Effects of fenbendazole on the murine humoral immune system

Abstract

Pinworms are highly contagious parasites that have been effectively treated in laboratory rodents with fenbendazole (FBZ). Whether FBZ has any detrimental side effects that may compromise experimental results is unknown. Here we asked whether the immune systems from young and aged mice are altered under FBZ treatment. We compared control and FBZ-treated groups of young (age, 2 to 4 mo) and old (age, 22 to 24 mo) BALB/cN mice. The treated mice received a total of 4 wk (alternating-week treatment regimen) of FBZ-medicated feed. Spleen and bone marrow were collected for immunologic assays, and heart, stomach, intestines, kidneys, and liver were evaluated by histopathology. Our results indicate that FBZ treatment has significant effects on the immune systems of mice; these effects are greater in aged mice. FBZ treatment adversely affected mRNA and protein expression of E2A (a transcription factor crucial for B lymphocytes) in activated precursor B lymphocytes obtained from the bone marrow of young and old mice. These effects were reversed by 6 wk on regular feed after the end of treatment. Activated B lymphocytes from the spleens of young and old mice showed decreased function (cell proliferation, E2A mRNA and protein expression) through the last time point of FBZ treatment but recovered by 2 to 4 wk after treatment. Our findings suggest that FBZ treatment may alter sensitive immune and molecular measures as presented here, and postponing the experimental use of mice until at least 6 wk after treatment should be considered.

Figure 1.

Experimental scheme for immunologic testing. T1 is after completion of the first week of treatment; T2 is after completion of the second week of treatment; T3 is after completion of the third week of treatment; T4 is after completion of the fourth week of treatment; T5, T6, and T7 are at 2, 4, and 6 wk after the fourth week of FBZ treatment. EMSA, electrophoretic mobility shift assay; LPS, lipopolysacharide.

Figure 2.

FBZ has no effect on proB cell expansion in both young and old mice. (A) ProB cells from untreated (white bars) or FBZ-treated (black bars) young mice were harvested, counted, and stained for membrane expression of B220 and CD43 both ex vivo and after IL7 stimulation for 7 d. Fold expansion of proB cells was calculated by dividing the percentage of proB cells after IL7 culture by the percentage found ex vivo. (B) ProB cells from untreated (white bars) or FBZ-treated (black bars) old mice were harvested, counted, and stained for membrane expression of B220, and CD43 both ex-vivo and after IL7 stimulation for 7 d. Fold expansion of proB cells was calculated as described for panel A. There were no significant differences between treated and untreated groups.

Figure 3.

FBZ affects E2A mRNA expression proB cells from in old but not young mice. (A) ProB cells from untreated (white bars) or FBZ-treated (black bars) young mice were stimulated with IL7 in culture for 7 d, harvested, counted, RNA-extracted, and used in RT-PCR assays. E2A:GAPDH mRNA ratios for young untreated mice were normalized to 100% (Y axis). (B) ProB cells from old mice untreated (white bars) or FBZ treated (black bars) were treated as described in panel A. Significant differences between untreated and treated groups were calculated by using 2-tailed Student t tests.

Figure 4.

FBZ decreases E47 protein expression in expanded proB cells from both young and old mice. E47:control (Ubc9) values for untreated young and old mice were normalized individually to 100%. (A) ProB cells from untreated (white bars) or FBZ-treated (black bars) young mice were stimulated with IL7 in culture for 7 d, harvested, counted, and used to produce nuclear extracts for Western blots. (B) ProB cells from untreated (white bars) or FBZ-treated (black bars) old mice were treated as described in panel A. Significant differences between untreated and treated groups were calculated by using 2-tailed Student t tests; there were no differences between any groups at T1 through T3 (data not shown).

Figure 5.

FBZ treatment decreases proliferation in both young and old splenic B cells only at T4. (A) Splenic B cells (10⁶ cells/mL) from untreated (white bars) or FBZ-treated (black bars) young mice were cultured either in the absence or presence of LPS (10 µg/mL) for 48 h. Results are expressed as stimulation indices (SI) of the untreated groups normalized to 100%. The SI is the counts per minute (cpm; mean ± SE) from triplicate cultures in the presence of stimulus divided by that from triplicate cultures in the absence of stimulus. (B) Splenic B cells from untreated (white bars) or FBZ-treated (black bars) old mice were processed as described in panel A. Significant differences between untreated and treated groups were calculated by using 2-tailed Student t tests.

Figure 6.

FBZ decreases DNA binding by E47 only in activated splenic B cells from old mice. (A) Splenic B cells (10⁶ cells/ml) from untreated (white bars) or FBZ-treated (black bars) young mice were cultured in the presence of LPS (10 μg/mL) for 48 h. Nuclear extracts from equal numbers of cultured B cells were prepared and run in EMSA (10 μg per lane). Results for the untreated groups were normalized to 100% as compared with a nonspecific band not supershifted by the antibody. (B) Splenic B cells from untreated (white bars) or FBZ-treated (black bars) old mice were treated as described in panel A. Significant differences between untreated and treated groups were calculated by using 2-tailed Student t tests.

Figure 7.

FBZ particularly decreases E2A mRNA levels in activated splenic B cells from old mice. (A) Splenic B cells (10⁶ cells/mL) from untreated (white bars) or FBZ-treated (black bars) young mice were cultured in the presence of LPS (10 μg/mL) for 24 h. After this time, mRNA was extracted from equal numbers of cultured B cells and RT-PCR reactions performed. E2A:GAPDH mRNA ratios for young untreated mice were normalized to 100%. (B) Splenic B cells from untreated (white bars) or FBZ-treated (black bars) were treated as described in panel A. Significant differences between untreated and treated groups were calculated by using 2-tailed Student t tests.