Dominant Negative Mutation Gene: In Vivo ...

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Cutting Edge: Functional Characterization of the Effect of the C3H/HeJ Defect in Mice that Lack an Lpsn Gene: In Vivo Evidence for a Dominant Negative Mutation Stefanie N. Vogel, Dabney Johnson, Pin-Yu Perera, Andrei Medvedev, Line Larivière, Salman T. Qureshi and Danielle Malo J Immunol 1999; 162:5666-5670; ; http://www.jimmunol.org/content/162/10/5666

This article cites 28 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/162/10/5666.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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Cutting Edge: Functional Characterization of the Effect of the C3H/HeJ Defect in Mice that Lack an Lpsn Gene: In Vivo Evidence for a Dominant Negative Mutation1 Stefanie N. Vogel,2* Dabney Johnson,† Pin-Yu Perera,* Andrei Medvedev,* Line Larivie`re,‡ Salman T. Qureshi,‡ and Danielle Malo‡§

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ollowing the initial characterization of the C3H/HeJ strain as LPS hyporesponsive ;30 years ago (1, 2), a large number of genetic analyses were conducted to identify the gene responsible for this trait (reviewed in Refs. 3 and 4). Analysis of F1, F2, and backcross hybrids from crosses between LPS-responsive and LPS-hyporesponsive mice showed that the defective LPS responses of C3H/HeJ mice in vivo and in vitro were controlled by a single autosomal gene (5–7). Two alleles were assigned to the Lps gene in inbred mouse strains: Lpsd (defective)3

*Uniformed Services University of the Health Sciences, Bethesda, MD 20814; †Oak Ridge National Laboratories, Oak Ridge, TN 37831; and ‡Department of Medicine and §Department of Human Genetics, McGill University, Montreal, Canada Received for publication January 28, 1999. Accepted for publication March 15, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grant AI-18797 (S.N.V.) and Medical Research Council of Canada Grant MT-12038 (D.M.). S.T.Q. is a recipient of a Clinician Scientist Award from Medical Research Council and a career award in the Biomedical Sciences from the Burroughs Wellcome Fund. D.M. is a scholar from Fonds de la Recherche en Sante´ du Que´bec. 2

Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, Uniformend Services University of Health Sciences, 4301 Jones Bridge Road, B103, Bethesda, MD 20814-4799. E-mail address: [email protected] Copyright © 1999 by The American Association of Immunologists

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and Lpsn (normal) (6). Lpsd mice also exhibit greatly increased susceptibility to bacterial infections (reviewed in Ref. 3). Although a number of knockout mice exhibit mitigated responses to LPS (8), a mutation within Lps leads to the most profound state of LPS hyporesponsiveness described to date. Lps was originally mapped to mouse mid-chromosome 4 by its linkage to Mup1 (major urinary protein-1) using BXH recombinant inbred strains (9 –11). Additional mapping data positioned Lps on chromosome 4, between Mup1 proximally and Ps (polysyndactyly) distally at 6 6 2 cM and 13 6 7 cM, respectively (6), with inferred linkage to b, the brown coat color locus. F1 progeny derived from Lpsn/Lpsn 3 C3H/HeJ (Lpsd/Lpsd) crosses exhibit intermediate (“codominant”) to fully responsive phenotypes, depending upon the particular measure of LPS sensitivity employed (12; reviewed in Ref. 3). The biological significance of a dominantly inherited response pattern (i.e., the F1 response is comparable to that of the Lpsn parental) vs one that is intermediate represents an area of controversy in the literature, yet both patterns of F1 responses have been reported, and at times even within the same study (12–14). In 1975, Coutinho et al. (14) suggested that an “intermediate” response pattern might suggest that: 1) half the cells can be activated by LPS and the other half lack a “triggering receptor,” or 2) all cells can be activated, but at the doses tested only half the cells achieve a threshold for activation. However, the latter possibility was dismissed because they observed intermediate F1 responses, even at very high LPS concentrations. McGhee et al. (13) also addressed LPS dosage, the contribution of background genes unrelated to Lps, and assay sensitivity as potentially contributory factors. The C57BL/10ScCR, and its progenitor strain, C57BL/10ScN, (15–17) were also identified as LPS hyporesponsive, and their defect mapped to the same chromosomal location on mouse chromosome 4 (16). The failure of C3H/HeJ 3 C57BL/10ScCR F1 mice to respond to LPS suggests that these two strains carry noncomplementary mutations (18). In contrast to the intermediate to full responsiveness seen in progeny from Lpsn/Lpsn 3 C3H/HeJ

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Abbreviations used in this paper: Lpsd, defective Lps gene; Lpsn, normal Lps gene; Mup1, major urinary protein-1 gene; Ps, polysyndactyly gene; b, brown coat color locus; Tyrp1, tyrosinase-related protein-1 gene; Bw, white-based brown b allele; TLR, Toll-like receptor; STS, sequence tag site; SSLP, simple sequence length polymorphism; SSCP, single-strand conformation polymorphism. 0022-1767/99/$02.00


A point mutation in the Tlr4 gene, which encodes Toll-like receptor 4, has recently been proposed to underlie LPS hyporesponsiveness in C3H/HeJ mice (Lpsd). The data presented herein demonstrate that F1 progeny from crosses between mice that carry a ;9-cM deletion of chromosome 4 (including deletion of LpsTlr4) and C3H/HeJ mice (i.e., Lps0 3 Lpsd F1 mice) exhibit a pattern of LPS sensitivity, measured by TNF activity, that is indistinguishable from that exhibited by Lpsn 3 Lpsd F1 progeny and whose average response is “intermediate” to parental responses. Thus, these data provide clear functional support for the hypothesis that the C3H/HeJ defect exerts a dominant negative effect on LPS sensitivity; however, expression of a normal Toll-like receptor 4 molecule is apparently not required. The Journal of Immunology, 1999, 162: 5666 – 5670.

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crosses, Lpsn/Lpsn 3 C57BL/10ScCR F1 progeny exhibit full Lpsn-like responsiveness only (16). The Lps gene was recently cloned (19, 20) and shown to encode Tlr4 (Toll-like receptor 4), a member of the IL-1 receptor family (21). C3H/HeJ mice carry a missense point mutation within Tlr4 that results in a substitution of a highly conserved proline at position 712 by an histidine, whereas, the C57BL/10ScCR and C57Bl/10ScN strains fail to express Tlr4 mRNA (19, 20) due to a chromosomal deletion of the gene (20). However, no functional data for the mechanism by which this defect exerts its profound effects have been published to date. The C3H/HeJ missense mutation within Tlr4 has been hypothesized to exert a dominant negative action on normal Tlr4 expressed in Lpsn 3 Lpsd F1 mice, resulting in “codominant” or “intermediate” LPS sensitivity (19). Our data provide the first direct functional evidence that the C3H/ HeJ defect indeed acts as a dominant negative mutation; however, the presence of a normal Tlr4 allele at Lps is not required for expression of a codominant response seen in F1 progeny.

Production of F1 mice and their genotyping C3H/HeJ and C3H/OuJ mice were purchased from Jackson Laboratories (Bar Harbor, ME). The brown (b) locus on mouse chromosome 4 is a recessive mutation of the fancy mouse that encodes for a tyrosinase-related protein-1 (Tyrp1) (22, 23). Multiple alleles at the b locus have been recovered from radiation mutagenesis experiments at Oak Ridge National Laboratory (Oak Ridge, TN); many of these alleles are homozygous-lethal deletions (24 –26). The two homozygous-lethal stocks used in this study were b11R30M, which carries a deletion encompassing both b and whirler [Df(b wi)], and b13R75M, which carries a more distal deletion including b and depilated [Df(b dep)]. The balancer chromosome for b11R30M carries the original b mutation incorporated into the Oak Ridge National Laboratory tester stock, which is homozygous for seven recessive mutations including nonagouti (a), brown (b), pink-eyed dilution ( p), chinchilla (cch), dilute (d) short ear (se) and piebald spotting (s) (24). The b allele carries a point mutation that creates a b-specific 5.2-kb TaqI fragment recognized by hybridization to a subfragment (MT4.Pv.25) of the pMT4 cDNA clone, which is the product of the Tyrp1 gene (22, 27). The balancer chromosome for b13R75M carries white-based brown (Bw), a b allele associated with an inversion at the 59 end of Tyrp1. The probe pBw3.6 detects a Bw-specific 4.3-kB PstI fragment (I. Jackson, unpublished observation). To produce F1 mice that carried one of the lethal b deletions opposite a C3H/HeJ chromosome, b/b1130RM or Bw/b13R75M males were mated to C3H/HeJ (1/1 for b) females, and offspring were genotyped using Southern blots of tail DNAs. For the b11R30M segregants, deletion carriers (1/b11R30M) were identified as those not carrying the b-specific 5.2-kb TaqI fragment recognized by MT4.Pv.25; 1/b13R75M segregants were those not carrying the 4.3-kb Bw-specific PstI fragment recognized by pBw3.6.

Haplotype mapping of DNA from b/b11R30M mice DNA markers (sequence tag site (STS), microsatellite, and microclone probes) used in this analysis were derived from a 1.7-Mb genomic DNA contig encompassing the Lps locus that was constructed between flanking markers D4Nds9 and D4 Mit178 (Ref. 20 and our unpublished observations). To discriminate between the Lpsn and Lpsd allele at Tlr4, Tlr4specific PCR primers (59-GTGCCCCGCTTTCACCTCTG-39 and (59TCTAGACACTACCACAATAA-39) flanking the region containing the C to A transversion resulting in the Pro712His mutation were used to amplify genomic DNA from F1 mice. PCR products were then analyzed by singlestrand conformation polymorphism (SSCP) (20). For PCR typing of simple sequence length polymorphism (SSLP) and SSCP markers, PCR reactions were performed in a total volume of 20 ml using a 20-ng aliquot of genomic DNA. One of the primers was end-labeled with (g-33P)ATP and T4 polynucleotide kinase. The cycling conditions were initial denaturation at 94°C for 3 min, 35 three-step cycles at 94°C for 1 min, 52°C for 30 s, and 72°C for 1 min, followed by a final cycle at 72°C for 7 min. RadiolabeledPCR products were denatured and electrophoresed on either 7% polyacrylamide gels containing 7 M urea (SSLP) or on 6% polyacrylamide gels containing 5% glycerol (SSCP).

FIGURE 1. Breeding strategy for propagation of F1 progeny resulting from crosses of brown deletion mutant, b/b11R30M, and C3H/HeJ parental strains.

Measurement of TNF bioactivity in vivo Parentals and F1 progeny were injected i.p. with 25 mg protein-free (,0.008%) Escherichia coli K235 LPS in 0.5 ml pyrogen-free saline. Mice were bled 90 min later. Serum TNF bioactivity was measured in a L929 cytotoxicity assay (28).

Measurement of NF-kB translocation in vitro Thioglycollate-elicited macrophages derived from C3H/HeJ and C3H/OuJ mice were cultured as described (29). Following stimulation with LPS or human rIL-1b (National Cancer Institute Biological Resources Branch, National Institutes of Health, Bethesda, MD), nuclear extracts were prepared and analyzed by EMSA (29).

Results and Discussion “Brown deletion mutants,” originally derived as a result of radiation mutagenesis studies, have large chromosome 4 deletions that encompass the brown coat color (b) gene (24 –26), which is linked to Lps (6). These mice were generated in F1 animals from Oak Ridge inbred strains, C3H/RL and 101/RL, and must be maintained in a heterozygotic state with a normal, “balancer” copy of chromosome 4 to insure viability. The deletion exhibited by the b11R30M strain (;9 cM in size) was predicted to eliminate the Lps gene based on mapping data (12, 24), whereas an equivalently large deletion carried by the b13R75M strain should not affect expression of Lps because the proximal breakpoint of the b13R75M deletion is distal to the proximal breakpoint in b11R30M mice (24). Hence, when b/b11R30M (Lpsn/Lps0) mice are crossed with C3H/ HeJ (Lpsd/Lpsd) mice, two genotypes at the Lps locus are predicted (Fig. 1): F1 progeny that are either heterozygotic (Lpsn/Lpsd) or hemizygous for expression of Lpsd (i.e., Lps0/Lpsd), due to a lack of an Lps allele on the deleted region of chromosome 4. Thus, half the progeny would be predicted to express intermediate to full LPS sensitivity (as reported for Lpsn 3 Lpsd F1 progeny; reviewed in


Materials and Methods

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Ref. 3), while the remaining progeny (Lps0/Lpsd) should be completely unresponsive due to the expression of a single, defective copy of Lpsd only. Conversely, in crosses with the control deletion mutant, b13R75M, all F1 mice would be expected to carry one copy of Lpsn, derived either from the “balancer” chromosome or from the chromosome that contained the deletion distal to Lps. To rule out the possibility that the b11R30M strain was a “skipper” mutation (i.e., a deletion mutation that retains a small segment of the original genome, and in this case, the Lpsn allele), additional fine mapping using 28 novel markers and four publicly available chromosome 4 probes was conducted to type DNA samples from the b/b11R30M strain (Fig. 2, A and B). All 32 DNA markers including Tlr4 were deleted in the b11R30M strain, but not in the b13R75M strain, suggesting that a “skipper” mutation was highly unlikely. In addition, additional F1 mice that were typed as Lpsd/Lps0 contained only the Pro to His mutation identified for the Lpsd allele, while Lpsn/Lpsd F1 mice expressed both alleles (Fig. 2C). This is evidenced by the fact that only two SSCP patterns, based on the distinctive migration of single stranded and double stranded PCR

products, are observed among F1 progeny: one that is C3H/HeJlike and one that is a combination of the C3H/HeJ and C3H/HeN or 101 patterns. Therefore, F1 progeny, generated by crossing the b/b11R30M or w 13R75M B /b strains with C3H/HeJ mice were genotyped for markers that distinguished “balancer” chromosome 4 from the copy of chromosome 4 that contained the deletion, as well as C3H/HeJ chromosome 4 markers. Parentals and F1 progeny were injected with LPS, and TNF bioactivity was measured in sera collected 90 min later. Fig. 3 shows that, as expected, the C3H/HeJ Lpsd parental mice failed to exhibit any TNF induction, while both brown deletion parents responded to LPS to make TNF. However, each group of F1 mice, including the Lps0/Lpsd (b11R30M/Lpsd) progeny, gave an “intermediate” response (as evidenced by the similarities of the distribution of individual TNF responses among all four groups of F1 mice and a comparison of average TNF levels). These data allow for several conclusions. First, the presence of a normal Lpsn allele (e.g., Tlr4) is apparently not required for some degree of LPS responsiveness, as evidenced by the fact that Lps0/


FIGURE 2. High-resolution mapping of the mouse chromosome 4 region containing the Lps locus. A, Linkage map of mouse mid-chromosome 4. The 0.9-cM region from D4Nds9 to D4 Mit178 is expanded for clarity. The recombinational distances between loci have been reported previously (20). The two brown deletions (11R30M and 13R75M), as defined by Bell et al. (26), are represented by boxes below the map. Dashes indicate that the distal end of the deletion is not represented on the map. B, Diagram of the 1.7-Mb chromosomal segment encompassing the Lps mutation. Informative DNA markers used in the analysis are ordered along the chromosome and consist of microsatellites (D4 Mit, D4Nds, and D4 Mcg markers), STSs (3f1, 3f3, 3b11, 3a4, 1h9, and 2b9), BAC-end clones (363o12-t7, 277i15-sp6, 277i15-t7, 256f23-t7), microclone (D4Rck137), and known gene (Tlr4). D4 Mcg47, D4 Mcg49, and D4 Mcg54 are located between 256f23-t7 and D4 Mcg6. C, SSCP analysis of Tlr4 genomic region carrying the C to A transversion resulting in the Pro to His substitution at position 712. Lane 1, C3H/HeJ; lane 2, C3H/HeN; lane 3, 101; lane 4, water; lanes 5 to 20 are F1 progeny issued from a b/b11R30M (His/-) and C3H/HeJ (Pro/Pro) cross. Bands identified by an arrow are C3H/HeJ-specific. Two SSCP patterns are observed among the F1 progeny and discriminate between His/Pro (lanes 5 to 13 and 15 to 18) and -/Pro (lanes 14, 19, and 20) genotypes.

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FIGURE 3. In vivo TNF responses of parental and F1 progeny to LPS challenge. Parental and F1 mice were challenged with LPS, and the sera were analyzed for TNF bioactivity. The genotypes of the mice with respect to the Lps locus can be summarized as follows: C3H/HeJ (Lpsd/Lpsd), b/b11R30M (Lpsn/Lps0), Bw/b13R75M (Lpsn/Lpsn), b/C3H/HeJ (Lpsn/Lpsd), b11R30M/C3H/HeJ (Lps0/Lpsd), Bw/C3H/HeJ (Lpsn/Lpsd), b13R75M/C3H/HeJ (Lpsn/Lpsd). The numbers in parentheses indicate the numbers of mice responding with the same response when .1. The horizontal bar indicates the geometric mean of the responses.

Lpsd F1 mice responded to LPS equivalently to Lpsn/Lpsd mice to produce TNF in vivo. These data raise the possibility that LPS signaling receptors in addition to TLR4 may exist and are consistent with the possible utilization of alternate TLR-like molecules as LPS signaling receptors. In this context, TLR2 has been shown in transfection studies to confer LPS-signaling to LPS-nonresponsive cells (30, 31). In addition, the wide range of LPS-induced TNF activity in both F1 crosses (Lpsn/Lpsd and Lps0/Lpsd) supports the presence of other segregating genes that might influence the TNF response to LPS. The finding that F1 progeny issued from crosses between mouse strains with Tlr4 deletions (e.g., C57BL/ 10ScN or C57BL/10ScCR) and C3H/HeJ mice fail to respond to LPS suggests that the genetic background of the brown deletion mice used in this study possess additional genes that compensate for the lack of Tlr4 or that their ;9-cM deletion eliminates other gene(s) that normally contribute to a suppressed TNF response (18). More importantly, the data suggest that the Lpsd mutation, when present as a single copy, in the absence or presence of the normal Lpsn allele, fails to exert the same degree of hyporesponsiveness as seen in mice that are homozygous for Lpsd, suggesting a gene dosage effect for inhibition of LPS responsiveness (e.g., Lpsd exhibits a dominant negative effect on normal LPS responsiveness.) Because the missense mutation lies in the intracytoplasmic domain of TLR4, it is possible that downstream adapter and signaling molecules (e.g., MyD88 and IRAK1 (21)), are sequestered by the defective molecule, precluding their engagement by alternate TLR molecules or other receptors that share these signaling components. Because TLR4 and the IL-1 receptor type I share signaling

components (21), we measured IL-1 responsiveness in C3H/HeJ macrophages. Fig. 4 shows that LPS induced NF-kB translocation in the C3H/OuJ (Lpsn) macrophages, but not in the C3H/HeJ macrophages, as previously reported (29, 32). However, the response to rIL-1b was comparable in the two strains over a broad concentration range, consistent with an earlier observation that administration of rIL-1a to C3H/HeJ mice induced circulating CSF levels comparable to those observed in Lpsn mice (33). Hence, the C3H/ HeJ defect in TLR4 does not preclude normal IL-1 responsiveness. Whether the defective TLR4 serves as a “decoy” receptor (i.e., it binds ligand, yet fails to signal in Lpsd/Lpsd cells), analogous to the type II IL-1 receptor (34), or exerts its inhibitory effect by forming nonsignaling complexes with functional homologous or heterologous TLR molecules, are other potential mechanisms.

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FIGURE 4. Comparison of NF-kB translocation induced in macrophages derived from Lpsn (C3H/OuJ) and Lpsd (C3H/HeJ) mice. Macrophages were stimulated with rIL-1b or LPS. NF-kB translocation was measured in nuclear extracts by EMSA. The results represent one of two separate experiments.

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