Analysis of the role of Bphs/Hrh1 in the genetic control of responsiveness to pertussis toxin

Abstract

In vivo intoxication with Bordetella pertussis toxin (PTX) elicits a variety of physiological responses including a marked leukocytosis, disruption of glucose regulation, adjuvant activity, alterations in vascular function, hypersensitivity to vasoactive agents, and death. We recently identified Bphs, the locus controlling PTX-induced hypersensitivity to the vasoactive amine histamine, as the histamine H(1) receptor (Hrh1). In this study Bphs congenic mice and mice with a disrupted Hrh1 gene were used to examine the role of Bphs/Hrh1 in the genetic control of susceptibility to a number of phenotypes elicited following in vivo intoxication. We report that the contribution of Bphs/Hrh1 to the overall genetic control of responsiveness to PTX is restricted to susceptibility to histamine hypersensitivity and enhancement of antigen-specific delayed-type hypersensitivity responses. Furthermore, the genetic contribution of Bphs/Hrh1 to vasoactive amine sensitization is specific for histamine, since hypersensitivity to serotonin was unaffected by Bphs/Hrh1. Bphs/Hrh1 also did not significantly influence susceptibility to the lethal effects, the leukocytosis response, disruption of glucose regulation, and histamine-independent increases in vascular permeability associated with in vivo intoxication. Nevertheless, significant interstrain differences in susceptibility to the lethal effects of PTX and leukocytosis response were observed. These results indicate that the phenotypic variation in responsiveness to PTX reflects the genetic control of distinct intermediate phenotypes rather than allelic variation in genes controlling overall susceptibility to intoxication.

FIG. 1.

Mortality of SJL/J (□), C3H/HeJ (▴), and C3H.SJL-Bphs^sD (▪) mice injected i.v. with 2.5 μg of PTX (List Biological Laboratories, Inc.) in 0.025 M Tris buffer containing 0.5 M NaCl and 0.017% Triton X-100, pH 7.6. Five mice were injected per test group. Mice were monitored daily for viability, and the data are expressed as the cumulative percent mortality versus the day postinjection.

FIG. 2.

Leukocytosis response in PTX-treated SJL/J, C3H/HeJ, C3H.SJL-Bphs^sD, C57BL/6J, and B6.129P-Hrh1^tm1Wat mice. Mice were injected with carrier (open bars) or 200.0 ng of PTX (solid bars) on day 0. Three days later the animals were killed, blood was collected in EDTA, and total WBC counts were determined. Units are expressed as 10⁻³/μl of blood ± standard deviations. Due to the unequal variances observed for the WBC counts among the 10 groups, a natural logarithm transformation was used to stabilize the variances. The two-way ANOVA indicated that there were significant differences between mouse strains (P < 0.0001) and between PTX- and carrier-treated mice (P < 0.0001). Multiple comparisons indicated that the WBC count for the SJL/J strain was higher than those for C3H/HeJ and C3H.SJL-Bphs^sD mice. Similarly, both C57BL/6J and B6.129P-Hrh1^tm1Wat mice also had higher counts than did C3H/HeJ and C3H.SJL-Bphs^sD mice. All other paired comparisons were not significant.

FIG. 3.

PTX-induced vascular permeability changes in striated thigh muscle (A) and brain (B) of SJL/J, C3H/HeJ, and C3H.SJL-Bphs^sD mice. Mice were injected i.v. with either carrier (open bars) or 200.0 ng of PTX (solid bars) on day 0. Three days later 0.5 ml (1.0 μCi) of ¹²⁵I-BSA in PBS at a concentration of approximately 2 mg/ml was administered by i.v. injection, and tissues were extracted 1 h later. Mean permeability indices ± standard deviations were determined by dividing the ¹²⁵I-BSA counts per minute per gram of tissue by the ¹²⁵I-BSA counts per minute per milliliter of blood (n = 5). There was an overall difference in permeability between striated smooth muscle and brain (P < 0.0001) with the muscle values being higher than the brain values. There were also differences between strains (P = 0.0013) and between PTX- and carrier-treated mice (P < 0.0001). Significant interaction with PTX or carrier treatment (P = 0.0394) was also seen among the strains. In particular, SJL/J mice showed the largest difference between PTX and carrier treatment while C3H/HeJ and C3H.SJL-Bphs^sD mice showed smaller and comparable differences.

FIG. 4.

Bphs/Hrh1 controls enhancement of DTH responses elicited by PTX to both foreign antigen (A) and self antigen MOG 35-55 (B). C3H/HeJ and C3H.SJL-Bphs^sD mice were sensitized with OVA while C57BL/6J and B6.129P-Hrh1^tm1Wat mice were injected with MOG 35-55 emulsified in CFA. Immediately thereafter, each animal received PTX or carrier by i.v. injection. Seven days later the average thickness of the right pinna was determined by taking three measurements with a spring-loaded micrometer. Subsequently, each pinna was injected with 10 μl of physiological saline containing 1.0 mg of OVA or MOG 35-55/ml. Ear thickness measurements were taken at various times postchallenge, and the corrected average thickness was determined. Data are plotted as micrometers ± standard deviations. (A) Open bars, C3H/HeJ; solid bars, C3H.SJL-Bphs^sD. The repeated-measure ANOVA for the DTH response to OVA revealed a statistically significant interaction (P < 0.0001) between the strain and the day postchallenge. In particular, C3H/HeJ and C3H.SJL-Bphs^sD groups showed significantly different postchallenge changes in thickness (P < 0.0001). (B) ▴, C57BL/6J mice immunized with MOG 35-55 emulsified in CFA plus PTX; ▪, B6.129P-Hrh1^tm1Wat mice immunized with MOG 35-55 emulsified in CFA plus PTX; □, C57BL/6J mice immunized with MOG 35-55 emulsified in CFA without PTX. The repeated-measure ANOVA for the DTH response to MOG 35-55 indicated that the three groups had significantly different responses over the 5 days postchallenge as reflected in an overall significant group by time interaction (P < 0.0001). Orthogonal decompositions of the time effects indicated that the linear effect (P < 0.0001) and quadratic effect (P = 0.0003) differed among the three groups with the C57BL/6J mice immunized with MOG 35-55 emulsified in CFA plus PTX showing a positive increase with a leveling out after day 1 while C57BL/6J mice immunized without PTX and B6.129P-Hrh1^tm1Wat mice treated with PTX had decreasing values over time.