Acid Stress Suppresses The Thioglycollate-Stimulated Inflammatory Response of Adult Rana pipiens

Matthew J. Colombo, Jaime L. Andrews, Emily J. Piddington, Muthuramanan Rameswaran, Elizabeth Fiorini, Itzick Vatnick, Marc A. Brodkin

Department of Biology, Widener University, Chester, PA 19013

ABSTRACT

Acidic environments function as a stressor for amphibians. Previous work in our lab showed that adult ranid frogs exhibit different levels of tolerance to mild acid conditions (pH 5.5). Adult Rana pipiens are more susceptible than other ranids and experienced 72% mortality after ten-day exposure to pH 5.5. We also demonstrated that acid exposure increases the permeability of the gut to the endogenous bacterial flora and results in a systemic infection. Inflammation is a natural protective defense against microbial infection, characterized by an influx of leukocytes to the site of infection. Thioglycollate medium is used experimentally to stimulate an inflammatory response in several vertebrates. Intra-peritoneal injection of thioglycollate causes a peritoneal exudate containing leukocytes. Our study shows that acid exposure (pH 5.5) greatly suppresses the inflammatory response in thioglycollate-induced R. pipiens. The number of leukocytes as well as their phagocytic efficiency was significantly reduced in acid-exposed thioglycollate-stimulated adult R. pipiens as compared to their controls. Frogs induced by thioglycollate injection and exposed to pH 5.5 had a 50% increase in cells that did not exhibit phagocytosis and a four-fold reduction in the number of highly efficient phagocytic cells.

INTRODUCTION

In the late 1970s, amphibians began to disappear in various locations around the globe. The causes of this global decline are not yet known, however pathogens seems to be involved in all instances of reported extinctions. In the Northeastern United States, R. pipiens enter hibernation in late October to early November and emerge from hibernation in early spring. Soon after their emergence, they mate and lay their eggs in late spring to early summer. These frogs emerge from hibernation with compromised immune systems (Cooper et al., 1992) to mate in surrounding streams and ponds. It is during this post-hibernation period when streams, ponds, and lakes in the northeastern United States suffer from episodic acidification due to snowmelt and rain events (Driscoll et al., 2001). Therefore, acidification of aquatic ecosystems may contribute to the declining populations of R. pipiens.

The effect of acid exposure on the inflammatory response was examined. Inflammation is a natural protective defense against microbial infection, characterized by an influx of leukocytes to the site of infection. Thioglycollate has been shown to cause this response in several other animal models (Menaszek et al., 1999; Melnicoff et al., 1989; Cadzinska et al., 1997). Using this experimental technique, we developed an assay to probe the mechanism of the effect of acid exposure on the inflammatory response of adult Rana pipiens. To further characterize this response, FITC-labeled 1.0 micron polystyrene beads were infused into the thioglycollate inoculation to serve as surrogates for bacteria. These allowed for the measurement of the phagocytic efficiency of activated leukocytes. METHODS

Experimental Groups and Conditions. Adult, post-hibernation Rana pipiens with a body weight of 15-30 grams were were randomly allocated into four groups of eight frogs each: thioglycollate-inoculated frogs at pH 5.5 and at pH 7.0, and buffer-inoculated frogs at pH 5.5 and at pH 7.0.  A sterile citric acid/sodium citrate buffer was used and changed daily. All frogs were placed individually in an environmental chamber (6 days, 25o C, 12/12 day-night cycle). On Day 5, each frog was injected intra-peritoneally with 2 ml of the appropriate solution. On Day 6, the frogs were sacrificed by ether asphyxiation, and peritoneal lavages were performed with 10 ml of an isotonic amphibian ringer solution.  

White Blood Cell Count. Cell counts were performed by pipetting the lavage fluid onto a hemacytometer and by multiplying the average number of leukocytes per 1 mm field by 104 (n = 31). Data for total leukocyte counts were analyzed by ANOVA and a t-test.

Fluorescent Beads. FITC-labeled beads were diluted into the inoculation medium to a final concentration of 2.5*107 beads per ml. The number of beads phagocytosed by each white blood cell was counted using a fluorescence microscope. In this experimental group, n = 24, one hundred leukocytes were counted per frog. Cells were placed into several categories: cells with 0 beads (classified as non-phagocytic cells, figure 3), 1-3 beads, 4-6 beads, 7-9 beads, and cells with >10 beads. Cells with >10 beads were classified as “highly efficient” phagocytic leukocytes (figure 4). Data for phagocytic activity were analyzed with a Chi square analysis and differences in phagocytic efficiency of non-phagocytic and highly efficient cells was discerned using a Mann-Whitney U-test.

Figure 3 - Non-phagocytic cells

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Figure 4 - Highly-efficient phagocytic cells

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Figure 4. Acid-stressed frogs with thioglycollate stimulation had significantly lower numbers of highly efficient phagocytic leukocytes compared to thioglycollate-stimulated frogs under neutral conditions. Moreover, thioglycollate stimulation does activate phagocytic activity, as there were significantly greater numbers of highly phagocytic leukocytes in thioglycollate-injected frogs at neutral conditions compared to control frogs injected with saline.

RESULTS

Thioglycollate-inoculated Rana pipiens exposed to a neutral pH (7.0) had 5.09E+05 + 1.8E+05 white blood cells per ml of lavage, a six-fold increase (figure 2) in the number of leukocytes as compared to 7.70E+04 + 2.9E+04 white blood cells per ml in acid-exposed frogs inoculated with thioglycollate, p < 0.00136 (F=6.89, df= 3). The bead count, demonstrating phagocytic efficiency, showed that when an inflammatory response was initiated by thioglycollate, acid-exposed frogs had 79.17% + 8.86 non-efficient (0 beads) leukocytes compared to 52.33% + 4.60, (U=6, p<0.05) in thioglycollate-injected frogs held at a neutral pH (figure 3). There was also a significant difference in the highly efficient phagocytic cells (figure 4). Thioglycollate-injected frogs exposed to pH 7.0 had 9.67% + 2.28 highly efficient phagocytic cells, while frogs injected with thioglycollate held at an acidic pH had 2.33% + 1.13 highly efficient cells.(U=33.5, p<0.01).

DISCUSSION

Vatnick et al. (1999) demonstrated that ten-day acid exposure results in 72% mortality among adult Rana pipiens (Figure 1). Moreover, cold-exposure followed by acid exposure resulted in 100% mortality during a ten day experiment (Vatnick et al. 1999). Frogs in the northeastern United States emerge from hibernation into ponds and streams that undergo seasonal acidification. At this time they have a compromised immune system due to prolonged cold exposure (Cooper et al., 1992). Acid exposure in combination with a suppressed immune system leave frogs vulnerable to microbial infection that may ultimately result in death.

We have demonstrated that an acidic environment weakens the frogs’ inflammatory response to thioglycollate resulting in decreased recruitment of peritoneal leukocytes to the inflammatory site, as well as diminishes their ability to phagocytose antigen.

Decreased phagocytic efficiency is exhibited by the shift in function of peritoneal exudate cells from highly efficient cells in frogs subjected to normal conditions to less efficient cells in frogs subjected to acidic conditions (Figs. 3, 4). This may be caused either by a decreased efficiency of phagocytic cells (i.e. macrophages, neutrophils) or by a reduction in the number of phagocytic cells. We are currently working on differential white blood cell counts in the peritoneal exudates of the frogs in these experiments in order to answer this question. The attenuation of the inflammatory response may compromise the frogs’ ability to fight off infection

We have recently shown that acid-stressed frogs may suffer from a systemic infection due to transit of endogenous gut bacteria across the intestinal epithelium into the vascular system (Brodkin et al., in review). These endogenous bacteria colonized the spleens of acid-exposed frogs. This data suggest that a compromised inflammatory response, in conjunction with an increased ability of gut bacteria to transit the intestinal epithelium, due to acid exposure may provide a partial explanation for the recent decline in Rana pipiens populations in the northeast United States.

Figure 2. The thioglycollate-induced inflammatory response is abolished to background levels after six-day acid stress.

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Figure 3. Acid-stressed frogs with thioglycollate stimulation had significantly greater numbers of non-phagocytic leukocytes compared to thioglycollate-stimulated frogs under neutral conditions.

Figure 1

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ACKNOWLEDGMENTS

This work was supported in part by Widener University Provost’s Grant and the Eppley Foundation.

REFERENCESCadizinska, M., Jozefowski, S., Gigaj, J., and Plytycz, B. 1977. Morphine modulation of thioglycollate elicited peritoneal inflammation in Goldfish. Cariasius auratus. Archivum Immunologiac Experimentalis 45:321-327.

Cooper EL, Wright RK, Klempau AE, Smith CT. 1992. Hibernation alters a frog’s immune system. Cryobiology, 29:616-631.

Driscoll CT, Lawrence GB, Bulgar AG, Butler TJ, Cronan CS, Eagar C, Lambert KF, Likens GE, Stoddart JL, Weathers KC. 2001. Acidic deposition in the northeastern United States: sources and inputs, ecosystem effects, and management strategies. Bioscience 51:180-198.

Melnicoff, M., Horan, P., and Morahan, P. 1989. Kinetics of changes in peritoneal cell populations following acute inflammation. Cellular Immunology 118: 178-91.

 Menaszek, E.Miskiewicz, K. and Plytycz, B. 1999. Comparative studies on experimental inflammations in anuran amphibians. Central-European Journal of Immunology 24:211-217.

Vatnick I, Brodkin MA, Simon MP, Grant BW, Conte CR, Gleave M, Myers R, Sadoff MM. 1999. The effects of exposure to mild acidic conditions on adult frogs (Rana pipiens and Rana clamitans): Mortality rates and pH preferences. J Herp 33:370-374. 

 

Figure 1. Ten-day acid exposure (pH = 5.5) results in 72%mortality in adult Rana pipiens, compared to 3.5% in frogs at pH = 7.0.