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Identification of Bphs, an Autoimmune Disease Locus, as Histamine Receptor H1

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Science  26 Jul 2002:
Vol. 297, Issue 5581, pp. 620-623
DOI: 10.1126/science.1072810

Abstract

Bphs controls Bordetella pertussis toxin (PTX)–induced vasoactive amine sensitization elicited by histamine (VAASH) and has an established role in autoimmunity. We report that congenic mapping links Bphs to the histamine H1 receptor gene (Hrh1/H1R) and that H1R differs at three amino acid residues in VAASH-susceptible and -resistant mice.Hrh1-/- mice are protected from VAASH, which can be restored by genetic complementation with a susceptibleBphs/Hrh1 allele, and experimental allergic encephalomyelitis and autoimmune orchitis due to immune deviation. Thus, natural alleles of Hrh1 control both the autoimmune T cell and vascular responses regulated by histamine after PTX sensitization.

PTX is a major virulence factor ofB. pertussis, the causative agent of Whooping Cough (1). The holotoxin is a hexameric protein that conforms to the αβ model of bacterial exotoxins (2). The α subunit is an adenosine diphosphate (ADP)-ribosyl transferase, which affects signal transduction by ribosylation of the α subunit of trimeric Gi proteins, whereas the β oligomer binds cell surface receptors on a variety of mammalian cells (2, 3). PTX, when administered in vivo, elicits a range of responses including disruption of glucose regulation, leukocytosis, adjuvant activity, increased vascular permeability associated with alteration of blood-tissue barrier functions, and sensitization to vasoactive amines (VAAS) (4, 5). The latter two phenotypes are the result of PTX-induced changes in vascular endothelial cells. Inbred strains of mice differ in susceptibility to vasoactive amine challenge after PTX sensitization in that genetically susceptible strains die from hypotensive and hypovolemic shock, whereas resistant strains do not (4). Additionally, the genetic control of susceptibility to lethal shock is vasoactive amine specific (4) and is mediated through a variety of mechanisms (6–9).

Hypersensitivity to histamine after PTX sensitization (VAASH) is controlled by an autosomal dominant locus known as Bphs(10). Bphs was one of the first nonmajor histocompatibility complex–linked genes shown to be involved in susceptibility to multiple autoimmune diseases (10). Previously, we mapped Bphs on mouse chromosome 6 using backcross populations generated with susceptible SJL/J and resistant C3H/HeJ and CBA/J mice (10). As the first step in positionally cloning Bphs, we generated a panel of recombinant, interval-specific congenic lines using marker-assisted selection to introgress the SJL/J Bphs allele (Bphss ) onto the C3H/HeJ background (11). These lines were tested for susceptibility to VAASH (Table 1). The results establish that Bphs resides within an interval ≤1 cM betweenD6Mit107 and D6Mit41, encompassingHrh1. Additionally, homozygous and heterozygous C3H.SJL-Bphs line D mice are as sensitive to VAASH as SJL/J, over a dose range of 6.25 to 100 mg/kg (table S1). This is consistent with dominance and a lack of gene dosage effect at this locus (10).

Table 1

Genotypes of C3H.SJL-Bphscongenic lines show colocalization of Bphs withHrh1. Animals were backcrossed and recombinant lines were identified and selected between N12 and N24. Mice from each line were sensitized with PTX on day 0 by intraperitoneal (i.p.) injection of 10.0 μg crude PTX (lines A to D) or by intravenous (i.v.) injection of 400 ng purified PTX (List Biological Laboratories, Inc.) (lines E to F). Control animals received carrier. Three days later, mice were challenged by either i.p. (lines A to D) or i.v. (lines E to F) injection of 50.0 mg/kg histamine in phosphate-buffered saline (dry weight free base). Results are expressed as the number of animals dead at 30 min over the number of animals studied. Marker map locations are in cM according to MGD (www.informatics.jax.org/searches/linkmap_form.shtml). S, SJL/J-derived alleles at the various marker loci; H, C3H/HeJ alleles; NS, not studied.

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It is known that the histamine H1 receptor antagonist mepyramine can block VAASH in rats (12), and, becauseHrh1 resides within the interval encoding Bphs,Hrh1 was a candidate gene for Bphs. Therefore, we cloned and sequenced the Hrh1 alleles from cDNA samples of 14 inbred strains of mice (table S2). With the exception of C3H/HeJ and CBA/J, all mouse strains are susceptible to VAASH. Hrh1sequences from susceptible and resistant mouse strains exhibited multiple, single nucleotide polymorphisms. However, among these polymorphisms, only three led to distinct and concordant amino acid changes in the predicted sequence (L263P, M313V, and S331P), all of which distinguish C3H/HeJ and CBA/J from all other strains of mice (fig. S1).

The identity of Hrh1 as Bphs was further verified using C57BL/6-Hrh1-/- (13) and C57BL/6-Hrh2-/- (14) mice.Hrh1-/- mice were completely resistant to VAASH, whereas Hrh2-/- mice were fully susceptible (Table 2). Genetic complementation experiments using F1 hybrids betweenHrh1-/- and mice bearing a resistantBphs allele (Bphsh ) or a susceptible allele (Bphss ) were performed. Susceptibility could be restored with a single Bphss allele from SJL/J, 129X1/SvJ, or C57BL/B6-Hrh2-/- mice but not with a resistant allele from C3H/HeJ mice (Table 2). Additionally, pretreatment of PTX-sensitized homozygous and heterozygous C3H.SJL-Bphs line D mice with histamine H1 receptor (H1R) selective antagonists before histamine challenge blocked VAASH, whereas H2 and H3selective antagonists did not (table S3). Taken together, our results establish that Hrh1 is Bphs and suggest that one or a combination of the critical amino acids at positions 263, 313, and 331 in the third intracellular loop, associated with signal transduction (15), underlies susceptibility and resistance to VAASH.

Table 2

Assessment of VAASH in C57BL/6-Hrh1 -/- and C57BL/6-Hrh2 -/- mice and F1 progeny between C57BL/6-Hrh1 -/- mice andBphs s/s and Bphs h/hstrains. Mice were sensitized with 400 ng purified PTX (List Biological Laboratories, Inc.) by i.v. injection on day 0. On day 3, mice were challenged with histamine (mg dry weight free base) by i.v. injection, and deaths were recorded at 30 min post challenge. The results are expressed as the number of animals dead over the number of animals studied.

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Next, we assessed the role of Bphs/Hrh1 in autoimmune disease. In the first study, (C3H/HeJ × SJL/J) × C3H/HeJ mice were phenotyped for susceptibility to experimental allergic encephalomyelitis (EAE), experimental allergic orchitis (EAO), and VAASH (11). Linkage of VAASH to EAE and EAO susceptibility was detected (χ2 = 6.42; P = 0.01 for EAE and χ2 = 5.60; P = 0.02 for EAO). When the populations were combined, the significance of the linkage was greater indicating that Bphs/Hrh1 alleles behaved in a similar fashion genetically in both autoimmune diseases (χ2 = 11.49; P = 0.00007). These results confirm that Bphs is linked to susceptibility to EAE and EAO and are consistent with previous results indicating thatBphs is one of several autoimmune disease modifying genes shared between these two models (16).

Because PTX sensitization at the time of immunization induces CD4+ T cells that are responsible for mediating enhanced and protracted delayed-type hypersensitivity responses (17) by producing extremely high levels of γ-interferon (IFNγ) after antigenic stimulation (18,19), and because histamine has recently been shown to regulate immune deviation through differential expression of histamine H1 and H2 receptors (20), we directly assessed the role of Hrh1 in susceptibility to both myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG 35-55)–induced EAE and testis antigen- induced EAO. With respect to MOG-induced EAE, the day of disease onset was delayed, and the severity of clinical signs markedly reduced in C57BL/6-Hrh1-/- compared with C57BL/6 wild-type mice (Fig. 1A). Similar results were seen with autoimmune orchitis (table S4). These results again indicate thatHrh1 fulfills the role of Bphs in both EAE and EAO.

Figure 1

(A) Mean clinical disease course for C57BL/6J (○) and C57BL/6-Hrh1-/- (•) mice immunized with MOG 35-55. Data are plotted as the mean clinical score ± SD. The Wilcoxon-Gehan test for time to event and the Friedman test for correlated observations indicated that both the onset (P = 0.04) and severity of clinical signs (P < 0.0001) seen in the C57BL/6-Hrh1-/- animals was significantly different from C57BL/6J mice. (B) Antigen specific ex vivo T cell proliferative responses for C57BL/6J (○) and C57BL/6-Hrh1-/- (•) mice immunized with MOG 35-55. Data are plotted as mean counts per minute (CPM) ± SD and were calculated from triplicate wells.

The examination of a variety of T cell parameters showed that proliferation of draining lymph node (DLN) cells did not differ between wild-type and Hrh1-/- mice immunized with the MOG 35-55 (Fig. 1B). In contrast, cytokine production by splenocytes differed considerably. IFNγ production was markedly decreased (Fig. 2A), whereas interleukin-4 (IL-4) production was increased in Hrh1-/- mice compared with wild-type mice (Fig. 2B). Tumor necrosis factor–α (TNFα), IL-2, and IL-5 cytokines were measured with no difference in cytokine production detected between the two groups (fig. S2). Additionally, there was no difference in the levels of total immunoglobulin G (IgG), IgG1, or IgG2a antibody to MOG (fig. S3) or in the brain and spinal cord histopathology betweenHrh1-/- and wild-type mice, with the exception that Hrh1-/- mice tended to have more eosinophilic infiltrates (fig. S4). These results provide a mechanistic basis for the genetic role of Bphs/Hrh1 in eliciting pathogenic T cells during the induction phase of autoimmune disease.

Figure 2

IFNγ (A) and IL-4 (B) production by MOG 35-55 stimulated splenocytes from C57BL/6J wild-type and C57BL/6-Hrh1-/- mice. Determination of statistical significance between groups was done using the Student'st test. (Asterisk indicates P < 0.001).

H1R has been reported to be essential for eliciting optimal T and B cell responses because Hrh1-/- mice exhibited reduced antigen-specific proliferative responses but still exhibited normal cytokine receptor–mediated signaling and toll receptor–mediated signaling (21). With respect to antigen-specific proliferation, our results differ from the earlier report by Banu and Watanabe. However, this difference is readily attributable to the different immunization protocols used. Subsequently, it was shown that histamine acting through H1R enhances T helper–1 cell (TH1) responses, whereas both TH1 and TH2 responses are suppressed by signaling through H2R. H1R is more abundant than H2R on TH1 cells. Thus the disruption of Hrh1 leads to immune deviation of T cell responses characterized by the suppression of IFNγ production and an up-regulation in the production of TH2-related cytokines (20). These findings are consistent with the role of Bphs/Hrh1 in MOG-induced EAE, in which the T cell response inHrh1-/- mice was strongly TH2 biased and associated with less severe disease.

We have identified Hrh1 as the general autoimmune disease susceptibility locus Bphs and have presumably delineated the critical amino acid residues that underlie the coupling of H1R to the Gq/11-second messenger pathway that mediates cell-specific responses (15, 21). To date, H1R has not been modeled with respect to its coupling to second messenger pathways. However, the cytoplasmic amino acids that are phosphorylated and are involved in H1R desensitization by phorbol esters have been identified, and they are distinct from the polymorphic residues identified in this study (22). In endothelial cells, H1R-mediated signaling responses include the ability of histamine to directly cause vasodilation and increased vascular permeability (5) and the release from Weibel Palade (WP) bodies (7) of stored vasoactive factors (IL-8, von Willebrand Factor, P-selectin, endothelin, and CD63) whose synthesis is induced by inflammatory stimuli such as PTX (8,9). The role of histamine as a secretagogue for the release of stored vasoactive factors from WP bodies (7–9) is consistent with the fact that VAASH persists upward of 60 days (4). Histamine and histamine-releasing agents also have dramatic effects on the blood-testis (23) and blood-brain barriers (24), and, in both EAO and EAE, tissue levels of histamine correlate with the onset of disease (25,26). It has long been known (27,28), and recently reaffirmed (29, 30), that H1R selective antagonists significantly inhibit susceptibility to EAE, and we have observed similar results in EAO (table S4). In T cells, signaling through H1R results in Ca2+ mobilization causing the translocation of nuclear factor of activated T cells (NFAT) to the nucleus and activation of the IL-2 gene (21) and, via an unknown mechanism, immune deviation of T cells (20). Our results demonstrate how the characterization of a monogenic, qualitative intermediate phenotype can result in the identification of a gene that mimics a quantitative trait locus due to its activity in multiple cell types. The identification ofHrh1 as Bphs also represents an example of an autoimmune disease susceptibility gene that functions at critical checkpoints during both the induction and effector phases of two different induced autoimmune diseases.

Supporting Online Material

www.sciencemag.org/cgi/content/full/297/5581/620/DC1

Materials and Methods

Figs. S1 to S4

Tables S1 to S4

References

  • * To whom correspondence should be addressed. E-mail: cteusche{at}zoo.uvm.edu

REFERENCES AND NOTES

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