Eun Young Choi,1Valeria V. Orlova,1Susanna C. Fagerholm,2,3Susanna M. Nurmi,2Li Zhang,4Christie M. Ballantyne,5
Carl G. Gahmberg,2and Triantafyllos Chavakis1
1Experimental Immunology Branch (EIB), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD;2Division of Biochemistry, Faculty of
Biosciences, University of Helsinki, Helsinki, Finland;3Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee,
Dundee, United Kingdom;4Department of Physiology, University of Maryland School of Medicine, Baltimore; and5Department of Medicine, Baylor College of
Medicine and Center for Cardiovascular Disease Prevention, Methodist DeBakey Heart Center, Houston, TX
Inside-out signaling regulation of the ?2-
integrin leukocyte function–associated
antigen-1 (LFA-1) by different cytoplas-
mic proteins, including 14-3-3 proteins, is
essential for adhesion and migration of
immune cells. Here, we identify a new
pathway for the regulation of LFA-1 activ-
ity by Cbl-b, an adapter molecule and
ubiquitin ligase that modulates several
signaling pathways. Cbl-b?/?mice dis-
played increased macrophage recruit-
ment in thioglycollate-induced peritoni-
deficiency in macrophages, as assessed
by bone marrow chimera experiments. In
vitro, Cbl-b?/?bone marrow–derived
mononuclear phagocytes (BMDMs) dis-
played increased adhesion to endothe-
lial cells. Activation of LFA-1 in Cbl-b–
deficient cells was responsible for their
increased endothelial adhesion in vitro
and peritoneal recruitment in vivo, as
the phenotype of Cbl-b deficiency was
reversed in Cbl-b?/?LFA-1?/?mice. Con-
sistently, LFA-1–mediated adhesion of
BMDM to ICAM-1 but not VLA-4–mediated
adhesion to VCAM-1 was enhanced by
Cbl-b deficiency. Cbl-b deficiency resulted
in increased phosphorylation of T758 in the
?2-chain of LFA-1 and thereby in enhanced
association of 14-3-3? protein with the ?2-
tently, disruption of the 14-3-3/?2-integrin
interaction abrogated the enhanced ICAM-1
mediated inflammatory cell recruitment by
stimulating the interaction between the
LFA-1 ?-chain and 14-3-3 proteins. (Blood.
Leukocyte extravasation to the site of infection or inflammation is a
well-organized cascade of adhesive events, including selectin-
dependent rolling, chemokine-dependent leukocyte activation, and
integrin-mediated firm adhesion and diapedesis.1Leukocyte func-
tion–associated antigen-1 (LFA-1; ?L?2; CD11a/CD18) is funda-
mental during firm endothelial adhesion of leukocytes by interact-
ing with endothelial counterligands such as ICAM-1.1-3
LFA-1 integrin activation is crucial for inflammatory cell
adhesion and is regulated by complementary mechanisms involv-
ing affinity alterations due to rapid conformational changes, as well
as affinity-independent mechanisms such as integrin lateral mobil-
ity, resulting in valency/avidity increases.4,5LFA-1 activation can
occur through inside-out signaling (ie, by intracellular signaling
pathways; eg, triggered by chemokines). These pathways include
protein kinases,6lipid kinases,7and small GTPases, such as Rap1
and its effector RAPL.8-10In addition, the actin cytoskeleton is
integral to LFA-1 activation (eg, the interaction of the cytoskeletal
protein talin with the cytoplasmic tail of the LFA-1 ?-chain
stimulates integrin conformational changes and activation).11
During inside-out signaling activation of LFA-1, phosphoryla-
tion of the cytoplasmic tails of the ? and ? chains of the integrin
can regulate their interactions with cytoplasmic factors.6,12,13
Constitutive phosphorylation of LFA-1 on the ?L-chain Ser-1140
regulates integrin affinity changes such as the ones mediated by
Rap1.13LFA-1 ?-chain phosphorylation occurs on several residues
upon cell stimulation. Phosphorylation of the TTT motif (residues
758-760) upon phorbol ester treatment or through T-cell receptor
activation14has been implicated in LFA-1–mediated cell adhesion
to ICAM-1 and modulation of cell spreading,15,16mainly by
mediating cytoskeletal interactions of the integrin and recruitment
tional adaptor proteins that recognize phosphoserine- or phospho-
threonine-containing motifs in proteins.17However, the functional
importance of the 14-3-3/LFA-1 interaction in inflammatory cell
recruitment has not been defined.
The Cbl family consists of 3 homologs, c-Cbl, Cbl-b, and Cbl-3,
that are adaptor molecules undergoing multiple interactions with
protein tyrosine kinases and SH2 and SH3 domain– containing
proteins.18They are E3 ubiquitin ligases, which function to
negatively regulate a diverse repertoire of surface receptors and
downstream signaling proteins.18,19c-Cbl and Cbl-b are predomi-
regulator in autoimmune diseases, as Cbl-b?/?mice were more suscep-
pathogenesis of autoimmune diseases; however, whether Cbl-b and/or
Cbl-b deficiency influence extravasation-related inflammatory cell
functions such as adhesion remains incompletely addressed. In a
recent report, Cbl-b–deficient T cells displayed increased Rap1
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
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3607BLOOD, 1APRIL 2008?VOLUME 111, NUMBER 7
activity and enhanced LFA-1–mediated adhesion of T cells to
ICAM-1 in vitro.23These findings prompted us to investigate
whether Cbl-b deficiency affects leukocyte recruitment in vivo as
well as extravasation-related processes such as integrin-mediated
adhesion of monocytes/macrophages in vitro and to analyze the
Reagents and antibodies
Thioglycollate broth, phorbol 12-myristate 13-acetate (PMA), and ?-actin
antibody were purchased from Sigma-Aldrich (St Louis, MO). Okadaic
acid and calyculin A were from LC Laboratories (Woburn, MA), and the
protein kinase C (PKC) inhibitor Go ¨6983 was from Calbiochem (San
Diego, CA). Recombinant mouse granulocyte-macrophage colony-
stimulating factor (GM-CSF) was obtained from Endogen (Rockford, IL).
Human ICAM-1, mouse ICAM-1, and mouse VCAM-1 were from R&D
Systems (Minneapolis, MN). The blocking monoclonal antibody (mAb) to
mouse LFA-1 (M17/4) and blocking mAb to mouse VLA-4 (R1.2) were
from Biolegend (San Diego, CA). The activating mAb to human CD11a,
MEM-83, and mAb C71/16 against CD18 were from Abcam (Cambridge,
United Kingdom). Phosphospecific polyclonal antibody against phospho-
threonine 758 in CD18 and monoclonal R2E7B against human ?2 subunit
were previously described.24Allophycocyanin (APC)–conjugated antibody
against mouse CD11b was obtained from BD Biosciences (San Jose, CA).
AlexaFluor488 anti-mouse CD18 (M18/2) and FITC-conjugated anti-
human CD11a (HI111) were purchased from Biolegend (San Diego, CA).
Anti-rabbit Alexa 488 and anti-rat Alexa 568 were from Molecular Probes
(Eugene, OR). Antibodies to 14-3-3? (K-19) and Cbl-b (G-1 and H-121)
were from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell lines and cDNA constructs
Mouse endothelioma b.End.3 cells and HEK293 cells were fromAmerican
Type Culture Collection (ATCC; Manassas, VA) and grown as described by
the supplier. 293 cells stably transfected with LFA-1 were previously
described.25All cell culture reagents were purchased from Invitrogen
(Carlsbad, CA) except otherwise described. The wild-type (WT) ?L-
integrin, WT ?2-integrin, and the mutant T758A ?2-integrin constructs
were previously described.13The EGFP-R18wt and EGFP-R18mut plas-
mids were gifts fromT. Pawson (University ofToronto, ON) and previously
described.17The EGFP-R18wt construct blocks 14-3-3 interactions with its
cellular ligands by binding to the phosphopeptide-binding groove in 14-3-3,
whereas the EGFP-R18mut construct does not bind to 14-3-3 proteins.
Isolation of BMDMs
Bone marrow–derived monocyte isolation was performed as described26
with some modification. Briefly, bone marrow cells were flushed from the
bones with ice-cold RPMI containing 10% fetal calf serum (FCS).After red
blood cell (RBC) lysis, the cells were washed and then resuspended in
complete RPMI 1640 medium. Cells were adjusted at a density of
106cells/mL and then cultivated on petri dishes in the presence of murine
GM-CSF (mGM-CSF; 10 ng/mL).After 4 days of incubation, floating cells
were removed and adherent cells were washed twice with phosphate-
buffered saline (PBS). The adherent bone marrow–derived mononuclear
phagocyte (BMDM) population was verified by fluorescence-activated cell
sorter (FACS) analysis as CD11b?cells. Cells were trypsinized for use in
siRNA-mediated knockdown and transfection
293 cells were transfected with siRNA against human Cbl-b or control
siRNA (Dharmacon, Lafayette, CO) using Lipofectamine 2000 (Invitro-
gen); after 24 hours, electroporation was performed (300V, 10 ms) using an
Electro Square Porator ECM 830 (BTX, San Diego, CA) for transient
transfection with human WT LFA-1 ?-chain together with either WT
?2-integrin or mutant T758A-?2 integrin. Western blotting was used to
confirm Cbl-b knockdown, whereas Western blotting and flow cytometric
analysis were used to analyze total and cell surface expression, respectively,
of the ?L- and ?2-chains.
The transfection of primary WT and Cbl-b?/?BMDM with EGFP-
R18wt and EGFP-R18mut was performed engaging a lentiviral system.The
EGFP-R18wt and EGFP-R18mut were amplified by polymerase chain
reaction (PCR) using the primers 5?-CCCGAATTCTTATCTAGATCCG-
GTGG-3? and 5?-CCCGAATTCTTATCTAGATCCGGTGG-3? containing
SpeI and EcoRI restriction sites, respectively. The PCR products were
ligated into XbaI and EcoRI sites of the lentivirus vector pLCMV-PL3 that
was kindly provided by P. Chumakov (Lerner Research Institute, The
Cleveland Clinic Foundation, OH). High titer stocks of recombinant
lentivirus were produced by cotransfection into 293FT cells (Invitrogen) of
pLCMV-PL3-R18wt and pLCMV-PL3-R18mut constructs together with
the packaging plasmids, pREV and pGagPol, and VSV-G (a kind gift from
P. Chumakov) using the Lipofectamine-Plus reagent (Invitrogen). Virus-
containing supernatants were collected 24 to 72 hours after transfection,
and virus was concentrated by PEG precipitation. Briefly, 40% PEG-8000
(Sigma-Aldrich) was added to the collected supernatants. Supernatants
were incubated on ice for at least 12 hours and then centrifuged at 1500g for
20 minutes. The pellets were resuspended in RPMI medium and filtered
before they were applied onto WT or Cbl-b?/?BMDMs in the presence of
8 ?g/mL Sequa-Brene (Sigma-Aldrich). Experiments were performed 3 to
4 days thereafter. The transfection efficiency of R18wt and R18mut was
assessed by immunofluorescence using the EGFP contained in both
constructs and was approximately 50% to 60%.
The generation of Cbl-b?/?and LFA-1?/?mice was described previ-
ously.21,27All mice were backcrossed for at least 7 generations to a
C57BL/6 background. Wild-type C57BL/6 mice were purchased from
Jackson Laboratory (Bar Harbor, ME). Protocols were approved by the NCI
Animal Care and Use Committee.
In vivo peritonitis model
Thioglycollate-induced peritonitis was performed with WT C57Bl/6,
Cbl-b?/?, and LFA-1?/?mice or with Cbl-b?/?LFA-1?/?, Cbl-b?/?LFA-
1?/?, Cbl-b?/?LFA-1?/?, or Cbl-b?/?LFA-1?/?littermates. A previously
described protocol was used.28–30To evaluate peritoneal neutrophil or
macrophage recruitment, mice were killed at 4 or 72 hours, respectively,
following injection of thioglycollate. Thereafter, the peritoneal lavage was
collected and the number of emigrated neutrophils or macrophages was
quantified by FACS analysis by staining for Gr-1 and CD11b.30,31Radiation
bone marrow chimeras were prepared as previously described.30WT and
Cbl-b?/?recipient mice were irradiated with 9.5 Gy (950 rad) and
reconstituted with 1.5 ? 107bone marrow cells fromWTmice (WT 3 WT
and WT 3 Cbl-b?/?).
Flow cytometric analysis
BMDMs as well as peritoneal cells were blocked with mAb 2.4G2 to Fc
receptors and subsequently incubated with APC-conjugated anti-mouse
CD11b antibodies in Hanks balanced salt solution (HBSS) buffer contain-
ing 0.1% bovine serum albumin (BSA) at 4°C for 1 hour. In other
experiments, mouse BMDMs were stained withAlexaFluor488-conjugated
anti-mouse CD18 (M18/2) and LFA-1–transfected 293 cells were incubated
with FITC anti-human CD11a (HI111) antibody. For intracellular 14-3-3?
staining, cultivated monocytes were fixed with 4% paraformaldehyde and
then permeabilized with 0.1% Triton X-100. The cells were then incubated
with antibody against 14-3-3? (K-19) for 1 hour at 4°C, washed with HBSS
buffer containing 0.1% BSA, and then labeled with fluorescein-conjugated
goat anti-rabbit IgG (dilution 1:40; BD Biosciences). After washing, cells
were resuspended in 500 ?L buffer and flow cytometry analysis was
performed using FACSCalibur (Becton Dickinson, Franklin Lakes, NJ).
3608 CHOI et al BLOOD, 1APRIL 2008?VOLUME 111, NUMBER 7
Cell adhesion assay
Adhesion of mouse BMDMs to mouse b.End.3 endothelial cells or
immobilized mouse ICAM-1 or VCAM-1 (each 10 ?g/mL; and to
immobilized BSA as a control) was performed as previously de-
scribed.29,30,32Briefly, mouse endothelial cells were cultivated onto microti-
ter plates until confluence. Alternatively, microplates were coated with
ICAM-1, VCAM-1, or BSA in PBS and blocked with 3% BSA. Fluores-
cence (BCECF)–labeled mouse BMDMs were washed twice in serum-free
medium and plated onto the endothelial cell monolayer or the precoated
wells (105/well) at 37°C for 60 minutes in the absence or presence of PMA
(50 ng/mL) and blocking antibodies. In other experiments, fluorescence-
labeled 293 transfectants were plated onto precoated human ICAM-1 in the
absence or presence of PMA or activating mAb to LFA-1, MEM-83.
Following the incubation period, the wells were washed and adhesion was
quantified using a fluorescence microplate reader (BIO-TEK, Winooksi,
VT). In the case of EGFP-R18– and EGFP-R18mut–transfected BMDMs,
no fluorescence labeling was used, and adhesion was quantified by crystal
violet staining and measuring absorbance at 590 nm.28
Analysis of cell spreading was performed as described.30Analysis of
BMDM transmigration through a monolayer of b.End.3 endothelial cells
cultivated on a transwell filter was performed as described.30
A previously described protocol was used with modifications.30BMDMs
were seeded onto ICAM-1–coated coverslips in culture medium and
incubated at 37°C for 5 minutes. The cells were washed and fixed with 2%
paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton
X-100 for 5 minutes. Cells were blocked with 1% BSAand 5% goat serum
in PBS for 1 hour at 22°C. Cells were then incubated with a combination of
monoclonal rat anti-CD18 (C71/16) and polyclonal anti–14-3-3 (K-19) for
1 hour at 22°C and washed, followed by the incubation of secondary goat
anti-rat Alexa 568 and goat anti-rabbit Alexa 488 in blocking buffer for
30 minutes at 22°C. Thereafter, cells were washed and mounted in
Fluoromount-G (SouthernBiotech, Hatfield, PA). Confocal fluorescence
images were captured using a Zeiss laser scanning microscope (Zeiss,
To assess active Rap1 in mouse BMDMs or mouse splenocytes, the
pull-down assay was performed with EZ-Detect Rap1 Activation Kit
(Pierce, Rockford, IL) according to the manufacturer’s instructions. Briefly,
GST-RalGDS-RBD was added to the Swellgel Immobilized Glutathione
Disc (Pierce) and 500 ?g cell lysates were immediately added.The reaction
mixture was incubated at 4°C for 1 hour with gentle rocking. Precipitates
were then washed, resuspended in 50 ?L of 2 ? sample buffer, boiled for
5 minutes at 100°C, and separated by 12% NuPAGE (Invitrogen). Rap1
antibody was used to detect active Rap1. Whole-cell lysates were used to
assess total levels of Rap1.
Immunoprecipitation and Western blot
Apreviously described protocol was used.13Briefly, BMDMs or peritoneal
macrophages were incubated with complete medium in the absence or
presence of PMA(120 ng/mL) without or with okadaic acid (1 ?M) at 37°C
for 30 minutes. The cells were washed with ice-cold PBS and lysed in a
lysis buffer containing 1% CHAPS, 150 mM NaCl, 1 mM CaCl2, 1 mM
MgCl2, 10 mM Tris-HCl (pH 7.5), 0.02% NaN3, 0.1% BSA, protease
inhibitors, and 0.5 ?M calyculin A. Cell lysates were incubated with
monoclonal anti-mouse CD18 (C71/16) at 4°C overnight with continuous
rotation and then added with UltraLink Immobilized Protein A/G (Pierce).
The complexes containing antibody-boundAg and coprecipitating proteins
were pelleted and washed 5 times with lysis buffer. The bound proteins
were eluted with LDS sample buffer (Invitrogen) containing ?-mercapto-
ethanol and analyzed by Western blotting with antibodies against 14-3-3?
(K-19), CD18 (C71/16), or phosphospecificT758 CD18 antibody.The blots
for phosphospecific T758 CD18 were stripped and reprobed with monoclo-
nal anti-CD18 (C71/16) antibody.
293 transfectants were washed once with ice-cold PBS and lysed in a
1% Triton X-100–containing lysis buffer, boiled, and separated by Nu-
PAGE, followed by protein transfer to nitrocellulose membranes. Mem-
branes were blocked for 1 hour at 22°C and incubated with antibodies
against Cbl-b (G-1), ?-actin, or CD18 (R2E7B). After washing, blots were
Carpinteria, CA) and developed with ECLPlus reagent (Pierce).
Data were compared using the Student t test and analysis of variance
(ANOVA) with post-hoc analysis as appropriate; P values less than .05
were regarded as significant.
Cbl-b deficiency increases LFA-1–dependent inflammatory cell
In order to study whether Cbl-b deficiency affects inflammatory
cell recruitment in vivo, the infiltration of macrophages to the
peritoneum was analyzed in WT and Cbl-b?/?mice after
intraperitoneal injection of thioglycollate. As compared with
WT mice, Cbl-b?/?mice displayed increased macrophage
recruitment into the peritoneal cavity (Figure 1A). No difference
in the baseline numbers of macrophages resident in the perito-
neum (ie, in mice that received PBS) between WT and Cbl-b?/?
mice was observed (Figure 1A). In addition, Cbl-b?/?mice
displayed increased neutrophil recruitment compared with WT
Figure 1. Cbl-b deficiency increases macrophage recruitment in vivo. (A) The
numbers of macrophages in WT (?) or Cbl-b?/?(f) mice are shown at 0 hours and
72 hours after intraperitoneal injection of thioglycollate solution. (B) Thioglycollate-
induced peritonitis in WT mice sublethally irradiated and reconstituted with bone
marrow cells from WT mice (WT 3 WT), or with bone marrow cells from Cbl-b?/?
mice (Cbl-b?/?3 WT). Data are expressed as absolute numbers of emigrated
macrophages into the peritoneum. Data are means plus or minus SD (n ? 3-9
mice/group).#P ? .01; *P ? .05; ns, nonsignificant.
LFA-1ACTIVATION BY Cbl-b DEFICIENCY 3609BLOOD, 1APRIL 2008?VOLUME 111, NUMBER 7
mice at 4 hours after thioglycollate injection (Figure S1A,
available on the Blood website; see the Supplemental Materials
link at the top of the online article).
The enhanced recruitment of macrophages into the peritoneum
associated with Cbl-b deficiency could be due to either intrinsic
differences in Cbl-b–deficient macrophages or due to differences in
the extent of peritonitis induced in Cbl-b–deficient versus WT
control mice. To address these possibilities, bone marrow chimera
experiments were performed. Irradiated WT recipient mice were
reconstituted with either WT or Cbl-b?/?bone marrow. As shown
in Figure 1B, WT mice reconstituted with Cbl-b–deficient bone
marrow displayed enhanced macrophage recruitment in thioglycol-
late-induced peritonitis, suggesting the conclusion that intrinsic
differences in Cbl-b–deficient macrophages account for their
enhanced recruitment into the inflamed peritoneum.
Leukocyte-endothelial adhesion is an integral component of
leukocyte recruitment. Nonstimulated or PMA-stimulated adhe-
sion of Cbl-b?/?BMDMs to endothelial cells was significantly
increased as compared with WT cells (Figure 2A). The 2 major
adhesive interactions involved in leukocyte endothelial adhesion
are the LFA-1/ICAM-1 and VLA-4/VCAM-1 systems.1We there-
fore investigated the adhesion of BMDMs to immobilized ICAM-1
and VCAM-1. Nonstimulated or PMA-stimulated LFA-1–depen-
dent adhesion of Cbl-b?/?BMDMs to immobilized ICAM-1 was
increased as compared with WT cells. In contrast, VLA-4–
dependent adhesion to VCAM-1 was not altered by Cbl-b defi-
ciency (Figure 2B,C; data with PMA stimulation not shown).
Moreover, spreading of Cbl-b?/?BMDMs onto ICAM-1 was
increased compared with WT cells (Figure 2D). Furthermore,
transmigration of BMDMs through an endothelial monolayer was
enhanced due to Cbl-b deficiency (Figure S1B).
To provide further evidence that Cbl-b deficiency specifically
activates LFA-1–mediated adhesion, we generated Cbl-b/LFA-1
double-deficient mice (Cbl-b?/?LFA-1?/?). As expected, LFA-
1?/?BMDMs displayed reduced endothelial adhesion (Figure 2A)
and reduced adhesion to ICAM-1 (Figure 2B) but not to VCAM-1
(Figure 2C). Interestingly, the increased adhesion of BMDMs to
endothelial cells or ICAM-1 due to Cbl-b deficiency was abolished
by LFA-1 deficiency (ie, Cbl-b deficiency failed to increase
adhesion of LFA-1?/?cells to endothelial cells or ICAM-1; Figure
2A,B). In order to verify that Cbl-b–induced LFA-1 activation was
the predominant pathway for the increased macrophage recruit-
ment in vivo, we performed thioglycollate-induced peritonitis
comparing WT, Cbl-b?/?, LFA-1?/?, and Cbl-b?/?LFA-1?/?mice.
Figure 2. Cbl-b deficiency increases LFA-1–dependent BMDM adhesion. (A) Adhesion of BMDMs to mouse endothelial cells is shown in the absence (?) or presence of
PMA(50 ng/mL; f). (B,C)Adhesion of BMDMs to immobilized ICAM-1 (B) or VCAM-1 (C) is shown in the absence (?) or in the presence of mAb to LFA-1 (20 ?g/mL; f) or mAb
to VLA-4 (20 ?g/mL; u). Cell adhesion is represented as percentage of adherent cells. Data are means plus or minus SD (n ? 3). Similar results were observed in 3 separate
experiments. (D) Analysis of spreading of WT and Cbl-b?/?BMDMs onto ICAM-1 was performed. Data are represented as percentage of spread cells. (E) The numbers of
macrophages at 72 hours after intraperitoneal injection of thioglycollate in Cbl-b?/?LFA-1?/?, Cbl-b?/?LFA-1?/?, Cbl-b?/?LFA-1?/?, or Cbl-b?/?LFA-1?/?mice are shown. Data
are expressed as absolute numbers of emigrated macrophages into the peritoneum. Data are means plus or minus SD (n ? 4-5 mice/group). *P ? .05;#P ? .01; ns,
3610CHOI et alBLOOD, 1APRIL 2008?VOLUME 111, NUMBER 7
LFA-1?/?mice had reduced macrophage recruitment (Figure 2E).
Moreover, the increased inflammatory cell recruitment in vivo due
to Cbl-b deficiency required the presence of LFA-1, as macrophage
recruitment in Cbl-b?/?LFA-1?/?mice equaled recruitment of
these cells in LFA-1?/?mice (Figure 2E). Taken together, Cbl-b
deficiency activates LFA-1–mediated macrophage adhesion in
vitro and recruitment in vivo.
Cbl-b deficiency activates LFA-1 by increasing the association
of 14-3-3? with the ?-chain of LFA-1
We then went on to address the underlying molecular mechanisms
of LFA-1 activation in inflammatory cells due to Cbl-b deficiency.
Several cytoplasmic factors, including the GTPase Rap1, talin, and
14-3-3 proteins, are involved in inside-out signaling activation of
LFA-1.8,11,13Previously, it was established that Cbl-b–deficient
T cells displayed increased Rap1 activity and thereby increased
LFA-1 activation.23However, although we found increased Rap1
activity in Cbl-b?/?splenocytes as compared with WT cells, we
observed no difference in Rap1 activity between WT and Cbl-b?/?
tation analysis, we found no difference in the association of talin
with the ?-chain of LFA-1 between WT and Cbl-b?/?BMDMs
(data not shown).
Association of 14-3-3? with the cytoplasmic tail of the ?-chain
of LFA-1, which is mediated by the phosphorylation of T758 of the
?2-chain in response to inside-out activating stimuli, such as
phorbol ester stimulation, can also contribute to LFA-1 activa-
tion.6,13The 14-3-3/LFA-1 interaction regulates LFA-1–mediated
cell adhesion and spreading, as it has been associated with changes
in the lateral mobility and clustering of the integrin, as well as with
effects of LFA-1 on actin reorganization, but not with affinity
changes.13,15,16As we observed increased LFA-1–mediated cell
adhesion and spreading to ICAM-1 of Cbl-b?/?BMDMs (Figure
2B,D), we assessed whether the 14-3-3/LFA-1 interaction is
affected by Cbl-b deficiency. Upon PMA-stimulated adhesion of
BMDMs to ICAM-1, we found colocalization of 14-3-3? with the
?2-chain of LFA-1 (Figure S3A), whereas no colocalization was
observed in nonadherent cells (data not shown). By quantifying the
number of ICAM-1–adherent BMDMs that displayed colocaliza-
tion between 14-3-3? and the ?2-chain, we found this number to be
significantly higher in Cbl-b?/?BMDMs compared with WT
BMDMs (Figure S3B). In order to provide biochemical evidence
about the increased 14-3-3/LFA-1 interaction due to Cbl-b defi-
cipitation of the integrin heterodimer was performed followed by
noprecipitation of LFA-1 with 14-3-3? was found upon PMA
stimulation and with pretreatment with a Ser/Thr phophatase
inhibitor, okadaic acid, consistent with previous observations.13,33
As shown in Figure 3A, increased association of 14-3-3? with
LFA-1 was observed in Cbl-b?/?BMDMs as compared with WT
cells. In contrast, the expression levels of total (Figure 3A) and
surface (Figure 3B) CD18 as well as the total expression of
14-3-3? (Figure 3B) were not different between WT and Cbl-b?/?
cells. The interaction of 14-3-3? with LFA-1 was also studied in
macrophages isolated from thioglycollate-induced peritonitis. WT
peritoneal macrophages displayed weak coimmunoprecipitation of
CD18 with 14-3-3?, especially in the presence of okadaic acid and
PMA. Association of CD18 with 14-3-3? in Cbl-b?/?peritoneal
macrophages was found under nonstimulated conditions, and this
interaction was further stimulated by PMA in the absence or
presence of okadaic acid (Figure S4). Together, these findings
suggest that the interaction of 14-3-3? with the ?2-chain of LFA-1
is enhanced upon Cbl-b deficiency.
In order to demonstrate that the enhanced interaction of 14-3-3?
with the ?2-chain of LFA-1 is responsible for the increased
LFA-1-mediated adhesiveness of Cbl-b?/?BMDMs, we interfered
with 14-3-3 activity.To do so, we engaged the R18wt construct that
binds to the phosphopeptide-binding groove in 14-3-3, thereby
preventing 14-3-3 interactions with its cellular partners, and the
LFA-1 interaction by the R18wt peptide abrogated the enhanced
ICAM-1 adhesion of Cbl-b?/?BMDMs (Figure 4A). These
findings suggest that the 14-3-3/LFA-1 interaction mediates the
activation of LFA-1 induced by Cbl-b deficiency.
Increased T758 phosphorylation in the ?-chain of LFA-1 due to
14-3-3 proteins recognize phosphoserine- or phosphothreonine-
containing motifs, and the interaction between 14-3-3? and the
?2-chain of LFA-1 is dependent on the phosphorylation of T758 in
the cytoplasmic tail of the ?2-chain.13By engaging a phosphospe-
cific antibody against the ?2-chain of LFA-1 that recognizes
phosphorylated T758,24we found that T758 phosphorylation of the
?2-chain was higher in Cbl-b?/?monocytes as compared with WT
Figure 3. Cbl-b deficiency stimulates the increased association of 14-3-3? with
the ?-chain of LFA-1 in BMDMs. (A) The association of ?2-integrin (CD18) with
14-3-3? in BMDMs from WT and Cbl-b?/?mice was studied. Immunoprecipitation of
CD18 was performed in cell lysates of PMA-treated WT and Cbl-b?/?BMDMs. The
presence of immune complexes was determined by Western blot analysis for 14-3-3?
(top panel). In addition, detection of CD18 by Western blot was also performed
(bottom panel). (B) Expression levels of surface ?2-integrin (CD18) and intracellular
14-3-3? (in fixed-permeabilized cells) were analyzed by FACS in WT and Cbl-b?/?
BMDMs. Nonspecific fluorescence was determined using secondary antibodies
LFA-1ACTIVATION BY Cbl-b DEFICIENCY 3611BLOOD, 1APRIL 2008?VOLUME 111, NUMBER 7
cells (Figure 4B). Since T758 can be phosphorylated by several
PKC isoforms,6,12we addressed whether the enhanced ICAM-1
adhesion of Cbl-b?/?BMDMs was affected by inhibition of PKC.
Treatment of Cbl-b?/?BMDMs with a relatively specific PKC
inhibitor, Go ¨6983, reversed their enhanced adhesiveness to immo-
bilized ICAM-1 (Figure 4C).
We then went on to verify in transfectants that the increased
association between 14-3-3 and the ?2-chain was responsible for
the LFA-1–mediated adhesion activated by Cbl-b deficiency. We
transfected 293 cells, which originally lack LFA-1, with WT-?L
together with either WT-?2 or with mutated T758A-?2. This
mutation prevents the interaction of LFA-1 with 14-3-3?.13We
performed siRNA-mediated Cbl-b knockdown in the transfected
293 cells. siRNA targeting Cbl-b but not control nontargeting
siRNA decreased specifically the expression of Cbl-b but not of
other proteins, including the expression of the transfected ?2-chain
of LFA-1 (Figure 5A). Surface expression ofWTLFA-1– or mutant
T758A-?2 LFA-1–transfected cells was comparable both under
control conditions (upon transfection of control siRNA) and upon
Cbl-b down-regulation by Cbl-b–specific siRNA (Figure S5). WT
LFA-1–transfected 293 cells adhered to ICAM-1; this adhesion
was further stimulated by PMA and by activating mAb to LFA-1,
MEM-83, that stimulates conformational changes in LFA-1 and
LFA-1 affinity.34Consistent with previous reports, the T758A
mutation in LFA-1 reduced the constitutive LFA-1–mediated
adhesion to ICAM-1 by 50%, and prevented the stimulating effect
of PMA on adhesion, whereas mAb MEM-83 could still signifi-
cantly stimulate adhesion of mutant T758A LFA-1–transfected
take place despite the T758A mutation. Upon Cbl-b knockdown,
constitutive adhesion of WT LFA-1–transfected cells to ICAM-1
was significantly up-regulated, as was PMA-stimulated adhesion,
albeit to a lower extent. In contrast, Cbl-b knockdown failed to
increase the constitutive or the PMA-stimulated adhesion of mutant
T758A LFA-1–transfected cells to ICAM-1 (Figure 5B). The
adhesion-stimulating effect of MEM-83 on both WT LFA-1–
transfected and on mutant T758A LFA-1–transfected cells re-
mained unaffected by Cbl-b knockdown (Figure 5B). Together,
these results suggest thatT758 in the ?2-chain of LFA-1 is essential
in mediating the activation of LFA-1 induced by Cbl-b
Figure 4. Increased T758 phosphorylation in the ?2-chain upon Cbl-b defi-
ciency mediates LFA-1 activation. (A) Disruption of the association of 14-3-3? with
the ?-chain of LFA-1 abrogates the enhanced ICAM-1 adhesion of Cbl-b?/?BMDMs.
WT(?) and Cbl-b?/?(f) BMDMs were transfected with R18wt or R18mut DNAs, and
allowed to adhere onto immobilized ICAM-1. Cell adhesion is represented as
percentage of control. The adhesion of R18mut-transfected WT BMDMs represents
the 100% control. Data are means plus or minus SD (n ? 3). (B) Increased T758
phosphorylation in the ?2-chain of LFA-1 due to Cbl-b deficiency. T758 phosphoryla-
tion of ?2-integrin (CD18) was determined in PMA-treated WT and Cbl-b?/?
monocytes. Immunoprecipitation of CD18 was performed followed by Western blot
using a phosphospecific T758 CD18 antibody or an antibody against total CD18.
(C) Adhesion of WT or Cbl-b?/?BMDMs to immobilized ICAM-1 is shown in the
absence (?) or in the presence (f) of the PKC inhibitor Go ¨6983 (50 nM). Cell
adhesion is represented as the percentage of adherent cells. Data are means plus or
minus SD. Similar results were observed in 3 separate experiments.#P ? .01; ns,
Figure 5. 293 cells were transfected with siRNA targeting human Cbl-b or with
control nontargeting siRNA. After 24 hours, the cells were transiently transfected
with either WT human LFA-1 or mutant human T758A ?2 LFA-1. The expression of
WTand mutant LFA-1 were comparable and not affected by Cbl-b knockdown (Figure
S5). (A) Cbl-b knockdown was verified by Western blot using an antibody to human
Cbl-b.As a control, Cbl-b knockdown did not affect the expression of ?-actin or of the
transfected ?2-integrin (CD18). (B) 293 cells were transfected with control siRNA or
siRNAtargeting Cbl-b and then with WT or mutant LFA-1 (?LDNAcombined with WT
?2 or T758A ?2) or mock transfected as control, and were allowed to adhere to
immobilized ICAM-1. Adhesion is shown in the absence (?) or presence (f) of PMA
(50 ng/mL) or LFA-1–activating mAb, MEM-83 (10 ?g/mL; u). Cell adhesion is
represented as the percentage of adherent cells. Data are means plus or minus SD.
*P ? .05; ns, nonsignificant.
3612 CHOI et al BLOOD, 1APRIL 2008?VOLUME 111, NUMBER 7
In the present report, we identified a novel, previously unappre-
ciated proinflammatory action of Cbl-b deficiency. LFA-1–
dependent inflammatory cell adhesion to endothelial cells in
vitro and recruitment in vivo are significantly enhanced upon
Cbl-b deficiency. Therefore, LFA-1–mediated inflammatory cell
adhesion joins the multitude of inflammatory pathways that are
negatively regulated by Cbl-b.19Inflammatory infiltrates are a
hallmark of autoimmune diseases. Thus, the increased inflamma-
tory cell recruitment due to Cbl-b deficiency, described here,
may contribute to the higher severity of autoimmunity observed
Cbl-b deficiency specifically induced activation of LFA-1 and
LFA-1–mediated adhesion by increasing phosphorylation of T758
in the ?2-chain of LFA-1 and thereby enhancing the association
between 14-3-3? and LFA-1. Consistently, LFA-1 deficiency
reversed the increased adhesion and recruitment of Cbl-b?/?
macrophages; also, disruption of the interaction between 14-3-3
and the cytoplasmic tail of the ?2-integrin abrogated the enhanced
as dimers, and the 14-3-3?? and ?? isoforms were previously
shown to associate with the cytoplasmic tail of the ?2-chain of
LFA-1 through phosphorylated T758.6Previous studies with trans-
fected cell lines and T cells provided evidence that the 14-3-3/
LFA-1 interaction participates in inside-out signaling activation of
LFA-1.13Thus, our present findings extend the importance of this
interaction to inflammatory cell adhesion and recruitment. To-
gether, Cbl-b deficiency unmasks the 14-3-3/LFA-1 interaction
resulting in LFA-1 activation.
Our experiments identified the interaction between 14-3-3 and
?2-chain as an intermediate step in the enhanced ability of
Cbl-b?/?macrophages to adhere and home to the inflamed tissue.
The affinity of the interaction between 14-3-3 and ?2-integrins is
poised at a low point, most likely due to the low stoichiometry of
T758 phopshorylation in the tail of the ?2-chain.6,33Therefore, the
phosphorylated tail of the ?2-chain and 14-3-3 protein may largely
dissociate during the washing procedures of immunoprecipitation,
thereby resulting in difficulties in detecting the interaction, as has
been previously reported for 14-3-3 interactions with other target
phosphoproteins.35However, the reversibility of the interaction
between the ?2-integrin and 14-3-3 protein make sense for a
dynamic regulatory interaction that has to respond to activation
An interaction between 14-3-3? with ?1-integrin has been
previously identified in nonhematopoetic cells to mediate cell
spreading, adhesion, and migration.36However, we found here no
increased activation ofVLA-4–mediated adhesion of inflammatory
cells due to Cbl-b deficiency. A potential explanation could be the
different nature of the 14-3-3/?1-integrin interaction which is
independent of ?1-integrin phosphorylation.36In contrast, the
14-3-3/LFA-1 interaction is dependent on T758 phosphorylation,
and this phosphorylation was increased by Cbl-b deficiency. T758
can be phosphorylated by several isoforms of the PKC in vitro,
such as PKCs ?, ?, ?, or ? (eg, upon PMA stimulation).6,12
Consistently, we found that treatment with a PKC inhibitor
reversed the higher LFA-1–dependent adhesion in Cbl-b?/?cells.
This result indicates that a PKC isoform may play a role in the
enhanced LFA-1–dependent adhesion due to Cbl-b deficiency,
although this issue requires further investigation.
Another member of the Cbl family, c-Cbl, has been impli-
cated as an intermediate in integrin-mediated outside-in signal-
ing. Ligation of ?2-integrins as well as of ?1-integrins in
neutrophils and monocytes can result in tyrosine phosphoryla-
tion of c-Cbl, mediated predominantly by src family kinases.37-41
c-Cbl thereby participates in the integrin-mediated adhesion-
dependent phosphatidylinositol-3-kinase activation, which is
important for cell-spreading events.37,38Distinct from these
previous reports showing a participation of c-Cbl in integrin-
mediated outside-in signaling, we provide here novel evidence
that Cbl-b is involved in inside-out signaling activation of the
?2-integrin LFA-1, but not of ?1-integrins. It is intriguing that
2 homologous ubiquitin ligases, Cbl-b and c-Cbl, regulate
inside-out signaling activation of ?2-integrins and integrin-
dependent outside-in signaling, respectively.
Previously, the T758-760 motif in the cytoplasmic tail of the
?2-integrin and its phosphorylation were found to be critical for
LFA-1–mediated adhesion and spreading to ICAM-1 through
regulation of integrin clustering rather than affinity changes in
LFA-1,15,16most likely because this motif mediates the interac-
tion of LFA-1 with 14-3-3 proteins.13Consistently, we found
here that Cbl-b deficiency increased constitutive and PMA-
stimulated LFA-1–dependent ICAM-1 adhesion and spreading,
in which integrin affinity changes are only marginally involved.
In contrast, LFA-1–dependent adhesion stimulated by an activat-
ing mAb (MEM-83) that is associated with affinity changes in
LFA-134was not much affected by Cbl-b deficiency. Thus, we
think that the pathway regulated by Cbl-b participates in events
that strengthen LFA-1–dependent adhesion rather than in the
rapid events leading to the initial affinity changes of the integrin.
Moreover, LFA-1 integrin affinity can be regulated by Rap1
activity changes, which are downstream of chemokines or the
T-cell receptor in T cells.3,8We found no changes in Rap1
activity in BMDMs due to Cbl-b deficiency, although we did
find increased Rap1 activity in Cbl-b?/?lymphocytes (data not
shown). This is consistent with a previous report that demon-
strated increased Rap1 activity in Cbl-b?/?T cells upon T-cell
receptor cross-linking.23It is likely that Cbl-b deficiency may
regulate different pathways in distinct cells. Although we found
no alteration of Rap1 activity in BMDMs in vitro, we cannot
exclude that Rap1 is not involved in the increased inflammatory
cell recruitment due to Cbl-b deficiency as observed in vivo.
Nevertheless, our present findings establish that Cbl-b defi-
ciency stimulates LFA-1–mediated inflammatory cell recruit-
ment in vivo, and this could be attributed to an increased LFA-1
activation due to an enhanced association of 14-3-3 with LFA-1
upon Cbl-b deficiency.
We thank Dr M. E. Kruhlak for help with confocal microscopy, D.
Winkler for genotyping, and S. Sharrow andT.Adams for help with
FACS analysis. We would also like to thank Dr J. Chiang and Dr R.
Hodes for providing the Cbl-b?/?mice and for helpful discussions,
Dr T. Pawson for the EGFP-R18 and EGFP-R18mut plasmids, Dr
P. Chumakov for the lentiviral construct, Dr S. Shaw for critically
reading the manuscript, and G. Sanchez-Howard and L. Stepanyan
(Bioqual, Rockville, MD) for help with bone marrow chimera
LFA-1ACTIVATION BY Cbl-b DEFICIENCY3613 BLOOD, 1APRIL 2008?VOLUME 111, NUMBER 7
This research was supported by the Intramural Research Download full-text
Program of the NIH, NCI (T.C.), the Sigrid Juselius Foundation,
and theAcademy of Finland (C.G.G.).
Contribution: E.Y.C. performed research, analyzed data, and wrote
manuscript; V.V.O. performed research; S.C.F. analyzed data and
provided analytical tools and vital reagents; S.M.N. and C.G.G.
provided analytical tools and vital reagents; L.Z. and C.M.B.
provided vital reagents; and T.C. designed and performed research
and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: T. Chavakis, EIB, NCI, NIH, 10 Center
Dr, Rm 4B17, Bethesda, MD 20892; e-mail: chavakist@
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