Autoimmune Disease-Associated Histamine Receptor H1 Alleles Exhibit Differential Protein Trafficking and Cell Surface Expression

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ExpressionProtein Trafficking and Cell Surface

Alleles Exhibit Differential1Receptor HAutoimmune Disease-Associated Histamine

TeuscherKantidakis, Graeme Milligan, Mercedes Rincon and CoryRoxana del Rio, Elizabeth P. Blankenhorn, Theodoros Rajkumar Noubade, Naresha Saligrama, Karen Spach,

http://www.jimmunol.org/content/180/11/7471doi: 10.4049/jimmunol.180.11.7471

2008; 180:7471-7479; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Autoimmune Disease-Associated Histamine Receptor H1

Alleles Exhibit Differential Protein Trafficking and CellSurface Expression1

Rajkumar Noubade,* Naresha Saligrama,* Karen Spach,* Roxana del Rio,*Elizabeth P. Blankenhorn,† Theodoros Kantidakis,‡ Graeme Milligan,‡ Mercedes Rincon,*and Cory Teuscher2*§

Structural polymorphisms (L263P, M313V, and S331P) in the third intracellular loop of the murine histamine receptor H1 (H1R)are candidates for Bphs, a shared autoimmune disease locus in experimental allergic encephalomyelitis and experimental allergicorchitis. The P-V-P haplotype is associated with increased disease susceptibility (H1RS) whereas the L-M-S haplotype is associatedwith less severe disease (H1RR). In this study, we show that selective re-expression of the H1RS allele in T cells fully complementsexperimental allergic encephalomyelitis susceptibility and the production of disease-associated cytokines while selective re-ex-pression of the H1RR allele does not. Mechanistically, we show that the two H1R alleles exhibit differential cell surface expressionand altered intracellular trafficking, with the H1RR allele being retained within the endoplasmic reticulum. Moreover, we show thatall three residues (L-M-S) comprising the H1RR haplotype are required for altered expression. These data are the first to dem-onstrate that structural polymorphisms influencing cell surface expression of a G protein-coupled receptor in T cells regulatesimmune functions and autoimmune disease susceptibility. The Journal of Immunology, 2008, 180: 7471–7479.

M ultiple sclerosis (MS)3 is the major demyelinating dis-ease of the CNS in humans, affecting �2.5 millionpeople worldwide (1). Both environmental and genetic

factors contribute to the immunopathologic etiology of the disease.A genetic component in disease susceptibility is supported by the20–30% concordance rate among monozygotic twins and 3–5%for dizygotic twins. Compared with the general population, MS is20–40 times more common in first-degree relatives and there is noexcess risk in adopted relatives of patients with MS (2). Evidenceof an environmental etiology in MS comes primarily from migra-tion studies and geographic distribution data. Migration studiesindicate that individuals moving from high-risk areas before pu-berty tend to adopt the lower risk of the native population and viceversa (3). Thus, susceptibility to MS is likely the result of envi-

ronmental triggers acting on a susceptible genetic background atthe population level.

Experimental allergic encephalomyelitis (EAE), the primary an-imal model of MS, is also a genetically determined inflammatorydisease of the CNS (4). EAE can be actively induced in geneticallysusceptible animals by immunization with either whole spinal cordhomogenate or encephalitogenic proteins/peptides and adjuvants(5). EAE, like MS, is a complex polygenic disease (6), with mul-tiple genes exerting a modest effect, thus making it difficult tostudy the contribution of individual loci to overall disease patho-genesis. However, reduction of complex disease states into inter-mediate or subphenotypes that are under the control of a singlelocus has the potential to facilitate mechanistic studies and geneidentification (6). One such phenotype associated with EAE is Bor-detella pertussis toxin-induced histamine sensitization, which iscontrolled by the single autosomal dominant locus known as Bphs(7). Previously, we identified Hrh1/H1R as the gene underlyingBphs (7) and as a shared autoimmune disease susceptibility gene inEAE (8) and experimental allergic orchitis (9). H1R is a seven-transmembrane spanning, G protein-coupled receptor (GPCR).Generally, ligation of H1R with histamine is believed to couple tosecond messenger signaling pathways via the activation of the het-erotrimeric G�q/11 family of G proteins and leads to a variety ofsignaling cascades depending on the cell type involved (10).

Compared with wild-type (WT) mice, H1R-deficient (H1RKO)mice exhibit significantly reduced EAE susceptibility (7). As adisease susceptibility gene, Hrh1/H1R can exert its effect in mul-tiple cell types involved in the disease process including endothe-lial cells, APCs, and T cells. Moreover, H1R may function at crit-ical checkpoints during both the induction and effector phases ofthe disease. In this regard, we recently demonstrated that selectivere-expression of the H1RS allele in T cells is sufficient to comple-ment EAE in H1RKO mice and that H1R signals are importantduring priming of naive T cells rather than during the effectorphase of the disease (11).

*Department of Medicine, University of Vermont, Burlington, VT 05405; †Depart-ment of Microbiology and Immunology, College of Medicine, Drexel University,Philadelphia, PA 19129; ‡Molecular Pharmacology Group, Institute of Biomedicaland Life Sciences, University of Glasgow, Glasgow, United Kingdom; and §Depart-ment of Pathology, University of Vermont, Burlington, VT 05405

Received for publication January 16, 2008. Accepted for publication March 25, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants AI041747,AI058052, AI045666, NS036526, and AI051454 and by National Multiple SclerosisSociety Grant RG-3575.2 Address correspondence and reprint requests to Dr. Cory Teuscher, C317 GivenMedical Building, University of Vermont, Burlington, VT 05405. E-mail address:[email protected] Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergicencephalomyelitis; GPCR, G protein-coupled receptor; WT, wild type; ER, endoplas-mic reticulum; HA, hemagglutinin; PTX, pertussis toxin; DO, day of onset; CDS,cumulative disease score; SI, severity index; PS, peak score; MOG35–55, myelin ol-igodendrocyte glycoprotein peptide 35–55; DLN, draining lymph node; SH3, Srchomology 3; DTH, delayed-type hypersensitivity.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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Hrh1/H1R-susceptible (Hrh1S/H1RS) and -resistant (Hrh1R/H1RR) alleles differ by three amino acids in their coding sequences(7). The H1RR haplotype possesses a L263, M313, and S331whereas the H1RS haplotype is characterized by P263, V313, andP331 (7). The mechanism whereby these polymorphic residuesinfluence EAE susceptibility is unknown but it was hypothesizedto be the result of differential coupling to second messenger sig-naling pathways, because the three residues reside within the thirdintracytoplasmic domain associated with G�q/11 activation (12). Inthis study, we show that, unlike the H1RS allele (11), expression ofthe H1RR allele in T cells does not complement EAE in H1RKOmice and that the polymorphic residues of the H1RR allele affectintracellular trafficking and retention in the endoplasmic reticulum(ER) rather than the inherent capacity to signal. Moreover, weshow that all three residues (L-M-S) comprising the H1RR haplo-type are required for altered cell surface expression. These data arethe first to demonstrate that structural polymorphisms influencingdifferential cell surface expression of a GPCR in T cells can reg-ulate immune functions and susceptibility to autoimmune disease.

Materials and MethodsMice

C57BL/6J mice were purchased from The Jackson Laboratory. B6.129P-Hrh1tm1Wat (H1RKO) (13) mice were maintained in the animal facility atthe University of Vermont (Burlington, VT). The experimental proceduresused in this study were approved by the Animal Care and Use Committeeof the University of Vermont.

For transgenic mouse generation, the HA-H1RS or HA-H1RR constructswere made by deleting the methionine of the Bphs-susceptible H1R allelefrom SJL/J and Bphs-resistant C3H/HeJ mice, respectively (7), and addinga hemagglutinin (HA) tag at the N terminus using TOPO cloning vector(Invitrogen). The HA-H1R was then subcloned downstream of the distal lckpromoter (14). The linear DNA fragment containing the distal lck pro-moter, the HA-H1R gene, and the human growth hormone intron and poly-adenylation signal was injected directly into fertilized C57BL/6J eggs atthe University of Vermont transgenic/knockout facility. Mice werescreened by DNA slot-blot testing using a BamHI-SacI 0.5-kb fragmentfrom the hGH gene as a probe. Two founders were generated for both theH1RS and H1RR alleles and each was crossed to H1RKO mice to establishtransgenic mouse lines on the H1RKO background (H1RKO-TgS1 andH1RKO-TgS2 and H1RKO-TgR1 and H1RKO-TgR2 mice). Mice from theH1RKO-TgS1 line expressed the transgene at comparable levels to the twolines expressing the H1RR allele, so it was used in all the experiments inthis study.

Cytokine production

For cytokine analysis, spleen and lymph nodes were obtained from miceimmunized 10 days earlier with either myelin oligodendrocyte glycopro-tein peptide 35–55 (MOG35–55)-CFA plus PTX or 2� MOG35–55-CFAsingle-cell suspensions prepared at a concentration of 1 � 106 cells/ml inRPMI 1640 medium and stimulated with 50 �g/ml MOG35–55. Cell culturesupernatants were recovered at 72 h and cytokine levels measured byELISA using anti-IFN-�, anti-IL-4, and anti-IL-17 mAbs and their corre-sponding biotinylated mAbs (BD Pharmingen). The TNF-� ELISA kit wasobtained from BD Pharmingen.

Proliferation assays

Mice were immunized with the 2� MOG35–55-CFA protocol: single-cellsuspensions were prepared at 2.5 � 105 cells/well in RPMI 1640 mediumand stimulated in a 96-well plate with different concentrations (0, 2, 10, and50 �g/ml) of MOG35–55 for 72 h and proliferation was determined by[3H]thymidine incorporation during the last 18 h of culture.

Cell surface expression studies

The pEGZ-HA vector plasmid was a gift from Dr. I. Berberich (Universityof Wurzburg, Wurzburg, Germany). Two restriction sites, BamHI andEcoRI, were inserted into H1RS or H1RR cDNA by PCR and cloned suchthat the second codon is in-frame with the HA tag of pEGZ generating anHA-H1R fusion protein. pEGZ is a bicistronic system with internal ribo-somal entry site-enhanced GFP. Enhanced GFP served as a marker fortransfected cells.

HEK293T cells were plated at 1.25 � 106 cells/plate and cultured inDMEM-F12 containing 10% FBS. When the cells were �50–80% con-fluent, they were transfected with 5 �g of pEGZ-HA-H1RS, pEGZ-HA-H1RR or the empty pEGZ vector using the calcium phosphate method.After 16–24 h, cells were scraped off the plate by rigorous pipetting with1% calf serum in PBS and stained with anti-HA mAb conjugated to PE(Miltenyi Biotec) according to the manufacturer’s guidelines. Cells wereanalyzed by flow cytometry using a FACSAria instrument (BD Pharmin-gen) and the data were further analyzed using FlowJo flow cytometry anal-ysis software (Tree Star).

Confocal microscopy

HEK293T cells were transfected with pEGA-HA-H1RS, pEGZ-HA-H1RR,or empty pEGZ control vector (5 �g of total DNA) using the calciumphosphate method. Cells were fixed, permeabilized, and stained using ananti-HA mAb (Cell Signaling Technologies) followed by an incubationwith Alexa-568 anti-mouse Ab (Molecular Probes). TOPRO-3 nuclearstain (Molecular Probes) was used as a nuclear marker. For nonpermeabi-lized cells, the transfected HEK293T cells were stained with the anti-HAmAb and were then fixed. Cells were examined by confocal microscopyusing the Zeiss LSM 510 META Confocal Laser Scanning Imaging System(Carl Zeiss Microimaging).

Cell lysates and Western blotting

Whole-cell lysates were prepared from HEK293T cells transfected withvarious pEGZ constructs in Triton lysis buffer and were then separated viaSDS-PAGE and transferred to nitrocellulose membranes as described pre-viously (11). Anti-HA mAb (Abcam) was used as primary Ab. Anti-actin(Santa Cruz Biotechnology) was used as a loading control.

[3H]Mepyramine-binding studies

[3H]Mepyramine-binding studies were conducted as described (15) andwere used to measure expression levels of H1R variants and the H1RS-G�q/11 and H1RR-G�q/11 fusion proteins.

[35S]GTP�S-binding assay

[35S]GTP�S-binding experiments to assess the capacity of H1R variants tocause activation of G�q/11 were initiated by the addition of cell membranescontaining 50 fmol H1R variant constructs to assay buffer (20 mM HEPES(pH 7.4), 3 mM MgCl2, 100 mM NaCl, 1 �M GDP, 0.2 mM ascorbic acid,and 100 nCi [35S]GTP�S) containing 100 �M histamine. Nonspecific bind-ing was determined in the above condition with the addition of 100 �MGTP�S. Reactions were incubated for 15 min at 30°C and were terminatedby the addition of 500 �l of ice-cold buffer containing 20 mM HEPES (pH7.4), 3 mM MgCl2, 100 mM NaCl, and 0.2 mM ascorbic acid. The sampleswere centrifuged at 16,000 � g for 10 min at 4°C. The resulting pelletswere resuspended in solubilization buffer (100 mM Tris, 200 mM NaCl, 1mM EDTA, and 1.25% Nonidet P-40) plus 0.2% SDS. Samples were pre-cleared with pansorbin for 1 h, followed by immunoprecipitation with aC-terminal anti-G�q/G�11 antiserum (16). Finally, the immune complexeswere washed with solubilization buffer and bound [35S]GTP�S was esti-mated by liquid-scintillation spectrometry.

Site-directed mutagenesis

pEGZ-HA-H1RS was used as template to generate single H1RS mutantswith each of the polymorphic residues replaced with the correspondingresidue of the H1RR allele using the Quickchange (Strategene) site-directedmutagenesis kit, according to the manufacturer’s guidelines. The forwardprimers used for the mutagenesis were: for P263L, 5�-GGGGGTCCAGAAGAGGCCGTCAAGAGACCCTACTGG-3�; for V312M, 5�-CATGCAGACACAGCCTGTGCCTGAGGGAGATGCCAGG-3�; for P330S, 5�-CCAGACCTTGAGCCAGCCCAAAATGGATGAGCAGAGC-3�. The reverseprimers were the complementary sequences of these primers. The altered nu-cleotides are shown in bold and underlined. The mutants were sequence con-firmed and were used as template for the generation of different combinationsof double H1RS mutants.

Conventional and quantitative real-time RT-PCR

Total RNA was extracted from CD4 T cells using RNeasy RNA isolationreagent (Qiagen) as recommended by the manufacturer. cDNA generatedfrom 1 �g of total RNA was used in conventional and quantitative real-time RT-PCR as described earlier (11).

Induction and evaluation of EAE

Mice were immunized for the induction of EAE using either 2�MOG35–55-CFA (17) or MOG35–55-CFA plus PTX protocol (18). For the

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2� MOG35–55-CFA induction protocol, mice are injected s.c. with anemulsion of 100 �g of MOG35–55 and an equal volume of CFA containing200 �g of Mycobacterium tuberculosis H37RA (Difco Laboratories) in theposterior right and left flank; 1 wk later, all mice were similarly injected attwo sites on the right and left flank anterior of the initial injection sites.Animals immunized using the MOG35–55-CFA plus PTX single-inocula-tion protocol received an emulsion of 200 �g of MOG35–55 and equalvolume of CFA containing 200 �g of M. tuberculosis H37RA by s.c.injections distributed equally in the posterior right and left flank and scruffof the neck. Immediately thereafter, each animal received 200 ng of PTX(List Biological Laboratories) by i.v. injection. Mice were scored dailystarting at day 5 postinjection as previously described (18). Clinical quan-titative trait variables including disease incidence and mean day of onset(DO), cumulative disease score (CDS), number of days affected, overallseverity index (SI), and the peak score (PS) were generated as previouslydescribed (17).

Statistical analysis

Statistical analyses, as detailed in the figure legends, were performed usingGraphPad Prism 4 software (GraphPad Software). A p value of 0.05 or lesswas considered significant.

ResultsExpression of H1RR does not complement EAE inH1R-deficient mice

Using transgenic complementation, we recently showed that ex-pression of the H1RS allele only in T cells of H1RKO mice was

sufficient to restore EAE severity to WT levels in these mice (11).To understand whether the H1RR allele would also complementEAE in H1RKO mice, we generated transgenic mice expressingthe N-terminal HA-tagged H1RR allele under the control of thedistal lck promoter, which drives expression in peripheral T cells(14). The transgenic founders were generated directly on theC57BL/6J background and were crossed to H1RKO mice to obtainH1RKO mice expressing the H1RR allele selectively in T cells. Theexpression of the transgene in CD4 T cells was assessed by RT-PCR using transgene-specific primers (Fig. 1A) and by real-timeRT-PCR using primers that recognize H1R (Fig. 1B). The twoestablished lines of H1RR (H1RKO-TgR1 and H1RKO-TgR2) ex-pressed the transgene mRNA at levels comparable to one of theH1RS allele transgenic mice (H1RKO-TgS) that we reported pre-viously (11).

We then examined the susceptibility of these transgenic mice toMOG35–55 induced EAE. We used two protocols to induce disease:MOG35–55-CFA plus PTX (Fig. 1C) and 2� MOG35–55-CFA (Fig.1D). Regression analysis revealed that the clinical disease courseselicited by both induction protocols fit a Sigmoidal curve and thatthe clinical course of disease in two independent lines of H1RKO-TgR mice was not different from that in H1RKO mice. However,as reported previously (11), the clinical course of EAE in H1RKO-TgS mice was significantly more severe than that of H1RKO mice

FIGURE 1. Transgenic expression of the H1RR allelein H1RKO T cells fails to complement EAE in H1RKOmice. H1R transgene expression was analyzed by (A)RT-PCR and (B) quantitative RT-PCR in CD4 T cellsfrom H1RKO mice and transgenic mice expressing theH1RS or H1RR allele that were crossed with H1RKOmice (H1RKO-TgS, H1RKO-TgR1, and H1RKO-TgR2). H1RKO-TgR1 and H1RKO-TgR2 represent twoindependent lines. C, Clinical EAE in WT (n � 19),H1RKO (n � 56), H1RKO-TgS (n � 24), and H1RKO-TgR (n � 17) mice that were immunized withMOG35–55-CFA plus PTX. Mice were scored dailystarting at day 5. Regression analysis revealed thatthe disease course elicited fits a Sigmoidal curve andthat the clinical disease course of the animals wassignificantly different among the strains. The clinicaldisease courses of WT and H1RKO-TgS mice wereboth significantly more severe than those of H1RKO-TgR and H1RKO mice (p � 0.0001 for all compari-sons). D, WT (n � 18), H1RKO (n � 33), H1RKO-TgS (n � 23), and H1RKO-TgR (n � 14) mice wereimmunized with 2� MOG35–55-CFA. EAE severitywas significantly different among the strains. Theclinical disease courses of WT and H1RKO-TgS micewere both significantly more severe than those ofH1RKO-TgR and H1RKO mice (p � 0.0001 for allcomparisons).

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and was equivalent to the disease course observed in WT mice.These results indicate that, unlike the H1RS allele, expression ofthe H1RR allele by H1RKO T cells does not complement EAEsusceptibility.

An analysis of EAE-associated clinical quantitative trait vari-ables from the two transgenic cohorts revealed that the mean dayof onset (DO), cumulative disease score (CDS), overall severityindex (SI), and the peak score (PS) were significantly differentamong the strains immunized with either MOG35–55-CFA plusPTX or 2� MOG35–55-CFA (Table I). Post hoc multiple compar-isons of each trait variable revealed that H1RKO-TgS mice wereequivalent to WT mice while H1RKO-TgR mice were equivalent toH1RKO mice. Furthermore, for each trait, H1RKO-TgS and WT micewere significantly greater than H1RKO-TgR and H1RKO mice.

We next analyzed the ex vivo MOG35–55-specific proliferativeresponse of spleen and draining lymph node (DLN) cells frommice immunized with 2� MOG35–55-CFA. Significant differencesin proliferative responses were not detected among WT, H1RKO,

H1RKO-TgS, and H1RKO-TgR mice (data not shown). BecauseMOG35–55-stimulated splenocytes from immunized-H1RKO miceexhibit an immune deviation from Th1 to Th2 response in ex vivorecall assays (7), we analyzed cytokine production by MOG35–55-stimulated spleen and DLN cells from mice immunized with bothEAE-induction protocols. With the classical MOG35–55-CFA plusPTX protocol, as we observed previously (11), Ag-stimulatedspleen and DLN cells from H1RKO-TgS mice produced signifi-cantly greater amounts of IFN-� compared with H1RKO mice andat levels comparable to WT mice (Fig. 2A). In contrast, the levelsof IFN-� produced by Ag-stimulated spleen and DLN cells fromthe two lines of H1RKO-TgR mice were equivalent to thoseproduced by H1RKO mice. Similarly, Ag-stimulated spleen andDLN cells from H1RKO-TgS mice produced IL-4 at levels com-parable to WT mice while those from H1RKO-TgR mice weresimilar to H1RKO mice (Fig. 2B). Similar results for IFN-�(Fig. 2D) and IL-4 (Fig. 2E) were observed for 2�MOG35–55-CFA-immunized mice.

FIGURE 2. Transgenic expression of H1RR inH1RKO T cells fails to complement cytokine produc-tion by H1RKO mice. A–C, Spleen and DLN cellswere isolated from MOG35–55-CFA plus PTX-immu-nized WT, H1RKO, H1RKO-TgS, and H1RKO-TgR

mice 10 days postimmunization and stimulated with50 �g/ml MOG35–55 for 72 h (n � 4–8 mice/group).Supernatants were collected and analyzed for the pro-duction of IFN-� (A), IL-4 (B), and IL-17 (C). Signif-icance of differences in cytokine production were as-sessed using the nonparametric Kruskal-Wallis testfollowed by Dunnett’s post hoc multiple comparisons(H � 25.73; p � 0.0001 for IFN-�, H � 31.34; p �0.0001 for IL-4, H � 0.514; p � 0.5 for IL-17. �, p �0.05; ��, p � 0.01; and ���, p � 0.001). D–F, Spleenand DLN cells from 2� MOG35–55-CFA immunizedmice were collected on day 10 postimmunization andwere activated with 50 �g/ml MOG35–55 for 72 h, su-pernatants were collected and analyzed for IFN-� (D),IL-4 (E), and IL-17 (F) by ELISA in triplicate. Sig-nificance of differences in cytokine production wereassessed using the nonparametric Kruskal-Wallis testfollowed by Dunnett’s post hoc multiple comparisons(H � 52.23; p � 0.0001 for IFN-�, H � 23.88; p �0.0001 for IL-4, H � 35.22; p � 0.0001 for IL-17. �,p � 0.05; ��, p � 0.01; and ���, p � 0.001). Data arepresented as the mean � SEM and are representativeof two independent experiments.

Table I. Clinical disease traits following immunization of mice with MOG35–55-CFA plus PTX or 2� MOG35–55-CFA

Strain Incidence DO CDS SI PS

MOG35–55-CFA plus PTXC57BL/6J 19/19 13.1 � 0.3 56.2 � 4.6 3.1 � 0.2 3.9 � 0.3H1RKO 55/56 15.7 � 0.4 32.1 � 1.4 2.1 � 0.1 3.0 � 0.1H1RKO-TgS 24/24 12.9 � 0.4 50.0 � 3.7 2.8 � 0.2 3.6 � 0.2H1RKO-TgR 16/17 13.3 � 0.3 28.6 � 2.3 1.7 � 0.1 2.4 � 0.2

�2 � 2.5 F � 13.5 21.2 19.2 14.4p � 0.5 p � 0.0001 �0.0001 �0.0001 �0.0001

C57BL/6J � H1RKO-TgS � H1RKO-TgR � H1RKO2� MOG35–55-CFA

C57BL/6J 18/18 16.6 � 0.7 37.6 � 2.9 2.6 � 0.1 3.2 � 0.2H1RKO 26/33 17.1 � 0.5 20.0 � 1.8 1.6 � 0.1 2.2 � 0.1H1RKO-TgS 22/23 16.2 � 0.6 36.4 � 3.8 2.5 � 0.2 3.2 � 0.2H1RKO-TgR 13/14 18.7 � 0.6 18.6 � 3.1 1.6 � 0.2 1.9 � 0.2

�2 � 7.4 F � 2.8 11.6 15.2 14.0p � 0.06 p � 0.05 �0.0001 �0.0001 �0.0001

C57BL/6J � H1RKO-TgS � H1RKO-TgR � H1RKO

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Because IL-17 is considered to be an important effector cytokinein EAE (19), we examined IL-17 production by spleen and DLNcells following ex vivo stimulation with MOG35–55. IL-17 produc-tion by WT, H1RKO, H1RKO-TgS, and H1RKO-TgR mice im-munized with MOG35–55-CFA and PTX was not significantly dif-ferent (Fig. 2C) among strains. In contrast, IL-17 production byMOG35–55-stimulated spleen and DLN cells from animals immu-nized with 2� MOG35–55-CFA differed significantly among thestrains (Fig. 2F). Compared with WT mice, H1RKO mice pro-duced significantly less IL-17, indicating that H1R signaling reg-ulates IL-17 production by T cells. Moreover, production of IL-17by H1RKO-TgS mice was not significantly different from WTmice and IL-17 production by H1RKO-TgR mice was not signif-icantly different from H1RKO mice (Fig. 2F). Taken together, likeEAE, H1RR expression in H1RKO T cells does not complementcytokine production by these cells.

H1R alleles activate G�q and G�11 equally well in vitro

To understand the mechanism by which the polymorphic residuesof the H1RS and H1RR alleles influence H1R function, we examinedthe predicted structural location for the three residues within H1R.The three polymorphic residues reside within the third intracyto-plasmic loop of H1R (Fig. 3A), which is the region frequentlyassociated with recruitment and activation of downstream G pro-teins (12). We, therefore, examined whether the polymorphic res-idues distinguishing the H1RS and H1RR alleles might result insignificant alterations in G protein activation. Because H1R is nor-mally coupled to G�q and/or G�11 proteins, we generated fusionproteins of the two H1R alleles with both G�q and G�11 by linking

in-frame the N terminus of G�q/11 with the C-terminal tail of H1RR

or H1RS.HEK293 cells were transfected with the H1RS-G�q/11 or H1RR-

G�q/11 fusion proteins, lysed and membrane fractions preparedfrom these cells. These were used initially to measure the levels ofexpression of each construct via the specific binding of the H1Rantagonist [3H]mepyramine. There were no differences in the lev-els of specific binding of [3H]mepyramine between the variousconstructs, indicating that the polymorphisms did not alter totalprotein expression. Also, the binding affinity of [3H]mepyraminewas not different between the two alleles (Fig. 3B). To study theirdifferential capacity to activate G�q and G�11, membrane amountscontaining exactly the same number of copies of each constructwere used in [35S]GTP�S-binding assays. A maximally effectiveconcentration of histamine stimulated binding of [35S]GTP�Sequally to G�q or G�11 when each G protein was linked to eitherthe H1RS or H1RR variants (Fig. 3, C and D). The dose-responsecurves to histamine indicated that the potency of histamine isequivalent for each receptor variant (data not shown). These dataindicate that the H1RS and H1RR alleles can activate these G pro-teins equally well and that the phenotypic difference associatedwith the H1R alleles is not inherently a function of differentialcapability to activate G�q or G�11.

H1R alleles are differentially expressed on the cell surface

Specific mutations in the signaling domain of several GPCRs (e.g.,vasopressin V2 receptor, rhodopsin) can interfere with their cellsurface expression and are associated with disease (20). To deter-mine whether the polymorphisms in H1R influence cell surface

FIGURE 3. H1RS and H1RR activate G�q/11 G pro-teins equally well. A, The amino acid sequence of themouse H1R is displayed with differences between theH1RR allele (red) and the H1RS allele (yellow) high-lighted. Each of the sites of variation is within the long,third intracellular loop. B, Saturation [3H]mepyraminebinding studies were performed on membranes ofHEK293T cells transfected to express a H1R-G�q fusionprotein (left side H1RR, right side H1RS). Nonspecificbinding was determined in the same manner but with theadditional presence of 1 �M mianserin. Data are pre-sented as the mean � SEM. These studies providedquantitation of construct expression levels. C and D,Membranes containing 50 fmol H1RS or H1RR linked toeither G�q (C) or G�11 (D) were used in [35S]GTP�S-binding studies conducted in the absence (basal, �) orpresence (histamine, f) of 100 �M histamine to assessthe capability of the two variants to activate the G pro-teins. H1RS and H1RR were equieffective in causing ac-tivation of each G protein. Representative data from fourindependent experiments are shown.

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expression of the receptor, HA-H1RS or HA-H1RR expression vec-tors were used to transfect HEK293T cells. The expression of thesereceptors at the cell surface was then examined by flow cytometricanalysis using an anti-HA mAb. HA-H1RS was expressed at higherlevels than HA-H1RR (Fig. 4A). The number of H1RS-positivecells (Fig. 4B) and the mean florescence intensity of H1RS wereconsiderably higher than those of H1RR (Fig. 4C), indicating thatthe two H1R alleles are differentially expressed on the cell surface.We observed similar results when the H1RS and H1RR constructswere transfected into 721.221 B cells (data not shown).

In parallel, we examined the cell surface expression of H1RS andH1RR by confocal microscopy using anti-HA mAb in cells stainedbefore permeabilization. The results confirmed higher expressionof H1RS on the surface than H1RR (Fig. 4D). However, Westernblot analysis of H1RS and H1RR expression in lysates of trans-fected HEK293T cells showed no difference in the amount of totalprotein present (Fig. 4E). Taken together, these data indicate thatthe polymorphic residues associated with the H1RS and H1RR hap-lotypes result in differential translocation of the receptor to the cellsurface.

H1RR is retained in the ER

The Western blot results described above (Fig. 4E) suggest that theH1RS and H1RR alleles are expressed at similar levels but that theH1RR allele is largely retained in intracellular compartments in-stead of being trafficked to the cell surface. To investigate thispossibility, HEK293T cells were transfected with HA-H1RS orHA-H1RR constructs. After 24 h, cells were fixed, permeabilized,stained with anti-HA mAb, and observed by confocal microscopy.A predominantly plasma membrane-staining pattern was observedfor the H1RS allele (Fig. 5A). In contrast, a large fraction of theH1RR allele appeared to localize intracellularly (Fig. 5A, rightpanel) indicating that H1RR is retained in the intracellular com-partments and fails to traffic efficiently to the cell surface. Thenetwork-like intracellular distribution of H1RR throughout the cell(Fig. 5A, right panel) resembled that of ER. Therefore, to deter-mine whether the H1RR allele is retained in this compartment, wetransiently cotransfected HEK293T cells with H1RS or H1RR con-structs and a plasmid expressing the dsRed fluorescent protein thattargets the ER. Colocalization of the two proteins was examined by

FIGURE 4. H1RS and H1RR alleles are differen-tially expressed on the cell surface. A, HEK293T cellswere transfected with empty pEGZ, pEGZ-HA-H1RS,or pEGZ-HA-H1RR plasmids. Cells were collected16–24 h later without trypisinization, stained with anti-HA mAb, and analyzed by flow cytometry. The thinline represents cells transfected with empty pEGZwhereas the thick line represents cells transfected withHA-H1RS and the filled area represents cells trans-fected with HA-H1RR. B and C, HEK293T cells wereanalyzed as in A and the percentage (B) and the MFI ofanti-HA on H1R

S-positive cells (C) were determined. D,HEK293T cells transfected with HA-H1R

S or HA-H1RR

plasmids and 24 h later cells were stained with anti-HAmAb (red) without permeabilization. Cells were visual-ized by confocal microscopy. GFP (green) is shown as amarker of transfected cells. E, HEK293T cells were trans-fected as in A; whole cell lysates were prepared and an-alyzed by Western blotting using anti-HA mAb. Actin isshown as loading control. Representative data from threeindependent experiments are shown.

FIGURE 5. H1RR is retained in the ER. A,HEK293T cells were transfected with HA-H1RS orHA-H1RR plasmids. Twenty-four hours later, cellswere fixed, permeabilized, stained with anti-HAmAb (red) and TOPRO-3 nuclear stain (green), andvisualized by confocal microscopy. B, HEK293Tcells were cotransfected with pdsRed plasmid thatexpresses ER-targeted florescent dsRed protein (red)and HA-H1RS or HA-H1RR. Twenty-four hours latercells were fixed, permeabilized, stained with anti-HA mAb (green), and the colocalization of HA-H1Rwith dsRed was visualized by confocal microscopy.Yellow color represents the colocalization of red andgreen colors. C, Quantification of HA-H1R colocal-ization with dsRed protein. Using Zeiss LSM 510META Confocal imaging software, the number ofpixels expressing both colors were determined in anumber of cells (n � 26) and the data are presented asthe average of number of pixels that coexpressdsRed and HA-H1R. Error bars represent SEM. Datawere analyzed using the nonparametric Mann-Whit-ney U test (U � 133.0; ����, p � 0.00001).

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confocal microscopy following staining the cells for HA-H1R. Themajority of H1RR was again expressed intracellularly and colocal-ized with the dsRed protein, while minimal colocalization of H1RS

with the ER-targeted dsRed protein was observed (Fig. 5B). UsingLSM5 image browser software, we quantified the number of pixelsthat express both dsRed protein and HA-H1R in multiple cells thatwere imaged under exactly the same settings. The results showeda significant difference in the colocalization of the H1RS and H1RR

alleles in ER (Fig. 5C), suggesting that the H1RR L-M-S haplotypeleads to its sequestration and retention in ER.

Retention of H1RR in the ER requires the L-M-S haplotype

To understand which of the three amino acids comprising the H1RR

L-M-S haplotype is responsible for the observed differential cellsurface expression of the allele, we generated single H1RS mu-tants, replacing each of the H1RS haplotype-associated residueswith the corresponding H1RR allele (P263L, V312M, and P330S),by site-directed mutagenesis. HEK293T cells were transfectedwith H1RS, H1RR, and each of the three H1RS mutant constructs.Cells were stained with anti-HA mAb, without permeabilization,and cell surface expression of H1R was analyzed by flow cytom-etry. Each of the single H1RS mutants was expressed at higherlevels on the cell surface than the H1RR allele (Fig. 6A) with thelevels comparable to those observed with the H1RS allele. This

indicates that the presence of a single H1RR polymorphism is notsufficient to induce its intracellular retention. We also generateddouble mutants of the H1RS allele wherein we replaced two resi-dues of the H1RS haplotype with the corresponding residues of theH1RR allele (P263L and V312M, P263L and P330S, V312M andP330S). Similar to the single H1RS mutants, the double H1RS mu-tants were expressed on the cell surface at levels comparable to theH1RS and at significantly higher levels than the H1RR allele (Fig.6B). We observed similar results in 721.221 B cells followingtransient transfection with H1RS, H1RS mutants, and H1RR con-structs (data not shown). Furthermore, when HEK293T cells werecotransfected with double H1RS mutants and the dsRed plasmid,each of the mutants showed a typical plasma membrane expressionpattern with very little colocalization with the ER-targeted dsRedprotein (Fig. 6C). Quantification of the number of pixels express-ing dsRed protein and HA-H1R confirmed that each of the doubleH1RS mutants behaved like H1RS and only H1RR was retained inER (Fig. 6D), confirming the flow cytometry data that all the poly-morphic residues are required for differential cell surface expres-sion of the H1R alleles. Taken together, these data indicate that allthree residues of the H1RR L-M-S haplotype are required for itsintracellular sequestration. Interestingly, we sequenced the H1Ralleles from �100 different inbred laboratory and wild-derivedmouse strains and did not identify any recombinant haplotypes

FIGURE 6. ER retention of the H1RR allele re-quires the L-M-S haplotype. A, HEK293T cells weretransfected with empty control, single HA-H1RS

mutants, or HA-H1RR plasmids. Cells were col-lected 16–24 h later without trypisinization, stainedwith anti-HA mAb, and analyzed by flow cytometry.Cells transfected with HA-H1RS are shown as pos-itive controls in the far right panel. The thin linerepresents cells transfected with empty pEGZ,whereas the thick line represents cells transfectedwith HA-H1RS, and the filled area represents cellstransfected with HA-H1RR. B, HEK293T cells wereanalyzed as in A and the mean florescence intensityof anti-HA on H1RS-positive cells was determined.The data presented are the average of triplicatetransfections. C, HEK293T cells were cotransfectedwith pdsRed plasmid that express ER-targeteddsRed protein (red) and HA-H1RS, mutants of HA-H1RS or HA-H1RR. Twenty-four hours later, cellswere fixed, permeabilized, stained with anti-HAmAb (green), and the colocalization of HA-H1Rwith dsRed (red) was visualized by confocal micros-copy. Yellow color represents the colocalization ofred and green colors. D, Quantification of HA-H1Rcolocalization with dsRed protein. Using Zeiss LSM510 META Confocal imaging software, the numberof pixels expressing both the colors was determinedin a number of cells (n � 16) and the data are pre-sented as the average number of pixels that coex-press dsRed and HA-H1R. Error bars indicate SEM.

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suggesting that the two alleles are evolutionarily conserved andmay have been selected functionally (data not shown).

DiscussionTo date, Hrh1/H1R is the only murine EAE and experimental al-lergic orchitis susceptibility gene that has been positionally cloned(7). In this study, using transgenic mouse models, we show thatpolymorphic variants in H1R regulate cytokine production by Tcells thereby influencing susceptibility to EAE. Furthermore, usingHEK293T cells, we show that the polymorphisms in H1R affect itsfunctions by modulating cell surface expression rather than inher-ently altering the capacity of the receptor to generate intracellularsignals.

Hrh1/H1R has long been implicated in EAE susceptibility (7, 8).As H1R is widely expressed (10), this suggested that it might actin different cell types and at multiple checkpoints. We recentlyshowed, however, that H1R expression in T cells is sufficient tocomplement EAE severity in H1RKO mice. In this study, we showthat the polymorphic residues of the H1RR allele interfere with itsability to complement EAE in H1RKO mice. This is in accordancewith genetic complementation studies in F1 hybrids betweenH1RKO and strains of mice expressing the H1RS or H1RR alleles.Susceptibility to histamine sensitivity could be restored in F1 hy-brids of H1RKO and SJL/J, 129X1/SvJ, or C57BL/6J that expressthe H1RS allele but not in F1 hybrids between H1RKO and C3H/HeJ or CBA/J mice that express H1RR (7).

Hrh1/H1R also controls delayed-type hypersensitivity (DTH)responses when PTX is used as an adjuvant. The DTH response ismediated by CD4 T cells that produce large amounts of IFN-�(21–23). Using C3H.BphsS congenic mice expressing the H1RS

allele from SJL/J mice on the resistant C3H/HeJ background, Gaoet al. (24) showed that polymorphisms in H1R regulate the OVA-specific DTH response elicited in mice immunized with OVA inCFA and PTX, indicating that the polymorphisms in H1R regulateIFN-� production by CD4 T cells. This study confirms the role ofH1R polymorphisms in regulating IFN-� production by these cells.Furthermore, the complementation of IFN-� production by spleno-cytes immunized using the 2� MOG35–55 model suggests thatH1R regulation of IFN-� production by T cells does notrequire PTX.

Recently, IL-17-producing Th17 CD4 T cells have been con-sidered more pathogenic in EAE (19). We show here, for the firsttime, that H1R signaling regulates IL-17 production and that H1Rpolymorphisms influence IL-17 production by T cells. How-ever, it is noteworthy that we did not observe differences inIL-17 production between WT and H1RKO mice immunizedwith MOG35–55-CFA plus PTX, nor in Th17 cells differentiatedin vitro in the presence of excessive amounts of IL-6. PTXpromotes the generation of Th17 cells, by inducing IL-6 pro-duction (25). Thus, it is possible that immunization with PTX (invivo) or addition of exogenous IL-6 (in vitro) enables CD4 T cellsto overcome the absence of H1R signals required for the optimalIL-6 production and generation of Th17 cells. Even though weobserved significant differences in IL-17 production by spleen andDLN cells from transgenic mice selectively expressing eitherH1RS or H1RR in T cells, we believe, based on in vitro differen-tiation data, that the H1R regulation of IL-6 and IL-17 is indepen-dent of H1R signals in T cells. In this regard, compared with WTmacrophages H1RKO macrophages produce significantly less IL-6(our unpublished data) and treatment of lung parenchymal macro-phages with H1R blockers results in decreased IL-6 production(26). Further studies are being conducted to elucidate the role ofH1R in the generation of Th17 CD4 T cells.

GPCRs, despite the diversity of their polypeptide sequences, asa family retain enough structural information to allow them to beproperly folded in the ER and adopt their highly conserved seventransmembrane confirmation (27). Several studies have identifiedcritical residues and motifs important in many of the functions ofGPCRs including ligand binding, G protein coupling, internaliza-tion, down-regulation, and intracellular trafficking (28). However,the three polymorphic residues distinguishing the H1RS and H1RR

alleles are located in the third intracytoplasmic loop and do notconstitute any known motif. Even though the exact PXXP motif isnot present, it is worth noting that two of the three polymorphicresidues associated with the H1RS haplotype are prolines, and thatproline-rich motifs are known to mediate protein-protein interac-tions with Src homology 3 (SH3) domains (29). In this regard,polymorphic residues containing polyproline motifs in the thirdintracytoplasmic loop of the dopamine D4 receptor and �1-adren-ergic receptor have been shown to interact with multiple SH3 do-main-containing proteins (30) and affect the trafficking of thesereceptors. However, at this point, we do not have any evidence tosuggest that H1R interacts with any of the known SH3 domain-containing proteins or that such interactions differ between H1RS

and H1RR alleles. Future studies will address this issue.GPCRs interact with numerous proteins that play a role in their

cellular trafficking (12). H1R has an unusually long third intracy-toplasmic loop, suggesting that the polymorphic residues may re-sult in improper folding of the receptor to a non-native conforma-tion in ER, which is then recognized by the quality controlmachinery of molecular chaperones and excluded from ER export.Several chaperone proteins (such as Nina (31, 32), ODR-4 (33,34), and a variety of receptor activity modifying proteins (35, 36))that support the trafficking of a range of GPCRs to their target sitehave been identified. Therefore, it is possible that polymorphicresidue-induced misfolding of H1RR could hinder its interactionwith an essential chaperone thereby affecting its trafficking.

Proper cell surface expression of GPCRs is required to accessthe requisite ligands and signal transduction machinery (12). Thefunctional importance of proper GPCR localization is emphasizedby several human diseases that result from receptor mutation andmislocalization, including X-linked nephogenic diabetes, retinitispigmentosa, and hypogonadotrophic hypogonadism, which resultfrom intracellular accumulation of mutant V2 vasopressin recep-tor, rhodopsin, and gonadotropin-releasing hormone receptor, re-spectively (20). In fact, mutations that lead to intracellular accu-mulation comprise the largest class of mutations in GPCRs thatresult in human diseases (12). Accordingly, our results are the firstto demonstrate that structural polymorphisms influencing differ-ential trafficking and cell surface expression of a GPCR in T cellscan regulate immune functions and susceptibility to autoimmunedisease.

DisclosuresThe authors have no financial conflict of interest.

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