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,
*
Department of Biochemistry, McGill University, Montreal, Canada;
Department of Biochemistry and Pediatrics, University of Ottawa, Ottawa, Canada; and
Apoptogen, Inc., Ottawa, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to their human and guinea pig counterparts, mouse
macrophages are not permissive to L. pneumophila replication
even though the bacteria still rapidly inhibit phagosome-lysosome
fusion soon after phagocytosis (reviewed in Ref. 9). The
A/J strain is an exception, however, as A/J inflammatory peritoneal
macrophages are highly permissive to L. pneumophila
replication in vitro, resulting in a 1000-fold increase in viable
bacteria during a 72-h infection, compared with macrophages from
nonpermissive mouse strains such as C57BL/6J, C3H, and DBA/2J
(10, 11). The permissiveness of A/J macrophages to
L. pneumophila replication provides a unique experimental
system to study the parallel human disease (9, 12).
Linkage studies have indicated that a single autosomal, recessive gene,
designated Lgn1 (11), determines macrophage
permissiveness to intracellular replication of L. pneumophila.
Lgn1 maps to the distal mouse chromosome 13 (13, 14, 15, 16, 17),
within a genetic interval of 0.32 centiMorgan (95% confidence
interval), defined distally by the genetic marker D13Die3
and proximally by D13Die6/D13Die26. Physical mapping studies
and assembly of a cloned contig of BAC and YAC clones for the region
suggest a minimal physical interval for Lgn1 of 
350 kb
(15, 16, 17, 18).
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 neuronal apoptosis inhibitory protein NAIP gene
(19, 20). There is one functional copy of NAIP
in the human genome and approximately two-thirds of type I SMA cases
are associated with its homozygous deletion (21). Although
it was later shown that the closely linked SMN gene is the
SMA-determining gene (22), NAIP remains a
strong candidate as a phenotypic modifier of SMN mutations.
The NAIP protein has been shown to inhibit apoptosis of neurons and
other cell types both in vitro and in vivo (23, 24). In
addition, NAIP has been shown to inhibit the proapoptotic cysteine
proteases known as caspases; in particular, caspases 3 and 7 have been
shown to interact with NAIP (A. MacKenzie, unpublished observations).
The mouse Lgn1 locus includes the Smn gene as
well as six copies of the Naip gene (Fig. 1
). Analysis of mouse brain RNA and other
tissues has revealed that at least three of the Naip copies
(Naip1, Naip2, and Naip3) encode
full-length mRNA and possibly functional proteins (25, 26). The tissue- and cell-specific expression of these
Naip mRNAs and proteins remain largely unknown.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred mouse strains A/J and C57BL/6J (B6) were initially purchased from The Jackson Laboratory (Bar Harbor, ME), and subsequently maintained as breeding colonies in our laboratories. Maintenance and experimental manipulation of the animals were performed according to the guidelines and regulations of the Canadian Council on Animal Care.
Isolation of thioglycolate(TGC)-elicited peritoneal macrophages
TGC-elicited inflammatory macrophages were obtained from the peritoneal cavity as previously described (10). Macrophages were elicited by i.p. injection of 1 ml of sterile 3% thioglycolate broth, and peritoneal exudate cells were obtained 72 h later by washing the peritoneal cavity with 10 ml of RPMI 1640 medium supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin (Life Technologies, Burlington, Canada). The TGC-elicited macrophages (2–3 x 107 cells) were plated in 80-cm2 flasks in RPMI 1640 medium supplemented with 10% heat-inactivated FBS (Life Technologies) and incubated for 16 h at 37°C, at which point nonadherent cells were removed by washing with PBS. Cells prepared in this way were used for RNA isolation, protein determination, and bacterial infections.
RNA expression
For Northern blotting experiments, a Clontech mouse multiple
tissue poly(A)+ RNA blot (2 µg/lane) was
hybridized with a partial Naip cDNA (clone ms6)
(25) from which 3'-untranslated sequences had been
removed. Clontech ExpressHyb solutions were used, and the hybridization
conditions were as recommended by the supplier. In other experiments
total cellular RNA was extracted from normal mouse tissues and
cultured cells using 6 M guanidium hydrochloride, and purified by
sequential ethanol precipitations and phenol-chloroform extractions.
Equal amounts of RNA (10 µg/lane) were separated on a 1% agarose gel
containing 0.66 M formaldehyde in MOPS buffer (40 mM
morpholinopropanesulfonic acid, 10 mM sodium acetate, and 10 mM EDTA,
pH 7.2) and blotted by capillarity onto a nylon membrane (GeneScreen
Plus, New England Nuclear) in 10x SSC (1x SSC is 0.15 M NaCl/0.015 M
sodium citrate). Following transfer, the RNA was cross-linked to the
blot by UV irradiation and by baking (2 h, 80°C). The blots were then
prehybridized overnight at 65°C in 0.75 M NaCl, 1% SDS, 10% dextran
sulfate, and denatured salmon sperm DNA (200 µg/ml). Hybridization
was performed for 20 h at 65°C in the same hybridization
solution without salmon sperm DNA. The probe used on total RNA blots
was a Naip cDNA subfragment (1.1-kb EcoRI
fragment encompassing exons 5–10 of Naip2, from 
cDNA
clone 8; see below). Hybridization probes were labeled with
[
-32P]dATP (sp. act., 3000 Ci/mmol;
DuPont-NEN, Boston, MA) by the random priming method (13).
Blots were washed at a final stringency of 0.5x SSC/0.1% SDS at
65°C for 30 min followed by autoradiography (Kodak Biomax MS film,
Eastman Kodak, Rochester, NY) at -80°C with an intensifying screen
for 1–8 days. Blots were stripped of probe by incubation in 0.1x
SSC/0.1% SDS (90°C, three times for 15 min each time) and
rehybridized to an actin cDNA control probe following the same
procedure.
For RT-PCR amplification of Naip cDNA sequences, cDNA synthesis and PCR amplification conditions were as previously described (30). Reverse transcriptase-directed first-strand cDNA synthesis was conducted using 2 mg of total cellular RNA, 100 ng of random hexamers, and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies). The hexamer/RNA mixture was first incubated for 5 min at 65°C, followed 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 corresponding to sequences in exon 2 that are conserved in all Naip isoforms, according to Scharf et al. (16) (exon 2F, 5'-GCTCTAGATCATGGACGCCACAGGAGATG-3'; exon 2R, 5'-CCGCTCGAGATGTCCCATGGGCATAAAATGGC-3'). Exon 4 sequences from all Naip transcripts were PCR-amplified using primer pairs corresponding to sequences in exons 3 and 5 that are conserved in all Naip isoforms (exon 3, 5'-GCTCTAGAGTAAAAGGGACACTGTGCAG-3' and a reverse primer on exon 5 5'-CCGCTCGAGTAATTCTCTTCTGACCCAGG-3'). Amplification products were gel-purified and subcloned in plasmid vector pBluescript, using restriction enzyme sites included in the oligonucleotide primers (underlined). The nucleotide sequence of 20 independent clones from each PCR amplification was determined and used to identify the Naip transcripts expressed in macrophages, using diagnostic sequence polymorphisms in exons 2 and 4 unique to each Naip copy and reported by Scharf et al. (16) and Yaraghi et al. (25).
The presence and identity of Naip mRNA transcripts expressed
in macrophages were also investigated by screening a cDNA library. For
this, a mouse macrophage cDNA library in bacteriophage vector gt11
(oligo(dT)-primed; Clontech, Palo Alto, CA) was screened with a
Naip cDNA (ms6 clone, without the 3'-untranslated region)
(25). Positive phage clones were plaque-purified, and
their inserts were characterized by restriction enzyme digestion, by
hybridization to different isoform-specific and nonspecific
Naip cDNA probes and ultimately by nucleotide sequencing of
cDNA inserts positive for exon 2 or exon 4.
Immunoblotting
Cultured cells and primary macrophages were collected and resuspended in a buffer consisting of 20 mM HEPES (pH 7.6), 150 mM NaCl, 0.5 mM DTT, 0.2 mM EDTA, 0.2 mM EGTA, 25% glycerol, and protease inhibitors (2 µg/ml aprotinin, 4 µg/ml leupeptin, 2 µg/ml pepstatin A, and 100 µg/ml PMSF) (31). Cells were then lysed by sonication (20 s, on ice), and unbroken cells and nuclei were eliminated by centrifugation (5 min, 2000 x g). The protein concentration was measured using a commercial reagent based on BCA staining (Pierce, Rockford, IL), using BSA as an internal standard. For protein extracts from tissues, organs were removed immediately after death, frozen in liquid nitrogen, and ground to a fine powder using mortar and pestle. The tissue powder was then resuspended in 10 ml/g of tissue of a solution consisting of 0.25 M sucrose and 0.03 M histidine (pH 7.2) supplemented with 2 mM EDTA and protease inhibitors. Tissues were homogenized using a glass potter with a tight-fitting Teflon pestle rotated at 1300 rpm. The homogenate was then centrifuged at 6000 x g for 15 min, and the supernatant corresponding to the soluble fraction was collected. Equal amounts of cellular protein were resolved on SDS-7.5% polyacrylamide gels, followed by electroblotting onto a nitrocellulose membrane (Schleicher & Schuell/Xymotech, Montreal, Canada). The blots were blocked overnight at 4°C in a solution containing 5% nonfat skim milk in 10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween-20. 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 specificity of the 1.7 antiserum has not yet been characterized, although high sequence conservation among Naip protein isoforms suggests that this antiserum may recognize several Naip isoforms (25). Alternatively, blots were analyzed with an anti-actin polyclonal antiserum (Sigm-Aldrich, Oakville, Ontario, Canada). Specific immune complexes were detected using a second goat anti-rabbit Ab (1/5000 dilution) coupled to peroxidase and were revealed by enhanced chemiluminescence (NEN). The intensity of the luminescent signal on Western blot was quantitated using a biological imaging system (BioImage, Ann Arbor, MI) and was standardized to the same signal obtained on each blot with the anti-actin Ab.
Immunoprecipitation
The TGC-elicited peritoneal macrophages were metabolically labeled with [35S]methionine, as we have previously described (31). Briefly, cells were incubated overnight in methionine-free medium (Life Technologies) containing 10% heat-inactivated dialyzed FBS, 5 mM L-glutamine, and [35S]methionine (sp. act., 1000 Ci/mmol; DuPont, Wilmington, DE) at a final concentration of 50 µCi/ml. Labeled cells were washed in cold PBS and lysed in 0.2 ml of 1% SDS/50 mM Tris, pH 7.5, followed by addition of 0.8 ml of 1% Triton X-100, 160 mM NaCl, 0.2% SDS, and 50 mM Tris, pH 7.5. For immunoprecipitation, labeled cell extracts (5–10 x 106 incorporated counts) were incubated for 16 h at 4°C in a 500-µl volume with polyclonal anti-Naip 1.7 antiserum or a rabbit preimmune serum (1/200 dilutions). Immune complexes were recovered by incubation for 2 h at 4°C with 1/1 mixture of protein A-Sepharose:protein G-Sepharose beads (Pharmacia Biotech, Piscataway, NJ), followed by five consecutive washes in a buffer containing 0.1% Triton X-100, 0.03% SDS, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and 5 mg/ml BSA and two washes in 150 mM NaCl. The final pellet was incubated at room temperature in Laemmli sample buffer for 10 min. Immune complexes were then analyzed by SDS-PAGE on a 7.5% polyacrylamide gel. Fluorography was performed using a commercially available amplifier (Amplify, Amersham) as recommended by the manufacturer. The gel was dried and exposed for 2 wk at -80°C.
Infection of macrophages in vitro
L. pneumophila Philadelphia-1 strain (serogroup 1, ATCC 33152, American Type Culture Collection, Manassas, VA) was used and was obtained from the Centers for Disease Control (Atlanta, GA). The organism was passaged once i.p. in guinea pigs (Harley strain) before it was used in this study. Fresh isolates were obtained from the spleen on day 3 postinoculation and were grown on buffered charcoal yeast extract agar plates, which contained Legionella agar base (Difco, Detroit, MI) supplemented with L-cysteine (0.4 g/L) and ferric pyrophosphate (0.25g/L), followed by further incubation at 37°C for 72 h. The bacteria were harvested by scraping the surface of the agar, resuspended, and stored at -80°C in tryptic soy broth (Difco) supplemented with 20% (v/v) glycerol until use. In other experiments, an avirulent dotA L. pneumophila mutant was used (provided by Dr. H. A. Shuman, Columbia University, New York, NY). This mutant was propagated under conditions similar to those used for wild-type L. pneumophila. The TGC-elicited peritoneal macrophages were infected with either wild-type or dotA transposon mutant of L. pneumophila at a multiplicity of infection (MOI) of two bacteria per macrophage in antibiotic-free medium, according to the protocol described by Yoshida et al. (11). At predetermined times after infection, infected macrophages were washed with PBS and scraped off the tissue culture flask. Macrophages were recovered by centrifugation and lysed, and protein extracts were analyzed by SDS-PAGE and immunoblotting.
For infection with Salmonella typhimurium, a
temperature-sensitive, replication-defective S. typhimurium
mutant TS
27 was used (gift from Dr. A.D. O’Brien, Uniformed
Services University of the Health Sciences, Bethesda, MD 29814 U.S.A.).
The protocols for propagation of this strain, preparation of the
infectious inoculum, and infection of macrophages were as recently
described by our group (32), with the following
modifications. The inoculum was from a S. typhimurium
TS27 culture in TSB (OD600, 0.15), and the infection was with a MOI
of 10. Phagocytosis was allowed to take place for 90 min at 37°C
followed by extensive washing of extracellular bacteria.
For phagocytosis of inert particles, TGC-elicited peritoneal macrophages were fed a meal of latex beads (3 µm in diameter, diluted 1/50 in warm RPMI medium from stock suspension; Sigma, St. Louis MO) for 2 h at 37°C. Macrophages were then washed of nonphagocytosed beads and either harvested immediately or after a further 24 h incubation period as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Lgn1 gene region on mouse chromosome 13 contains a
minimum of six closely homologous copies of the Naip gene
(designated copies 1–6; Fig. 1
) (16, 25).To date,
full-length cDNA sequences have been reported for copies 1, 2, and 3
(GenBank accession nos. AF007769, AF102871, and AF135492), while
partial sequences of single exons have been reported for additional
Naip copies (16, 17, 25, 26). Recent
hybridization studies and sequencing of genomic clones suggest that
only three of the six Naip gene copies (copies 1, 2, and 3)
encode mRNAs that have the 5' sequences required for translation in
normal tissues (25).
To examine a possible association between Naip and the
Lgn1 locus, we first investigated possible Naip
mRNA expression in macrophages, the cell population known to
phenotypically express the genetic difference at Lgn1
(18). As isoform-specific Naip hybridization
probes have not been described and as full-length sequence data are not
yet available for the six Naip loci, hybridization probes
that are expected to cross-react with most, if not all, Naip
copies were used for this Northern blotting analysis (Fig. 2). The two hybridization probes used
overlap the BIR domains, which are highly conserved in the sequenced
Naip copies (93% identity) (25). The
Naip mRNA expression was most abundant in the intestinal
tract (Fig. 2B). Hybridization of a Northern blot containing
poly(A)+ mRNA identified readily detectable
Naip expression in macrophage-rich tissues (spleen, lung,
and liver), with lower expression in kidney and testis, while
expression was below detection levels in brain, heart, and skeletal
muscle. Using Northern blots containing total cellular RNA,
Naip expression was easily detected in primary, TGC-elicited
macrophages (Fig. 2B). The Naip mRNA was
expressed in macrophage cell lines J774A and RAW264.7 and was also
present in two mouse fibroblast cell lines (L and LTA; Fig. 2B). Treatment of RAW264.7 macrophages with IFN-
did not
affect Naip mRNA expression levels. Taken together, these
results indicate that Naip mRNA is expressed at readily
detectable levels in macrophage-rich organs, in elicited (TGC)
macrophages, and in two murine macrophage cell lines. In these cells,
Naip is detected as a 5- to 5.5-kb hybridizing species, a
size compatible with the known full-length sequence of Naip
copies 1, 2, and 3 (25, 26, 33). Interestingly, we
consistently noted on independent Northern blots a lower level of
Naip RNA expression in peritoneal macrophages from A/J mice
(Lgns) compared with B6
(Lgnr) mice; this was by a factor of
2.5-fold (Fig. 2B).
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Naip protein expression in tissues and macrophages
The Naip protein expression was next analyzed in tissues and cell
types positive for Naip mRNA expression. For this, we used a
rabbit anti-Naip polyclonal antiserum (antiserum 1.7) directed
against a fusion protein consisting of GST fused to a large central
portion of the predicted Naip1 protein. The immunoblotting results in
Fig. 3 show that this antiserum detects
abundant Naip protein expression in intestinal extracts prepared from
either the ileum or the colon (sites of known mRNA expression; Fig. 2).
The Naip protein is also expressed in soluble tissue extracts from
spleen and is enriched in similar extracts from mature macrophages
(Fig. 3). The immunoreactive Naip species migrates at 150 kDa, a
molecular mass compatible with that expected from the predicted amino
acid sequence of Naip cDNAs (33). The relative
levels of Naip protein detected in Fig. 3 by immunoblotting are
comparable to the levels of Naip mRNAs detected in the same
tissues by Northern blotting (Fig. 2, A and
B).
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-6). Results from immunoblotting
experiments showed that A/J macrophages express considerably less Naip
protein than their B6 counterparts (Fig. 4B). This
difference was not due to uneven loading of proteins on the gel, as
very similar immunoreactive signals were obtained in these samples with
an anti-actin antiserum (Fig. 4B, bottom
panel). Independently, immunoprecipitation using metabolically
labeled macrophage extracts also showed lower Naip protein expression
in A/J compared with B6 macrophages (Fig. 4A). Thirdly,
several 2-fold dilutions of A/J and B6 macrophage extracts (10, 20, and
40 µg) were separated by SDS-PAGE and analyzed by Western blotting
(Fig. 5A), and the intensity
of the immunoreactive band (indexed optical density, IOD) was
quantitated using an Imaging station (BioImage). The IOD of the Naip
band vs the IOD of actin band was calculated for each sample (Fig. 5B) and was used to calculate a relative Naip expression
ratio in A/J and B6 populations. A 4-fold difference in Naip protein
expression was observed for B6 vs A/J macrophages (six independent
experiments; p < 0.01, by Student’s t
test). Finally, the reduced levels of Naip protein seen in A/J
macrophages compared with B6 cells are in agreement with differences in
Naip mRNA expression levels detected for these two
populations by Northern blotting (Fig. 2B).
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Modulation of Naip protein expression in macrophages
The level of Naip protein expression was monitored in macrophages
after phagocytic events and during infection with intracellular
parasites. In the first experiment, macrophages from A/J and B6 mice
were infected in vitro with a wild-type strain of L.
pneumophila (MOI of 2) for 2 h at 37°C. Following extensive
washing, cells were harvested at predetermined time points, and soluble
protein extracts were prepared and analyzed for Naip protein expression
by Western blotting. A representative experiment is shown in Fig. 6, but similar results were obtained in
four independent experiments. During L. pneumophila
infection, Naip protein expression was increased, with a progressive
increase during the first 6–12 h, at which point it peaked and
remained constant over the 48-h observation period. A maximum induction
of 4.5- to 5-fold was seen in B6 macrophages, as quantitated by imaging
and comparison to control immunoreactive signals obtained for actin.
The induction was specifically due to L. pneumophila
infection, because this increased Naip expression was not seen in
control, noninfected cultures similarly incubated for 48 h.
Finally, although the absolute level of Naip expression was lower in
A/J than in B6 macrophages at all time points, we noted a comparable
induction of Naip expression in A/J macrophages. These results indicate
that Naip protein expression in macrophages is increased following
L. pneumophila infection. To determine whether this
modulation of Naip expression was an active process mediated by live,
intracellular, and replicating L. pneumophila cells, similar
experiments were performed with an avirulent dotA L.
pneumophila mutant that does not inhibit phagosome maturation and
thus does not replicate intracellularly (34). The results
shown in Fig. 7A show that
Naip protein expression was also up-regulated in B6 macrophages after
infection with the avirulent dotA mutant by a factor of
4-fold. These results suggest that increased Naip expression in
macrophages is not in response to active intracellular replication of
L. pneumophila.
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27)
that does not replicate in primary macrophages or macrophage cell lines
at 37°C (32). Twenty-four hours after infection of B6
macrophages with the TS27 mutant, Naip induction was readily
detected in these cells, and after normalizing to actin expression
level, this induction was 3-fold (Fig. 7A). Finally, we
also tried to determine whether increased Naip expression detected
during S. typhimurium and L. pneumophila
infection was specific to intracellular bacteria or whether it may be
part of a more general macrophage response to phagocytic events. Thus,
B6 macrophages were fed a meal of inert Latex beads, and the level of
Naip expression was monitored at 0 and 24 h postphagocytosis (Fig. 7B). We noted little if any Naip induction immediately
following phagocytosis, while increased expression was detected after
24 h (Fig. 7B) by a factor of 3-fold. Together, these results indicate that Naip protein expression can be further increased in macrophages in response to ingestion of live bacteria or inert particles.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pathway (37). Induction of
apoptosis by L. pneumophila is through the activation of
caspase 3, which is detectable 2 h after infection and is maximal
at 3 h (9-fold increase in activity) (29). Avirulent
L. pneumophila mutants cannot induce either apoptosis or
caspase 3 activation. Specific inhibition of caspase 3 activity can
block both L. pneumophila-induced apoptosis and
cytopathogenicity (29). Whether the nonpermissive nature
of mouse macrophages (vs human cells) to L. pneumophila
infection is linked to resistance of murine cells to L.
pneumophila-induced apoptosis is currently not known.
Likewise, it remains to be determined whether the differential response
of susceptible A/J and resistant B6 macrophages to L.
pneumophila infection involves different macrophage apoptotic
responses in these two strains.
We have used a positional cloning approach to clone the Lgn1
locus on mouse chromosome 13. Combined genetic and physical mapping
studies by us (14, 18, 33) and others
(15, 16, 17) have narrowed the interval for Lgn1 to
a chromosome segment that includes up to six copies of the
Naip gene (Fig. 1). Naip is a very interesting
candidate for Lgn1 for the following reasons. 1) The
Naip gene cluster does not recombine with Lgn1;
2) infection and replication of L. pneumophila in permissive
human cells are associated with activation of caspase 3
(29) and induction of apoptosis (27, 28); 3)
Naip protein expression prevents apoptosis in a number of cell types
(23, 24); and 4) we have shown NAIP to be a potent
inhibitor of apoptosis largely and possibly exclusively through the
direct inhibition of caspase 3 with an IC50 in
the range of 20 nM (A. Mackenzie et al., unpublished observations).
Thus, we have studied the possible expression of Naip mRNA
and protein 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 sequencing, and
cDNA cloning from macrophages indicate that Naip2 is the
most abundantly expressed Naip copy (>50%) followed 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 difference at Lgn1, strengthening the candidacy
of Naip for Lgn1. In addition, we have observed
that Naip protein expression in macrophages can be further up-regulated
during a 48-h infection with L. pneumophila (Fig. 6) at low
MOI. This induction of Naip expression does not require intracellular
bacterial replication, because 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
macrophages. Should Naip also act as an inhibitor of apoptosis in
macrophages, then this response may increase the lifespan of
macrophages, possibly enhancing their net antimicrobial activity. Thus,
the genetic mapping data, the known function of Naip, and the role
proposed for apoptosis in L. pneumophila infection together
with the expression of Naip protein detected in cells phenotypically
expressing the genetic difference at Lgn1 and the modulation
of Naip protein expression observed in macrophages during phagocytosis
of inert particles or live bacteria combine to make Naip an attractive
candidate for Lgn1. In such a model, successful infection of
macrophages by L. pneumophila is dependent on the induction
of apoptosis. In mouse macrophages, constitutive or inducible Naip
expression may play a protective role by preventing induction of
apoptosis. This Naip-mediated inhibition of L. pneumophila
replication would be lost in A/J cells by a loss-of-function
mutation.
Southern blotting analyses of genomic DNA and RT-PCR analysis 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 mice that would result in the absence of expression of
individual Naip copies. We did detect a small, but
reproducible, 2- to 3-fold reduction in Naip mRNA levels in
A/J compared with B6 macrophages. It is difficult to conclude with
certainty that this difference is due to reduced expression of a
specific Naip copy in A/J as opposed to lower transcription of the
whole locus. The levels of constitutive and inducible Naip protein
expression were also analyzed in A/J
(Lgn1s) and B6
(Lgn1r) macrophages. It was consistently
observed that both constitutive and inducible Naip protein expression
levels were reduced by at least 4-fold in macrophages from A/J vs B6
mice (Figs. 5 and 6). The reduced protein expression in A/J macrophages
may be a result of decreased protein expression of a single or multiple
Naip isoforms in A/J macrophages compared with B6. This reduced Naip
expression may result in enhanced ability of L. pneumophila
to induce apoptosis (activation of caspase 3) and thus increased
permissiveness to infection. Additional experiments are required to
resolve this issue.
In conclusion, the expression of Naip protein in macrophages, both at rest and after phagocytosis, reported in this study suggests that Naip may play a key role in macrophage function, possibly by contributing to the apoptotic response of these cells. The possible participation of Naip in this process opens a new window for understanding the regulation of the apoptotic response in macrophages. These results also make the Naip cluster an attractive candidate for the host resistance locus Lgn1. A formal demonstration of this point awaits the creation of mouse mutant strains bearing loss- or gain-of-function mutations at this locus.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Philippe Gros, Department of Biochemistry, McGill University, Montreal, Canada H3G 1Y6. E-mail address:
3 Abbreviations used in this paper: SMA, spinal muscular atrophy; BAC, bacterial artificial chromosome; BIR, baculovirus inhibition of apoptosis protein repeat; IOD, indexed optical density; MOI, multiplicity of infection; NAIP, neuronal apoptosis inhibitory protein; SMN, survival motor neuron; TGC, thioglycolate; YAC, yeast artificial chromosome.
Received for publication August 13, 1999. Accepted for publication November 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection. Proc. Natl. Acad. Sci. USA 95:4646.This article has been cited by other articles:
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 A. Hebb, C. Moore, V Bhan, T Campbell, J. Fisk, H. Robertson, M Thorne, E Lacasse, M Holcik, J Gillard, et al. Expression of the inhibitor of apoptosis protein family in multiple sclerosis reveals a potential immunomodulatory role during autoimmune mediated demyelination Multiple Sclerosis, June 1, 2008; 14(5): 577 - 594. [Abstract] [PDF]
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 J. K.X. Maier, S. Balabanian, C. R. Coffill, A. Stewart, L. Pelletier, D. J. Franks, N. H. Gendron, and A. E. MacKenzie Distribution of Neuronal Apoptosis Inhibitory Protein in Human Tissues J. Histochem. Cytochem., September 1, 2007; 55(9): 911 - 923. [Abstract] [Full Text] [PDF]
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R. Asare, M. Santic, I. Gobin, M. Doric, J. Suttles, J. E. Graham, C. D. Price, and Y. Abu Kwaik Genetic Susceptibility and Caspase Activation in Mouse and Human Macrophages Are Distinct for Legionella longbeachae and L. pneumophila Infect. Immun., April 1, 2007; 75(4): 1933 - 1945. [Abstract] [Full Text] [PDF]
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K. A. Archer and C. R. Roy MyD88-Dependent Responses Involving Toll-Like Receptor 2 Are Important for Protection and Clearance of Legionella pneumophila in a Mouse Model of Legionnaires' Disease. Infect. Immun., June 1, 2006; 74(6): 3325 - 3333. [Abstract] [Full Text] [PDF]
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 A. Fortier, G. Min-Oo, J. Forbes, S. Lam-Yuk-Tseung, and P. Gros Single gene effects in mouse models of host: pathogen interactions J. Leukoc. Biol., June 1, 2005; 77(6): 868 - 877. [Abstract] [Full Text] [PDF]
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 M. Watarai, I. Derre, J. Kirby, J. D. Growney, W. F. Dietrich, and R. R. Isberg Legionella pneumophila Is Internalized by a Macropinocytotic Uptake Pathway Controlled by the Dot/Icm System and the Mouse Lgn1 Locus J. Exp. Med., October 15, 2001; 194(8): 1081 - 1096. [Abstract] [Full Text] [PDF]
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 W. F. Dietrich Using Mouse Genetics to Understand Infectious Disease Pathogenesis Genome Res., March 1, 2001; 11(3): 325 - 331. [Full Text]
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 L. Aravind, V. M. Dixit, and E. V. Koonin Apoptotic Molecular Machinery: Vastly Increased Complexity in Vertebrates Revealed by Genome Comparisons Science, February 16, 2001; 291(5507): 1279 - 1284. [Abstract] [Full Text]
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M. G. Endrizzi, V. Hadinoto, J. D. Growney, W. Miller, and W. F. Dietrich Genomic Sequence Analysis of the Mouse Naip Gene Array Genome Res., August 1, 2000; 10(8): 1095 - 1102. [Abstract] [Full Text]
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J. D. Growney and W. F. Dietrich High-resolution Genetic and Physical Map of the Lgn1 Interval in C57BL/6J Implicates Naip2 or Naip5 in Legionella pneumophila Pathogenesis Genome Res., August 1, 2000; 10(8): 1158 - 1171. [Abstract] [Full Text]
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