The Inhibitory Receptor PIR-B Negatively
Regulates Neutrophil and Macrophage
Integrin Signaling
This information is current as
of June 16, 2017.
Shalini Pereira, Hong Zhang, Toshiyuki Takai and Clifford
A. Lowell
J Immunol 2004; 173:5757-5765; ;
doi: 10.4049/jimmunol.173.9.5757
http://www.jimmunol.org/content/173/9/5757
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References
The Journal of Immunology
The Inhibitory Receptor PIR-B Negatively Regulates
Neutrophil and Macrophage Integrin Signaling1
Shalini Pereira,* Hong Zhang,* Toshiyuki Takai,† and Clifford A. Lowell2*
M
igration of polymorphonuclear neutrophils (PMNs)3
and macrophages to sites of acute inflammation requires the concerted interaction of cellular adhesion
receptors on these leukocytes with endothelial and extravascular
tissue components. Integrin receptors expressed on the leukocyte
surface play a central role in these interactions, mediating linkages
between the cytoskeleton and the external environment (1–3).
Cross-linking of integrins in the presence of soluble inflammatory
mediators such as TNF-␣ and fMLP induces both an enhancement
in the affinity and avidity of integrins for their ligands (“inside-out”
signaling), as well as rearrangement of the actin cytoskeleton leading to the formation of focal adhesion-like structures and cell
spreading (3–5). Additionally, the recognition and adhesion of leukocyte integrins regulates many of the inflammatory responses of
activated neutrophils (“outside-in” signaling; Refs. 6 and 7). Indeed, the firm arrest of neutrophils via integrin-extracellular matrix
(ECM) interactions at sites of inflammation is a prerequisite for the
sustained release of reactive oxygen intermediates and granule
components, leading to the elimination of pathogens (3, 8, 9).
The Src family kinases play an essential role in integrin signaling. Loss of c-Src, in genetically deficient mouse models, leads to
defects in ␤3 integrin signaling in platelets (10). For ␣v␤3 integrin
*Department of Laboratory Medicine, University of California, San Francisco, CA
94143; and †Department of Experimental Immunology, Institute of Development,
Aging and Cancer, Tohoku University, Sendai, Japan
Received for publication April 20, 2004. Accepted for publication August 26, 2004.
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 paper was supported by National Institutes of Health Grant DK58066 (to
C.A.L.) and by the CREST Program of Japan Science and Technology Corporation.
C.A.L. is a Scholar of the Leukemia and Lymphoma Society.
2
Address correspondence and reprint requests to Dr. Clifford A. Lowell, Department
of Laboratory Medicine, University of California, 513 Parnassus Avenue, San Francisco, CA 94143. E-mail address: [email protected]
3
Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; ECM, extracellular matrix; m, murine; BMDM, bone marrow-derived macrophage; SHP-1, Src
homology region 2 domain-containing phosphatase 1; RIPA, radioimmunoprecipitation assay; SIRP, signal regulatory protein; MHC I, MHC class I.
Copyright © 2004 by The American Association of Immunologists, Inc.
signaling, the mechanism by which c-Src becomes activated during integrin signaling involves a direct interaction between the
c-Src Src homology 3 domain and the C-terminal tail of the ␤3
chain (11). Clustering of ␤3 integrins in vivo activates c-Src leading to adhesion-dependent phosphorylation of Syk and other
downstream signaling events. Myeloid leukocytes lacking the Src
family kinases, Hck, Fgr, and Lyn are defective in integrin-mediated effector functions in vitro (5, 7, 12). In vivo experiments conducted with mice lacking Hck and Fgr revealed decreased accumulation of PMNs in tissues during endotoxemia (13). Although
loss of myeloid Src family kinases leads to reduced leukocyte integrin function, constitutive activation of Hck in mice results in
exaggerated integrin signaling responses in vitro and diffuse pulmonary inflammation in vivo (14). Similarly, myeloid-specific deletion of the Src family kinase regulator, Csk, results in constitutively activated Src family kinases, increased neutrophil integrin
signaling, and pulmonary inflammation (15). Although it is clear
that the Src family kinases Hck and Fgr play a positive role in
leukocyte integrin signaling, it is also evident that Lyn functions
predominately as an inhibitor of this pathway. PMNs derived from
lyn⫺/⫺ mice have enhanced respiratory burst, secondary granule
release, and a hyperadhesive phenotype when adherent to surfaces
coated with integrin ligands (16). The mechanism by which Lyn
down-modulates integrin signaling may be mediated by phosphorylation of inhibitory receptors such as PIR-B.
PIR-B is an Ig-like receptor containing cytoplasmic ITIM motifs
(17, 18). It is expressed on B lymphocytes, myeloid cells, and
dendritic cells. Phosphorylation of ITIM domains by Src family
kinases leads to recruitment of phosphatases, such as Src homology region 2 domain-containing phosphatase 1 (SHP-1) or SHIP,
which inhibit downstream signaling responses. Studies with mouse
splenic B cells demonstrated that Ab-mediated engagement of
PIR-B inhibits BCR-induced Ca2⫹ mobilization from intracellular
stores via recruitment of SHP-1 but not SHIP to its tyrosine-phosphorylated ITIMs (19). Furthermore, splenic pir-b⫺/⫺ B2 cells are
constitutively activated and proliferate much more than those from
wild-type mice upon BCR ligation (20). In DT40 cells, PIR-B
ligation inhibits the BCR-induced tyrosine phosphorylation of Ig␣/
0022-1767/04/$02.00
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The Ig-like receptor family member, PIR-B, has been shown to play an inhibitory role in receptor signaling within B cells, mast
cells, and dendritic cells. As it has been implicated in integrin-mediated responses, we investigated the effect of loss of the PIR-B
protein on integrin-mediated signaling in primary murine myeloid cells. The pir-bⴚ/ⴚ neutrophils displayed enhanced respiratory
burst, secondary granule release, and a hyperadhesive phenotype when plated on surfaces coated with either extracellular matrix
proteins or cellular adhesion molecules in the presence or absence of the soluble inflammatory agonist TNF-␣. The pir-bⴚ/ⴚ and
wild-type cells responded equivalently when stimulated with TNF-␣ in suspension, indicating that the hyperresponsive phenotype
of the pir-bⴚ/ⴚ cells during adhesion was due to enhanced integrin signaling. Both wild-type and pir-bⴚ/ⴚ neutrophils expressed
similar levels of integrin subunits. Primary bone marrow-derived macrophages from pir-bⴚ/ⴚ mice were also hyperadhesive and
spread more rapidly than wild-type cells following plating on surfaces that cross-linked cellular ␤2 integrins. Biochemical analysis
of macrophages from pir-bⴚ/ⴚ mice revealed enhanced phosphorylation and activation of proteins involved in integrin signaling.
These observations point to a nonredundant role for PIR-B in the regulation of leukocyte integrin signaling. The Journal of
Immunology, 2004, 173: 5757–5765.
5758
Superoxide release assays
Varying concentrations of ECM proteins, rmICAM-1, or poly-RGD were
used to coat the wells of a 96-well Immulon-4 plate (Dynex Technologies,
Chantilly, VA), which were subsequently blocked with 20% FCS (5). FCSblocked wells, with pretreatment with ECM proteins, served as negative
controls. Purified bone marrow neutrophils (2.5 ⫻ 105 cells per well) were
added to the wells and stimulated with either 20 ng/ml TNF-␣ or 100 nM
PMA in HBSS, 10 mM HEPES, 0.5 mM CaCl2, and 1 mM MgCl2, or with
medium alone. Respiratory burst was measured using the cytochrome c
reduction test as described (5, 7). Adhesion-independent superoxide production was measured by plating cells in 0.5% milk-coated wells, to which
they do not adhere, then stimulating them with 3 ␮M fMLP. The data are
presented as a cumulative assay, and all time points were performed in
triplicate, and the results were averaged. Statistical differences were calculated using InStat software (GraphPad, San Diego, CA).
Secondary granule release assays
Materials and Methods
Secondary granule release was monitored by assaying for secretion of lactoferrin into the cell medium following cross-linking of integrins on the
surface of neutrophils (5). One hundred microliters of cells at 2 ⫻ 106/ml
were incubated in either ECM or rmICAM-1-coated wells of a microtiter
tissue culture plate at 37°C for 45– 60 min, and 20 ng/ml TNF-␣, 100 nM
PMA or HBSS, 10 mM HEPES, 0.5 mM CaCl2, and 1 mM MgCl2 were
added as indicated. The contents of individual wells were transferred to a
polypropylene 96-well V-bottom plate and were centrifuged at 2000 rpm
for 10 min to remove nonadherent cells. Twenty-five microliters of supernatant per sample was diluted 4-fold in carbonate buffer (pH 9.6) and
incubated overnight at 4°C in an Immulon-4 microtiter plate. After incubation, the samples were processed as described (12). Assays were performed in triplicate, and the results were averaged. Statistical differences
were calculated using InStat software (GraphPad).
Isolation of bone marrow PMNs and culture of bone marrowderived macrophages (BMDMs)
Cell adhesion assays
Bone marrow neutrophils were obtained from C57B6 mice (Charles River
Laboratories, Wilmington, MA) or pir-b⫺/⫺ mice backcrossed to C57B6 for at
least 10 generations to ensure congenicity. The isolation procedure was essentially as described in Ref. 5. Purified neutrophils were preincubated in HBSS
containing 10 mM HEPES, 0.5 mM CaCl2 and 1 mM MgCl2 for 10 min at
room temperature before use in integrin-dependent assays in vitro. BMDMs
were obtained by culturing bone marrow leukocytes isolated from the same
animals in DME H-21, 4.5 g/L glucose medium (University of California San
Francisco Cell Culture Facility) containing 10% FCS (Invitrogen Life Technologies, Grand Island, NY), 20 mM HEPES, and 10% CSF-1-conditioned
medium (obtained from CMG1 cells, which are fibroblasts transfected with the
murine (m) CSF-1 cDNA, kindly provided by E. Brown, University of California, San Francisco, CA). Adherent primary macrophages were used between days 6 and 9 of culture.
Reagents
The following Abs against integrins were used in these experiments. Biotinylated Abs against mCD49d (R1-2), CD49e (5H10-27), CD11a (2D7),
CD11b (M1/70), CD11c (HL3), ␤7 (M293), CD29 (Ha2/5), CD18 (M18/2
used for routine staining and C71/16 used for TNF-␣-induced up-regulation), CD61 (2C9.G2), rat IgG2b (A95-1), and rat IgG2a (R35-95; BD
Pharmingen, San Diego, CA). The L-selectin-specific Ab (MEL-14) was
conjugated to PE and the anti-Gr1 Ab (11-26c.2a) was conjugated to FITC
(BD Pharmingen). The anti-PIR-B mAb, 6C1, which recognizes an extracellular domain common to both PIR-A and PIR-B (25) was a kind gift
from M. Cooper (University of Alabama, Birmingham, AL). The antiphosphotyrosine Ab 4G10 (Upstate Biotechnology, Lake Placid, NY),
polyclonal anti-PIR-B, anti-Cbl, anti-Vav, anti-Pyk2, and anti-talin Abs
(Santa Cruz Biotechnology, Santa Cruz, CA) were used in immunoprecipitation and Western blotting experiments. The polyclonal anti-PIR-B is
generated against epitopes in the C-terminal region of the protein that are
not present in PIR-A, and hence, recognizes only PIR-B. Murine ICAM-1
was a kind gift from C. M. Vines (University of New Mexico School of
Medicine, Albuquerque, NM). The ECM proteins used were rat collagen
type I, fibrinogen from mouse plasma factor I, and fibronectin from bovine
plasma (Sigma-Aldrich, St. Louis, MO). Recombinant E-selectin/Fc (IgM)
fusion protein was a kind gift from S. D. Rosen (University of California,
San Francisco, CA). The fusion protein was generated as described (26).
The poly-RGD peptide (Sigma-Aldrich) was also used as an adhesive ligand. Murine TNF-␣ (PeproTech, Rocky Hill, NJ) and PMA (SigmaAldrich) were used as stimulants in the adhesion-mediated assays, while
fMLP (Sigma-Aldrich) was used as an agonist in suspension assays.
A total of 100 ␮l of purified neutrophils at 4 ⫻ 106/ml were incubated with
the indicated stimulants in ECM protein, rmICAM-1, or E-selectin/Fc
(IgM)-coated wells of a 96-well Immulon-4 plate at 37°C for 10 – 45 min
(only 30-min data are shown). The percentage of cells that remained tightly
adherent to the coated surfaces following four washes with warm (37°C)
PBS and two 30-s centrifugations at 50 ⫻ g force was determined by
measuring cellular membrane acid phosphatase activity. The data were
plotted relative to the number of input cells, which were defined as 100%.
All wells were done in triplicate, and the results were averaged. Specificity
of PMN binding to E-selectin was demonstrated by incubation of cells in
0.5 mM EGTA, which completely inhibited binding. Statistical differences
were calculated using InStat software (GraphPad).
FACS analysis of neutrophils and macrophages
Bone marrow leukocytes were incubated with the indicated biotinylated or
directly conjugated Abs, followed by incubation with streptavidin conjugated to PE (BD Pharmingen), and analyzed by flow cytometry. The
mPMN population was defined on the basis of forward and side scatter and
Gr-1 staining. Stimulation-dependent changes in the expression levels of
CD18 and L-selectin were assessed by incubating the cells with 20 ng/ml
TNF-␣ for 15 min at 4°C before incubation with specific Ab. Surface
expression of the PIR-B protein was examined by incubating either bone
marrow leukocytes or day 5– 6 adherent cultured BMDMs with the antiPIR-A/B mAb, 6C1, and then counterstaining with a PE-conjugated antirat secondary Ab before flow cytometry analysis.
Adhesion and photomicroscopy of BMDMs
Adhesive responses of BMDMs were assayed as follows. Day 5– 6, adherent cultured BMDMs were removed from culture dishes and suspended in
DME H-21, 4.5 g/L glucose medium supplemented with 3% FCS, 20 mM
HEPES at 2 ⫻ 105 cells/ml. The cells were rested in suspension at 37°C for
2–3 h before transfer to 60 ⫻ 15 mm bacterial plates (KORD/VALMARK;
Midwest Scientific, St. Louis, MO) and incubated at 37°C for a further 45
min. Adherent cells were visualized on a Nikon microscope (Melville, NY)
under phase contrast at ⫻10 magnification and digital images of at least 20
random fields were captured using the IPLab Spectrum P program (Scanlytics, Fairfax, VA). The average cell area occupied by adherent macrophages was quantified using NIH Image software (version 1.63). A minimum of 300 –350 adherent cells was analyzed, and the results averaged.
Data were plotted as a function of the average area per cell in square
millimeters for each genotype.
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Ig␤, Syk, Btk, and phospholipase C␥2 (21). PIR-B also plays a
role in modifying IgE Ab-mediated allergic responses in mouse
bone marrow-derived mast cells, where it is found to be constitutively tyrosine phosphorylated and associated with intracellular
SHP-1 (22). Coligation of PIR-B with the IgE receptor (Fc⑀RI)
inhibits IgE-mediated mast cell activation and release of serotonin
(23). In BaF/3 cells, stimulation with IL-3 leads to an increased
recruitment of SHP1 to PIR-B, suggesting a functional link between PIR-B and cytokine receptor signaling (24). PIR-B also
plays a role in DC maturation because knockout mice lacking
PIR-B displayed perturbations in DC development and altered Th1
and Th2 immune responses (20).
Although PIR-B has been postulated to be involved in integrin
signaling in studies using lyn⫺/⫺ myeloid cells (16), a direct role for
this receptor has not been established. In this work, we directly tested
the function of PIR-B in integrin signaling using primary neutrophils
and macrophages from pir-b⫺/⫺ mice. We demonstrate that pir-b⫺/⫺
cells are hyperresponsive to integrin cross-linking. They display enhanced adhesion, superoxide, and secondary granule release when
plated on surfaces coated with integrin ligands. Examination of cellular signaling events in pir-b⫺/⫺ leukocytes following integrin engagement, revealed enhanced phosphorylation of molecules involved
in integrin signaling. These results provide the first direct evidence
that PIR-B functions as a negative regulator of integrin-mediated signaling in primary myeloid cells.
REGULATION OF INTEGRIN SIGNALING BY PIR-B
The Journal of Immunology
FIGURE 1. Murine PMNs and BMDMs express much higher levels of
PIR-B than PIR-A. Neutrophils and BMDMs from wild-type (WT) and
pir-b⫺/⫺ mice were stained with mAb 6C1, then counterstained with PEconjugated anti-rat, and then analyzed by flow cytometry. The light gray
graph indicates staining of WT cells with the secondary anti-rat Ab alone.
Immunoprecipitation and Western blotting
FIGURE 2. PIR-B-deficient PMNs display increased superoxide production following plating on
ECM-coated surfaces. A, WT and pir-b⫺/⫺ PMNs
were plated in microtiter wells, in the presence or
absence of TNF-␣, that had been precoated with
20% FCS, murine fibrinogen (mFbg), bovine fibronectin (BoFN), rat collagen (rColl), or rmICAM-1 at the indicated concentrations. The development of respiratory burst over a 2-h period was
monitored, and the peak amount of superoxide released at the 60-min time point was determined for
each genotype. B, PMNs were plated in wells, with
and without TNF-␣, precoated with poly-RGD peptide (10 ␮g/ml) from human fibronectin (FN Pol),
and respiratory burst over the entire 2-h period is
shown. Data are representative of three independent
experiments. C, PMNs were plated in mFbg-coated
wells and stimulated with PMA. All assays were
done in triplicate and data shown in A and C are the
average of at least three independent experiments ⫾
the SD. ⴱⴱ, p ⬍ 0.01, and ⴱ, p ⬍ 0.04 between WT vs
pir-b⫺/⫺ cells (either resting or TNF-␣ stimulated).
For immunoprecipitation studies, RIPA lysates were precleared using
recombinant protein G-agarose (Invitrogen Life Technologies) and incubated with anti-PIR-B anti-Cbl, anti-Vav, anti-Pyk2, and anti-talin polyclonal Abs (Santa Cruz Biotechnology). Immune complexes were captured
with recombinant protein G-agarose, washed, and resuspended in sample
buffer. Samples were run on an SDS-PAGE gel, transferred to nitrocellulose membranes, and blotted with either 4G10 or the precipitating Abs,
followed by HRP-conjugated secondary Ab (Amersham Biosciences, Piscataway, NJ, or Jackson ImmunoResearch Laboratories, West Grove, PA).
Immunoblots were developed using the ECL system (Amersham Biosciences) and quantitated by densitometry. The phosphotyrosine index was
calculated by determining the ratio of the phosphotyrosine band to the
loading control for each lane, and then normalizing the nonadherent wildtype sample to 1.0.
Results
PIR-B expression in mPMNs and BMDMs
PIR-B is abundantly expressed on the surface of B lymphocytes
and various myeloid cells (dendritic cells, macrophages, and mast
cells); however, its expression has not been demonstrated on
PMNs. Using a mAb that recognizes an extracellular region of
PIR-B that is shared with PIR-A (the activating version of the
receptor which contains an intracellular ITAM motif, see Ref. 25),
we compared the relative expression of these receptors in PMNs
and BMDMs. PIR-B expression was easily detected in PMNs and
BMDMs from wild-type mice by flow cytometry (Fig. 1). In cells
derived from pir-b⫺/⫺ mice, the remaining staining with this mAb
is attributed to PIR-A (20). In both PMNs and BMDMs, the relative expression of the inhibitory receptor, PIR-B, is much higher
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Adherent-cultured BMDMs were removed from culture dishes and suspended in DME H-21, 4.5 g/l glucose medium supplemented with 3% FCS,
20 mM HEPES at 1 ⫻ 106 cells/ml. The cells were rested in suspension at
37°C for 2 h before transfer to 100 ⫻ 15-mm bacterial plates (KORD/
VALMARK; Midwest Scientific) and incubated for a further 20 min. Cells
in suspension were maintained at 37°C over the same time period as negative controls. Adherent and nonadherent cells from the bacterial plates
were pooled and lysed in radioimmunoprecipitation assay (RIPA) buffer
alongside cells from the suspension controls, and the lysates were cleared
of insoluble material.
5759
5760
REGULATION OF INTEGRIN SIGNALING BY PIR-B
than the activating receptor, PIR-A, as has been previously reported in B lymphocytes and dendritic cells (20); however, this
analysis does not exclude slightly decreased PIR-A expression in
the pir-b⫺/⫺ cells compared with wild type.
pir-b⫺/⫺ PMNs are hyperresponsive to integrin-mediated stimuli
FIGURE 3. PIR-B-deficient PMNs are more efficient at adhesion-induced degranulation. A, WT and pir-b⫺/⫺ PMNs were plated in microtiter
wells, in the presence or absence of TNF-␣, that had been precoated with
the indicated concentrations of FCS, rat collagen (rColl), murine fibronigen
(mFbg), or bovine fibronectin (BoFN), for 60 min and the release of the
secondary granule marker lactoferrin was determined by ELISA. B, Degranulation responses of WT vs pir-b⫺/⫺ PMNs on surfaces coated with
rmICAM-1. C, Degranulation responses of WT vs pir-b⫺/⫺ PMNs stimulated with PMA. All assays were done in at least triplicate, and data shown
are the average of three independent experiments ⫾ SD. ⴱⴱ, p ⬍ 0.01, and
ⴱ, p ⬍ 0.04 between WT vs pir-b⫺/⫺ cells (TNF-␣ stimulated).
tion, and downstream effector function. To determine whether the
hyperresponsiveness of the pir-b⫺/⫺ PMNs to integrin cross-linking could be ascribed to an increased ability to firmly adhere to
surfaces coated with integrin ligands, we plated cells onto surfaces
precoated with either ECM proteins or mICAM-1, and evaluated
their ability to withstand three washes with PBS and two applications of a force of 50 – 60 ⫻ g. As observed in Fig. 4, A and B,
PIR-B-deficient cells displayed enhanced tight adhesion when
plated on either ECM proteins or mICAM-1, for 30 min, even in
the absence of TNF-␣. Adhesion of pir-b⫺/⫺ neutrophils to 20%
FCS-coated surfaces was not significantly higher than wild-type
cells. PIR-B-deficient PMNs were also hyperadhesive following
shorter (15-min) and longer (45-min) periods of incubation on fibrinogen, indicating that the mutant cells displayed both more
rapid and tighter adhesion (data not shown). Although there was a
significant increase in adhesion to ECM proteins of both wild-type
and pir-b⫺/⫺ PMNs when stimulated with TNF-␣, the general response of pir-b⫺/⫺ cells was much more robust. To determine
whether the hyperadhesive phenotype of pir-b⫺/⫺ was specific to
integrins or more general in nature, we performed neutrophil adhesion assays to recombinant E-selectin. In these assays, adhesion
to E-selectin was appreciably lower than adhesion to ECM proteins and was not augmented by TNF-␣ stimulation but was completely blocked by incubation with EGTA (Fig. 4C). PIR-B-deficient cells did show a significantly higher binding to E-selectin
compared with wild-type cells, indicating that selectin-mediated
adhesion is also up-regulated in these cells. However, examination
of the pir-b⫺/⫺ PMNs in parallel-plate flow chambers indicates
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The engagement of integrins on the surface of mPMNs by specific
ligands results in cytoskeletal rearrangements and the activation of
effector functions. Acquisition of full effector function, which results in the assembly of the subunits of the NADPH oxidase complex at the plasma membrane leading to respiratory burst and release of granule constituents, usually requires costimulation by a
soluble proinflammatory agonist such as TNF-␣ (3). To investigate
the role played by PIR-B in integrin-mediated signaling, we used
an in vitro assay that provides cells with an integrin-ligand-coated
adhesive surface together with soluble costimulation and measures
cellular responses when surface integrins are engaged. Bone marrow neutrophils derived from pir-b⫺/⫺ and wild-type mice were
added to microtiter wells coated with increasing concentrations of
the ECM proteins, fibrinogen, fibronectin, and collagen, or the cellular adhesion molecule, mICAM-1, and the nanomoles of superoxide released by adherent PMNs in the presence or absence of
TNF-␣ over time was monitored. As shown in Fig. 2A, PIR-Bdeficient neutrophils released elevated levels of superoxide compared with wild-type cells over a range of concentrations with all
the integrin ligands tested. Although TNF-␣ was required to
achieve maximal superoxide release, the pir-b⫺/⫺ cells were responsive to integrin cross-linking when plated on high-dose ligands even in the absence of the soluble agonist. Similarly, the
pir-b⫺/⫺ neutrophils produced significantly elevated levels of superoxide even when they were plated on surfaces coated with only
20% FCS, likely due to the presence of low levels of ECM proteins
(fibrinogen) in serum. When a synthetic ligand, consisting of multiple copies of the integrin-binding RGD motif of human fibronectin (fibronectin polymer), was used to coat wells, the requirement
for costimulation was circumvented and both wild-type and pirb⫺/⫺ neutrophils were equally responsive (Fig. 2B). This no doubt
reflects the greater cross-linking efficiency of the polymer as compared with the ECM proteins. Treatment of cells with PMA, which
bypasses signaling through surface receptors, and maximally stimulates neutrophils, revealed similar levels of respiratory burst activity by both wild-type and pir-b⫺/⫺ cells (Fig. 2C). This is in
concordance with the results obtained with fibronectin polymer
cross-linking (Fig. 2B), and further confirms that the PIR-B-deficient neutrophils do not contain elevated levels of the NADPH
oxidase complex.
To ascertain whether the degranulation responses of the pirb⫺/⫺ PMNs were similarly enhanced, we examined the release of
the secondary granule marker, lactoferrin, into the surrounding
medium following cross-linking of integrins on the surface of the
cells when adherent to ECM proteins or mICAM-1. In agreement
with the results obtained in the respiratory burst assay, engagement
of integrins by either ECM proteins or mICAM-1 elicited higher
levels of lactoferrin secretion by pir-b⫺/⫺ cells following TNF-␣
stimulation when compared with the wild type (Fig. 3, A and B).
As seen in the respiratory burst experiments, pir-b⫺/⫺ cells also
released significant amounts of lactoferrin when plated on 20%
FCS-coated plates. Similar responses by both cell types on treatment with PMA, which elicits maximal secondary granule release,
confirmed that the PIR-B-deficient cells did not contain elevated
concentrations of lactoferrin (Fig. 3C).
The initial recognition of integrins on the surface of PMNs for
their cognate ligands is followed by an increase in the affinity and
avidity of integrin binding culminating in firm adhesion, activa-
The Journal of Immunology
5761
that these cells have the same rolling velocity on P-selectin/ICAM1-transfected mouse L cells as do wild-type cells (data not shown),
suggesting that the increased adhesiveness of pir-b⫺/⫺ cells to
selectin ligands may be only apparent in static assays. Following
stimulation with PMA, 100% of both wild-type and pir-b⫺/⫺
PMNs were tightly adherent to ECM proteins (Fig. 4D), indicating
that membrane-proximal signaling events were primarily responsible for the observed differences in adhesion between the two cell
types. To summarize, the pir-b⫺/⫺ mutation enhances the integrindependent activation of neutrophils regardless of the integrin ligand tested or the response examined. This results in increased
adhesion, respiratory burst, and degranulation by the pir-b⫺/⫺
cells.
pir-b⫺/⫺ PMNs do not express elevated levels of integrin
subunits
One potential explanation for the enhanced integrin responses of
the PIR-B-deficient PMNs is that they express higher levels of
integrin subunits. We examined the expression of several integrin
subunits on the surface of bone marrow-derived PMNs from wildtype and pir-b⫺/⫺ mice using flow cytometry. Surface expression
of the major integrin subunits known to be involved in adhesion to
fibrinogen, fibronectin, collagen, and mICAM-1 were found to be
equivalent between wild-type and pir-b⫺/⫺ cells (Fig. 5). Therefore, increased expression of integrin subunits is not responsible
for the hyperresponsive phenotype of pir-b⫺/⫺ PMNs in integrinmediated reactions.
pir-b⫺/⫺ PMNs respond normally to TNF-␣ and fMLP
Engagement of integrins on the surface of cells is usually insufficient to promote activation, especially if the ligand is of low valency. Costimulation by a soluble inflammatory agonist such as
TNF-␣ is necessary for full functional responses to occur. The
accepted hypothesis is that the soluble agonist serves to promote
an increase in the affinity and avidity (inside-out signaling) of the
integrin molecules for their ligand leading to rearrangements of the
cytoskeleton and the activation of a signaling cascade (outside-in
signaling), the end result of which is respiratory burst and granule
release. To determine whether the hyperresponsiveness of pirb⫺/⫺ PMNs in adhesion-mediated signaling was integrin-specific
vs due to an enhanced responsiveness to TNF-␣ stimulation, we
examined neutrophil responses in suspension in the absence of any
contribution by integrins.
Stimulation of neutrophils in suspension with the proinflammatory ligands IL-1 or TNF-␣ leads to immediate up-regulation of
surface levels of CD18 and concomitant shedding of L-selectin (4,
16, 27). To determine whether pir-b⫺/⫺ PMNs showed enhanced
responses to TNF-␣ stimulation alone, we examined the CD18 and
L-selectin responses of these cells. As shown in Fig. 6, both wildtype and pir-b⫺/⫺ PMNs displayed strong and equivalent up-regulation of CD18 expression with concomitant shedding of L-selectin from the cell surface following a 15-min stimulation with
TNF-␣. These data indicate that the enhanced responses of pirb⫺/⫺ PMNs to adhesion are not due to exaggerated TNF-␣
signaling.
As a further control to examine the responses of pir-b⫺/⫺ PMNs
to other adhesion-independent agonists, we examined the response
of these cells to fMLP stimulation. Treatment of PMNs with fMLP
will cause abundant superoxide production within several minutes
in suspended cells (compared with adhesion-mediated activation of respiratory burst which develops over 15–30 min; see
Refs. 16 and 28). As shown in Fig. 6B, both pir-b⫺/⫺ and
wild-type PMNs mounted a robust and equivalent respiratory
burst response to fMLP when cells were plated in wells to
which they do not adhere. Together, these data indicate that the
hyperresponsive phenotype of pir-b⫺/⫺ PMNs is restricted to
adhesion-dependent activation and is not seen with agonists that
activate cells in suspension.
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FIGURE 4. Pir-b⫺/⫺ PMNs are hyperadhesive to
ECM and ICAM-1-coated surfaces. A, WT and pirb⫺/⫺ PMNs were plated in microtiter wells, in the
presence or absence of TNF-␣, that had been precoated with the indicated concentrations of FCS, rat
collagen (rColl), murine fibronigen (mFbg), or bovine fibronectin (BoFN) for 30 min, and the percentage of cells adherent to the surface, following washing and two low-speed centrifugation steps, was
determined. B, Adhesion of WT vs pir-b⫺/⫺ PMNs to
rICAM-1-coated surfaces. C, Adhesion of WT vs pirb⫺/⫺ PMNs to rE-selectin/IgM fusion protein, in the
presence or absence of TNF-␣ or 0.5 mM EGTA. D,
Adhesion following stimulation with PMA. All assays were done in triplicate, and data shown are the
average of three independent experiments ⫾ SD. ⴱⴱ,
p ⬍ 0.01, and ⴱ, p ⬍ 0.04 between WT vs pirb-b⫺/⫺
cells (either resting or TNF-␣ stimulated).
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Primary BMDMs from pir-b⫺/⫺ mice are hyperadhesive and
display enhanced spreading on the cross-linking of surface
integrins
The above observations point to a nonredundant role for PIR-B in
the regulation of neutrophil integrin signaling. To determine
whether PIR-B plays a similar role in integrin signaling of primary
BMDMs, we examined the adhesion and spreading responses of
pir-b⫺/⫺ macrophages when exposed to a plastic surface (VALMARK) that has been demonstrated to cross-link ␤2 integrins (16,
29). To ensure that only integrin-specific interactions were being
analyzed, BMDMs from wild-type and pir-b⫺/⫺ mice were rested
in suspension in the absence of growth factor for 2–3 h at 37°C
before plating onto VALMARK plates. As observed in Fig. 7,
during the initial 45 min following plating, there was a significant
increase in the rate at which the pir-b⫺/⫺ macrophages adhered
and spread on the surface of the VALMARK plates. Quantitation
of the surface area occupied by single cells on spreading demonstrated that the average size of the spread by pir-b⫺/⫺ macrophages was nearly twice that of the wild-type cells. This hyperadhesion and enhanced spreading by pir-b⫺/⫺ macrophages was
observed only during the initial spreading response; within 2–3 h
FIGURE 6. PIR-B-deficient PMNs display normal responses to TNF-␣
and fMLP stimulation. A, WT vs pir-b⫺/⫺ PMNs were incubated in suspension with or without TNF-␣ for 15 min, and then stained for L-selectin
or CD18, and then analyzed by flow cytometry. In the L-selectin panels, the
dashed line indicates staining in the absence of the mAb, the dark line
indicates resting cells, and the gray line indicates TNF-␣-stimulated cells.
In the CD18 panels, the dark line indicates resting cells, the gray line
indicates the TNF-␣-stimulated cells. Anti-CD18 mAb C71/16 was used at
0.2 ␮g/ml to maximize staining differences between resting and TNF-␣stimulated cells. Data shown are representative of three independent experiments. B, WT and pir-b⫺/⫺ PMNs were plated in microtiter wells
blocked with 0.5% milk (to which they do not adhere) and stimulated with
3 ␮M fMLP. Superoxide production was monitored over the indicated time
course (30 min compared with 120 min in Fig. 2).
following plating on VALMARK, the wild-type cells were able to
catch up to the pir-b⫺/⫺ cells and no significant differences in total
cell area were observed (data not shown), indicating that deficiency of PIR-B affected the rate at which BMDMs spread. This
observation demonstrates that PIR-B also plays a role in the negative regulation of integrin-mediated signaling in primary
macrophages.
Intracellular signaling events following integrin cross-linking in
primary BMDMs from pir-b⫺/⫺ mice
All of the above data support a role for PIR-B in the negative
regulation of integrin signaling. To ascertain the effects of loss of
PIR-B on signal transduction events that follow integrin engagement, we examined the phosphorylation state of PIR-B and several
downstream signaling molecules that are known to participate in
integrin signaling. Primary BMDMs were used in this study because they were found to respond to integrin signaling in a manner
similar to PMNs and because they allowed us to circumvent the
difficulties faced in obtaining large enough numbers of mature murine neutrophils to perform biochemical analyses.
Because the effect of the PIR-B mutation on macrophage
spreading was most significant in the initial time points following
plating of macrophages on VALMARK plates, we examined the
effect on signaling events at 20 min following plating. As previously reported, following 15 min of adhesion, PIR-B tyrosine
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FIGURE 5. pir-b⫺/⫺ PMNs express normal levels of surface integrin
subunits. Resting bone marrow PMNs from WT vs pir-b⫺/⫺ mice were
stained with the indicated integrin-specific mAbs and analyzed by flow
cytometry.
REGULATION OF INTEGRIN SIGNALING BY PIR-B
The Journal of Immunology
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phosphorylation increased significantly (Fig. 8) resulting in the
recruitment of SHP-1 to the receptor (see data in Ref. 16). Several
cytoskeletal-associated proteins are known to be tyrosine phosphorylated following cell adhesion (30). Because FAK, a major
component of the focal adhesion complex, is expressed very
poorly in myeloid cells (Ref. 31; S. Pereira and C. A. Lowell,
unpublished observation) we examined the FAK-related protein,
Pyk2, which is highly expressed in macrophages. We also examined the actin binding protein, talin, which is proposed to provide
the link between integrins and the actin cytoskeleton (32, 33). As
is observed in Fig. 8, integrin-mediated tyrosine phosphorylation
of both the cytoskeleton-associated proteins, Pyk2 and talin, was
enhanced in pir-b⫺/⫺ macrophages as compared with wild-type
cells, although the relative increase in talin phosphorylation was
significantly less than Pyk2. This elevated phosphorylation response correlates very well with the observed hyperresponsive
phenotype of the pir-b⫺/⫺ myeloid cells in adhesion-mediated
activation.
The Cbl protein has been shown to become tyrosine phosphorylated in several hemopoietic cell lines, and to translocate to the
membrane in primary BMDMs following integrin-mediated cell
adhesion (31, 34, 35). In keeping with the results obtained with
Pyk2 and talin, Cbl was also hyperphosphorylated in adhering pirb⫺/⫺ macrophages.
Vav, which functions as a guanine exchange factor of the Rac
small GTPase family, is phosphorylated during ␤2 integrin-mediated cell adhesion and plays a direct role in actin cytoskeletal reorganization (36, 37). Examination of Vav following surface integrin cross-linking of macrophages revealed enhanced tyrosine
phosphorylation of the protein in pir-b⫺/⫺ cells. Taken together,
these results indicate that the ITIM-containing membrane protein,
PIR-B, functions as a negative regulator of integrin signaling, and
that loss of the protein leads to an enhancement in the biochemical
and functional responses during integrin-mediated adhesion.
Discussion
The cell surface receptor, PIR-B, has immunoreceptor tyrosinebased inhibition motifs (ITIMs) in its cytoplasmic domain that,
upon tyrosine phosphorylation, recruit the tyrosine protein phosphatase, SHP-1. PIR-B has been found to regulate immunoreceptor
signaling or ITAM-dependent pathways, such as the BCR and
Fc⑀R receptors, via its ability to recruit phosphatases and downmodulate signaling (21, 22). In this study, we demonstrate that
PIR-B plays a role in regulating signal strength in adhesion-mediated activation of myeloid cells. pir-b⫺/⫺ neutrophils are hyperadhesive to a variety of ligand-coated surfaces that cross-link
multiple integrins. Their downstream adhesion-mediated effector
responses such as respiratory burst and secondary granule release
are also enhanced. In macrophages, deficiency of PIR-B results in
an enhanced rate of spreading and increased downstream tyrosine
phosphorylation of a number of substrates implicated in integrin
signaling. Combined with the observation that PIR-B phosphorylation is induced in adhering leukocytes (16), these data indicate
that PIR-B, via its recruitment of phosphatases, plays an essential
role in down-modulating the signaling cascade initiated by integrin
ligation. Although the role played by PIR-B in the negative regulation of immunoreceptor signaling has been documented, this is
the first direct evidence using primary myeloid cells from pir-b⫺/⫺
mice, that this receptor can regulate a nonimmunoreceptor signaling pathway such as integrins.
Of the three myeloid Src family kinases, Lyn has been demonstrated to play a dual role in multiple signaling pathways. It functions predominantly as a negative regulator in BCR, Fc⑀R, integrin, and cytokine signaling (16, 38 – 41). PIR-B has been
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FIGURE 7. PIR-B-deficient BMDMs show increased spreading responses. WT vs pir-b⫺/⫺ BMDMs
were rested in suspension in 3% FBS medium, and
then plated on VALMARK plates and photographed
under phase contrast microscopy 45 min later. Panels
shown are representative of 20 random fields. The area
of the spread by wild-type vs pir-b⫺/⫺ cells was determined using NIH Image software and plotted. Data
are from cells photographed in 20 random fields, in two
independent experiments. ⴱ, p ⬍ 0.05 between pirbb⫺/⫺ and WT BMDMs.
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demonstrated to be a substrate of Lyn, because PIR-B phosphorylation is dramatically reduced in B cells and macrophages isolated from lyn⫺/⫺ mice (16, 42). There are striking similarities
between the responses of pir-b⫺/⫺ and lyn⫺/⫺ PMNs and macrophages to integrin cross-linking. Similar to our observations with
the pir-b⫺/⫺ leukocytes, lyn⫺/⫺ PMNs are hyperadhesive to surfaces coated with integrin ligands and manifest increased integrindependent functional responses (16). However, Lyn also phosphorylates other myeloid inhibitory receptors, such as signal
regulatory protein (SIRP)1␣, which may also contribute to the negative regulation of integrin signaling. Loss of Lyn in macrophages
leads to a decrease in adhesion-mediated phosphorylation and recruitment of SHP-1 by both SIRP1␣ and PIR-B (16). The overall
similarity of the PIR-B- and Lyn-deficient myeloid cells supports
the idea that PIR-B is the principal receptor that Lyn uses for
negative regulation of integrin signaling, at least in PMNs, where
the functional assays are more quantitative. Indeed, in PMNs,
SIRP1␣ is not significantly phosphorylated, in either resting or
adherent cells (H. Zhang and C. A. Lowell, unpublished observations), again suggesting PIR-B is the major regulatory receptor in
these cells. Examination of the integrin-mediated responses of
sirp1␣⫺/⫺ PMNs will eventually be required to sort out the relative contribution of this receptor vs PIR-B to regulation of integrin
signaling.
There appear to be differences between the way PIR-B functions
in B cells as compared with macrophages. PIR-B is constitutively
phosphorylated in resting B cells, and stimulation of those cells by
Ag does not further enhance its phosphorylation (42). In contrast,
in resting, nonadherent primary macrophages, we find that PIR-B
is negligibly phosphorylated, and its phosphorylation can be inducibly increased by integrin engagement (16). Part of this difference may be due to the fact that we used mainly serum-starved
(3% FBS) BMDMs to examine PIR-B phosphorylation in completely resting cells. This allows us to maximize the change in
PIR-B phosphorylation following adhesion. PIR-B phosphorylation is detectable in actively growing, CSF-1-stimulated BMDMs
(S. Pereira and C. A. Lowell, unpublished observations). Coassociation studies have demonstrated that the increase in integrinmediated tyrosine phosphorylation of the PIR-B ITIMs is accompanied by increased recruitment of the tyrosine phosphatase SHP-1
(16). This would suggest that PIR-B serves as a constitutive negative regulator in B lymphocytes, such that the propagation of a
positive signal would necessitate dephosphorylation of its ITIMs
and the subsequent dissociation of recruited phosphatases. In contrast, in macrophages, PIR-B may function as an inducible regulator. However, in both B cells and macrophages and possibly mast
cells, it appears that Lyn is the kinase that regulates PIR-B phosphorylation (16, 42).
One of the major ligands for PIR-B appears to be MHC class I
(MHC I) molecules (43). The fact that PIR-B contributes to negative regulation of adhesion signaling would suggest that corecognition of MHC I by PIR-B, in concert with recognition of integrin
ligands on vascular endothelium (such as ICAM-1), may play an
important role in neutrophil extravasation out of the vasculature
and into the tissues. Binding of MHC I on endothelial cells by
PIR-B may prevent hyperadhesion or hyperactivation of leukocytes by integrin-mediated signals. In this fashion, PIR-B may facilitate leukocyte migration in certain inflammatory reactions and
limit premature activation of the cells by endothelial cell integrin
ligands. In contrast, within tissue sites, loss of MHC I binding
would relieve the inhibition, allowing maximal neutrophil activation by integrin-mediated signals. This model would assume that
PIR-B associates with integrins during recognition of MHC I in
migrating cells. Although it would seem likely that PIR-B and ␤2
(or other) integrin molecules must be in close proximity in cells for
PIR-B to be able to regulate integrin signaling, such an association
has yet to be experimentally demonstrated.
The issue of whether PIR-B is regulating integrin inside-out vs
outside-in signaling may be difficult to distinguish. The hyperadhesive phenotype of the pir-b⫺/⫺ neutrophils, even without costimulation by inflammatory mediators (Fig. 4), suggests that
PIR-B deficiency affects the inside-out pathway, which regulates
the transition of integrins from a low affinity to a high affinity state.
The fact that PIR-B-deficient PMNs also produced superoxide
when plated on high-density ECM-coated surfaces without costimulation by TNF-␣ (Fig. 2) suggests that the ␤2 integrins on
pir-b⫺/⫺ PMNs may constitutively exist in a higher affinity state,
even in resting cells, as compared with wild-type PMNs. However,
the observation that the receptor is inducibly phosphorylated during integrin-mediated adhesion (16) and that the signaling events
downstream of adhesion are enhanced in pir-b⫺/⫺ macrophages,
would suggest that the regulation occurs during outside-in signaling events, with PIR-B regulating post-integrin receptor signaling
thresholds. It is possible that loss of PIR-B might affect both inside-out and outside-in integrin signaling. Distinguishing between
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FIGURE 8. pir-b⫺/⫺ BMDMs manifest increased intracellular signaling
responses following adhesion. WT and pir-b⫺/⫺ BMDMs were rested in
suspension, and then plated on VALMARK plates for 20 min. The adherent and nonadherent cells were collected and lysed in RIPA buffer. Equal
amounts of lysates from BMDMs maintained in suspension or adherent
cells were used for immunoprecipitation (IP) with the indicated Abs, and
then subjected to PAGE and immunoblotted (IB) with antiphosphotyrosine mAbs. Following ECL development, filters were stripped
and reprobed with the precipitating Abs to demonstrate equal loading of
proteins. Data shown are representative of two to three independent experiments for each protein. Gels were quantitated by densitometry, and the
relative phosphotyrosine index was calculated as described in Materials
and Methods.
REGULATION OF INTEGRIN SIGNALING BY PIR-B
The Journal of Immunology
Acknowledgments
We thank Yongmei Hu and Hong Yu for excellent support with maintenance of the mouse strains used in this study. We also thank
Dr. Eric Brown (University of California, San Francisco, CA) and his
laboratory colleagues for thoughtful discussions related to this project.
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the two possibilities, as well as determining where PIR-B’s influence predominates, will necessitate the development of activationspecific mAbs directed against murine integrin subunits.
Overall, these data provide a novel functional role for PIR-B.
Negative regulation of ITAM-dependent immunoreceptor type signaling pathways by ITIM-containing receptors has been extensively documented in a number of different pathways. It is unusual
for an ITIM-containing receptor such as PIR-B to be directly implicated in regulating a nonimmunoreceptor pathway, such as leukocyte integrins. Given that neutrophil integrin signaling is dependent upon Src family kinases, Syk and SLP-76 (4, 7, 44), just as
are many immunoreceptors, it is possible that activation of leukocytes by integrin-mediated adhesion may actually involve ITAMlike sequences. The possible ITAM-like sequences present in the
ERM proteins, which are involved in the actin cytoskeletal rearrangements that follow integrin ligation, may fulfill this role (45).
Clearly, further investigation of the proximal events in integrin
signaling are required to define how activation of a Src-SykSLP-76 pathway is achieved, and how this pathway may be regulated by PIR-B/SHP-1.
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