Identification of alternative pathway serum complement activity in the blood of the American alligator (Alligator mississippiensis)

https://doi.org/10.1016/j.cbpc.2005.03.009Get rights and content

Abstract

Incubation of different dilutions of alligator serum with sheep red blood cells (SRBCs) that had not been sensitized with antibodies resulted in concentration-dependent hemolytic activity. This hemolytic activity was not affected by the presence of ammonium hydroxide and methylamine, known inactivators of the classical complement cascade. However, the hemolytic activities were inhibited by EDTA and salicylaldoxime, indicating that the alternate pathway is primarily responsible for these activities. Immunofixation of electrophoretically-resolved alligator serum proteins with antihuman C3 polyclonal antibodies resulted in detection of a protein antigenically similar to human C3 in alligator serum. SDS-PAGE, followed by Western blot analysis, revealed the presence of two alligator serum proteins with nearly identical molecular weights as human C3α and C3β. SRBC hemolysis and antibacterial activity by alligator serum was significantly reduced in the presence of antihuman C3 antibodies. The hemolytic effect of alligator serum was shown to occur rapidly, with significant activity within 5 min and maximal activity occurring at 15 min. SRBC hemolysis was also temperature-dependent, with reduced activity below 15 °C and above 30 °C. These data suggest that the antibiotic properties of alligator serum are partially due to the presence of a complement-facilitated humoral immune response analogous to that described in mammalian systems.

Introduction

Eukaryotic organisms must continuously defend themselves against infiltration and colonization by microorganisms. The humoral immune response comprises a significant portion of the immune system and acts as an initial defense mechanism against microbial growth shortly after infection occurs. The serum complement system, an important component of the humoral immune response, is composed of 25–30 proteins that can be activated to initiate the inflammatory response, recruit leukocytes to the site of infection, mediate opsinization of particulate foreign materials and 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). Because of the immunological importance of the serum complement system, a deficiency or mutation in any complement protein is almost always associated with multiple recurring infections (Morgan and Walport, 1991, Pascual and French, 1995).

Complement proteins are expressed and circulated as inactive precursor proteins that can be activated in a very precise and highly coordinated fashion (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 results in the modulation of immune function. The serum complement system has been fully characterized in humans as all of the proteins have been purified to homogeneity, their functions in each pathway identified, and their genes isolated (Campbell et al., 1988). Although several studies have reported the presence of complement components in a variety of reptiles (Koppenheffer, 1986), the serum complement system is not well characterized in reptilian systems. The results from this study strongly suggest a potent complement system exists in the serum of the American alligator.

Section snippets

Chemicals and biochemicals

Nutrient broth (cat. # G-3055-50) and nutrient agar (cat. # G-3056-40) were purchased from ISC Bioexpress (Kaysville, UT, USA). Lyophilized ATCC cultures (Escherichia coli, ATCC 25922) were purchased from Remel (Lenexa, KS, USA). An ATCC-registered strain of protease derived from Streptomyces griseus was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Lyophilized goat antihuman C3 polyclonal antibodies (F(ab)2) were obtained from ICN Biomedicals, Inc. (Irvine, CA, USA, cat.

Results

Sheep red blood cell (SRBC) hemolytic assays have been used for years to assess the functionality of serum complement in the clinical laboratory (Mayer, 1967). The data listed in Table 1 display the ability of alligator serum to disrupt SRBCs in vitro. Incubation of 1% SRBCs (v / v) with alligator serum for 30 min at room temperature resulted in a 94 ± 4% increase (relative to veronal buffer controls) in optical density at 525 nm. Pretreatment of alligator serum samples for 30 min at 56 °C, prior

Discussion

Several investigators have reported the presence of an active serum complement system in a variety of reptilian species (Koppenheffer, 1987, Sunyer and Lambris, 1998, Sunyer et al., 1998). For instance, Kuo et al. (2000) described the complement-mediated killing of the Lyme disease spirochete (Borrelia burgdorferi) in the western fence lizard. Koppenheffer (1986) has demonstrated the presence of both classical and alternative pathways in turtle serum. Other studies have focused on the

Acknowledgements

The authors wish to acknowledge the contributions of Dr. William Taylor and Mrs. Jan Prudomme of the Department of Biological and Environmental Sciences at McNeese State University. In addition, we wish to thank Phillip “Scooter” Trosclair, III, with the Louisiana Department of Wildlife and Fisheries, for technical assistance. This research project was supported by the Shearman Research Initiative Fund, Pfizer Global Research, and the J. Bennett Johnston Research Foundation.

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