The Neuronal Apoptosis Inhibitory Protein (Naip) Is Expressed in Macrophages and Is Modulated After Phagocytosis and During Intracellular Infection with Legionella This information is current as pneumophila of September 24, 2021. Eduardo Diez, Zahra Yaraghi, Alex MacKenzie and Philippe Gros J Immunol 2000; 164:1470-1477; ; doi: 10.4049/jimmunol.164.3.1470 Downloaded from http://www.jimmunol.org/content/164/3/1470

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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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Neuronal Apoptosis Inhibitory Protein (Naip) Is Expressed in Macrophages and Is Modulated After Phagocytosis and During Intracellular Infection with Legionella pneumophila1

Eduardo Diez,* Zahra Yaraghi,† Alex MacKenzie,†‡ and Philippe Gros2*

Legionella pneumophila is an intracellular pathogen that causes Legionnaires’ disease in humans. Inbred mouse strains are uniformly resistant to L. pneumophila infection with the notable exception of A/J, where the chromosome 13 locus Lgn1 renders A/J macrophages permissive to L. pneumophila replication. The mouse Lgn1 region is syntenic with the spinal muscular atrophy (SMA) locus on human chromosome 5 and includes several copies of the neuronal apoptosis inhibitory protein (Naip) gene. We have analyzed a possible link among Lgn1, Naip, and macrophage function. RNA expression studies show that Naip (mostly copy 2) mRNA transcripts are expressed in macrophage-rich tissues, such as spleen, lung, and liver and are abundant in primary Downloaded from macrophages. Immunoblotting and immunoprecipitation analyses identify Naip protein expression in mouse macrophages and in macrophage cell lines RAW 264.7 and J774A. Interestingly, macrophages from permissive A/J mice express significantly less Naip protein than their nonpermissive C57BL/6J counterpart. Naip protein expression is increased after phagocytic events. Naip protein levels during infection with either virulent or avirulent strains of L. pneumophila increase during the first 6 h postinfection and remain elevated during the 48-h observation period. This enhanced expression is also observed in macrophages infected with

Salmonella typhimurium. Likewise, an increase in Naip protein levels in macrophages is observed 24 h after phagocytosis of Latex http://www.jimmunol.org/ beads. The cosegregation of Lgn1 and Naip together with the detected Naip protein expression in macrophages as well as its modulation after phagocytic events and during intracellular infection make it an attractive candidate for the Lgn1 locus. The Journal of Immunology, 2000, 164: 1470–1477.

egionella pneumophila is a facultative intracellular parasite is an exception, however, as A/J inflammatory peritoneal macro- that in humans can cause an acute form of pneumonia called phages are highly permissive to L. pneumophila replication in L Legionnaires’ disease (1). L. pneumophila enters macro- vitro, resulting in a 1000-fold increase in viable during a phages through a unique coiling phagocytosis mechanism (2) and 72-h infection, compared with macrophages from nonpermissive replicates within maturation-defective phagosomes (3), which do mouse strains such as C57BL/6J, C3H, and DBA/2J (10, 11). The by guest on September 24, 2021 not fuse to endosomes or lysosomes (4). These replicative phago- permissiveness of A/J macrophages to L. pneumophila replication somes are morphologically distinct and are associated with endo- provides a unique experimental system to study the parallel human plasmic reticulum membranes and dotted with ribosomes (5). Al- disease (9, 12). Linkage studies have indicated that a single auto- though certain Legionella proteins (dot/icm) have recently been somal, recessive gene, designated Lgn1 (11), determines macro- shown to play an important role in the inhibition of phagosome- phage permissiveness to intracellular replication of L. pneumo- lysosome fusion (6–8), the molecular mechanisms underlying suc- phila. Lgn1 maps to the distal mouse chromosome 13 (13–17), cessful intracellular survival and replication of L. pneumophila,in within a genetic interval of 0.32 centiMorgan (95% confidence particular the host proteins targeted for inhibition, remain largely interval), defined distally by the genetic marker D13Die3 and unknown. proximally by D13Die6/D13Die26. Physical mapping studies and In contrast to their human and guinea pig counterparts, mouse assembly of a cloned contig of BAC and YAC clones for the macrophages are not permissive to L. pneumophila replication region suggest a minimal physical interval for Lgn1 of ϳ350 kb even though the bacteria still rapidly inhibit phagosome-lysosome (15–18). fusion soon after phagocytosis (reviewed in Ref. 9). The A/J strain The murine chromosome 13 Lgn1 region is syntenic with the spinal muscular atrophy (SMA)3 locus on human chromosome 5, which includes the survival motor neuron SMN gene and the neu- *Department of Biochemistry, McGill University, Montreal, Canada; †Department of ronal apoptosis inhibitory protein NAIP gene (19, 20). There is one Biochemistry and Pediatrics, University of Ottawa, Ottawa, Canada; and ‡Apoptogen, Inc., Ottawa, Ontario, Canada functional copy of NAIP in the human genome and approximately Received for publication August 13, 1999. Accepted for publication November two-thirds of type I SMA cases are associated with its homozygous 16, 1999. deletion (21). Although it was later shown that the closely linked The costs of publication of this article were defrayed in part by the payment of page SMN gene is the SMA-determining gene (22), NAIP remains a charges. This article must therefore be hereby marked advertisement in accordance strong candidate as a phenotypic modifier of SMN mutations. The with 18 U.S.C. Section 1734 solely to indicate this fact. NAIP protein has been shown to inhibit apoptosis of neurons and 1 This work was supported by grants (to P.G.) from the Network of Centers of Ex- cellence (Canadian Genetic Diseases Network). P.G. is supported by a Senior Scien- tist award, and E.D. is supported by a studentship from the Medical Research Council of Canada. P.G. is an International Research Scholar of the Howard Hughes Medical 3 Abbreviations used in this paper: SMA, spinal muscular atrophy; BAC, bacterial Institute. artificial chromosome; BIR, baculovirus inhibition of apoptosis protein repeat; IOD, 2 Address correspondence and reprint requests to Dr. Philippe Gros, Department of indexed optical density; MOI, multiplicity of infection; NAIP, neuronal apoptosis Biochemistry, McGill University, Montreal, Canada H3G 1Y6. E-mail address: inhibitory protein; SMN, survival motor neuron; TGC, thioglycolate; YAC, yeast [email protected] artificial chromosome.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 1471

FIGURE 1. Schematic representation of the Lgn1 region on distal mouse chromosome 13. Genetic mapping has defined a 0.32-centiMorgan minimal interval for Lgn1 delineated proximally by Dl3Die6 (Die6) and distally by D13Die3 (Die3; 2/1270 recombinations each). Physical mapping has suggested that the size of the Lgn1 locus is between 125–350 kb and contains six copies of the Naip gene (mNaip, shown as squares). Copy 2 is the most closely linked to Smn, followed distally by copy 5 (25). Although a preliminary order has been proposed for five of the Naip copies by Scharf et al. in 1996 (16), the order of the remaining four Naip copies has not been established with certainty. Smn and M4f5 have recently been segregated from Lgn1 (17). The asterisk identifies the Naip2 copy most abundantly expressed in macrophages. The data shown were obtained from maps in the reports by Scharf et al. (16), Diez et al. (18), Yaraghi et al. (25), and Endrizzi et al. (17).

other cell types both in vitro and in vivo (23, 24). In addition, conditions were as recommended by the supplier. In other experiments

NAIP has been shown to inhibit the proapoptotic cysteine pro- total cellular RNA was extracted from normal mouse tissues and cultured Downloaded from teases known as caspases; in particular, caspases 3 and 7 have been cells using 6 M guanidium hydrochloride, and purified by sequential eth- anol precipitations and phenol-chloroform extractions. Equal amounts of shown to interact with NAIP (A. MacKenzie, unpublished obser- RNA (10 ␮g/lane) were separated on a 1% agarose gel containing 0.66 M vations). The mouse Lgn1 locus includes the Smn gene as well as formaldehyde in MOPS buffer (40 mM morpholinopropanesulfonic acid, six copies of the Naip gene (Fig. 1). Analysis of mouse brain RNA 10 mM sodium acetate, and 10 mM EDTA, pH 7.2) and blotted by cap- and other tissues has revealed that at least three of the Naip copies illarity onto a nylon membrane (GeneScreen Plus, New England Nuclear) in 10ϫ SSC (1ϫ SSC is 0.15 M NaCl/0.015 M sodium citrate). Following

(Naip1, Naip2, and Naip3) encode full-length mRNA and possibly http://www.jimmunol.org/ transfer, the RNA was cross-linked to the blot by UV irradiation and by functional proteins (25, 26). The tissue- and cell-specific expres- baking (2 h, 80°C). The blots were then prehybridized overnight at 65°C in sion of these Naip mRNAs and proteins remain largely unknown. 0.75 M NaCl, 1% SDS, 10% dextran sulfate, and denatured salmon sperm It has recently been observed that live L. pneumophila induce DNA (200 ␮g/ml). Hybridization was performed for 20 h at 65°C in the apoptosis in human macrophages in vitro, whereas heat-killed bac- same hybridization solution without salmon sperm DNA. The probe used teria or avirulent mutants do not (27–29). Of particular interest, in on total RNA blots was a Naip cDNA subfragment (1.1-kb EcoRI fragment ␭ ␭ vitro and in vivo studies have shown that L. pneumophila-induced encompassing exons 5–10 of Naip2, from cDNA clone 8; see below). Hybridization probes were labeled with [␣-32P]dATP (sp. act., 3000 Ci/ apoptosis is mediated by activation of caspase 3 (29). These stud- mmol; DuPont-NEN, Boston, MA) by the random priming method (13). ies have suggested that induction of apoptosis may be an important Blots were washed at a final stringency of 0.5ϫ SSC/0.1% SDS at 65°C for

pathogenicity determinant of L. pneumophila for intracellular sur- 30 min followed by autoradiography (Kodak Biomax MS film, Eastman by guest on September 24, 2021 vival in host macrophages. These observations together with the Kodak, Rochester, NY) at Ϫ80°C with an intensifying screen for 1–8 days. ϫ genetic and physical colocalization of Naip genes within the min- Blots were stripped of probe by incubation in 0.1 SSC/0.1% SDS (90°C, three times for 15 min each time) and rehybridized to an actin cDNA imal interval of Lgn1 and the demonstrated role of Naip proteins in control probe following the same procedure. inhibition of apoptosis in neuronal tissues have prompted us to For RT-PCR amplification of Naip cDNA sequences, cDNA synthesis analyze a possible link among Lgn1, Naip protein, and macro- and PCR amplification conditions were as previously described (30). Re- phage function. verse transcriptase-directed first-strand cDNA synthesis was conducted us- ing 2 mg of total cellular RNA, 100 ng of random hexamers, and 200 U of Moloney murine leukemia reverse transcriptase (Life Technologies). Materials and Methods The hexamer/RNA mixture was first incubated for 5 min at 65°C, followed Animals by addition of enzyme and further incubation at 37°C for 90 min. Exon 2 sequences from all Naip transcripts were PCR-amplified using primer pairs Inbred mouse strains A/J and C57BL/6J (B6) were initially purchased from corresponding to sequences in exon 2 that are conserved in all Naip isoforms, The Jackson Laboratory (Bar Harbor, ME), and subsequently maintained according to Scharf et al. (16) (exon 2F, 5Ј-GCTCTAGATCATGGACGC as breeding colonies in our laboratories. Maintenance and experimental CACAGGAGATG-3Ј; exon 2R, 5Ј-CCGCTCGAGATGTCCCAT manipulation of the animals were performed according to the guidelines Ј and regulations of the Canadian Council on Animal Care. GGGCATAAAATGGC-3 ). Exon 4 sequences from all Naip transcripts were PCR-amplified using primer pairs corresponding to sequences in exons 3 and Isolation of thioglycolate(TGC)-elicited peritoneal macrophages 5 that are conserved in all Naip isoforms (exon 3, 5Ј-GCTCTAGAGTA AAAGGGACACTGTGCAG-3Ј and a reverse primer on exon 5 5Ј- TGC-elicited inflammatory macrophages were obtained from the perito- CCGCTCGAGTAATTCTCTTCTGACCCAGG-3Ј). Amplification prod- neal cavity as previously described (10). Macrophages were elicited by i.p. ucts were gel-purified and subcloned in plasmid vector pBluescript, using injection of 1 ml of sterile 3% thioglycolate broth, and peritoneal exudate restriction enzyme sites included in the oligonucleotide primers (under- cells were obtained 72 h later by washing the peritoneal cavity with 10 ml lined). The nucleotide sequence of 20 independent clones from each PCR of RPMI 1640 medium supplemented with 100 U/ml penicillin and 100 amplification was determined and used to identify the Naip transcripts mg/ml streptomycin (Life Technologies, Burlington, Canada). The TGC- expressed in macrophages, using diagnostic sequence polymorphisms in ϫ 7 2 elicited macrophages (2–3 10 cells) were plated in 80-cm flasks in exons 2 and 4 unique to each Naip copy and reported by Scharf et al. (16) RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Life and Yaraghi et al. (25). Technologies) and incubated for 16 h at 37°C, at which point nonadherent The presence and identity of Naip mRNA transcripts expressed in mac- cells were removed by washing with PBS. Cells prepared in this way were rophages were also investigated by screening a cDNA library. For this, a used for RNA isolation, protein determination, and bacterial infections. mouse macrophage cDNA library in bacteriophage vector ␭gt11 (oli- RNA expression go(dT)-primed; Clontech, Palo Alto, CA) was screened with a Naip cDNA (ms6 clone, without the 3Ј-untranslated region) (25). Positive phage clones For Northern blotting experiments, a Clontech mouse multiple tissue were plaque-purified, and their inserts were characterized by restriction poly(A)ϩ RNA blot (ϳ2 ␮g/lane) was hybridized with a partial Naip enzyme digestion, by hybridization to different isoform-specific and non- cDNA (clone ms6) (25) from which 3Ј-untranslated sequences had been specific Naip cDNA probes and ultimately by nucleotide sequencing of removed. Clontech ExpressHyb solutions were used, and the hybridization cDNA inserts positive for exon 2 or exon 4. 1472 EXPRESSION OF THE APOPTOSIS INHIBITOR PROTEIN NAIP IN MACROPHAGES

Immunoblotting with either wild-type or dotA transposon mutant of L. pneumophila at a multiplicity of infection (MOI) of two bacteria per macrophage in antibi- Cultured cells and primary macrophages were collected and resuspended in otic-free medium, according to the protocol described by Yoshida et al. a buffer consisting of 20 mM HEPES (pH 7.6), 150 mM NaCl, 0.5 mM (11). At predetermined times after infection, infected macrophages were DTT, 0.2 mM EDTA, 0.2 mM EGTA, 25% glycerol, and protease inhib- washed with PBS and scraped off the tissue culture flask. Macrophages ␮ ␮ ␮ itors (2 g/ml aprotinin, 4 g/ml leupeptin, 2 g/ml pepstatin A, and 100 were recovered by centrifugation and lysed, and protein extracts were an- ␮ g/ml PMSF) (31). Cells were then lysed by sonication (20 s, on ice), and alyzed by SDS-PAGE and immunoblotting. unbroken cells and nuclei were eliminated by centrifugation (5 min, For infection with Salmonella typhimurium, a temperature-sensitive, ϫ 2000 g). The protein concentration was measured using a commercial replication-defective S. typhimurium mutant TS⌬27 was used (gift from reagent based on BCA staining (Pierce, Rockford, IL), using BSA as an Dr. A.D. O’Brien, Uniformed Services University of the Health Sciences, internal standard. For protein extracts from tissues, organs were removed Bethesda, MD 29814 U.S.A.). The protocols for propagation of this strain, immediately after death, frozen in liquid nitrogen, and ground to a fine preparation of the infectious inoculum, and infection of macrophages were powder using mortar and pestle. The tissue powder was then resuspended as recently described by our group (32), with the following modifications. in 10 ml/g of tissue of a solution consisting of 0.25 M sucrose and 0.03 M The inoculum was from a S. typhimurium TS⌬27 culture in TSB (OD600, histidine (pH 7.2) supplemented with 2 mM EDTA and protease inhibitors. 0.15), and the infection was with a MOI of 10. Phagocytosis was allowed Tissues were homogenized using a glass potter with a tight-fitting Teflon to take place for 90 min at 37°C followed by extensive washing of extra- pestle rotated at 1300 rpm. The homogenate was then centrifuged at cellular bacteria. ϫ 6000 g for 15 min, and the supernatant corresponding to the soluble For phagocytosis of inert particles, TGC-elicited peritoneal macro- fraction was collected. Equal amounts of cellular protein were resolved on phages were fed a meal of latex beads (3 ␮m in diameter, diluted 1/50 in SDS-7.5% polyacrylamide gels, followed by electroblotting onto a nitro- warm RPMI medium from stock suspension; Sigma, St. Louis MO) for 2 h cellulose membrane (Schleicher & Schuell/Xymotech, Montreal, Canada). at 37°C. Macrophages were then washed of nonphagocytosed beads and The blots were blocked overnight at 4°C in a solution containing 5% nonfat either harvested immediately or after a further 24 h incubation period as

skim milk in 10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween-20. described above. Downloaded from Membranes were then probed with the polyclonal anti-Naip antiserum 1.7 (used at a 1/2000 dilution). This rabbit polyclonal antiserum is directed against a mouse Naip/GST fusion protein containing 1.7 kb (nucleotides 2000–3660) of the Naip1 cDNA from clone ms6 (25). The isoform spec- Results ificity of the 1.7 antiserum has not yet been characterized, although high Naip mRNA expression in macrophages sequence conservation among Naip protein isoforms suggests that this an- The Lgn1 gene region on mouse chromosome 13 contains a min- tiserum may recognize several Naip isoforms (25). Alternatively, blots were analyzed with an anti-actin polyclonal antiserum (Sigm-Aldrich, imum of six closely homologous copies of the Naip gene (desig- http://www.jimmunol.org/ Oakville, Ontario, Canada). Specific immune complexes were detected us- nated copies 1–6; Fig. 1) (16, 25).To date, full-length cDNA se- ing a second goat anti-rabbit Ab (1/5000 dilution) coupled to peroxidase quences have been reported for copies 1, 2, and 3 (GenBank and were revealed by enhanced chemiluminescence (NEN). The intensity accession nos. AF007769, AF102871, and AF135492), while par- of the luminescent signal on Western blot was quantitated using a biolog- ical imaging system (BioImage, Ann Arbor, MI) and was standardized to tial sequences of single exons have been reported for additional the same signal obtained on each blot with the anti-actin Ab. Naip copies (16, 17, 25, 26). Recent hybridization studies and sequencing of genomic clones suggest that only three of the six Immunoprecipitation Naip gene copies (copies 1, 2, and 3) encode mRNAs that have the The TGC-elicited peritoneal macrophages were metabolically labeled with 5Ј sequences required for translation in normal tissues (25). [35S]methionine, as we have previously described (31). Briefly, cells were To examine a possible association between Naip and the Lgn1 by guest on September 24, 2021 incubated overnight in methionine-free medium (Life Technologies) con- locus, we first investigated possible Naip mRNA expression in taining 10% heat-inactivated dialyzed FBS, 5 mM L-glutamine, and macrophages, the cell population known to phenotypically express [35S]methionine (sp. act., 1000 Ci/mmol; DuPont, Wilmington, DE) at a final concentration of 50 ␮Ci/ml. Labeled cells were washed in cold PBS the genetic difference at Lgn1 (18). As isoform-specific Naip hy- and lysed in 0.2 ml of 1% SDS/50 mM Tris, pH 7.5, followed by addition bridization probes have not been described and as full-length se- of 0.8 ml of 1% Triton X-100, 160 mM NaCl, 0.2% SDS, and 50 mM Tris, quence data are not yet available for the six Naip loci, hybridiza- 6 pH 7.5. For immunoprecipitation, labeled cell extracts (5–10 ϫ 10 incor- tion probes that are expected to cross-react with most, if not all, porated counts) were incubated for 16 h at 4°C in a 500-␮l volume with polyclonal anti-Naip 1.7 antiserum or a rabbit preimmune serum (1/200 Naip copies were used for this Northern blotting analysis (Fig. 2). dilutions). Immune complexes were recovered by incubation for2hat4°C The two hybridization probes used overlap the BIR domains, with 1/1 mixture of protein A-Sepharose:protein G-Sepharose beads (Phar- which are highly conserved in the sequenced Naip copies (93% macia Biotech, Piscataway, NJ), followed by five consecutive washes in a identity) (25). The Naip mRNA expression was most abundant in buffer containing 0.1% Triton X-100, 0.03% SDS, 150 mM NaCl, 50 mM the intestinal tract (Fig. 2B). Hybridization of a Northern blot con- Tris-HCl (pH 7.5), and 5 mg/ml BSA and two washes in 150 mM NaCl. ϩ The final pellet was incubated at room temperature in Laemmli sample taining poly(A) mRNA identified readily detectable Naip expres- buffer for 10 min. Immune complexes were then analyzed by SDS-PAGE sion in macrophage-rich tissues (spleen, lung, and liver), with on a 7.5% polyacrylamide gel. Fluorography was performed using a com- lower expression in kidney and testis, while expression was below mercially available amplifier (Amplify, Amersham) as recommended by detection levels in brain, heart, and skeletal muscle. Using North- the manufacturer. The gel was dried and exposed for 2 wk at Ϫ80°C. ern blots containing total cellular RNA, Naip expression was easily Infection of macrophages in vitro detected in primary, TGC-elicited macrophages (Fig. 2B). The Naip mRNA was expressed in macrophage cell lines J774A and L. pneumophila Philadelphia-1 strain (serogroup 1, ATCC 33152, Ameri- can Type Culture Collection, Manassas, VA) was used and was obtained RAW264.7 and was also present in two mouse fibroblast cell lines from the Centers for Disease Control (Atlanta, GA). The organism was (L and LTA; Fig. 2B). Treatment of RAW264.7 macrophages with passaged once i.p. in guinea pigs (Harley strain) before it was used in this IFN-␥ did not affect Naip mRNA expression levels. Taken to- study. Fresh isolates were obtained from the spleen on day 3 postinocula- gether, these results indicate that Naip mRNA is expressed at tion and were grown on buffered charcoal yeast extract agar plates, which readily detectable levels in macrophage-rich organs, in elicited contained Legionella agar base (Difco, Detroit, MI) supplemented with L-cysteine (0.4 g/L) and ferric pyrophosphate (0.25g/L), followed by fur- (TGC) macrophages, and in two murine macrophage cell lines. In ther incubation at 37°C for 72 h. The bacteria were harvested by scraping these cells, Naip is detected as a 5- to 5.5-kb hybridizing species, the surface of the agar, resuspended, and stored at Ϫ80°C in tryptic soy a size compatible with the known full-length sequence of Naip broth (Difco) supplemented with 20% (v/v) glycerol until use. In other copies 1, 2, and 3 (25, 26, 33). Interestingly, we consistently noted experiments, an avirulent dotA L. pneumophila mutant was used (provided on independent Northern blots a lower level of Naip RNA expres- by Dr. H. A. Shuman, Columbia University, New York, NY). This mutant s was propagated under conditions similar to those used for wild-type L. sion in peritoneal macrophages from A/J mice (Lgn ) compared pneumophila. The TGC-elicited peritoneal macrophages were infected with B6 (Lgnr) mice; this was by a factor of ϳ2.5-fold (Fig. 2B). The Journal of Immunology 1473 Downloaded from FIGURE 2. Northern blot analysis of Naip mRNA expression. A, A blot containing polyadenylated RNA (2 ␮g/lane) from different mouse tissues was hybridized to a 32P-labeled Naip cDNA subfragment (Naip1; see Materials and Methods) under high stringency conditions (upper panel). The same blot was rehybridized to an actin cDNA probe (bottom panel). The hybridizing Naip (5 kb) and actin species (2 kb) are identified, and the positions of molecular size markers (in kilobases) is indicated to the left of the blot. B, Total cellular RNA (10 ␮g/lane) from mouse fibroblast cell lines (L and LTA), from macrophage cell lines RAW 264.7 (treated or not with IFN-␥), and J774 and from spleen, intestine, and TGC-elicited peritoneal macrophages from mouse strains A/J and C57BL/6J (B6) were separated on a denaturing formaldehyde gel and transferred to a hybridization membrane. The blot was then probed with a Naip2 cDNA probe overlapping exon 2 to exon 5 as described in Materials and Methods (top panel). The same blot was then rehybridized to a control http://www.jimmunol.org/ actin cDNA probe (bottom panel). The position of molecular size markers (in kilobases) is indicated to the left of the blot.

RT-PCR was used to determine which of the Naip copy mRNAs The immunoreactive Naip species migrates at ϳ150 kDa, a mo- are expressed in macrophages. The following strategy was applied. lecular mass compatible with that expected from the predicted Macrophage RNA was transcribed into total cDNA using random sequence of Naip cDNAs (33). The relative levels of hexamers and reverse transcriptase. Oligonucleotide primers cor- Naip protein detected in Fig. 3 by immunoblotting are comparable responding to perfectly conserved sequences in Naip copies 1–6 to the levels of Naip mRNAs detected in the same tissues by

flanking exon 2 and exon 4 were then used to amplify these por- Northern blotting (Fig. 2, A and B). by guest on September 24, 2021 tions of all Naip transcripts present. These products were eluted from gel as a single band and cloned, and the nucleotide sequences of 20 such clones were determined; previously published copy- specific single nucleotide polymorphisms (16, 25) were then used to identify which Naip isoforms are expressed in macrophages. This analysis revealed that 10 of the clones sequenced corre- sponded to Naip2 (50%), five to Naip1 (25%), and two to Naip3 (10%), with additional single clones corresponding to Naip4, Naip5, and Naip6. We also screened a macrophage cDNA library with a highly conserved Naip hybridization probe and character- ized the positive clones by restriction mapping and partial nucle- otide sequencing. Using this approach, we also noted that the ma- jority of clones analyzed corresponded to Naip2 (data not shown). Together, these results suggest that although multiple Naip RNA isoforms are expressed by macrophages, Naip2 appears to repre- sent the majority of Naip transcripts produced in these cells. This is in keeping with the tissue expression results of Yaraghi et al. (33) and the cDNA cloning experiments of Huang et al. (26). FIGURE 3. Naip protein expression in mouse tissues. Mouse intestinal Naip protein expression in tissues and macrophages segments corresponding to the ileum and colon as well as spleen were The Naip protein expression was next analyzed in tissues and cell dissected and homogenized to isolate a total soluble protein fraction. Like- types positive for Naip mRNA expression. For this, we used a wise, TGC-elicited mouse peritoneal macrophages were harvested and rabbit anti-Naip polyclonal antiserum (antiserum 1.7) directed lysed, and a total soluble protein extract was prepared. Proteins (60 ␮g) against a fusion protein consisting of GST fused to a large central were separated by SDS-PAGE on a 7.5% acrylamide gel and transferred to a nylon membrane. The immunoblot was incubated with a polyclonal rab- portion of the predicted Naip1 protein. The immunoblotting results bit anti-Naip antiserum (1.7; used at a 1/2000 dilution) and was revealed by in Fig. 3 show that this antiserum detects abundant Naip protein a secondary goat anti-rabbit antiserum. The size of the major immunore- expression in intestinal extracts prepared from either the ileum or active band detected (150 kDa) is in agreement with the predicted size of the colon (sites of known mRNA expression; Fig. 2). The Naip the Naip protein (GenBank accession nos. AF007769, AF102871, and protein is also expressed in soluble tissue extracts from spleen and AF135492). The positions of the molecular mass markers are indicated to is enriched in similar extracts from mature macrophages (Fig. 3). the left of the blot. 1474 EXPRESSION OF THE APOPTOSIS INHIBITOR PROTEIN NAIP IN MACROPHAGES Downloaded from

FIGURE 4. Naip protein expression in mouse macrophages. A, Mouse TGC-elicited peritoneal macrophages from A/J and B6 strains were met- abolically labeled with [35S]methionine for 16 h in methionine-free me- http://www.jimmunol.org/ dium supplemented with 10% dialyzed FBS, and total cell lysates were prepared in detergent-containing immunoprecipitation buffer (see Materi- als and Methods). Lysates were precleared by incubation with preimmune rabbit serum, followed by incubation with anti-Naip polyclonal Ab 1.7 (used at a 1/200 dilution). Immune complexes were recovered by incuba- FIGURE 5. Comparison of Naip protein expression in A/J and B6 mac- tion with protein A/protein G-Sepharose beads and were separated by SDS- rophages. A, Immunoblotting of 2-fold serial dilutions of soluble protein PAGE on a 7.5% acrylamide gel, followed by autoradiography. The im- extracts from A/J and B6 macrophages (10, 20, and 40 ␮g/lane), using munoreactive Naip protein migrates as a single band of apparent molecular either polyclonal anti-Naip ab (top panel) or anti-actin Ab (bottom panel). mass 150 kDa. B, Total cell lysates from TGC-elicited peritoneal macro- Conditions for immunoblotting were described in Fig. 3 and Materials and phages from A/J and B6 and from macrophage cell lines RAW 264.7 and Methods. B, The intensities of the immunoreactive Naip and actin signals by guest on September 24, 2021 J774A (60 ␮g/lane) were separated by SDS-PAGE and analyzed by im- were quantitated using a Bioimaging station. An IOD value was deter- munoblotting as described in Fig. 3. The top panel shows the immunoblot mined for each lane by calculating the ratios of the Naip to actin signals. probed with the anti-Naip Ab, and the bottom panel shows the same blot Results from six independent experiments were pooled and used to calcu- probed with an anti-actin Ab. The positions and sizes of protein molecular late an average of the relative Naip expression value for B6 compared with mass markers are indicated to the left of the blot. A/J macrophages (set at 1). The mean and SE are shown for B6, indicating that Naip protein levels are significantly higher in B6 than in A/J macro- phages (by Student’s t test, p Ͻ 0.01). The relative expression of Naip protein was compared in peri- toneal macrophages from susceptible A/J (Lgns) and resistant B6 (Lgnr) mice (Figs. 4-6). Results from immunoblotting experiments showed that A/J macrophages express considerably less Naip pro- Together, these results indicate that Naip is expressed at signif- tein than their B6 counterparts (Fig. 4B). This difference was not icant levels in mouse macrophages and macrophage cell lines. In- due to uneven loading of proteins on the gel, as very similar im- terestingly, macrophages from susceptible A/J mice express less munoreactive signals were obtained in these samples with an anti- Naip protein than their resistant B6 counterpart. actin antiserum (Fig. 4B, bottom panel). Independently, immuno- precipitation using metabolically labeled macrophage extracts also Modulation of Naip protein expression in macrophages showed lower Naip protein expression in A/J compared with B6 The level of Naip protein expression was monitored in macro- macrophages (Fig. 4A). Thirdly, several 2-fold dilutions of A/J and phages after phagocytic events and during infection with intracel- B6 macrophage extracts (10, 20, and 40 ␮g) were separated by lular parasites. In the first experiment, macrophages from A/J and SDS-PAGE and analyzed by Western blotting (Fig. 5A), and the B6 mice were infected in vitro with a wild-type strain of L. pneu- intensity of the immunoreactive band (indexed optical density, mophila (MOI of 2) for2hat37°C. Following extensive washing, IOD) was quantitated using an Imaging station (BioImage). The cells were harvested at predetermined time points, and soluble pro- IOD of the Naip band vs the IOD of actin band was calculated for tein extracts were prepared and analyzed for Naip protein expres- each sample (Fig. 5B) and was used to calculate a relative Naip sion by Western blotting. A representative experiment is shown in expression ratio in A/J and B6 populations. A 4-fold difference in Fig. 6, but similar results were obtained in four independent ex- Naip protein expression was observed for B6 vs A/J macrophages periments. During L. pneumophila infection, Naip protein expres- (six independent experiments; p Ͻ 0.01, by Student’s t test). Fi- sion was increased, with a progressive increase during the first nally, the reduced levels of Naip protein seen in A/J macrophages 6–12 h, at which point it peaked and remained constant over the compared with B6 cells are in agreement with differences in Naip 48-h observation period. A maximum induction of 4.5- to 5-fold mRNA expression levels detected for these two populations by was seen in B6 macrophages, as quantitated by imaging and com- Northern blotting (Fig. 2B). parison to control immunoreactive signals obtained for actin. The The Journal of Immunology 1475

FIGURE 6. Naip protein expression in A/J and B6 macrophages during infection with L. pneumophila. Peritoneal macrophages from A/J and B6 mice were infected with L. pneu- mophila Philadelphia 1 with an MOI of two bacteria per cell (see Materials and Methods). At 2, 4, 6, 12, 24, and 48 h postinfection, protein extracts were prepared, separated by SDS-PAGE, and analyzed by immunoblot- ting for Naip protein expression (top panel). The relative Naip expression level was cal- culated as described in Fig. 5, using actin as an internal standard (bottom panel). The rel- ative expression is further expressed as the increase above the Naip expression level (ϮSE) measured in A/J macrophages at the zero time point, before infection. These data

represent four independent experiments. Sta- Downloaded from tistically significant differences are indicated: †, p Ͻ 0.001 vs uninfected macrophages of the same strain. http://www.jimmunol.org/ induction was specifically due to L. pneumophila infection, be- and 24 h postphagocytosis (Fig. 7B). We noted little if any Naip cause this increased Naip expression was not seen in control, non- induction immediately following phagocytosis, while increased infected cultures similarly incubated for 48 h. Finally, although the expression was detected after 24 h (Fig. 7B) by a factor of ϳ3-fold. absolute level of Naip expression was lower in A/J than in B6 Together, these results indicate that Naip protein expression can macrophages at all time points, we noted a comparable induction be further increased in macrophages in response to ingestion of of Naip expression in A/J macrophages. These results indicate that live bacteria or inert particles.

Naip protein expression in macrophages is increased following L. by guest on September 24, 2021 pneumophila infection. To determine whether this modulation of Naip expression was an active process mediated by live, intracel- Discussion lular, and replicating L. pneumophila cells, similar experiments Apoptosis of phagocytes in response to intracellular infection has were performed with an avirulent dotA L. pneumophila mutant that been described for a number of pathogens (35). Apoptosis of in- does not inhibit phagosome maturation and thus does not replicate fected macrophages may be an advantageous strategy for a mul- intracellularly (34). The results shown in Fig. 7A show that Naip ticellular host, where this would have a net effect of limiting in- protein expression was also up-regulated in B6 macrophages after fection; on the other hand, from the parasite’s perspective, host cell infection with the avirulent dotA mutant by a factor of ϳ4-fold. death may be required to release intracellular organisms. Thus, a These results suggest that increased Naip expression in macro- number of intracellular pathogens have developed intracellular phages is not in response to active intracellular replication of L. survival strategies that are based on activation (Shigella flexneri) pneumophila. or inhibition (Chlamydia trachomatis, rickettsii) of host Additional experiments were conducted to determine whether macrophage apoptotic responses (35, 36). Successful intracellular enhanced Naip expression during L. pneumophila infection was survival and replication of L. pneumophila also appear to involve specific to this bacterium or was also seen with another unrelated modulation of host macrophage apoptosis. L. pneumophila induces intracellular parasite, S. typhimurium.AsS. typhimurium causes a apoptosis during infection of permissive, HL-60-derived human severe destructive infection in primary macrophages from B6 macrophages (27), but also in the human macrophage line U937 mice, it was not possible to assess the effect of wild-type S. typhi- and the alveolar epithelial cell line WI-26 (28). L. pneumophila- murium infection on protein expression at 24 or 48 h. Thus, for induced apoptosis occurs within 1–2 h of infection, can take place these experiments we used a temperature-sensitive, replication-de- in the absence of intracellular replication, and can also be induced fective mutant of S. typhimurium (TS⌬27) that does not replicate by extracellular bacteria. Induction of apoptosis in L. pneumo- in primary macrophages or macrophage cell lines at 37°C (32). phila-infected macrophages is mediated by activation of the Twenty-four hours after infection of B6 macrophages with the caspase pathway (28) and does not require a functional TNF-␣ TS⌬27 mutant, Naip induction was readily detected in these cells, pathway (37). Induction of apoptosis by L. pneumophila is through and after normalizing to actin expression level, this induction was the activation of caspase 3, which is detectable 2 h after infection ϳ3-fold (Fig. 7A). Finally, we also tried to determine whether and is maximal at 3 h (9-fold increase in activity) (29). Avirulent increased Naip expression detected during S. typhimurium and L. L. pneumophila mutants cannot induce either apoptosis or caspase pneumophila infection was specific to intracellular bacteria or 3 activation. Specific inhibition of caspase 3 activity can block whether it may be part of a more general macrophage response to both L. pneumophila-induced apoptosis and cytopathogenicity phagocytic events. Thus, B6 macrophages were fed a meal of inert (29). Whether the nonpermissive nature of mouse macrophages (vs Latex beads, and the level of Naip expression was monitored at 0 human cells) to L. pneumophila infection is linked to resistance of 1476 EXPRESSION OF THE APOPTOSIS INHIBITOR PROTEIN NAIP IN MACROPHAGES

In the current study we have shown that Naip mRNA is indeed expressed in macrophage-rich tissues, in particular in primary macrophages derived from them as well as in macrophage cell lines. Screening of a macrophage cDNA library with a Naip cDNA probe suggests a frequency of 0.03% of total cellular mRNA (data not shown), suggesting that Naip mRNA is actually quite abundant in macrophages. Results from RT-PCR studies, nucleotide se- quencing, and cDNA cloning from macrophages indicate that Naip2 is the most abundantly expressed Naip copy (Ͼ50%) fol- lowed by Naip1. These results are in agreement with recent tissue expression studies of the mouse Naip isoforms in normal tissues and macrophages (26, 33). Using a polyclonal anti-Naip antiserum, we show that Naip protein is expressed in macrophages (Figs. 3–7), the cell population phenotypically expressing the genetic dif- ference at Lgn1, strengthening the candidacy of Naip for Lgn1.In addition, we have observed that Naip protein expression in mac- rophages can be further up-regulated during a 48-h infection with L. pneumophila (Fig. 6) at low MOI. This induction of Naip ex- pression does not require intracellular bacterial replication, be- Downloaded from cause it still occurs when an avirulent dotA L. pneumophila mutant or an unrelated replication-defective S. typhimurium mutant is used for infection (Fig. 7). Interestingly, this Naip protein induction is also seen 24 h after phagocytosis of inert Latex particles by mac- rophages. Should Naip also act as an inhibitor of apoptosis in macrophages, then this response may increase the lifespan of mac- http://www.jimmunol.org/ FIGURE 7. Naip protein expression after infection with avirulent bac- rophages, possibly enhancing their net antimicrobial activity. teria and after phagocytosis of inert particles. A, Peritoneal macrophages Thus, the genetic mapping data, the known function of Naip, and from B6 mice were infected with either a temperature-sensitive replication the role proposed for apoptosis in L. pneumophila infection to- defective mutant of S. typhimurium (TS⌬27) or an avirulent dotA mutant of gether with the expression of Naip protein detected in cells phe- L. pneumophila, as described in Materials and Methods. Twenty-four hours after infection, cell extracts were prepared, separated by SDS-PAGE, notypically expressing the genetic difference at Lgn1 and the mod- and analyzed for Naip (top panel) and actin (middle panel) protein expres- ulation of Naip protein expression observed in macrophages sion by immunoblotting. The relative Naip expression was quantitated as during phagocytosis of inert particles or live bacteria combine to

described in Fig. 5, using the actin signal as an internal standard, and this make Naip an attractive candidate for Lgn1. In such a model, suc- by guest on September 24, 2021 is shown in the bottom panel. The Naip expression levels are expressed as cessful infection of macrophages by L. pneumophila is dependent a mean compiled from two independent experiments Ϯ SE. The asterisk on the induction of apoptosis. In mouse macrophages, constitutive Ͻ denotes a statistically significant increase (p 0.001) over levels in un- or inducible Naip expression may play a protective role by pre- infected macrophages. B, Peritoneal macrophages from B6 mice were fed venting induction of apoptosis. This Naip-mediated inhibition of L. a meal of Latex beads for2hat37°C, and protein extracts were prepared at that point or after washing the cells and further incubation for 24 h. The pneumophila replication would be lost in A/J cells by a loss-of- results are presented as described in A. function mutation. Southern blotting analyses of genomic DNA and RT-PCR anal- ysis of Naip transcripts from A/J and B6 macrophages failed to detect a major genomic deletion of part of the Naip cluster in A/J murine cells to L. pneumophila-induced apoptosis is currently not mice that would result in the absence of expression of individual known. Likewise, it remains to be determined whether the differ- Naip copies. We did detect a small, but reproducible, 2- to 3-fold ential response of susceptible A/J and resistant B6 macrophages to reduction in Naip mRNA levels in A/J compared with B6 macro- L. pneumophila infection involves different macrophage apoptotic phages. It is difficult to conclude with certainty that this difference responses in these two strains. is due to reduced expression of a specific Naip copy in A/J as We have used a positional cloning approach to clone the Lgn1 opposed to lower transcription of the whole locus. The levels of locus on mouse chromosome 13. Combined genetic and physical constitutive and inducible Naip protein expression were also ana- s r mapping studies by us (14, 18, 33) and others (15–17) have nar- lyzed in A/J (Lgn1 ) and B6 (Lgn1 ) macrophages. It was consis- rowed the interval for Lgn1 to a chromosome segment that in- tently observed that both constitutive and inducible Naip protein cludes up to six copies of the Naip gene (Fig. 1). Naip is a very expression levels were reduced by at least 4-fold in macrophages interesting candidate for Lgn1 for the following reasons. 1) The from A/J vs B6 mice (Figs. 5 and 6). The reduced protein expres- Naip gene cluster does not recombine with Lgn1; 2) infection and sion in A/J macrophages may be a result of decreased protein replication of L. pneumophila in permissive human cells are asso- expression of a single or multiple Naip isoforms in A/J macro- ciated with activation of caspase 3 (29) and induction of apoptosis phages compared with B6. This reduced Naip expression may re- (27, 28); 3) Naip protein expression prevents apoptosis in a num- sult in enhanced ability of L. pneumophila to induce apoptosis ber of cell types (23, 24); and 4) we have shown NAIP to be a (activation of caspase 3) and thus increased permissiveness to in- potent inhibitor of apoptosis largely and possibly exclusively fection. Additional experiments are required to resolve this issue. through the direct inhibition of caspase 3 with an IC50 in the range In conclusion, the expression of Naip protein in macrophages, of 20 nM (A. Mackenzie et al., unpublished observations). Thus, both at rest and after phagocytosis, reported in this study suggests we have studied the possible expression of Naip mRNA and pro- that Naip may play a key role in macrophage function, possibly by tein in macrophages. contributing to the apoptotic response of these cells. 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