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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
Figure 1
ABSTRACT
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 10 4
(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.
REFERENCES
Cadizinska, 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.
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Figure 4 - Highly-efficient phagocytic cells
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4
0
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pH 5.5/ Thioglycollate
pH 7.0/ Thioglycollate
pH 5.5/ Buffer
pH 7.0/ Buffer
8.00E+05
WBC / ml
6
pH 7.0
Figure 2
6.00E+05
4.00E+05
2.00E+05
1
Figure 2. The thioglycollate-induced inflammatory response is abolished to background
levels after six-day acid stress.
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DA
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).
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.
Figure 3 - Non-phagocytic cells
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
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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 acidexposed 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.
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DISCUSSION
0.00E+00
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8
2
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.
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10
20
pH 5.5
Number of cells
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, 25 o C, 12/12 daynight 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.
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0
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
Number of cells
% mortality
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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.
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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. Intraperitoneal 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.
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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.
ACKNOWLEDGMENTS
This work was supported in part by Widener University Provost’s Grant and the Eppley
Foundation.