Download Infection Salmonella PIR-B-Deficient Mice Are Susceptible to

PIR-B-Deficient Mice Are Susceptible to
Salmonella Infection
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of June 17, 2017.
Ikuko Torii, Satoshi Oka, Muneki Hotomi, William H.
Benjamin, Jr, Toshiyuki Takai, John F. Kearney, David E.
Briles and Hiromi Kubagawa
J Immunol 2008; 181:4229-4239; ;
doi: 10.4049/jimmunol.181.6.4229
http://www.jimmunol.org/content/181/6/4229
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References
The Journal of Immunology
PIR-B-Deficient Mice Are Susceptible to Salmonella Infection1
Ikuko Torii,2* Satoshi Oka,2* Muneki Hotomi,† William H. Benjamin, Jr.,* Toshiyuki Takai,‡
John F. Kearney,† David E. Briles,† and Hiromi Kubagawa3*
D
uring the last decade, many genes have been identified
that encode novel pairs of immunoreceptors which have
similar ectodomains but function to produce opposing
signals (1, 2). The pairing of activating and inhibitory receptors is
thought to be necessary for the initiation, amplification, and termination of immune responses. Paired Ig-like receptors of activating (PIR-A)4 and inhibitory (PIR-B) isoforms in rodents are
among the earliest paired receptors (3, 4). PIR-A associates noncovalently with the Fc receptor common ␥-chain, a transmembrane
signal transducer containing ITAM motifs in the cytoplasmic tail,
*Department of Pathology and †Department of Microbiology, University of Alabama
at Birmingham, Birmingham, AL 35294-3300, and ‡Department of Experimental Immunology, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan
Received for publication March 19, 2008. Accepted for publication July 15, 2008.
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 in part supported by National Institutes of Health grants AI14782
(J.F.K.), AI21548 (D.E.B.), AI42127 (H.K.). I.T. was an overseas Research Scholar
of the Ministry of Education, Culture, Sports, Science and Technology in Japan.
I.T. and M.H. initiated this study and developed the in vivo results (Figs. 1 and 2).
Serological (Fig. 3) and histological analyses (Fig. 4) were performed by S.O. and I.T.
and by I.T and H.K., respectively. S.O. performed the flow cytometric analysis (Fig.
5), the ex vivo studies (Fig. 6) and the statistical evaluation. T.T provided PIR-B⫺/⫺
mice. J.F.K. conducted the immunohistochemical analysis. W.H.B., D.E.B., and H.K.
designed the research and H.K. wrote the paper.
2
I.T. and S.O. contributed equally to this study.
3
Address correspondence and reprint requests to Dr. Hiromi Kubagawa, Department
of Pathology, University of Alabama at Birmingham, SHEL Room 506, 1825
University Boulevard, Birmingham, AL 35294-2182. E-mail address:
[email protected]
4
Abbreviations used in this paper: PIR-A, paired Ig-like receptors of activating isoform; PIR-B, paired Ig-like receptor of inhibitory isoform; BMM␸, bone marrowderived macrophages; BMPMN, bone marrow polymorphonuclear leukocytes; DC,
dendritic cells; GVH, graft-versus host; WT, wild type; ITSM, immunoreceptor tyrosine-based switch motif; LILR, leukocyte Ig-like receptors; LSEC, liver sinusoidal
endothelial cell; MFI, mean fluorescence intensity; MNC, mononuclear cells; M-CSF,
macrophage CSF; MOI, multiplicity of infection; SCV, Salmonella-containing vacuoles; SHP, Src homology region 2 domain-containing phosphatase.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
to form a cell activation complex (5– 8). In contrast, PIR-B contains three functional ITIM motifs in its cytoplasmic tail, which
negatively regulate cellular activity via Src homology region 2
domain-containing phosphatase 1 (SHP-1) and 2 (SHP-2) (9 –12).
PIR-A and PIR-B are expressed by many hematopoietic cell types,
including B cells, dendritic cells (DC), monocyte/macrophages,
neutrophils, eosinophils, mast cells, and megakaryocyte/platelets
(7, 12, 13). PIR are not expressed by T cells, NK cells, or erythrocytes, a feature that distinguishes mouse PIR from the human
PIR homologues, leukocyte Ig-like receptors (LILR) (originally
called the Ig-like transcripts, monocyte-macrophage Ig-like receptors, or CD85), some of which are expressed by T cells and NK
cells as well (14 –17). PIR is also expressed by lymphoid progenitors committed to differentiation to T cells, NK cells, and DC, but
not to B-lineage cells, suggesting a complex pattern of PIR expression during hematopoietic differentiation (18). In addition to
the hematopoietic cells, it has been recently shown that PIR-B is
expressed by neurons throughout the brain using in situ hybridization and protein blot analyses (19).
Several interesting findings regarding the PIR ligands and disruption of the Pirb gene (PIR-B⫺/⫺) have been demonstrated (20).
Like human LILR, both PIR-B and PIR-A react in surface plasmon
resonance assays with various MHC class I molecules at relatively
high affinity (21). Furthermore, the interaction between PIR and
MHC class I is found to occur at cis (i.e., on the same cell) and not
at trans (i.e., between different cells; Ref. 22). In addition to endogenous MHC class I, both PIR-B and PIR-A are found to recognize cell wall components of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, suggesting
multiple ligand recognition by PIR (23). Although PIR-B⫺/⫺ mice
exhibit normal T and B cell development except for slightly
higher levels of peritoneal B-1 cells, PIR-B⫺/⫺ B cells are hyperresponsive upon BCR ligation. PIR-B⫺/⫺ mice show significantly augmented IgG1 and IgE responses to T cell dependent
Ags and produce more IL-4 and IFN-␥ than wild-type (WT)
control mice, suggesting an enhanced Th2 response attributable
to the immaturity of PIR-B⫺/⫺ DC (24). PIR-B⫺/⫺ mice also
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Paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by many hematopoietic cells, including B lymphocytes and myeloid cells. To determine the functional roles of PIR-A and PIR-B in primary bacterial infection,
PIR-B-deficient (PIR-Bⴚ/ⴚ) and wild-type (WT) control mice were injected i.v. with an attenuated strain of Salmonella enterica
Typhimurium (WB335). PIR-Bⴚ/ⴚ mice were found to be more susceptible to Salmonella infection than WT mice, as evidenced
by high mortality rate, high bacterial loads in the liver and spleen, and a failure to clear bacteria from the circulation. Although
blood levels of major cytokines and Salmonella-specific Abs were mostly comparable in the two groups of mice, distinct patterns
of inflammatory lesions were found in their livers at 7–14 days postinfection: diffuse spreading along the sinusoids in PIR-Bⴚ/ⴚ
mice vs nodular restricted localization in WT mice. PIR-Bⴚ/ⴚ mice have more inflammatory cells in the liver but fewer B cells and
CD8ⴙ T cells in the spleen than WT mice at 14 days postinfection. PIR-Bⴚ/ⴚ bone marrow-derived macrophages (BMM␸) failed
to control intracellular replication of Salmonella in vitro, in part due to inefficient phagosomal oxidant production, when compared
with WT BMM␸. PIR-Bⴚ/ⴚ BMM␸ also produced more nitrite and TNF-␣ upon exposure to Salmonella than WT BMM␸ did.
These findings suggest that the disruption of PIR-A and PIR-B balance affects their regulatory roles in host defense to bacterial
infection. The Journal of Immunology, 2008, 181: 4229 – 4239.
4230
exhibit an exaggerated graft-vs-host (GVH) reaction, possibly
due to the interaction between PIR-A on PIR-B⫺/⫺ DC and
allogeneic MHC class I on donor T cells that leads to increased
production of IFN-␥, a critical cytokine in lethal GVH as well
as to increased proliferation of CTLs (21). PIR-B⫺/⫺ phagocytic cells are also shown to be hyperresponsive to integrin
ligation (25, 26). In the present study, we have determined the
influence of PIR-B deficiency on the pathogenesis of Salmonella infection in mice.
Materials and Methods
Mice
Bacterial strain, preparation, and inoculation
WB335, an LT2 strain of Salmonella enterica serovar Typhimurium (S.
typhimurium) attenuated due to a spontaneous mutation of rpoS, an RNA
polymerase ␴ factor (27, 28), was used in the present studies. This attenuated strain was used because C57BL/6 mice are hypersusceptible to WT
Salmonella (27, 28). WB335 was stored at ⫺80°C and a single colony of
WB335 from a blood agar plate was picked to LB broth and grown to an
OD of 0.5 at 600 nm and the resultant bacterial suspensions were frozen in
the presence of 10% glycerol at ⫺80°C as frozen infection stocks. For all
infection experiments, the bacterial dose of frozen stocks was re-evaluated
by thawing a vial and plating onto LB agar plates 1 day before inoculation.
Based on this estimation, enough vials to obtain needed CFU were thawed,
washed in pyrogen-free, sterile Ringer’s solution (Abbot Laboratories),
resuspended in the desired concentrations of bacteria, and injected in a 200
␮l of volume into the lateral tail vein of mice. The numbers of inoculated
bacteria were confirmed by CFU plate counts. The survival status of
WB335-infected mice was monitored at least twice a day during the course
of infection. In some experiments, WB335 (4 ⫻ 108 CFU) were heat-killed
by incubating at 60°C for 30 min, labeled with Alexa Fluor 488 fluorochrome (Invitrogen) according to the manufacturer’s instructions and
stored at ⫺80°C before use for phagocytic assays.
Enumeration of bacteria in organs
To determine the bacterial loads in blood and major organs (liver, spleen,
kidneys, and lungs), the blood was drawn into a heparin-coated syringe by
cardiac puncture from mice euthanized with CO2 and the organs were
removed aseptically and weighed. Serial dilutions of a blood aliquot in cold
sterile Ringer’s solution were plated on blood agar plates (Becton Dickinson Microbiology System) and incubated at 37°C overnight. The remaining blood samples were subjected to centrifugation at 3000 rpm for 10 min
at 4°C for plasma preparation. A portion of each removed organ was
weighed and homogenized in 1 ml of cold sterile Ringer’s solution and 50
␮l aliquots of the homogenate and its serial dilutions were plated on blood
agar plates. Visible CFU were determined after overnight incubation at
37°C and the bacterial load was expressed as CFU per the entire organ.
Assays for cytokines and Abs
The concentrations of cytokines (TNF-␣, IFN-␥, and IL-10) in plasma
were determined by using specific ELISA kits (Pierce Biotechnology) according to the manufacturer’s instructions. The titers of Salmonella-specific Abs of IgM, IgG, and IgA classes in the plasma collected on day 14
from infected mice were determined by ELISA. In brief, 96-well flat-bottom plates were precoated with 100 ␮l of heat-killed WB335 suspension
(108 CFU/ml) by centrifugation at 1500 rpm for 15 min at 4°C, washed,
blocked with 1% BSA/0.1% Tween 20 in PBS, and incubated sequentially
with serial dilutions of plasma samples and with AP-labeled goat Abs
specific for mouse IgM, IgG, or IgA (Southern Biotechnology Associates)
before incubation with the substrate p-nitrophenyl phosphate solution as
described previously (29). A pool of normal mouse sera and antisera were
used as standards and the results were expressed as the fold-increase over
the normal sera.
Histopathological and immunohistological analyses
A portion of each organ removed from each WB335-infected mouse was
fixed in neutral buffered, 10% formalin (Sigma-Aldrich) for at least 1 day,
dehydrated, embedded in paraffin, sectioned, and stained with H&E for
histopathological analysis. Another portion of each organ was embedded in
OCT compound, snap-frozen in liquid nitrogen, cut in 4 – 6 ␮m thickness
with a cryostat, fixed in acetone, and rehydrated in PBS. Sections were
incubated with appropriately diluted rabbit antisera against WB335 before
developing with FITC-labeled goat anti-rabbit Ig Ab without cross-reactivity to mouse Ig (Southern Biotechnology Associates). The immunostained tissue sections were examined under a Leica/Leitz DMRB fluorescence microscope equipped with appropriate filter cubes (Chroma
Technology). Images were acquired with a C5810 series digital color camera (Hamamatsu Photonic System). Images were processed with Adobe
PhotoShop and IP LAB Spectrum software (Signal Analytics Software).
Immunofluorescence analysis of cells
Liver and splenic tissues were removed during the course of infection at the
indicated days and cut into small pieces before digestion with 0.1% collagenase (Sigma-Aldrich)/0.1% DNase I in HBSS containing 5% FCS. The
resultant liver cell suspension was first centrifuged at 700 rpm for 2 min at
4°C to remove hepatocytes and then at 1200 rpm for 10 min. The cell
pellets were resuspended in HBSS/5% FCS, overlaid onto Percoll gradient
(2 ml of cell suspension/6 ml of 25% Percoll/4 ml of 50% Percoll), and
centrifuged at 800 ⫻ g for 30 min at 4°C, before collecting the mononuclear cells (MNC) at the 25%/50% interface. Erythrocytes in the collagenase-treated splenic cell suspension were lysed in 0.15 M ammonium chloride buffer at pH 7.4. Both cell suspensions were passed through nylon
meshes to remove tissue debris and subjected to cell surface immunostaining as described previously (7). Briefly, cells were first incubated with
Ab93 mAb (rat ␥2a␭ isotype) to block the Fc␥RII/III (30) and then with the
following reagents: FITC- or PE-labeled mAbs specific for mouse CD4
(H129.19, rat ␥2a), CD8 (53– 6.7, rat ␥2a), CD11c (HL3, hamster ␥1), or
CD11b (M1/70, rat ␥2b) (BD Pharmingen), PE-labeled rat anti-mouse PIR
mAb (6C1, rat ␥1␬; Ref. 7), or biotin-labeled rat mAbs specific for mouse
CD19 (1D3, ␥2a) (BD Pharmingen) or liver sinusoidal endothelial cell
(LSEC; ME-9F1, ␥2a) (Miltenyi Biotec). Streptavidin-allophycocyanin
was used as a developing reagent for biotinylated mAbs. Rat anti-mouse
endoglin/CD105 mAb (clone 209701, ␥2a) (R&D Systems) was labeled
with Alexa Fluoro 488 (Invitrogen Molecular Probe). The controls included the isotype-matched irrelevant mAbs labeled with the corresponding fluorochromes or biotin. The stained cells were analyzed by the FACScalibur or FACSort flow cytometer with CellQuest software (BD
Pharmingen).
Phagocytosis assay
Bone marrow cells were obtained from the femurs of PIR-B⫺/⫺ and WT
C57BL/6 mice of the same age and sex. After lysing erythrocytes, bone
marrow cells were cultured at 3 ⫻ 106 cells/ml in RPMI 1640 containing
10% FCS and 10% macrophage CSF (M-CSF) conditioned medium, the
culture supernatant of the CMG1 cell line which was transfected with
mouse M-CSF cDNA and was kindly provided by Dr. Eric Brown (University of California at San Francisco, San Francisco, CA), for 3 days.
After removing nonadherent cells, the adherent cells were cultured in the
above fresh medium for an additional two days and harvested to be used as
bone-marrow-derived macrophages (BMM␸). Bone marrow polymorphonuclear leukocytes (BMPMN) were enriched by centrifugation at 1200 ⫻
g for 30 min at 25°C over Percoll gradients (3 ml of cell suspension/3 ml
of 62% Percoll/3 ml of 81% Percoll). BMM␸ or BMPMN were incubated
with the heat-killed, Alexa 488-labeled, opsonized WB335 at different ratios of bacteria/cell numbers from 10/1 to 1/10 for 30 min with shaking at
37°C. All opsonization procedures in this study were performed using normal fresh mouse sera. After quenching the fluorescence of the extracellular
WB335 with trypan blue at the final concentration of 125 ␮g/ml, the intracellular fluorescence of phagocytosed WB335 was assessed by FACSort
(31, 32).
Intracellular killing assay
For in vitro infection, exponential phase WB335 bacteria, which were opsonized and resuspended in DMEM containing 10% FCS without antibiotics, were added in triplicate at various multiplicity of infection (MOI)
into 96-well plates containing 3 ⫻ 105 BMM␸ or BMPMN per well, centrifuged briefly, and incubated at 37°C for 25 min under 5% CO2 before
addition of gentamicin at the final concentration of 100 ␮g/ml to kill the
extracellular WB335 for 1 h. After replacing the medium with DMEM/
10% FCS containing gentamicin (10 ␮g/ml), infected cells were cultured
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C57BL/6 mice and RAG-2-deficient mice on a C57BL/6 background
(RAG-2⫺/⫺) were purchased from The Jackson Laboratory. PIR-B⫺/⫺
mice on a C57BL/6 background were generated by one of us (T.T.; Ref.
23) and were bred and maintained in filter-topped isolator cages at our
animal facility. PIR-B⫺/⫺ mice were backcrossed with C57BL/6 mice for
at least nine generations. All animals of both sexes were used at 6 –12 wk
of age. All studies involving animals were conducted in accordance with
and after approval of the University of Alabama at Birmingham Institutional Animal Care and Use Committee.
ROLE OF PAIRED Ig-LIKE RECEPTORS IN INFECTION
The Journal of Immunology
4231
for another 2 or 24 h, washed, and lysed in 100 ␮l Triton X-100 before
CFU plate counts (33).
Assays for superoxide, nitrite, and TNF-␣ release
Phagosomal oxidant production
The above assay determines primarily extracellular superoxide as the 12
kDa cytochrome C molecule is likely excluded from the interior of the cell
due to its size (35). To determine oxidant production inside the phagosome
(36, 37), heat-killed WB335 bacteria (109 cells) were labeled with the
oxidant sensitive fluorescent dye OxyBURST Green H2DCFDA (2⬘,7⬘-
A
Statistical analysis
Data are recorded as the mean ⫾ 1 SD. Differences in group survival were
analyzed using Mantel Cox Logrank p test. All other simple comparisons
were performed with Student’s t test, with p ⱕ 0.05 considered to represent
statistical significance.
Results
PIR-B⫺/⫺ mice are more susceptible to Salmonella infection
than WT mice
To determine the role of PIR-B in the bacterium/host interaction,
we used the Salmonella enteric fever mouse model. S. typhimurium is known as a Gram-negative, facultative intracellular
pathogen capable of replication both outside and inside host cells.
The WB335 strain of S. typhimurium, which has a mutation of
RNA polymerase ␴ factor (rpoS) resulting in attenuated virulence
in susceptible strains of mice such as C57BL/6 (27, 28), was selected for this purpose. Both PIR-B⫺/⫺ and WT C57BL/6 mice of
the same age and sex were infected i.v. with various doses of the
attenuated WB335 organisms (103 to 6 ⫻ 106 CFU/mouse) in a
group of 10 –15 mice per each dose and their survival status was
monitored for three to four weeks after infection. When inoculated
with the high dose of bacteria (6 ⫻ 106 CFU/mouse), both groups
B
C
6×106 CFU
6×105 CFU
6×104 CFU
100
100
100
80
80
80
60
60
40
40
40
20
20
20
0
60
p=0.55
0
1
3
5
0
1
3
5
7
9
11 13 15 17 19 21
D
1
3
5
7
9
11 13
15
17
19 21
E
5×103 CFU
survival (%)
p=0.06
1×103 CFU
100
100
80
80
WT
60
60
p=0.01
p<0.01
40
40
20
20
PIR-B KO
0
0
1
3
5
7
9
11 13 15 17 19 21 23 25 27 29
1
3
5
7
9
11 13 15 17 19 21 23 25 27 29
days post-infection
FIGURE 1. Survival of mice from Salmonella infection. Age-matched, PIR-B⫺/⫺ (F) and WT (E) C57BL/6 male mice (n ⫽ 12 for A and B, n ⫽ 10
for C and D, and n ⫽ 15 for E) were infected i.v. with the indicated doses of attenuated strain (WB335) of S. typhimurium and survival was monitored
for 21 to 30 days. The y-axis indicates survival (%) and the x-axis indicates days after infection.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
BMM␸ (5 ⫻ 105 cells) or BMPMN (5 ⫻ 105 cells) were resuspended in
250 ␮l of HBSS containing 10 mM HEPES, 0.5 mM CaCl2, 1 mM MgCl2,
and 120 ␮M cytochrome C; plated in triplicate into polypropylene tubes;
and stimulated with or without live serum-opsonized WB335 at various
MOI or 162 nM PMA for 2 h (for BMM␸) or 15 min (for BMPMN) in
37°C shaking water bath. The respiratory burst reaction as measured by the
cytochrome C reduction was stopped by incubation on an ice bath for 10
min, followed by centrifugation at 2000 rpm for 5 min at 4°C and assessment of the supernatant absorbance at 550 nm. The OD values were converted to the nmoles of the reduced cytochrome C by using the extinction
coefficient of E550 nm ⫽ 2.1 ⫻ 104 M⫺1cm⫺1 (34). For nitrite production,
BMM␸ (105 cells) or BMPMN (5 ⫻ 105 cells) were plated in triplicate into
96-well plates and stimulated with or without heat-killed opsonized
WB335 at different concentrations or LPS (1 ␮g/ml) for 48 h (for BMM␸)
or 24 h (for BMPMN), before collection of the supernatants. The concentration of nitrite in the resultant culture supernatants was measured as an
index of NO synthase activity by the Griess Reagent system (100 –1.56 ␮M
for sensitivity; Promega) according to the manufacturer’s instructions. For
TNF-␣ release, BMM␸ (2 ⫻ 105 cells) and BMPMN (5 ⫻ 105 cells) were
plated in triplicate into 24-well and 96-well plates, respectively, and stimulated for 24 h with heat-killed opsonized WB335 at the bacteria/cells ratio
of 10. The TNF-␣ in the culture supernatants was measured by ELISA as
described above.
dichlorodihydrofluorescein diacetate; Molecular Probes) according to the
manufacturer’s instructions. Labeled bacteria were washed twice, resuspended in PBS and opsonized before incubation with BMM␸ at different
ratios of bacteria/BMM␸ numbers for 2 h at 37°C with shaking. After
stopping the reaction by incubation iced bath for 10 min, the fluorescence
of BMM␸ that engulfed H2DCFDA-WB335 bacteria was analyzed by
FACSort.
4232
ROLE OF PAIRED Ig-LIKE RECEPTORS IN INFECTION
A
6×106 CFU day2
Bacterial loads (Log10 CFU / organ)
10
B
PIR-B⫺/⫺ mice are incapable of controlling bacteria replication
in vivo
To determine the tissue localization of inoculated bacteria, three to
five mice in each dose group were sacrificed during the course of
infection and the bacterial loads in various organs were assessed.
In mice receiving higher doses of bacteria (⬎6 ⫻ 104 CFU/
mouse), both PIR-B⫺/⫺ and WT mice exhibited similar high bacterial burdens at 2 and 3 days postinfection in all tissues examined
including the blood (Fig. 2, A–C). By contrast, in mice receiving
low doses of bacteria (5 ⫻ 103 CFU), PIR-B⫺/⫺ mice were found
to have more bacteria than WT mice in the liver and spleen at 7
days postinfection (Fig. 2E). Furthermore, the bacterial burden in
those tissues of PIR-B⫺/⫺ mice reached ⬃5 ⫻ 107 CFU, close to
the maximum number of bacteria recovered from live mice. Bacteremia was detected at 3 days (2/5 mice) and 7 days (2/3 mice)
postinfection in PIR-B⫺/⫺ mice, but not in WT mice, consistent
with the high mortality of PIR-B⫺/⫺ mice. In the one PIR-B⫺/⫺
mouse that survived for the entire 30 day period, bacteria were
undetectable in the blood (Fig. 2F). These findings suggest that the
high susceptibility of PIR-B⫺/⫺ mice to Salmonella infection is
associated with a failure to control bacterial replication in target
organs.
Mostly comparable blood levels of cytokines and Abs in both
groups of mice
The ability of Salmonella to survive and/or replicate inside macrophages is dependent on the activation state of the host cells that
are affected by host cytokines. To determine the role of cytokines
in the susceptibility of PIR-B⫺/⫺ mice to Salmonella infection, we
C
6×105 CFU day3
10
9
10
9
8
8
8
7
7
7
6
6
6
5
5
5
4
4
4
3
3
3
2
2
2
1
1
1
0
0
Liver
D
Spleen Blood Kidneys Lungs
5×103 CFU day3
9
0
Liver
E
Spleen Blood Kidneys Lungs
Liver
F
5×103 CFU day7
10
10
9
9
8
8
8
7
7
7
6
6
6
5
5
5
4
4
4
3
3
3
2
2
2
1
1
1
0
0
Liver
Spleen
Blood Kidneys Lungs
6×104 CFU day3
Spleen Blood Kidneys Lungs
5×103 CFU day30
10
p=0.04
p<0.01
9
0
Liver
Spleen Blood Kidneys Lungs
Liver
⫺/⫺
Spleen Blood Kidneys Lungs
FIGURE 2. Bacterial titers in various tissues of mice infected with S. typhimurium WB335. PIR-B
(F) and WT (E) mice were infected i.v. with the
indicated CFU of WB335 organisms. Three (A–C, E, F) or five (D) mice were sacrificed at the indicated days after infection except for one PIR-B⫺/⫺ mouse
at 30-day postinfection. The bacterial loads (CFU) in the indicated tissues were assessed as described in Materials and Methods. The Log10 values of
WB335 CFU per organ represent individual mice. The geometric mean values (O) and the detection limits are indicated (䡠䡠䡠䡠). The total blood volume (ml)
was determined as one thirteenth of the body weight (gm).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
of mice died within 3 days postinfection (Fig. 1A). When injected
with 10- or 100-fold fewer bacteria, all PIR-B⫺/⫺ mice and most
WT mice died within two weeks after infection (Fig. 1, B and C).
However, a small proportion (10 –20%) of the WT mice survived
for the entire 3 wk test period. A more striking survival difference
was observed in the mouse groups receiving lower doses of bacteria (ⱕ 5 ⫻ 103 CFU/mouse). With one exception, PIR-B⫺/⫺
mice receiving 5 ⫻ 103 CFU died over a 4-wk period starting at 4
days postinfection, whereas many control mice survived during
this time period (Fig. 1D). Furthermore, many of the infected PIRB⫺/⫺ mice showed signs of morbidity much sooner than the infected WT mice during the course of infection. When inoculated
with 103 CFU/mouse, most PIR-B⫺/⫺ mice survived for the first 2
wk but started to die in the third week, which resulted in a much
sharper slope of the mortality curve than that with 5 ⫻ 103 CFU
(Fig. 1E). Most WT mice survived during this 4-wk period. Although these survival experiments were conducted using male
mice, the same results were obtained with female mice, indicating
no sexual differences in the susceptibility to Salmonella infection
of PIR-B⫺/⫺ mice (data not shown). Notably, when the RAG-2⫺/⫺
mice, which are totally deficient in mature T and B cells, were
infected with the same WB335 strain at 5 ⫻ 103 CFU/mouse, all
five mice survived for the first 14 days after infection and then died
during the next 2 days, suggesting that similar to other strains of S.
typhimurium, both innate and adaptive immunity are important for
full protection against infection with the attenuated WB335 strain
(data not shown). Collectively, these findings indicate that PIRB⫺/⫺ mice are more susceptible than WT mice to infection with S.
typhimurium.
The Journal of Immunology
4233
p<0.01
40
IFNγ
20
16
3
2
4
1
0
B
day3
day7 day14 day30
5
8
20
day1
IL-10
4
12
0
(ng/ml)
TNFα
60
(ng/ml)
Plasma Concentration (pg/ml)
A
0
day1
day3
day7 day14 day30
day1
day3
day7 day14 day30
6
4
2
0
IgM
IgG
IgA
FIGURE 3. Plasma levels of cytokines and Abs in mice infected with S. typhimurium WB335. Plasma were prepared from PIR-B⫺/⫺ (f) or WT (䡺)
mice at the indicated days after infection with WB335 (5 ⫻ 103 CFU) and subjected to the assessments of cytokines (A) and anti-Salmonella Ab titers (B).
Results are expressed as arithmetic means ⫾ SD from five to six mice at day 1, five to ten mice at days 3 and 7, six to fifteen mice at day 14, and three
WT mice and one PIR-B⫺/⫺ mouse at day 30. The anti-Salmonella Abs in plasma samples from the infected animals are expressed as the fold increase
over the value obtained from pooled uninfected plasma.
determined the plasma levels of representative proinflammatory
(TNF-␣ and INF␥) and anti-inflammatory (IL-10) cytokines during the course of infection by ELISA. The concentrations of these
cytokines in the blood of infected animals were indistinguishable
between PIR-B⫺/⫺ and WT mice, except for the TNF-␣ level at 14
days postinfection where PIR-B⫺/⫺ mice showed significantly
higher than WT mice (Fig. 3, A). Notably, there was a large SD of
the concentrations of IL-10 in PIR-B⫺/⫺ mice at 14 days postinfection, suggesting considerable individual variability among the
six mice examined. Because Ab is shown to be important for full
protection against Salmonella infection, we measured the titer of
Abs to the WB335 strain in the plasma at 14 days postinfection.
The titers of three major classes of anti-Salmonella Abs did not
differ significantly between PIR-B⫺/⫺ and WT control mice (Fig.
3B). Thus, blood levels of cytokines and Abs could not be the
explanation for the susceptibility of PIR-B⫺/⫺ mice to infection
with Salmonella.
Distinct patterns of inflammatory foci and bacteria spreading in
liver between PIR-B⫺/⫺ and WT mice
To obtain further insight into the progression of Salmonella infection in PIR-B⫺/⫺ and WT mice, we examined the histology of the
major organs. Macroscopically, as expected, the spleen size markedly increased following infection and the extent of splenomegaly
was comparable in both groups of mice except at 30 days postinfection. The sizes of other organs after infection were also comparable in both groups (Fig. 4A). Microscopically, the white pulp
in the spleen was markedly expanded at 1 day postinfection. Numerous nodular inflammatory foci called typhoid nodules, which
were characterized by cellular aggregates of PMN (in early stage)
and macrophages (in later stage), were scattered predominantly in
the red pulp at day 3 of both the WT and PIR-B⫺/⫺ mice. By day
7, in addition to numerous granulomatous typhoid nodules, we
observed the obliteration of follicular margins and the marked pro-
liferation of sinus lining cells and sinus macrophages in both
groups of mice (Fig. 4B). These granulomatous typhoid nodules
and histological changes in the sinus were still prominent at 14
days and 30 days postinfection in both groups.
In the liver, small typhoid nodules consisting of PMN and
Kupffer cells or macrophages were visible in hepatic lobules as
early as 1 day postinfection and their incidence increased in the
next six days in both groups of mice. Quite distinctive pathological
changes were observed, however, at 7 days postinfection. In the
WT mice, the inflammatory cellular lesions were localized and
exhibited nodular distribution, whereas in the PIR-B⫺/⫺ mice, in
addition to granulomatous typhoid nodules, inflammatory mononuclear cells infiltrated along the sinusoids, resulting in a spreading
pattern and the dilatation of sinusoidal spaces accompanied with
narrowing of hepatic cords (Fig. 4C). These sinusoidal changes
were still observed in most of the PIR-B⫺/⫺ mice (4/5), but only
in a minority of the WT mice (1/5) at 14 days postinfection. At 30
days postinfection the inflammatory foci, including the granulomatous typhoid nodules, became smaller and infrequent in WT
mice, but were still frequently observed along with mild sinusoidal
infiltration in the surviving PIR-B⫺/⫺ mouse, indicating that the effects of Salmonella infection were more readily controlled in the WT
mice than in the PIR-B⫺/⫺ mice. In addition to the above changes,
fibrin deposition, thrombosis formation, and parenchymal necrosis
were also observed in both groups after 3 days postinfection.
To determine the bacterial distribution in livers, we performed
immunofluorescence analysis using Salmonella-specific Abs. Salmonella were found more diffusely along the sinusoids in PIRB⫺/⫺ mice, but were more localized in the WT mice (data not
shown), consistent with the patterns of inflammatory cellular infiltration on H&E stained slides. In lungs and kidneys, interstitial
inflammatory cell infiltration was observed in both groups after 7
days postinfection. Collectively, these findings suggest that while
the splenic histological changes following Salmonella infection are
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Antibody titer (Unit)
8
4234
ROLE OF PAIRED Ig-LIKE RECEPTORS IN INFECTION
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 4. Pathological changes in mice infected with S. typhimurium WB335. A, PIR-B⫺/⫺ (F) and WT (E) mice infected with WB335 Salmonella
(5 ⫻ 103 CFU) were sacrificed at the indicated days of postinfection and the weight index of each organ was estimated as: (the organ weight from infected
animals) ⫼ (the organ weight from uninfected animals). Results are expressed as arithmetic means ⫾ SD. B and C, H&E staining of spleen and liver at
3 and 7 days postinfection with 5 ⫻ 103 CFU.
The Journal of Immunology
4235
indistinguishable between the PIR-B⫺/⫺ and WT mice, the liver
exhibits quite distinctive inflammatory lesions with diffuse spreading along the sinusoids in PIR-B⫺/⫺ mice vs nodular localization
in WT mice.
Salmonella-infected PIR-B⫺/⫺ mice have more inflammatory
cells in liver, but fewer B cells and CD8⫹ T cells in spleen than
WT infected mice
A
To explore the cellular basis for high susceptibility of PIR-B⫺/⫺
mice to Salmonella infection, BMM␸ and BMPMN from PIRB⫺/⫺ and WT mice were examined for their in vitro responses to
B
6
4
R2
R1
R1
R1
4
Mac1
Mac1
Mac1+/Gr1- cells (R1)
CD4
CD19 Mac1+/Gr1- Mac1+/Gr1+
CD8
2
WT
PIR-B KO
0
Count
P<0.0 1
Mac1+/Gr1+ cells (R2)
WT
PIR-B KO
Count
2
0
LSEC
C
PIR
3
PIR
WT
PIR-B KO
2
p<0.01
1
p=0.02
400
300
200
100
0
p<0.01
WT
PIR-B KO
PIR-MFI of Mac1+/Gr1+ cells
WT uninfected
KO uninfected
WT infected
KO infected
PIR-MFI of Mac1+/Gr1- cells
Total number of cells (x108)
PIR-B KO
R2
Gr1
WT
PIR-B KO
WT
(x105)
P<0.0 1
Gr1
6
Total number of cells (x108)
PIR-B⫺/⫺ macrophages are unable to control intracellular
Salmonella growth ex vivo
1000
500
0
0
CD4
CD8
CD19
Mac1
Gr1
FIGURE 5. Immunofluorescence analysis of liver mononuclear cells and splenocytes from mice infected with S. typhimurium. A, Liver MNC from
PIR-B⫺/⫺ (f) or WT (䡺) mice at 7 days postinfection with WB335 (5 ⫻ 103 CFU) were incubated with fluorochrome-labeled mAbs specific for the
indicated Ags as well as with isotype-matched control mAbs before analysis with FACSort flow cytometer. Results are expressed as total number of cells
with the indicated phenotype per liver from four different mice in each group. Note two- to threefold increase in the number of Mac-1⫹/Gr-1⫺ M␸/Kupffer
cells and of Mac-1⫹/Gr-1⫹ PMN in PIR-B⫺/⫺ mice in compared with WT mice. B, Gates of Mac-1⫹/Gr-1⫺ (R1) and Mac-1⫹/Gr-1⫹ (R2) cell populations
in WT and PIR-B⫺/⫺ mice (top panel). Cell surface PIR intensity on the population of Mac-1⫹/Gr-1⫺ M␸/Kupffer cells (left) and of Mac-1⫹/Gr-1⫹ PMN
(right) from PIR-B⫺/⫺ (solid histogram) and WT (open histogram) mice (middle panel). MFI of PIR on M␸/Kupffer cells (left) and PMN (right) in WT
(䡺) and PIR-B⫺/⫺ (f) mice (bottom panel). Note twofold increase of cell surface PIR intensity on PIR-B⫺/⫺ M␸/Kupffer cells. C, Splenic MNC from
PIR-B⫺/⫺ (f) or WT (䡺) mice at 14 days postinfection with WB335 (5 ⫻ 103 CFU) or from uninfected PIR-B⫺/⫺ (0) or WT (u) mice were similarly
examined for the indicated cell populations by immunofluorescence. Results are expressed as mean ⫾ SD of the total number of cells per spleen from five
different mice in each group. Note no significant increase in the number of CD19⫹ B cells and CD8⫹ T cells in PIR-B⫺/⫺ mice after Salmonella infection.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Because the histological changes of liver were evident at 7 days
postinfection (see Fig. 4C), we examined by flow cytometry the
infiltrating cells, resident Kupffer cells, and sinusoidal endothelial
cells in the 7-day liver samples. As shown in Fig. 5A, the total
numbers of Mac-1⫹/Gr-1⫺ macrophage/Kupffer cells and of Mac1⫹/Gr-1⫹ PMN in the liver are two- to three-fold greater in PIRB⫺/⫺ mice as compared to WT mice. The cell surface PIR levels
were also compared among these cell populations using the 6C1
mAb, which recognizes both PIR-A and PIR-B. The surface PIR-A
level in the PIR-B⫺/⫺ macrophage/Kupffer cell, but not PMN,
population was enhanced two-fold in comparison with the surface
PIR-A and PIR-B levels in the WT cell populations, indicating in
turn the enhanced expression of PIR-A on PIR-B⫺/⫺ macrophage/
Kupffer cells (Fig. 5B). The total numbers of CD4⫹ T, CD8⫹ T,
and CD19⫹ B cells in the liver were comparable in both groups of
mice. Despite the pathological findings of bacterial spreading
along the liver sinusoids in PIR-B⫺/⫺ mice, the sinusoidal endothelial cell population defined by the expression of endoglin/
CD105 and LSEC Ag was also indistinguishable between the PIRB⫺/⫺ and WT mice.
In spleen, the total numbers of CD19⫹ B cells, CD4⫹ T cells,
CD8⫹ T cells, Mac-1⫹ macrophages, GR-1⫹ PMN, and CD11c⫹
dendritic cells were comparable between PIR-B⫺/⫺ and WT mice
at 7 days postinfection (data not shown). At 14 days postinfection,
however, the number of CD19⫹ B cells and, to a lesser extent, of
CD8⫹ T cells in PIR-B⫺/⫺ mice did not increase as markedly as
in WT mice after Salmonella infection (Fig. 5C). As expected,
Mac-1⫹ macrophages and Gr-1⫹ PMN were markedly increased
in both groups of mice. Other cell subsets were comparable in both
groups of mice. Collectively, these findings suggest that systemic
infection of Salmonella results in more inflammatory infiltrating
cells in liver and/or Kupffer cell reactions, enhanced expression of
PIR-A by macrophage/Kupffer cells, and insufficient increase of
splenic B cells and CD8⫹ T cells in PIR-B⫺/⫺ mice.
4236
ROLE OF PAIRED Ig-LIKE RECEPTORS IN INFECTION
WB335 bacteria. The ability to engulf fluorochrome-labeled, heatkilled WB335 by BMM␸ and BMPMN was comparable between
PIR-B⫺/⫺ and WT mice at different ratios of bacteria/phagocytic
cells (Fig. 6A). Upon in vitro infection with live WB335, bacterial
replication in BMPMN was indistinguishable in both groups of
mice at both 2- and 24-h postinfection at different MOI. In the
PIR-B⫺/⫺ BMM␸, however, more bacterial growth was evident at
A
24 h postinfection with a high MOI (i.e., 10.8) when compared
with WT BMM␸. These results show that PIR-B⫺/⫺ BMM␸ are
unable to control intracellular Salmonella replication, consistent
with the in vivo findings (Fig. 6B). The extracellular superoxide
anion release by phagocytes from WT and PIR-B⫺/⫺ mice upon in
vitro infection with heat-killed (not shown) or live WB335 or stimulation with 12-O-tetradecanoylphorbol-13-acetate or fMLP was
B
BMPMN
60
75
40
50
20
25
0
0
2
0.2
20
2
0.2
20
Salmonella / Phagocytic cells
8
24hr
p<0.01
WT
PIR-B KO
6
4
20
2
0
0
3.6
1.2
10.8
3.6
1.2
10.8
MOI
BMPMN
2hr
16
12
24hr
16
WT
PIR-B KO
12
8
8
4
4
WT
PIR-B KO
0
3.6
1.2
10.8
1.2
3.6
10.8
MOI
BMPMN
WT
PIR-B KO
48
4
D
3
%
100
PIR-B KO
32
% of control
BMMφ
WT
2
16
1
0
0
200
HBSS TPA
20
fMLP
HBSS TPA
Salmonella/Mφ
200
20
F
E
BMMφ
BMPMN
p<0.01
p<0.01
p<0.01
80
p=0.01
50
25
0
fMLP
60
3
2
40
p<0.01
BMMφ
0
1
10
100
Salmonella / Mφ ratio
BMPMN
1.0
1.0
WT
PIR-B KO
p=0.02
75
Salmonella/PMN
WT
PIR-B KO
TNFα (ng/ml)
Superoxide anion release (nmol/106 cells)
10
WT
PIR-B KO
0
C
Nitrite production (µM)
2hr
40
Number of bacteria (x103) / 3x105 cells
BMMφ
100
BMMφ
60
WT
PIR-B KO
p<0.01
0.5
0.5
1
20
0
0
0
1
10
100
Salmonella/Mφ ratio
LPS
(1µg/ml)
0
1
10
100
Salmonella/PMN ratio
⫺/⫺
LPS
(1µg/ml)
Untreated
HK-WB335
Untreated
HK-WB335
FIGURE 6. Ex vivo functions of phagocytes from PIR-B
and WT mice. A, Phagocytic activity. BMM␸ (left) and BMPMN (right) from PIR-B⫺/⫺
(f) and WT (䡺) mice were incubated with heat-killed, Alexa 488-labeled, opsonized WB335 at the indicated ratios of bacteria/phagocytic cells. After
quenching the extracellular fluorescence, the intracellular fluorescence of engulfed WB335 was determined by flow cytometry. B, Bactericidal activity.
Phagocytes described in A were infected with live opsonized WB335 bacteria at the indicated MOI, and the number of intracellular bacteria was enumerated
by CFU plate counts at the indicated time periods. C–F, Cells described in A were incubated with the indicated stimuli and the superoxide anion release
(C), the nitrite production (E) and the TNF-␣ production (F) were assessed as described in Materials and Methods. For phagosomal oxidant production
(D), BMM␸ from PIR-B⫺/⫺ and WT mice were incubated with oxidant sensitive dye-labeled WB335 bacteria at the indicated ratios and the fluorescence
of BMM␸ engulfing the dye-labeled WB335 was analyzed by FACSort. A typical profile at the Salmonella/BMM␸ ratio of 100 is shown (left panel) and
the values of [100 ⫻ (MFI of PIR-B⫺/⫺ BMM␸/MFI of WT BMM␸)] at different ratios are plotted (right panel). Results are expressed as means ⫾ SD
from three different experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Frequency (%) of cells engulfing bacteria
WT
PIR-B KO
The Journal of Immunology
Discussion
Using a mouse model of Salmonella infection with an attenuated
strain of S. typhimurium (WB335), we showed that PIR-B⫺/⫺ mice
were more susceptible to Salmonella infection than the WT mice,
as evidenced by increased mortality rate, high bacterial loads in the
liver and spleen, and a failure to clear bacteria from the circulation.
The blood levels of cytokines (TNF-␣, IFN-␥ and IL-10) and Salmonella-specific Abs (IgM, IgG, and IgA) were mostly comparable between these two groups of mice. However, distinct bacterial
spreading patterns were notable in the liver at 7–14 days postinfection and a diffuse spreading along the sinusoids was observed in
PIR-B⫺/⫺ mice vs a nodular restricted localization in WT mice.
PIR-B⫺/⫺ mice had more inflammatory cells in the liver, but fewer
B cells and CD8⫹ T cells in the spleen than WT mice. These in
vivo differences were substantiated by the ex vivo findings where
BMM␸ in PIR-B⫺/⫺ mice failed to control intracellular replication
of Salmonella due to inefficient phagosomal oxidant production
compared with those in WT mice. PIR-B⫺/⫺ BMM␸ also produced more nitrite and TNF-␣ upon exposure to Salmonella than
control macrophages.
Although PIR-A and PIR-B are among the first paired receptors
with opposing signaling capabilities to be identified, there are now
more than 20 such related immunoreceptors reported, implying
that the pairing of activation and inhibition is essential in modulators for initiation, amplification, and termination of immune responses (1, 2). Because PIR is expressed by many hematopoietic
cell types (B cells, monocyte/macrophages, DC, PMN, mast cells,
and megakaryocyte/platelets), it has been postulated that the disruption of PIR-A and PIR-B balance may affect their regulatory
roles in host defense, including humoral immune, inflammatory,
Ag-presenting, allergic, and coagulative responses. Indeed, several
functional alterations have been reported for the PIR-B⫺/⫺ mice.
These include hyperreactivity of B cells to BCR ligation and to T
cell independent Ags (24), enhanced Th2 response due to immature DC (24), exaggerated GVH reactions when sublethally irradiated PIR-B⫺/⫺ mice receive allogeneic splenocytes (21), and
hyperresponsiveness of macrophages and PMN to integrin ligation
ex vivo (25, 26). When we initially set up the present experiments,
it was difficult to anticipate what the outcomes would be. The
expected enhanced inflammatory responses in the PIR-B⫺/⫺ mice
would render them more resistant to Salmonella infection. In
contrast, their diminished ability to attenuate the inflammatory reactions might have pathological sequelae.
We have now shown that the PIR-B⫺/⫺ mice are more susceptible to the primary infection of WB335 than the WT control mice.
Interestingly, the slopes of the morbidity curves of 5 ⫻ 103 vs 103
CFU dose are different; in the former higher dose PIR-B⫺/⫺ mice
died gradually beginning at day 4 postinfection over the next 3 wk,
whereas in the latter lower dose most PIR-B⫺/⫺ mice survived for
two weeks but began to die in the third week postinfection, similar
to Rag-2⫺/⫺ mice receiving 5 ⫻ 103 CFU/mouse. It has been
shown that the innate immune system can restrict replication of S.
typhimurium to a certain degree, but that acquired immunity is
essential for effective control and eradication of bacteria (38 – 40).
These survival data suggest that the disruption of the Pirb gene
affects both innate and acquired immunity to a primary infection
with S. typhimurium.
The high susceptibility of PIR-B⫺/⫺ mice to Salmonella infection appears to be associated with their inability to control bacteria
replication in target organs as evidenced by high bacterial loads in
the liver, spleen, and lung tissues; diffuse spreading pattern of bacteria along the liver sinusoids; failure to efficiently clear bacteria
from circulation; and more intracellular bacterial growth inside the
marrow-derived macrophages ex vivo. It has recently been shown
that PIR-B can recognize S. aureus as well as some Gram-negative
bacteria such as H. pylori and E. coli, suggesting that PIR-B may
recognize pathogen-associated molecular patterns (23). In this regard, we found by immunofluorescence that neither PIR-B nor
PIR-A could directly bind live or heat-killed WB335 (S. Oka, unpublished observations).
PIR-B⫺/⫺ and WT phagocytes ingested opsonized WB335 and
subsequently released superoxide anion into culture supernatants
at comparable levels. It has been shown that Salmonella survive
and replicate in phagocytes within unique Salmonella-containing
vacuoles (SCV) inaccessible to the host defense mechanisms (41–
43). Delayed acidification of SCV and their incomplete fusion with
lysosomes are thought to promote intracellular survival of Salmonella in these vacuoles. Phagosomal oxidant production by PIRB⫺/⫺ macrophages was indeed less than by WT macrophages.
This reduced production of oxidants may permit increased replication of bacteria inside PIR-B⫺/⫺ macrophages and leads to the
inability to control Salmonella replication in the reticuloendothelial system of PIR-B⫺/⫺ mice.
The precise mechanism by which the PIR-B deficiency leads to
reduction of phagosomal oxidation is presently unknown. However, the extracellular superoxide release by both macrophages and
PMN and the phagosomal oxidation by PMN were comparable
between PIR-B⫺/⫺ and WT mice, suggesting that the assembly
and activation processes of the NADPH oxidase subunits might
not be impaired in PIR-B-deficient phagocytes. It should also be
noted that heat-killed bacteria were used for phagosomal oxidation
assays because the live bacteria could not be labeled with an oxidation-sensitive dye; hence, the evading strategies of Salmonella
from host defense could be excluded from our consideration of the
mechanism. Furthermore, the reduced phagosomal oxidation in
PIR-B-deficient macrophages was a generalized phenomenon as
observed with several other heat-killed microorganisms. Collectively, the processes of phagosomal development and subsequent
oxidant production appear to be indirectly impaired by disruption
of the PIR-A and PIR-B balance in macrophages, possibly through
differences in the phagocytosing cellular status, the stimuli via
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
similar (Fig. 6C). However, oxidant production inside the phagosome engulfing WB335 was clearly diminished in PIR-B⫺/⫺
BMM␸ compared with WT BMM␸ (Fig. 6D). Unlike BMM␸, the
phagosomal oxidation in BMPMN was indistinguishable between
WT and PIR-B⫺/⫺ mice (not shown). It should be noted that similar reduction in phagosomal oxidation by PIR-B⫺/⫺ BMM␸ was
also observed with other heat-killed microorganisms (e.g., E. coli,
S. pneumonia, S. aureus, and C. albicans), suggesting a generalized phenomenon related to the PIR-B-deficient macrophages and
not to a particular pathogen (not shown). These findings are thus
consistent with the inability of PIR-B⫺/⫺ macrophages to control
intracellular bacterial growth.
PIR-B⫺/⫺ BMM␸ were also found to produce more nitrite than
the WT BMM␸ when exposed to heat-killed WB335 at different
ratios of bacteria/phagocytes (Fig. 6E). This was also observed
with LPS stimulation. Unlike BMM␸, the BMPMN, irrespective
of the mouse genotype, did not produce significant amounts of
nitrites by either WB335 or LPS stimulation. Furthermore, the
PIR-B⫺/⫺ BMM␸ were found to produce more TNF-␣ upon exposure to the heat-killed WB355 than the WT BMM␸ (Fig. 6F). In
contrast, BMPMN produced comparable amounts of TNF-␣ upon
stimulation with heat-killed WB335. Collectively, these findings
suggest that PIR-B⫺/⫺ macrophages are unable to control the intracellular growth of Salmonella, but are hyperresponsive to the
heat-killed Salmonella as evidenced by enhanced production of
nitrite and TNF-␣ ex vivo.
4237
4238
nella virulence factors or evading systems. Inbred strains of mice
vary considerably in resistance or susceptibility to Salmonella infection and several major resistance genes have been identified in
mice (51). These include MHC class I and II, natural resistance
associated macrophage protein, TLR4, Bruton’s tyrosine kinase,
LPS-binding protein and CD14, NADPH oxidase and inducible
NO synthase, and cytokines (TNF-␣, INF-␥). Many immunodeficient mouse strains are also unable to control the in vivo replication of attenuated Salmonella strains. For example, T cell deficient
nu/nu, MHC class II⫺/⫺, TCR␤⫺/⫺, INF␥R⫺/⫺, TNF-␣ p55R⫺/⫺,
CD28⫺/⫺, CD40L/CD154⫺/⫺ and MD-2⫺/⫺ mice all succumb to
infection with attenuated strains of Salmonella that are normally
eradicated in WT mice (52–58). PIR-A and PIR-B are one pair of
the family members of paired immunoreceptors which share similar ectodomains but exhibit opposing signaling capabilities and
are postulated to play important regulatory roles in host defense
based on their wide cellular distribution. In this study, we show
clear evidence that the balance of PIR-A and PIR-B functional
activities is important for immune responses to bacterial infection.
Acknowledgments
We thank J. Yvette Hale, Janice D. King, Dong-Won Kang, and Lisa Jia for
their excellent technical assistance; Dr. Eric J. Brown for providing CMG1
cell line; Dr. Melissa Swiecki for technical advice; Drs. Richard
B. Johnston, Jr., Masataka Sasada, Victor Darley-Usmar, and Louis B.
Justement for helpful discussions and suggestions; and Jacquelin
B. Bennett for manuscript preparation.
Disclosures
The authors have no financial conflict of interest.
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multiple phagocytic receptors, the cytokine milieu, and the time
after onset of exposure to pathogens. Alternatively, PIR-B may
have a dual function of both inhibitory and activating activities as
suggested by the presence of an additional Src homology region
2-binding motif called the immunoreceptor tyrosine-based switch
motif (ITSM) “T/SxYxxV/I” (where x represents any amino acid)
(44). In this regard, it has recently been shown that while PIR-B
negatively regulates the eotaxin-dependent eosinophil chemotaxis
by recruiting the SHP-1 protein tyrosine phosphatase, PIR-B also
positively regulates the leukotriene B4-induced eosinophil chemotaxis by recruiting activating kinases (JAK1, JAK2, Shc, and Crk)
(13). It is thus conceivable that PIR-B may recruit some kinases,
such as Src family kinases, Syk, and PI3 kinase p85 known to be
involved in activation of NADPH oxidase (45, 46), through its
ITSM during phagocytosis of bacteria.
Another clear difference in Salmonella-infected animals is the
distinct patterns of inflammatory lesions in the liver after infection.
Although in WT mice such lesions were more localized and exhibited nodular distribution, in PIR-B⫺/⫺ mice inflammatory cells
infiltrated diffusely along the sinusoids and exhibited a spreading
pattern. PIR-B⫺/⫺ mice also had more macrophages and PMN in
the liver than WT mice, as determined by flow cytometry. These
differences were observed as early as 7 days postinfection. Notably, the cell surface PIR-A levels on PIR-B⫺/⫺ macrophages, but
not PMN, were enhanced 2-fold in comparison with those on WT
macrophages which were contributed by PIR-A and PIR-B. In our
previous study, cell surface levels of PIR on WT splenic B cells
and macrophages were found to be up-regulated by ⬃33% after
LPS stimulation for 3 days in vitro (7). The observed enhancement
of surface PIR-A levels seen in Salmonella-infected PIR-B⫺/⫺
mice might result from long exposure to Salmonella, leading to
dysregulated macrophage responses against bacterial infection.
The increased production of NO and TNF-␣ by these PIR-B⫺/⫺
macrophages may have detrimental, rather than protective, effects
locally on the surrounding hepatic tissues. Similar association of
enhanced PIR-A expression with enhanced host reaction was also
demonstrated in DC populations of PIR-B⫺/⫺ mice that develop an
exaggerated acute GVH reaction (21). One of the unique features
of PIR is that PIR-B on resting B cells, DC, and myeloid cells is
constitutively tyrosine-phosphorylated by Src family tyrosine kinases (Lyn or Fgr and Hck) and associates constitutively with tyrosine phosphatases (SHP-1 and SHP-2), thereby restraining potential cellular activation via BCR, integrin, and chemokine
receptors (25, 26, 47, 48). This tonic inhibition is apparently absent
in PIR-B⫺/⫺ mice and the present studies clearly indicate that the
disruption of PIR-A and PIR-B balance also affects their regulatory
roles in host defense to bacterial infection.
The finding that the total numbers of splenic B cells and, to a
lesser extent, CD8⫹ T cells in PIR-B⫺/⫺ mice did not increase as
markedly as those in WT mice at 14 days postinfection was unexpected. Sepsis or endotoxemia is known to accelerate lymphocyte apoptosis in both animals and humans (49, 50). In our studies,
LPS-induced apoptosis was frequently observed in the splenic
white pulp of PIR-B⫺/⫺, but not WT, mice and B cells appeared to
be more prone to this cell death (I. Torii and H. Kubagawa, unpublished observations). It is thus reasonable to suggest that PIR-B
deficiency leads to increased sensitivity of B cells to LPS-induced
apoptosis during Salmonella infection. As T cells do not express
PIR, the inability of PIR-B⫺/⫺ mice to increase CD8⫹ T cells two
weeks after Salmonella infection results most likely from an indirect effect of PIR-B deficiency.
The molecular mechanisms of resistance and susceptibility to
Salmonella infection are extremely complex and multifactorial
with host innate and adaptive immune responses vs with Salmo-
ROLE OF PAIRED Ig-LIKE RECEPTORS IN INFECTION
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