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,**
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* Cellular and Molecular Biology Division, Toronto Western Research Institute, and
Toronto General Research Institute, University Health Network, Toronto, Canada; Departments of
Immunology,
Biochemistry,
¶ Laboratory Medicine and Pathobiology, and
|| Medicine, University of Toronto, Toronto, Canada;
# Cell Biology Program, Hospital for Sick Children, Toronto, Canada;
** Department of Pathology, The Fourth Military Medical University, Xi’an, China; and
Department of Immunology, University of Alberta, Edmonton, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The emigration of circulating neutrophils from blood to inflamed tissue
is a complex process dependent upon the coordination of many cellular
functions that culminate in directed cell movement (4, 5, 6).
Adhesion receptors are key to several of these processes.
Transendothelial migration is initiated by neutrophil rolling mediated
by the selectin family of adhesion receptors. Rolling neutrophils
arrest on endothelium and undergo firm adhesion, characterized by
spreading and the formation of focal adhesion-like structures mediated
by integrin receptors. Integrins are heterodimeric transmembrane
proteins consisting of noncovalently bound 
- and 
-chains
(7). In neutrophils, the 1
integrins comprise a common -chain (CD29) and distinct -chains
(CD49) (8, 9). The 2 family of
integrins share their -chain (CD18) with distinct -chains (CD11).
The importance of the 2 integrins in adhesion
and transmigration is evident by the analysis of patients with
leukocyte adhesion deficiency type I syndrome which results from
mutations in CD18 that lead to severe deficiency of the CD11/CD18
integrins and profound reduction in neutrophil recruitment during
inflammation (10). The 2
integrins are critical for firm adhesion of rolling neutrophils on
vascular surfaces and transendothelial migration (11, 12).
LFA-1 (L2,
CD11a/CD18) and Mac-1 (M2, CD11b/CD18)
are the most abundant 2 integrins on neutrophils.
Although these integrins share ICAM-1 as a ligand, the regulation of
their surface expression and their function during neutrophil migration
appear distinct. Neutrophil activation results in significant
up-regulation of surface Mac-1 levels, but not LFA-1 (13).
Although the importance of both integrin receptors in firm adhesion of
neutrophils to ICAM-1 or to endothelial surfaces was demonstrated, the
role of Mac-1 in recruitment of neutrophils into inflammatory sites is
not completely understood. Although a number of studies showed
inhibition of neutrophil migration by Mac-1 blocking Abs, LFA-1
appeared to play a more important role than Mac-1 in recruitment to
inflammatory sites (13, 14, 15). Interestingly, increased
accumulation of neutrophils was found at inflammatory sites in
Mac-1-deficient mice which correlated with their increased survival
(16). Thus, distinct or additional roles for Mac-1 have
been demonstrated as compared with LFA-1 in inflammatory response.
The tight adhesion of neutrophils to extracellular matrix proteins is accompanied by polarization toward a chemotactic gradient that mediates migration to the inflamed site. Polarization is achieved by the formations of lamellipodia (flat sheet-like structures) and filopodia (thin needle-like projections) at the leading edge and uropod at the rear of the cell (17). Cycles of attachment to the extracellular substrate at the front and detachment from the rear are mediated by temporal and spatial changes in the localization and strength of adhesive forces through integrin receptors (18, 19). Lamellipodia and filopodia are devoid of cytoplasm but contain mainly microfilaments that are formed by local filamentous actin (F-actin)3 polymerization. F-actin filaments are bundled in filopodia and cross-linked in lamellipodia (20). Thus, proteins which promote F-actin polymerization, or bundle or cross-link actin filaments, are key to provide the rigidity required for these structures to extend, and modulations in their expression have major impact on motile properties of cells (21, 22, 23, 24, 25).
The mouse leukocyte-specific protein 1 (LSP1; formerly lymphocyte-specific protein 1 but renamed to reflect its expression in macrophages and neutrophils) (26, 27) is a 52-kDa intracellular F-actin binding protein which accumulates on the cortical cytoskeleton (28, 29, 30). LSP1 mRNA and protein is expressed in mature and immature B and T cells, macrophages, and neutrophils (31, 32, 33). Human and mouse LSP1 share the same expression patterns (26, 34, 35, 36, 37) and the amino acid identity between the two species is 85% for the basic C-terminal half containing multiple F-actin binding domains and 53% for the acidic N-terminal half. LSP1 has two putative EF-hand motifs near the N terminus and rLSP1 binds Ca2+. LSP1 is a substrate for mitogen-activated protein kinase activated protein kinase 2 (37) and for protein kinase C (38).
Adult Lsp1-/- mice generated in our laboratory show no significant differences from wild-type (wt) mice in the development of the lymphoid and myeloid lineage (27). They display a 3-fold increase in resident peritoneal macrophages but normal proportions of B and T cells within the lymphoid population. Thioglycollate-induced influx of monocytes and neutrophils into the peritoneum is increased in Lsp1-/- mice. In vitro transmigration assays using elicited neutrophils demonstrated increased chemoattractant-induced motility of Lsp1-/- neutrophils. These results suggest that LSP1 is a negative regulator of neutrophil motility in vivo and in vitro. Studies by Howard and colleagues (39, 40) support this conclusion. LSP1 is over-expressed and a 89-kDa protein is under-expressed in patients with neutrophil actin dysfunction (NAD) disorder 47/89. Neutrophils from these patients and transfectants with high expression of LSP1 display formations of large F-actin bundles branching into hair-like cell surface projections and impaired motility (41, 42).
In this paper, we evaluated the in vivo role for LSP1 in regulating neutrophil influx to acute inflammation in the joint and investigated the requirement for LSP1 in various processes which culminate in fMLP-induced neutrophil motility with a long-term goal of elucidating the mechanism(s) by which LSP1 functions as a negative regulator of neutrophil migration during inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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129/SvJ mice (wt and Lsp1-/-) were bred and housed in microisolators in the Toronto Western Research Institute Animal facility (Toronto, Ontario, Canada). Eight- to 12-wk-old female or male mice were used. All protocols were approved by the Animal Care Committee of the University Health Network.
Reagents and Abs
Zymosan, fMLP, PMA, fibrinogen, fibronectin (human plasma), poly-L-lysine, BSA (IgG-free, low endotoxin), and HBSS with or without Ca2+/Mg2+ were purchased from Sigma-Aldrich (St. Louis, MO); Immulon 2HB microtiter plates, used in adhesion assays, were obtained from Dynex Technologies (Chantilly, VA). Mouse ICAM-1 was purified from NS-1 B lymphoma cell membranes by affinity chromatography (43). Human chimeric VCAM-1/Fc and ICAM-2/Fc were purchased from R&D Systems (Minneapolis, MN). FITC-conjugated fMLP, Calcein-AM, and Alexa Fluor 488 Phalloidin were obtained from Molecular Probes (Eugene, OR).
For flow cytometry, mAbs against Mac-1 (FITC-anti-CD11b, M1/70;
Serotec, Oxford, U.K.), Gr-1 (PE-conjugated), corresponding isotype
controls (Cedarlane Laboratories, Hornby, Ontario, Canada), or LFA-1
(FD441.8; a kind gift from Dr. F. Takei, Terry Fox Laboratory,
Vancouver, British Columbia, Canada), 4 or
5 (PS/2 or BMA-5, respectively; a kind gift
from Dr. B. Chan, University of London, Ontario, Canada) in conjunction
with biotinylated anti-rat Abs and streptavidin-PE (Cedarlane
Laboratories) were used. F(ab')2 of goat
anti-rabbit IgG (Fc-specific) and anti-FITC Abs were obtained
from Caltag Laboratories (Burlingame, CA), unconjugated blocking Abs
against mouse Mac-1 (M1/70), LFA-1 (M17/5.2), and CD18 (2E6) were
purified from corresponding hybridoma cultures (American Type Culture
Collection, Manassas, VA), isotype control rat IgG2b (low endotoxin)
was obtained from BD PharMingen (San Diego, CA).
Zymosan-induced acute inflammation in mouse knee joints
Intra-articular injection of 180 µg zymosan was performed along the suprapatellar ligament into the joint cavity of anesthetized mice (44). The contralateral knee was injected similarly with an equal volume of nonpyrogenic saline. Both knee injections were completed by injection of the dye fast green (1.5 µl) separated from zymosan by a small air bubble (0.1 µl) to assess the success of the injection. The intra-articular injections were performed using a microinjector equipped with an operating microscope (Zeiss, Toronto, Ontario, Canada) attached to a MedVet anesthetic system. After 24 h, swelling of the knees was determined by using a microcaliper (Fisher Scientific, Whitby, Ontario, Canada), knee joints were dissected, fixed in 10% buffered formalin and decalcified in a 25% formic acid, 0.4 M sodium citrate solution. H&E staining was performed by Cell Path Technologies (Toronto, Canada). Inflammation was evaluated by two independent scientists in a blinded fashion by scoring the influx of inflammatory cells into the subsynovium and joint cavity (synovial fluid).
Isolation of bone marrow (BM) neutrophils
Mouse BM cells were flushed from the femur and tibia of wt and Lsp1-/- male mice with Ca2+/Mg2+-free HBSS. Cells were washed and resuspended in 45% Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) and neutrophils were purified from Percoll density gradients (45). RBC were lysed with E-lyse solution (Cardinal Associates, Santa Fe, NM). Cells were washed with ice-cold Ca2+/Mg2+-free HBSS (for Calcein-AM labeling) or HBSS with Ca2+/Mg2+ and 0.1% BSA (HB, for all other studies). Flow cytometry revealed high expression of the murine granulocytic marker Gr-1 typically on 75–80% of the isolated cells.
Chemotaxis and chemokinesis assays
Chemokinesis was performed by adding fMLP directly to BM neutrophils and chemotaxis was performed by using a point source of fMLP derived from a micropipette. Micropipettes with tips 30–100 µm in diameter were filled with 10-3 M fMLP in 1% low-melt agarose (FMC Bioproducts, Rockland, ME). Neutrophils (0.4 x 106 in HB) were plated on fibrinogen or fibronectin-coated coverslips and were incubated for 10 min in a 37°C heated chamber to allow for adhesion. Nonadherent cells were removed by several washes with HB. Time-lapse video microscopy was performed by capturing differential interference contrast images every 15 s under a Leica DM IRB microscope using MetaFluor software (Universal Imaging, West Chester, PA). Image acquisition was initiated 1 min before the placement of the micropipette or addition of fMLP into the chamber to obtain resting neutrophil images. The average speed was calculated from the total migration distance determined by MetaMorph software (Universal Imaging) and the elapsed time. In chemokinesis experiments the response criterion was cell movement at least over a distance corresponding to one cell body.
Coating with extracellular matrix proteins
Fibrinogen (1 mg/ml), fibronectin (20 µg/ml), or ICAM-1 (1 µg/ml) in PBS were added to microtiter wells or to acid-washed coverslips and incubated for 2 h at 37°C. Unbound proteins were removed, plates coated with ICAM-1 were blocked with 2% BSA (IgG free, low endotoxin; Sigma-Aldrich) in PBS for 30 min at room temperature (RT). For coating with VCAM-1/Fc and ICAM-2/Fc, goat anti-human IgG (Fc-specific F(ab')2, 100 µg/ml) were added to each well for 60 min at RT, followed by blocking with 2% BSA before the addition of 20 µg/ml VCAM-1/Fc or ICAM-2/Fc overnight at 4°C. All coating steps were followed by three washes with PBS. Poly-L-lysine was coated (10 µg/ml) as described by the supplier.
For adhesion assays under shear flow, 20 µl goat anti-rabbit IgG (Fc-specific F(ab')2, 50 µg/ml) and fibrinogen (100 µg/ml) were adsorbed onto the center of a 35-mm dish for 60 min in a humidified atmosphere at RT; 10 µl rabbit anti-FITC Abs (20 µg/ml) were subsequently added for 60 min. After blocking with 0.5% BSA overnight at 4°C, 10 µl fMLP-FITC (20 µg/ml) were added at RT.
Neutrophil adhesion assays
For static adhesion assays, Calcein-AM-labeled (27) BM neutrophils in HB (106/100 µl) were plated into precoated microtiter wells (in triplicate) and incubated at 37°C with 7% CO2 for 60 min following the addition of 100 µl agonists. Plates were centrifuged upside-down as described (45) and a CytoFluor 2300 fluorometric plate reader (Millipore, Nepean, Ontario, Canada) was used to calculate the percentage of adhered cells. A standard curve was constructed with increasing amounts of labeled cells added into a separate plate.
For assays under shear flow, BM neutrophils (106/ml) settled for 2 min in a parallel flow chamber as previously described (46). Shear stress was applied at 4 dynes/cm2 for 30 s. Cells were observed under an inverted phase contrast microscope, videotaped, and analyzed (46).
fMLP-induced Mac-1 up-regulation and F-actin polymerization
For Mac-1 up-regulation, BM neutrophils in HB (0.5 x 106 cells) were prewarmed for 4 min at 37°C and stimulated with different concentrations of fMLP for 10 min, and stained with FITC-conjugated anti-Mac-1, PE-conjugated anti-Gr-1, or isotype controls (47). Mac-1+ live cells were analyzed within the gated Gr-1high neutrophils by flow cytometry using FACSCalibur and CellQuest Software (BD Biosciences, San Jose, CA).
For F-actin measures, BM neutrophils in HB (2.5 x 106/ml) were prewarmed at 25°C and incubated with or without 10-6 M fMLP for different times, fixed with 3.7% paraformaldehyde in PBS for 20 min at RT, washed, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. After washing, cells were stained with Alexa Fluor 488 phalloidin for 30 min and analyzed by flow cytometry as above.
Fluorescence microscopy
BM neutrophils (0.4 x 106 cells) in HB were preincubated on fibrinogen- or fibronectin-coated coverslips for 10 min before incubation with or without 10-5 M fMLP for 30 min at 37°C with 5% CO2. After washing briefly with prewarmed HB, the adhered cells were fixed with 4% paraformaldehyde solution (Canemco, Quebec, Canada) for 30 min at RT. Cells were stained for F-actin as described above. Coverslips were mounted with DAKO fluorescent mounting medium (Carpinteria, CA). Slides were examined with a Zeiss LSM 510 scanning laser confocal mounted on a Zeiss Axiovert 100 microscope with a 100x oil immersion objective. Alexa Fluor 488 phalloidin-stained neutrophils were visualized with a fluorescein filter set (515 nm dichroic mirror, 530 nm long-pass barrier filter) using Argon laser (488 nm) illumination. Digital images were prepared using Adobe Photoshop 6.0 software (Adobe Systems, Mountain View, CA). Two hundred cells were counted under a Leica DMRB immunofluorescence microscope to determine the proportion of cells with head-to-tail morphology. The overall F-actin levels in adhered cells was determined from merged stacked confocal images of cells, acquired under nonsaturating laser intensities, using NIH Image 1.62 software. Cell outlines were manually traced on images and pixel intensities in the Alexa Fluor 488 phalloidin-stained areas were expressed as relative fluorescent intensity (RFI).
Statistical Analysis
All data are expressed as mean values ± 1 SEM. Unpaired Student’s t tests or Welch’s t tests were performed using InStat software (GraphPad Software, San Diego).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported increased accumulation of Lsp1-/- neutrophils as compared with wt during thioglycollate-induced peritonitis. Lsp1-/- mice exhibit increased resident macrophage levels in the peritoneum but not in the lung, indicating that LSP1 deficiency does not affect all resident macrophage populations. Because, in our former study, we did not eliminate the possibility that the increased level of resident macrophages contribute to the increased influx of neutrophils to the peritoneum, we chose to induce acute inflammation in another tissue where resident cell levels would be unaffected by LSP1-deficiency.
The thin cell layer of resident synoviocytes along the synovial
membrane are composed of macrophages and fibroblasts (48, 49). To verify whether the unstimulated synovial lining displays
a similar number of resident cells in the knee joints from wt vs
Lsp1-/- mice, we counted 200–500 total
cells/mouse synovial lining and determined the average cell number per
unit length. We found no differences between five mice from each
genotype (not shown), confirming that the resident cells of
unstimulated knee joints are equivalent. To determine the accumulation
rate of Lsp1-/- vs wt neutrophils in this
system, we injected zymosan into the knee joints. Within 24 h,
zymosan induced a typical acute inflammation characterized by swelling
of the joints and the influx of inflammatory cells (50, 51). Swelling which is a manifestation of edema due to increased
vascular permeability was not affected by LSP1-deficiency (Table I
). Neutrophils, identified by their
typical multilobed morphology by H&E staining, were the predominant
cells accumulating in the joint at this early time point of
inflammation. Fig. 1 illustrates an
example for each genotype analyzed and Table I summarizes the average
scores for inflammation. Fig. 1, A and B, shows
control joints injected with saline, which exhibit similar morphology
in both genotypes. Zymosan-induced accumulation of neutrophils was
significantly increased in the synovial cavity of
Lsp1-/- mice (Fig. 1, D and
F) as compared with wt (Fig. 1, C and
E) (Table I). Although increased levels of infiltration into
the subsynovium were also found in
Lsp1-/- mice, the differences were not
statistically significant. These results demonstrate that LSP1
negatively regulates neutrophil recruitment into the joint during acute
inflammation independently from changes in resident cell numbers.
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We previously demonstrated that in the absence of LSP1, there is
increased in vitro transmigration of thioglycollate-elicited
neutrophils in response to chemoattractants, fMLP, or KC. These
findings raised the question of whether the increased chemotaxis of
Lsp1-/- neutrophils depends on their
activation state since we used elicited neutrophils. Furthermore, the
transmigration assays do not distinguish between increased speed per
cell or an increased number of cells responding. To better elucidate
the role of LSP1 in neutrophil motility, we performed time-lapse in
vitro fMLP-induced chemokinesis and chemotaxis assays on fibrinogen or
fibronectin using BM neutrophils purified from
Lsp1-/- and wt mice. Chemokinesis assays,
in which a uniform concentration of fMLP (10-4 or
10-5 M) induced undirectional movement, revealed similar
number of cells responding (75–80 of 100 cells/genotype, not shown) on
either substrate. Chemotaxis assays were used to determine the average
migration rate toward a gradient of fMLP per cell. Table II shows that in the absence of LSP1,
neutrophils gained a significant increase in their average speed during
chemotaxis on fibrinogen-coated coverslips. Interestingly, the average
speed of wt and Lsp1-/- neutrophils was
similar on fibronectin-coated coverslips. These data raised the
question of whether the negative regulatory role of LSP1 in neutrophil
motility is related to its role in adhesion and whether such a role
would be specific for a particular class of integrins.
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2 integrin-mediated
adhesion
Neutrophils require an optimal level of adhesion via
1 and 2 integrins on
activated endothelial surfaces to mediate spreading and chemotaxis
(19, 52). The 1 or
2 family of integrins on neutrophils mediate
binding to extracellular matrix proteins or their counterreceptors such
as fibronectin (41,
51), VCAM-1
(41,
91) or fibrinogen
(M2,
X2), ICAM-1
(L2,
M2) or ICAM-2
(L2).
We performed in vitro adhesion assays with BM neutrophils using various
substrates which serve as ligands for 2 (Fig. 2) or 1 (Fig. 3) integrins. Fig. 2, A,
B, and D, illustrates fMLP and PMA-induced tight
adhesion to ICAM-1, fibrinogen, or ICAM-2, respectively, which are
mediated by 2 integrins. Adhesion of
unstimulated wt and Lsp1-/- neutrophils
is similar with each substrate tested. Upon fMLP stimulation, we
observed a significant increase in tight adhesion of wt neutrophils to
fibrinogen, but not to ICAM-1, while fMLP-stimulated
Lsp1-/- neutrophils adhered more tightly
(1.5- to 2-fold) to both extracellular proteins (Fig. 2, A
and B). We obtained a similar increase in fMLP-induced
adhesion of Lsp1-/- neutrophils when
cells were layered on wells coated with five times less fibrinogen (0.2
mg/ml): wt 8.90 ± 0.83%; Lsp1-/- 12.56
± 1.05% (n = 5, p < 0.03),
indicating that saturating amounts of fibrinogen have been used
throughout this study. Because under physiological conditions
neutrophils sense and respond to stimulus under shear flow, we repeated
the fMLP-induced adhesion experiments to fibrinogen under conditions
where neutrophils were subjected to a shear force of 4
dyn/cm2 (Fig. 2C). Under these
conditions, 58.2 ± 1.6% of wt neutrophils and 86.9 ± 1.4% of
Lsp1-/- neutrophils remained adhered to
fibrinogen. A shear force of 10 dyn/cm2 yielded
similar results (not shown). We have thus confirmed by two different
assays that LSP1 deficiency results in enhanced fMLP-induced adhesion
to fibrinogen. These results, together with significantly increased
adhesion to ICAM-1, suggest strongly that LSP1 negatively regulates
2 integrin-mediated adhesion. Although
fibrinogen is a ligand for Mac-1, the increased binding of
Lsp1-/- neutrophils to ICAM-1 may be the
result of binding to both Mac-1 and LFA-1. To verify the capacity of
neutrophil adhesion to LFA-1 in the presence or absence of LSP1, we
conducted adhesion assays to the specific LFA-1 ligand ICAM-2.
Although, fMLP did not induce adhesion to ICAM-2 in wt or in
LSP1-deficient neutrophils (Fig. 2D), PMA induced similar
adhesion to ICAM-2 in neutrophils from both genotypes showing that
murine neutrophils are capable of adhering to this extracellular
protein. Similar levels of PMA-induced adhesion were also observed with
ICAM-1 (Fig. 2A) while adhesion of
Lsp1-/- neutrophils to fibrinogen was
significantly increased as compared with wt in the presence of PMA. The
lack of difference in PMA-induced adhesion to ICAM-1 of wt and
Lsp1-/- neutrophils may be due to their
similar binding via LFA-1 as was shown for ICAM-2 (Fig. 2D).
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1
integrin-mediated firm adhesion, we performed adhesion assays with
extracellular matrix proteins VCAM-1 or fibronectin. Fig. 3
, A and B, shows that unstimulated neutrophils of
both genotypes bind similarly at background levels to VCAM-1 or
fibronectin. Interestingly, the increase in adhesion by fMLP or PMA to
these substrates is indistinguishable between wt and
Lsp1-/- neutrophils. Neutrophil adhesion
was similar between the two genotypes when lower (5 µg/ml) or higher
(100 µg/ml) concentrations of fibronectin were used to coat the
wells. The fMLP-induced adhesion on 100 µg/ml fibronectin was: wt
19.93% ± 1.60; Lsp1-/- 18.67% ± 2.86
(n = 3), indicating that the 20 µg/ml fibronectin
used throughout this paper is saturating.
Our attempts to confirm the identity of the 2
integrin for which LSP1 is a negative regulator of adhesion by use of
blocking Abs against murine integrin receptors have not been
successful. We have used anti-CD11b (M1/70), anti-LFA-1
(M17/5.2, FD441.8), and anti-CD18 (2E6) Abs which we purified from
hybridoma supernatants to block adhesion (13). However,
these and the control IgG2b Abs (low endotoxin) stimulated, rather than
blocked, adhesion of BM neutrophils from both genotypes even when
F(ab')2 were used. As a test for the involvement
of integrins in the increased adhesion of LSP1-deficient neutrophils,
we repeated the adhesion assays in the absence of
Ca2+ and Mg2+ which are
required for integrin function (7, 53, 54). Table III illustrates that unstimulated, fMLP-,
or PMA-induced neutrophils of either genotype were unable to bind to
ICAM-1 in the absence of the divalent ions and consequently, the
increase in binding of Lsp1-/- neutrophils to
ICAM-1 as compared with their wt counterparts was abolished. Similarly,
PMA-induced adhesion to ICAM-2 was also abolished in the absence of
Ca2+ and Mg2+ (not shown).
In contrast, adhesion of unstimulated or stimulated neutrophils of
either genotype to poly-L-lysine, which does not involve integrins, was
not significantly affected by the absence of Ca2+
and Mg2+. It is notable that LSP1 deficiency did
not affect adhesion to poly-L-lysine under any of the conditions
tested.
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2 integrins,
and among them most likely Mac-1, rather than LFA-1, as one of the
integrins being regulated by LSP1. LSP1 is not required for fMLP-induced Mac-1 up-regulation and F-actin polymerization
Neutrophils up-regulate their surface Mac-1 levels within minutes
of agonist stimulation (55, 56). We asked whether the
negative regulatory role of LSP1 in Mac-1-mediated adhesion could be at
the level of Mac-1 expression in fMLP-stimulated neutrophils. Fig. 4A shows a robust increase in
surface Mac-1 levels (3- to 4-fold) 10 min after fMLP stimulation which
was similar for wt and Lsp1-/-
neutrophils. This demonstrates that LSP1 is not involved in the
up-regulation of Mac-1 surface expression in stimulated neutrophils.
Surface expression of other integrins, LFA-1,
41 or
51, which do not
significantly change after fMLP stimulation, were similar in
neutrophils from both genotypes (not shown).
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B). Neutrophils stimulated with
10-6 M fMLP displayed a maximal F-actin
polymerization at 30 s which declined and reached a plateau level
after 1 min. LSP1 deficiency did not substantially change this kinetics
except at 2 min where a small (14.3%) but statistically significant
decrease in F-actin content in Lsp1-/-
neutrophils was observed. No differences in F-actin content were
observed with 10-5 M fMLP in any of the time
points tested (not shown). Taken together, these results demonstrate a
lack of LSP1 involvement in F-actin polymerization in fMLP-induced
neutrophils. LSP1-deficiency leads to increased polarization of neutrophils on fibrinogen
Because LSP1 exhibits F-actin bundling capacity (29, 41), it was logical to determine whether the specific negative
regulatory role for LSP1 in fMLP-induced neutrophil adhesion to
fibrinogen, but not to fibronectin, would be accompanied by similar
differences in F-actin polarization on these substrates. Adhesion was
induced by fMLP on fibrinogen- or fibronectin-coated coverslips and
neutrophils were stained for F-actin. When analyzed by confocal
microscopy, a small number of unstimulated neutrophils with typical
unpolarized spherical appearance were found on coverslips coated with
either substrate (see insets in Fig. 6, A1 and
B1). With fMLP, both wt and
Lsp1-/- neutrophils underwent
polarization on either substrate (Figs. 5 and 6). On fibrinogen, these changes were
much more pronounced in Lsp1-/-
neutrophils than in wt, while they were similar between the two
genotypes on fibronectin. As shown in Fig. 5, A1,
A2, and B, wt neutrophils on fibrinogen displayed
polarized F-actin patches at the anterior end (lamellipodia, white
arrows) which were lost in regions further away from the substratum
(Fig. 5A, 3–5). Polarized wt neutrophils displayed one or
two trailing filopodia at the posterior end (Fig. 5A, open
arrowheads; Fig. 5B, inset).
Lsp1-/- neutrophils on fibrinogen (Fig. 5C) displayed larger areas of intense F-actin caps as
compared with wt (white arrows). These caps were thicker, as they could
be seen in optical sections away from the substratum (see Fig. 5C, 1–5). Some neutrophils from both genotypes
had more than one lamellipodia (insets), but again the
Lsp1-/- neutrophils displayed more
polarized structures. We also found a significant number of
Lsp1-/- neutrophils with bright
actin-stained focal contacts at the proximity of lamellipodia which
often had more extensive trailing filopodia formations than in wt (Fig. 5D, arrowheads). Quantitatively, on fibrinogen, there was a
2-fold increase (2.06 ± 0.19, n = 3) in the
proportion of Lsp1-/- neutrophils which
exhibit highly polarized F-actin with head-to-tail morphology as
compared with wt. On fibronectin, the extent of head-to-tail
polarization of F-actin in wt neutrophils was similar to that of
Lsp1-/- neutrophils (Fig. 6, A
and B, and inset B). Lamellipodia could be
observed throughout the different optical sections of both genotypes
(Fig. 6, A or B, 1–6). The ratio of
polarized wt vs Lsp1-/- neutrophils was
1.11 ± 0.16, n = 3.
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),
these data show that LSP1-deficiency does not affect the overall
F-actin levels stimulated by fMLP even when neutrophils exhibit
increased adhesion and F-actin polarization on fibrinogen. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, GM-CSF, IL-8, and other chemokines) that activate neutrophils
(57, 58). We reported previously that neutrophil influx
into the peritoneum is increased in response to thioglycollate in
Lsp1-/- mice which harbor increased
levels of resident peritoneal macrophages. Therefore, it was important
to study the role of LSP1 in neutrophil migration in vivo,
independently from changes in resident cell levels to distinguish
between intrinsic vs environmental factors influencing the migration of
activated neutrophils. In this study, we found that in unstimulated
knee joints the number of the synoviocytes was indistinguishable
between the two genotypes while the accumulation in the synovial fluid
of Lsp1-/- neutrophils was significantly
increased in response to zymosan. These findings demonstrate that the
increased neutrophil recruitment in
Lsp1-/- mice is independent from the
resident cell numbers and is likely due to the changes in the
chemotactic characteristics of neutrophils.
Our in vitro studies using single cell analysis by time-lapse video
microscopy of BM neutrophils further confirmed the notion that LSP1
deficiency affects the locomotive machinery of neutrophils because we
showed that the average migration rate per cell toward a gradient of
fMLP is greater in the absence of LSP1 and this does not require
previous activation of neutrophils. Interestingly, we also found that
this was dependent on the substrate on which the neutrophils migrate.
Accelerated migration of LSP1-deficient neutrophils occurred on
fibrinogen, but not fibronectin, raising the possibility that the role
of LSP1 on neutrophil migration stems from a specific role for this
protein in adhesion via certain integrins. In vitro adhesion assays
using different extracellular substrate proteins indeed showed that
fMLP-stimulated Lsp1-/- neutrophils
exhibit increased adhesion to the 2 integrin
ligands, fibrinogen (Mac-1), or to ICAM-1 (LFA-1 and Mac-1) but not to
ICAM-2 (LFA-1). Adhesion to 1 integrin ligands
VCAM-1 or fibronectin were similar. These results suggest strongly for
the first time that the negative regulatory role of LSP1 in cell
migration is dependent on its role in adhesion via specific integrins.
Although adhesion to fibrinogen may not exclusively occur via Mac-1,
the fact that increased adhesion of
Lsp1-/- neutrophils occurs both to ICAM-1
and fibrinogen and not to ICAM-2, fibronectin, and VCAM-1 strongly
suggests that at least one integrin modulated by LSP1 is Mac-1, while
LFA-1 and 1 integrins are not negatively
regulated by this protein. The tight adhesion of wt neutrophils to
fibronectin or VCAM-1 was stronger than to fibrinogen or ICAM-1
and similarly the extent of polarization and average migration rate of
wt neutrophils were higher on fibronectin than fibrinogen. Thus, at
least in the case of 1 integrins, the strength
of adhesion may compensate for the negative regulatory role of
LSP1.
During chemotaxis, neutrophils undergo transient shape changes by sending forward lamellipodia in the direction of migration. These attach to the extracellular matrix and allow the cell body to move toward this new attachment. Cells also release attachments of their posterior ends (6, 59). In this study, we observed striking differences in fMLP-induced F-actin polarization between wt and LSP1-deficient neutrophils when they were induced to adhere on fibrinogen, while adhesion to fibronectin did not result in such differences. It is notable that in suspended neutrophils, in the absence of any attachment, fMLP-stimulated F-actin polarization, as measured by percent-polarized cells or the area occupied by F-actin caps was similar between wt and Lsp1-/- neutrophils (not shown). These findings support the notion that LSP1 is not involved directly in fMLP-induced polarization of F-actin but rather emphasized the function of LSP1 in regulating the gradient of adhesive strength which develops during chemotaxis.
Hannigan et al. (60) recently reported that LSP1-deficient blood neutrophils exhibit impaired chemotaxis toward a gradient of the chemokine KC on glass coverslips, which they attributed to undirectional protrusions produced in the absence of LSP1. Our data show the importance of the substrate used for understanding the role of LSP1 in chemotaxis. They also reported that LSP1 is not required for adhesion to fibrinogen in response to KC or PMA. Besides the differences in the experimental conditions, these authors used total bone marrow cells as opposed to purified neutrophils, which may have masked the increased adhesion of neutrophils.
A specific role of LSP1 in the functioning of Mac-1 rather than LFA-1 can be considered if LSP1 interacts with Mac-1 directly or through an adapter protein and provides a linkage to F-actin in unstimulated neutrophils. Given that LSP1 is an abundant protein with F-actin bundling capacity, we suggest that LSP1 deficiency results in a more flexible actin-cytoskeleton architecture which undergoes more efficient rearrangements during adhesion. We envisage that in wt cells, LSP1 undergoes modification by phosphorylation and dissociates from F-actin to allow for cytoskeletal rearrangement as has been described for the macrophage-enriched myristoylated alanine-rich C kinase substrate or L-plastin, two other F-actin bundling proteins (61, 62). LSP1 could be phosphorylated by mitogen-activated protein kinase activated protein kinase 2 of the p38 pathway (37) which has been implicated in fMLP-induced chemotaxis and adhesion of neutrophils (63). The regulatory function of LSP1 in adhesion may also involve its recently demonstrated role in modulating extracellular signal-regulated kinase activity (64), because fMLP-induced extracellular signal-regulated kinase activation contributes to Mac-1-mediated adhesion of neutrophils (65, 66, 67).
In stimulated neutrophils, new expression of Mac-1 receptors occurs on the surface by exocytosis of the secretory vesicles storing these integrins. We showed that the basal and fMLP-up-regulated surface levels of Mac-1 are not LSP1-dependent. There is evidence that activation of neutrophils induces the trafficking of existing Mac-1 to uropod while newly expressed Mac-1 receptors are found at the anterior of the cell forming new adhesion sites (68, 69). In addition, activation of surface integrins through avidity change occurs through signals generated by chemotactic receptors in a cytoskeleton-dependent fashion. There is increasing evidence that integrins need to be relieved from the cytoskeletal constraints to move along the plane of the membrane for clustering (70, 71, 72). Thus, lack of LSP1 and the resulting flexibility of the actin filaments may promote the movement of Mac-1 integrins within the different polarized compartments of adhered neutrophils or along the plane of the plasma membrane to induce high avidity binding to the ligand.
LSP1 is overexpressed in neutrophils obtained from patients with NAD 47/89 who suffer from recurrent infections (39, 40). In U937 or melanoma cell transfectants, overexpression of LSP1 inhibits cell migration (42). These findings are in agreement with our results that LSP1 is a negative regulator of neutrophil chemotaxis. LSP1 overexpression in neutrophils may lead to an abnormally rigid cytoskeleton that impedes dynamic actin rearrangement during adhesion and migration. Indeed, aberrant hair-like protrusions with extensive F-actin bundles were found in NAD 47/89 neutrophils, which may account for their inability to spread in vitro and emigrate into sites of infection in vivo (39).
In conclusion, we showed that LSP1 negatively regulates neutrophil chemotaxis and polarization through its regulatory function on adhesion via specific integrins such as Mac-1. We suggest that these findings account for our in vivo observations that LSP1 modulates neutrophil accumulation during acute inflammation. Future studies will focus on the mechanisms by which LSP1 regulates Mac-1 function in adhesion. It also remains to be investigated whether this negative regulatory role of LSP1 affects other neutrophil functions involving Mac-1, i.e., phagocytosis and cytotoxicity (72, 73, 74).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jenny Jongstra-Bilen, Toronto Western Hospital, Main Pavilion, Room 13-312A, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada. E-mail address: jbilen{at}uhnres.utoronto.ca
3 Abbreviations used in this paper: F-actin, filamentous actin; LSP1, leukocyte-specific protein 1; wt, wild type; BM, bone marrow; RT, room temperature; RFI, relative fluorescent intensity.
Received for publication October 23, 2001. Accepted for publication April 29, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 integrins are critically involved in neutrophil locomotion in extravascular tissue in vivo. J. Exp. Med. 187:2091.91 integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers. J. Leukocyte Biol. 66:809.[Abstract]
2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity 5:653.[Medline]
-actinin levels affects cell motility and confers tumorigenicity on 3T3 cells. J. Cell Sci. 107:1773.[Abstract]
4 integrin affinity for vascular cell adhesion molecule 1 which mediates arrest: an early step in the process of emigration. J. Exp. Med. 193:1149.2 integrin molecules. J. Biol. Chem. 275:20217. or FMLP stimulation. J. Immunol. 160:1982.I inhibits ERK2 activation and increases apoptosis. J. Biol. Chem. 276:24506.M2-integrin glycoprotein. Crit. Rev. Immunol. 20:197.[Medline]
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) or
Lsp1-/- (
) neutrophils to ICAM-1
(A), fibrinogen (B and C),
or ICAM-2/Fc (D) in response to 10-4 M
fMLP, 25 ng/ml PMA, or the carrier, 0.1% DMSO (A,
B, and D), and as described in
Materials and Methods for C. *,
p < 0.04; **, p <
0.0001.
