Production of superoxide ions by leukocytes of the American alligator (Alligator mississippiensis)

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Abstract

This study was conducted to characterize the production of superoxide ions by leukocytes in whole blood of the American alligator (Alligator mississippiensis). We used WST-1, a tetrazolium salt which can be reduced to a water-soluble formazan compound with high molar absorptivity at 438 nm, to probe the production of superoxide by alligator leukocytes. Incubation of alligator whole blood with WST-1 resulted in a time- and concentration-dependent increase in absorbance of the plasma at 438 nm. The reduction of WST-1 was inhibited in a concentration-dependent manner by superoxide dismutase, an enzyme that catalyzes the reduction of superoxide to peroxide, confirming that the reduction of WST-1 was due to the presence of superoxide. Treatment of whole blood with nitrotetrazolium blue (NBT) resulted in the staining of heterophils and monocytes, enforcing the idea that that the production of superoxide is due to the presence of leukocytes, and not other blood cell components. It is interesting to note that the production of superoxide by the alligator leukocytes required no external stimulation while human leukocytes must be stimulated with an immunological challenge before producing superoxide. This is the first report of the production of superoxide as an innate immune mechanism in crocodilians.

Introduction

Expression of NADPH oxidase, an enzyme that generates superoxide radical anion, plays a critical role in host defense (Bayne, 2003). Production of superoxide is the initial reaction in a complex pathway referred to as “respiratory burst”. The final reactive oxygen intermediates, generated as a result of the initiation of the respiratory burst pathway, are largely dictated by the phyla and tissue in which the reactions occur (Bayne, 2003). The activation of NADPH oxidase is a complex process, requiring at least four protein components (Chanock et al., 1994).

The enzymatic conversion of molecular oxygen to several toxic reactive oxygen species, including hydrogen peroxide and superoxide anion, comprises a critical innate host defense mechanism (Babior, 1978). Superoxide anion is an unstable radical that can be measured by techniques such as electron spin resonance (Kasazaki et al., 2003) and fluorescence spectroscopy (Remans et al., 2005). However, recent advances have made it easy to measure superoxide by spectrophotometric means (Peskin and Winterbourn, 2000). Therefore, WST-1 can be used as an effective radical trap to measure the accumulative production of superoxide in biological systems. WST-1 is oxidized from a tetrazolium compound to a soluble formazan product, with a high molar extinction coefficient, by superoxide, and thus can be easily measured spectrophotometrically.

Leukocytic production of superoxide is an ancient innate immune response observed in birds (Rodriguez et al., 2001, Terron et al., 2004), reptiles (Mccoll and Daniels, 1988), and teleost fish (Panigrahi et al., 2007, Selvaraj et al., 2006), as well as in invertebrates such as insects (Koike et al., 1991, Bergin et al., 2005). To date, no report exists on the production of superoxide by crocodilians. Previous studies in our laboratory have shown that it is exceedingly difficult to isolate and maintain healthy leukocyte cultures from crocodilian blood. Therefore, we adopted an assay for measuring superoxide activity in the whole blood of the alligator. This study was conducted to characterize the production of superoxide by leukocytes of the American alligator (Alligator mississippiensis).

Section snippets

Chemicals and biochemicals

WST-1 was purchased from Dojindo Molecular Technologies, Inc (Gaithersburg, MD, USA). Phorbol myristate acetate (PMA), ionomycin, and superoxide dismutase were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).

Treatment of animals

Juvenile alligators (135–152 cm) were housed in outdoor 10 m × 10 m fenced pens each with approximately 1.25 m2 of water surface area. They were fed a dried pelletized food (Burris Mill and Feed, Franklinton, LA, USA) formulation ad libitum. Water tanks were cleaned five

Results

Fig. 1 shows the concentration-dependent production of superoxide by whole blood of the American alligator. Incubation of alligator whole blood with 100 mM WST-1 for 2 h at 30 °C resulted in 77.4 ±3.1 μM superoxide. Dilution of the whole blood with PBS to 1:2, 1:4, and 1:8 resulted in 41.8 ± 2.2, 24.8 ± 1.4, and 15.6 ± 2.2 μM superoxide, respectively. Linear regression of the data resulted in a correlation coefficient of greater than 0.999. In addition, the y-intercept of the line was 3.3 μM,

Discussion

The production of superoxide is an ancient innate defense mechanism. A wide variety of invertebrates have been shown to have the capacity for cell-mediated generation of superoxide. For instance, Bergin et al. (2005) demonstrated that hemocytes from the greater wax moth (Galleria mellonella) produce superoxide. In addition, Bergin and coworkers (2005) demonstrated the presence of several insect proteins that were shown to be homologous to NADPH oxidase system proteins in humans. Other studies

Acknowledgement

This project was supported by an undergraduate research grant from Merck/AAAS awarded to M. Merchant.

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