thibodeaux, D., Loubser, K., Elsey, R.M., 2004. Amoebacidal effects of serum from the American alligator (Alligator mississippiensis). J. Parasitol. 90, 1480–1483. Merchant, M., Roche', C., Thibodeaux, D., Elsey, R., 2005a. Identification of alternative serum complement activity in the blood of the American alligator (Alligator mississippiensis). Comp. Biochem. Physiol., B 141, 281–288. Merchant, M., Pallansch, M., Paulman, R., Wells, J., Nalca, A., Ptak, R., 2005b. Antiviral activity of serum from the American alligator (Alligator mississippiensis). Antiviral Res. 66 (1), 35–38. Merchant, M., Verret, B., Elsey, R.M., 2005c. Divalent metal requirements for serum complement activity in the American alligator (Alligator mississippiensis). Comp. Biochem. Physiol. B. 141, 289–293. Merchant et al., 2006. Comparisons of innate immune activity of all known living crocodylian species. Comp. Biochem. Physiol. B 143, 133–137. Muller-Eberhard, H.J., 1986. The membrane attack complex of complement. Annu. Rev. Immunol. 4, 503. Nakao, M., Mutsuro, J., Obo, R., Fujiki, K., Nonaka, M., Yano, T., 2000. Molecular cloning and protein analysis of divergent forms of the complement component C3 from a bony fish, the common carp (Cyprinus carpio): presence of variants lacking the catalytic histidine. Eur. J. Immunol. 30, 858–866. Nonaka, M., 2001. Evolution of the complement system. Curr. Opin. Immunol. 13, 69–73. Nonaka, M., Smith, S.L., 2000. Complement system of bony and cartilaginous fish. Fish Shellfish Immunol. 10, 215–228. Olson, G., Hessler, J., Faith, R., 1977. Technics for the blood collection and intravascular infusion of reptiles. Lab. Anim. Sci. 25 (6), 783–786. Seebacher, F., Grigg, G.C., 1997. Patterns of body temperature in wild freshwater crocodiles, Crocodylus johnstoni: thermoregulation versus thermoconformity, seasonal acclimatization and the effect of social interactions. Copeia 1997 (3), 549–557. Shaharabany, M., Gollop, N., Ravin, S., Golomb, E., DeMarco, L., Ferreira, P.C., Boson, W.L., Friedman, E., 1999. Naturally occurring antibacterial activities of avian and crocodile tissues. J. Antimicrob. Chemother. 44 (3), 416–418. Smith, S.L., 1998. Shark complement: an assessment. Immunol. Rev. 166, 67–78.
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Characterization of serum complement activity of saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles Mark Merchant a,⁎, Adam Britton b a
Department of Chemistry, McNeese State University, Box 90455, Lake Charles, LA, 70609, USA b Wildlife Management Incorporated, Darwin, Northern Territory, Australia Received 12 October 2005; received in revised form 3 January 2006; accepted 8 January 2006 Available online 17 February 2006
Abstract We employed a spectroscopic assay, based on the hemolysis of sheep red blood cells (SRBCs), to assess the innate immune function of saltwater and freshwater crocodiles in vitro. Incubation of serum from freshwater and saltwater crocodiles with SRBCs resulted in concentrationdependent increases in SRBC hemolysis. The hemolytic activity occurred rapidly, with detectable activity within 2 min and maximum activity at 20 min. These activities, in both crocodilian species, were heat sensitive, unaffected by 20 mM methylamine, and completely inhibited by low concentrations of EDTA, suggesting that the alternative serum complement cascade is responsible for the observed effects. The hemolytic activities of the sera were inhibited by other chelators of divalent metal ions, such as phosphate and citrate. The inhibition of SRBC hemolysis by EDTA could be completely restored by the addition of 10 mM Ca2+ or Mg2+, but not Ba2+, Cu2+ or Fe2+, indicating specificity for these metal ions. The serum complement activities of both crocodilians were temperature-dependent, with peak activities occurring at 25–30 °C and reduced activities below 25 °C and above 35 °C. © 2006 Elsevier Inc. All rights reserved. Keywords: Crocodilian; Immunology; Innate immunity; Humoral immunity
1. Introduction The serum complement system, an important portion of the innate immune system, is composed of nine proteins that can be activated to initiate the inflammatory response, recruit leukocytes to the site of infection, mediate opsinization of particulate foreign materials and to kill microorganisms directly by the assembly of a multiprotein membrane attack complex in the outer membrane of microbes (Muller-Eberhard, 1986; Dalmasso et al., 1989). Complement proteins are expressed and circulated as inactive precursor proteins that can be activated in a very precise and highly coordinated manner (Campbell et al., 1988). The complement cascade can be initiated by three distinct mechanisms: an antibody-dependent classical pathway, an antibody-independent alternative pathway, and a lectin pathway, that result in the modulation of immune function.
⁎ Corresponding author. Tel.: +1 337 475 5773; fax: +1 337 475 5950. E-mail address: [email protected] (M. Merchant). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.01.009
The serum complement system is an ancient mechanism of innate immunity that developed prior to the appearance of vertebrates, and is found in early invertebrate species, including deuterostomes. The development of the alternative pathway of complement activation probably predated the classical and lectin-mediated pathways, since the alternative pathway involves direct interaction of complement proteins with microbial membranes and does not require the presence of a specialized recognition molecule (Zhu et al., 2005). The alternative and the lectin pathways have been characterized in echinoderms (AlSharif et al., 1998; Smith et al., 2001) and tunicates (Smith et al., 1999; Nonaka, 2001), while the classical pathway seems to have appeared for the first time in jawed fishes. Both cartilaginous and teleost fish contain complement components that participate in the alternative, lectin, and classical pathways of complement activation. Serum complement proteins C3, C4, and C5 have been identified, cloned, and purified in teleost fish (Smith, 1998; Nonaka and Smith, 2000). Thus far, teleost fish are the only animals in which multiple forms of functionally active C3 proteins, that are the products of different genes, have been
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found (Sunyer et al., 1996, 1997a,b, 1998; Kuroda et al., 2000; Nakao et al., 2000). The mode of activation of the classical pathway in this teleost fish is very similar to that of mammals (Boshra et al., 2004). The results from several recent studies in our laboratory have indicated that crocodilians exhibit serum complement activities (Merchant et al., 2005a,b). The results from this study strongly suggest a potent complement system exists in the serum of the saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles.
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4. Statistics and controls For each SRBC hemolysis assay, a complete lysis positive control was acquired by rapidly syringing a solution of 1% SRBCs through a 31 ga needle in the presence of 1% (v / v) Triton X-100. This action resulted in 100% lysis of the SRBCs, as inspected by phase contrast microscopy at 400× magnification. This control sample was used as a comparison for all other samples. Each sample was analyzed in triplicate so that valid statistical results could be obtained. All results represent the means ± standard deviations.
2. Materials and methods 5. Results 2.1. Chemicals
2.2. Treatment of animals Captive saltwater crocodiles (1.6–1.7 m) were housed in outdoor 15 × 15 m pens. They were fed chicken heads ad libitum 4 times per week. Captive freshwater crocodiles (1.0–1.5 m) were housed in a 10 × 8 m enclosure and fed chicken heads ad libitum 3 times per week. Wild saltwater crocodiles were captured from a boat at night with the aid of a spotlight. The crocodiles were captured from the South Alligator River, Kakadu National Park in the Northern Territory of Australia. The animals were captured using a harpoon with three 1.9-cm barbs. Blood was harvested from the spinal vein (Olson et al., 1977; Zippel et al., 2003) using an 18 ga needle and a 60 mL syringe. The blood was allowed to clot at room temperature for at least 4 h before centrifugation at 3000 ×g for 15 min. The serum was removed and pooled. 3. SRBC hemolysis assay One milliliter of crocodile serum was incubated with 1 mL of 2% unsensitized sheep red blood cells (SRBCs). The samples were incubated for 20 min, except during the kinetic study, and centrifuged at 2000 ×g for 2 min. The resulting supernatant was transferred to a plastic cuvette and the optical density was measured at 540 nm. For the kinetic hemolysis analysis, 30 mL of 25% crocodile serum was added to 30 mL of 2% SRBCs (v / v). Two milliliter aliquots were removed at various times. The samples were centrifuged at 2000 ×g and the resulting supernatant was removed and the optical density measured at 540 nm. For the kinetic study, 30 mL of crocodile serum was mixed with 30 mL of 2% SRBCs (v / v) and aliquots were removed and centrifuged (2000 ×g) at various time points. The temperature-dependent study was conducted by preincubating aliquots of SRBCs and crocodile serum at specific temperatures for 10 min. The samples and SRBCs were then mixed and incubated for 20 min at the respective temperatures.
The data in Fig. 1 show the concentration-dependent hemolysis of unsensitized SRBCs by serum from saltwater and freshwater crocodiles. Incubation of increasing concentrations of serum from both crocodilian species with SRBCs in vitro resulted in increased hemolytic activities. The calculated CH50 values were 473 and 451 mL for serum derived from the saltwater and freshwater crocodiles, respectively. The CH50 values derived from these data represent the concentrations of serum required to produce 50% of maximum hemolysis of 1% SRBCs in a 1 mL reaction volume. This value is used clinically to determine the relative complement activity of a serum sample. The data in Table 1 reveal the effects of heat, protease, EDTA, phosphate, citrate, and methylamine on the hemolysis of SRBCs by crocodile serum. Mild heat treatment of serum (56 °C, 30 min) completely obliterated the SRBC hemolysis by serum from both crocodile species. In addition, treatment of the sera samples with 10 units of a protease derived from Streptomyces griseus (Sigma Chemical Co, St. Louis, MO, USA) for 30 min at 37 °C also reduced the hemolysis to baseline levels. Treatment of 110
Crocodylus porosus Crocodylus johnstoni
100
SRBC Hemolysis (% maximum)
Fresh 10% sheep red blood cells (v / v) were purchased from IMVS Veterinary Services Biological Products, South Australia. EDTA, MgCl2, CaCl2, CuCl2, Fe SO4, BaCl2, and Triton X-100 were purchased from Sigma Chemical Company (St. Louis, MO).
90 80 70 60 50 40 30 20 10 0 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Serum titer (mL) Fig. 1. Concentration-dependent hemolysis of SRBCs by saltwater and freshwater crocodile serum. SRBCs were incubated with different concentrations of sera from freshwater and saltwater crocodiles for 20 min. The samples were centrifuged and the optical densities of the supernatants were measured at 450 nm. Each sample was analyzed in triplicate and the results represent the means ± standard deviations.
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Table 1 Effects of heat, protease and EDTA treatment on the hemolysis of SRBCs by serum from the saltwater and freshwater crocodiles Hemolysis (% maximum)
None Heat-treated (56 °C, 30 min) Protease (10 U, 30 min, 37 °C) EDTA (10 mM) Sodium phosphate (30 mM) Potassium citrate (30 mM) Methylamine (20 mM)
Freshwater crocodile
Saltwater crocodile
98 ± 2 4±1 2±1 2±0 7±3 5±1 95 ± 4
101 ± 4 6±3 3±2 2±1 7±2 9±4 98 ± 4
Undiluted crocodile serum was heat-treated, or treated with protease, EDTA, phosphate, citrate, or methylamine, and then incubated with 1% SRBCs for 30 min at room temperature. The samples were then centrifuged at 2000 ×g and the optical densities of the supernatants were measured at 540 nm. The results represent the means ± standard deviations for three independent determinations.
Hemolysis (% maximum)
sera with divalent metal chelators Na2 EDTA (10 mM, pH 8.0), Na2HPO4 (30 mM), or K3 citrate (30 mM) for just 1 min at ambient temperature resulted in substantial reductions in the ability of the sera to disrupt the integrity of the SRBCs. However, treatment of the serum samples with 30 mM Na2HSO4 or KCl did not affect the hemolysis of SRBCs by the crocodile sera (data not shown), eliminating the possibility that the observed inhibition of the SRBC hemolysis by the chelators was due to elevated ionic strength of the solution. The kinetic analyses of the hemolysis of SRBCs by serum from the saltwater and freshwater crocodiles are depicted in Fig. 2. The sera showed little activity at 5 min exposure to SRBCs. However, the saltwater and freshwater crocodile sera exhibited 56.3% and 65.6% of maximal activity, respectively, after 10 min. The hemolytic response was maximal (98.2%) for the saltwater crocodile and near maximal (88.2%) for the freshwater crocodile at 15 min. Fig. 3 displays the concentration-dependent effects of EDTA on the hemolysis of SRBCs by crocodile serum. SRBC hemolysis was not affected in the presence of 1 mM EDTA. However, the hemolytic activities for serum from both crocodile species
Hemolysis (% maximum)
Serum treatment
Crocodylus porosus Crocodylus johnstoni
100
80
60
40
20
0 0
1
2
3
4
5
6
7
8
9
10
[EDTA] (mM) Fig. 3. Concentration-dependent effects of EDTA on the hemolysis of SRBCs by sera from freshwater and saltwater crocodiles. SRBCs were incubated with sera from freshwater and saltwater crocodiles in the presence of varying amounts of EDTA for 20 min. The samples were centrifuged and the optical densities of the supernatants were measured at 450 nm. Each sample was analyzed in triplicate and the results represent the means ± standard deviations.
were reduced to 77.6% and 15.4% in the presence of 2 mM EDTA, respectively. The ability of the sera from both crocodiles to hemolyze SRBCs was completely inhibited by 3 mM EDTA. The inhibition of the hemolytic activities of sera from both crocodiles could be completely reversed by the addition of either 30 mM Ca2+ or Mg2+ (Table 2). However, 30 mM Ba2+, Cu2+, or Fe2+ did not affect the inhibition of innate immune activity by serum from either crocodile species. The data in Fig. 4 illustrate the temperature-dependent effects of the hemolysis of SRBCs by serum from the two crocodiles. Peak serum complement activity was observed at 25–30 °C in the freshwater crocodile and 20–35 °C in the saltwater crocodile. However, the complement activity was reduced at temperatures below 20 °C. The hemolysis of SRBCs by the freshwater crocodile serum was reduced to 47.3%, 13.5%, and 9.2% of maximum at temperatures of 15, 10, and
100 Table 2 Effects of different divalent metal ions on EDTA-inhibited hemolysis of SRBCs by crocodile serum
80 60
Crocodylus porosus Crocodylus johnstoni
40 20 0 0
10
20
30
40
50
60
Time (min) Fig. 2. Kinetic analyses of serum complement activities of sera from saltwater and freshwater crocodiles. SRBCs were incubated with an equal volume of 25% sera from freshwater and saltwater crocodiles for various amounts of time. The samples were centrifuged and the optical densities of the supernatants were measured at 450 nm. Each sample was analyzed in triplicate and the results represent the means ± standard deviations.
Serum treatment
Hemolysis (% maximum) Freshwater crocodile
Saltwater crocodile
None 5 mM EDTA 5 mM EDTA + 10 5 mM EDTA + 10 5 mM EDTA + 10 5 mM EDTA + 10 5 mM EDTA + 10
102 ± 4 3±1 97 ± 4 99 ± 3 2±2 3±2 5±3
98 ± 6 2±1 100 ± 2 97 ± 2 0±0 2±1 2±1
mM Mg2+ mM Ca2+ mM Cu2+ mM Ba2+ mM Fe2+
Crocodile serum samples were treated with 5 mM EDTA, while other samples were treated with EDTA plus 10 mM Ca2+, Mg2+, Ba2+, Cu2+, or Fe2+. The samples were incubated with 1% SRBCs for 30 min at ambient temperature and then centrifuged at 2000 ×g and the optical densities of the supernatants were measured at 540 nm. The results represent the means ± standard deviations for three independent determinations.
SRBC Hemolysis (% maximum)
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Crocodylus porosus Crocodylus johnstoni
100 80 60 40 20 0
5
10
15
20
25
30
35
40
o
Temperature ( C) Fig. 4. Temperature-dependent hemolysis of SRBCs by sera from freshwater and saltwater crocodiles. SRBCs were incubated with sera from freshwater and saltwater crocodiles at different temperatures for 20 min. The samples were centrifuged and the optical densities of the supernatants were measured at 450 nm. Each sample was analyzed in triplicate and the results represent the means ± standard deviations.
5 °C, respectively. A parallel reduction of activity for serum from the saltwater crocodile was observed (Fig. 4). For the freshwater crocodile, activity was reduced slightly to 92.4% at 35 °C, and further reduced to 69.4% at 40 °C. The saltwater crocodile serum exhibited maximal activity (96.4%) at 35 °C, but was reduced to 74.9% at 40 °C. 6. Discussion Several studies have focused on the antimicrobial properties of serum from crocodilians. Shaharabany et al. (1999) showed that crude extracts from the tissues of the Nile crocodile (Crocodylus niloticus) exhibit antimicrobial properties. More recent studies in our laboratory showed that serum from the American alligator (Alligator mississippiensis) exhibits potent antibacterial (Merchant et al., 2003), amoebacidal (Merchant et al., 2004), and antiviral (Merchant et al., 2005c) properties. Further studies showed that the antimicrobial activities of alligator serum were due to the alternative serum complement pathway (Merchant et al., 2005a). Still more recent studies in our laboratory have demonstrated innate immunity in all 23 extant crocodilian species, and also showed that these activities follow distinct taxonomical crocodilian lineages (Merchant et al., 2006). The ability of serum to hemolyze SRBCs has been linked to serum complement activity in several reptilian species (Dias de Silva et al., 1984; Mansour et al., 1980). We have employed this assay to characterize the serum complement activity of the saltwater (C. porosus) and freshwater (C. johnstoni) crocodiles. The data displayed in Fig. 1 reveal that the lysis of SRBCs by serum from the saltwater and freshwater crocodiles occurs in a concentration-dependent manner. The incubation of serum derived from both saltwater and freshwater crocodiles with 1% SRBCs resulted in strong hemolytic activity, as determined by an increase in optical density at 540 nm due to the release of hemoglobin by the disrupted erythrocytes. The hemolytic activities are similar to each other, and also comparable to that of the American alligator (Merchant et al., 2005a).
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The results from mechanistic experiments displayed in Table 2 show that the ability of the sera samples' ability to disrupt SRBCs is severely limited by modest heat treatment (56 °C, 30 min). These conditions are classical complement inactivation conditions. Human complement contains heatlabile proteins that are thermally inactivated under these conditions. The fact that the crocodilian hemolytic activities were obliterated by mild heat treatment eliminates the possibility that these activities are due to the presence of heat-stable antimicrobial peptides. In addition, the inhibition of hemolytic activities by protease indicates that the molecules responsible for the SRBC hemolysis are proteinaceous in nature. The data in Table 2 also illustrate that the hemolytic activities are severely limited in the presence of 10 mM EDTA. It is long been known that human serum requires the presence of divalent metal ions, specifically Mg2+ and Ca2+, to hemolyze SRBCs in vitro (Levine et al., 1953a; Levine et al., 1953b). Other studies have shown that divalent metal ions are required for alternative pathway-mediated complement activity, but not for classical pathway activation (Vogt et al., 1977). Furthermore, the addition of methylamine had no effect on the hemolysis of SRBCs by sera from either crocodilian species (Table 2). It is known that primary amines inhibit activation of the classical complement cascade by interacting with the thioester in the C4 protein, which is not involved in the alternative pathway (Gorski and Howard, 1980). The fact that the hemolysis of unsensitized SRBCs by crocodile serum is heat sensitive, requires divalent metal ions, and is unaffected by methylamine strongly suggests that the alternative pathway of complement activation is responsible for the hemolytic action. In addition, these results suggest similarities in the molecular mechanisms of complement activation between these crocodilians and that of humans. The kinetic profiles of serum from the saltwater and freshwater crocodiles are very similar (Fig. 2). These data show that the hemolysis of SRBCs occurs very rapidly when exposed to the serum of both crocodilian species. The maximum activities are reached within 15–20 min, which is remarkably similar to the kinetics of complement-mediated antibacterial activity of human serum (Wright and Levine, 1981). In addition, these kinetic curves are extremely similar to that previously reported for the American alligator (Merchant et al., 2005a). The hemolytic activities of serum from both crocodile species require divalent metals ions for activity (Fig. 3, Tables 1 and 2). These SRBC hemolytic activities are completely inhibited by EDTA concentrations as low as 3 mM. The hemolytic activity can also be inhibited by other chelators of divalent metal ions, such as phosphate, and citrate. In addition, the inhibition of SRBC hemolysis by EDTA can be restored by the addition of excess (30 mM) Ca2+ or Mg2+. These data are in contrast to the need of human serum for both Ca2+ and Mg2+ (Levine et al., 1953a,b), but is consistent with the fact that alligator serum requires either Ca2+ or Mg2+ (Merchant et al., 2005a) for complement activity. However, the crocodile serum complement activation does not show a complete lack of specificity, as addition of 30 mM Ba2+, Cu2+, or Fe2+ did not restore the EDTA-inhibited hemolysis of SRBCs. These data
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indicate that the mechanisms of complement activities for diverse crocodilian species are similar. Since crocodiles are ectothermic vertebrates whose body temperatures exhibit marked season and diurnal variations, we investigated the effectiveness of the serum complement system of the saltwater and freshwater crocodiles at a broad spectrum of temperatures in vitro. Grigg et al. (1998) described the thermal regulation of free-ranging saltwater crocodiles (C. porosus) throughout the year and reported body temperatures ranging from 20 to 35 °C. These animals maintained body temperatures that were near or at the optimal temperatures for humoral immune activity. In another study, Seebacher and Grigg (1997) measured body temperature of wild freshwater crocodiles (C. johnstoni) and found that their body temperatures ranged from 16 to 36 °C. Our data (Fig. 4) show that the serum complement activity of the freshwater crocodile falls to 85% at 20 °C and is further reduced to 47.3% at 15 °C, indicating that the freshwater crocodiles may be somewhat immunocompromised during the winter months when their body temperatures fall below 20 °C. However, this potentially depressed innate immunity may not be biologically relevant as the animals are not as active, and microbial growth is not as abundant during the winter months. The data presented in this study strongly suggest that the freshwater and saltwater crocodiles exhibit alternative serum complement activities. However, this study does rule out the presence of a classical complement cascade in these crocodilians. It is interesting to note that previous studies have shown that the spectrum of antibacterial activity of serum from A. mississippiensis is substantially different from than that of both C. johnstoni and C. porosus (Merchant et al., 2005b). However, the present study reveals striking similarities in the serum complement mechanisms of these crocodilians. We propose that, although the mechanisms of complement activation may be similar, the proteins that act to stimulate the complement cascade are activated by different microorganisms. Alternatively, the proteins that form the membrane attack complex may show different specificities for different types of microbial membrane compositions. Acknowledgements The authors wish to thank Tommy Nichols, Service Ranger with the Northern Territory Parks and Wildlife Service, and Garry Lindner, Park Ranger at Kakadu National Park, for their help in the capture of wild saltwater crocodiles used in this study. We also wish to acknowledge Ms. Erin O'Brien for her help in the capture and bleeding of crocodiles. References Al-Sharif, W.Z., Sunyer, J.O., Lambris, J.D., Smith, L.C., 1998. Sea urchin coelomocytes specifically express a homologue of the complement component C3. J. Immunol. 160, 2983–2997. Boshra, H., Gelman, A.E., Sunyer, J.O., 2004. Structural and functional characterization of complement C4 and C1s-like molecules in teleost fish: insights into the evolution of classical and alternative pathways. J. Immunol. 173, 349–359.
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