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Departments of
*
Immunology and
Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263; and
Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL 60611
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-actinin, strongly implicate the
actin-based cytoskeleton in dynamically controlling L-selectin
adhesion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Linkages between cell surface adhesion molecules and the actin-based
cytoskeletal framework have been strongly implicated in controlling the
avidity of ß1 and ß2 integrins in
leukocytes 10, 11, 12, 13, 14 . Similarly, a functional relationship between
L-selectin and the cytoskeleton is suggested by evidence that specific
inhibitors of actin polymerization, i.e., cytochalasins B and D,
prevent L-selectin-mediated adhesion of lymphocytes to vascular
endothelium in vitro and inhibit leukocyte rolling on endothelium in
vivo 15, 16, 17 . Moreover, a highly conserved region comprising the
COOH-terminal 11 amino acids of the L-selectin cytoplasmic tail binds
directly to the rod domain of -actinin, an actin binding
cytoskeletal linker protein 18, 19 . L-selectin coprecipitates with a
complex of cytoskeletal proteins including -actinin and vinculin in
the detergent-soluble fraction of cells transfected with full-length
L-selectin 18 , indicating a constitutive association between
L-selectin and cytoskeletal proteins in the cytosol of unstimulated
cells. A deletion mutant of L-selectin lacking the COOH-terminal 11
amino acids of the cytoplasmic tail fails to associate with -actinin
and is unable to support adhesion either in vitro or in vivo, although
this truncation of the L-selectin cytoplasmic domain does not affect
cell surface expression, carbohydrate recognition, or positioning on
microvilli 16, 18 . Taken together, these findings suggest that the
constitutive association of L-selectin with -actinin plays an
important role in controlling cell adhesion by L-selectin. However,
because -actinin/L-selectin interactions were demonstrated in
detergent-soluble cellular extracts 18 , these studies did not address
the issue of whether L-selectin associates with the detergent-insoluble
actin-based cytoskeletal framework. Moreover, the functional importance
of L-selectin association with the actin cytoskeleton for
leukocyte-endothelial cell interactions has not been fully elucidated.
In this report we demonstrate a dynamic interaction between L-selectin and the cortical cytoskeleton underlying the lymphocyte plasma membrane. While constitutive association of L-selectin with the detergent-insoluble cytoskeleton could not be detected in resting lymphocytes, L-selectin-cytoskeletal interactions requiring the L-selectin cytoplasmic domain were shown to be inducible by multiple independent stimuli including L-selectin cross-linking by Abs, engagement by a physiologic ligand, GlyCAM-1, and direct exposure of lymphocytes to fever-range hyperthermia. This inducible attachment of L-selectin to the cytoskeletal matrix was further associated with enhanced cell adhesion. The results support a model in which L-selectin adhesion and avidity are positively regulated as a consequence of ligand-triggered physical interactions between L-selectin and the lymphocyte structural cytoskeletal matrix.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBL were isolated from human normal donor buffy coat leukocyte
concentrates (American Red Cross, Buffalo, NY) by Ficoll/Hypaque
centrifugation as described previously 8, 20 . Following removal of
adherent cells, the PBL population was cultured at a final
concentration of 4 x 106 cells/ml in RPMI 1640 medium
(Life Technologies, Grand Island, NY) containing 10% FCS (Life
Technologies), 2 mM L-glutamine, 100 U/ml penicillin, and
50 µg/ml streptomycin (complete medium). Hyperthermia treatment of
human PBL was performed by culturing cells for 12 h at 40°C in a
5% CO2 incubator. An IFN-sensitive subclone of the
Burkitt’s lymphoma-derived Daudi B cell line was maintained in
logarithmic growth (5–9 x 105 cells/ml) in complete
medium containing 10-5 M 2-ME (Sigma, St. Louis, MO).
Since Daudi cells constitutively express low levels of L-selectin, it
was necessary to preincubate these cells with recombinant human IFN-
(500 IU/ml for 24 h; Schering, Bloomfield, NJ) to up-regulate
L-selectin levels 9, 20, 21 . The mouse pre-B 300.19 cell lines that
are stably transfected with either full-length human L-selectin cDNA
(300.19/L-selectin) or a deletion mutant lacking the carboxyl-terminal
11 residues of the cytoplasmic domain (300.19/L
cyto) have
been previously described 7, 16, 18 . Stable transfectants were
maintained in suspension culture in complete medium containing 7
x 10-6 M 2-ME.
mAbs and reagents
The following L-selectin-specific, noncross-blocking murine mAb were used that recognize distinct epitopes within the lectin domain 22, 23, 24 : anti-Leu 8-FITC (IgG2a, Becton Dickinson, Sunnyvale, CA), DREG-56 24 (IgG1, kindly provided by Dr. T. Kishimoto, Boehringer Ingelheim, Ridgefield, CT), and TQ1 (IgG1, Coulter, Hialeah, FL). Anti-human LFA-1 (CD11a; an IgG1 clone TS1/22) was from the American Type Culture Collection (Manassas, VA), and anti-ICAM-1 (IgG1, clone 84H10) was obtained from Immunotech (Westbrook, ME). Anti-ß1 integrin (IgG1, clone 1F11) 25, 26 was kindly provided by Dr. Richard Bankert (Roswell Park Cancer Institute, Buffalo, NY). FITC- or phycoerythrin-labeled isotype-matched control Abs were obtained from Becton Dickinson. Goat F(ab')2 anti-mouse IgG-FITC was purchased from Cappel Products (Durham, NC), goat F(ab') anti-mouse IgG-phycoerythrin was obtained from R&D Systems (Minneapolis, MN), and sheep F(ab')2 anti-mouse IgG was obtained from Cappel Organon Teknika (West Chester, PA). Goat anti-mouse IgG bound to 20 nm colloidal gold was purchased from Goldmark Biologicals (Phillipsburg, NJ). Purified GlyCAM-1 31 was a generous gift of Dr. Steven Rosen (University of California, San Francisco). Cytochalasin B, colchicine, and nocodozole were obtained from Sigma; staurosporine was obtained from Kamiya Biomedical (Thousand Oaks, CA); genistein was purchased from Life Technologies; and calphostin C was obtained from Calbiochem (San Diego, CA).
Immunofluorescence staining
Immunofluorescence staining of cells was performed either before or immediately after detergent extraction as indicated in the text. A total of 2 x 106 cells were incubated with 0.1 mg/ml mouse Ig (Sigma) for 10 min at 4°C to block Fc receptor sites, followed by saturating concentrations of FITC-conjugated mAb directed against specific adhesion molecules or isotype-matched control Abs for 30 min at 4°C 20, 26 . Cells were then incubated with FITC-labeled goat anti-mouse polyclonal IgG cross-linking Ab for 30 min at 4°C and were fixed in 1% formaldehyde/PBS. Cross-linking experiments were performed at 4°C to prevent mAb-induced L-selectin shedding 20 . FITC-conjugated primary and secondary Ab were used to increase the intensity of the fluorescent signal; however, equivalent results were obtained using either unconjugated primary mAb in combination with FITC-labeled secondary Ab or FITC-conjugated primary mAb in combination with unconjugated secondary Ab. In some instances only primary fluorochrome-labeled anti-L-selectin mAb were used to examine the association of this adhesion molecule with the cytoskeleton in the absence of extensive cross-linking.
Detergent extraction of cellular proteins
Cells were transferred to polystyrene tubes that were precoated with 1% BSA/PBS (to minimize cell loss during the detergent extraction procedure) and incubated with a cytoskeletal stabilizing buffer (CSB) that minimizes disruption of the cytoskeletal network 27, 28 . For confocal immunofluorescence microscopy, cells were extracted in 0.5% Triton X-100 in CSB-1 (10 mM PIPES (pH 6.8), 100 mM KCl, 300 mM sucrose, 2.5 mM MgCl2, 1 mM CaCl2, 2 mM PMSF, and 0.4 mM leupeptin) 28 . For flow cytometry, cells were extracted in 0.5% Nonidet P-40 in CSB-2 buffer (50 mM NaCl, 2 mM MgCl2, 0.22 mM EGTA, 13 mM Tris (pH 8.0), 1 mM PMSF, 10 mM iodoacetamide, and 2% FCS), which stabilizes cellular extracts for analysis during flow cytometry procedures 27 . Detergent extraction was conducted either at 4°C for 2 min (confocal microscopy) or at room temperature for 30 min (flow cytometry). The detergent-insoluble fractions were then washed in CSB without detergent and fixed in 1% formaldehyde/PBS. Fluorescence associated with the detergent-insoluble fraction was analyzed by confocal laser scanning microscopy or flow cytometry. Identical punctate L-selectin staining patterns were observed in detergent-insoluble extracts prepared using procedures for both confocal microscopy and flow cytometry.
Confocal immunofluorescence analysis
Confocal laser scanning microscopy was performed using a Bio-Rad MRC-600 system (Bio-Rad, Palo Alto, CA) and an OPTIPHOT Nikon microscope (Nikon, Melville, NY) as described previously 21 . An argon ion laser adjusted at 488-nm wavelength was used for the analysis of fluorescein, and images were recorded using a 60x oil immersion Plan Apo60 objective lens (Nikon; numerical aperture of 1.4). The adjustment of the confocal system allows a field depth of about 1 µm. The emitted signal was digitalized by Kalman filter (Bio-Rad) collection, and each section was scanned five times.
Flow cytometric analysis
Flow cytometric analysis of the association of L-selectin with the detergent-insoluble cytoskeleton was determined by a technique described by Geppert and Lipsky 27 . Immunofluorescence associated with the detergent-insoluble cell fraction was analyzed using a FACScan (Becton Dickinson) in the Roswell Park Cancer Institute flow cytometry facility. PBL and detergent extracts were analyzed using a lymphocyte gate that excludes monocytes and neutrophils 26 . The FL1 detector was adjusted to optimize the discrimination of positively and negatively stained populations. A total of 2000–5000 events were collected, and analysis was performed using Winlist 1.0 (Verity Software House, Topsham, ME).
Quantitative lymphocyte adhesion assay
Lymphocyte binding to HEV was assessed as previously described 8, 16, 20, 26 . Lymphocytes (5 x 106 cells in 100 µl) were overlaid onto 12-µm-thick cryosections of BALB/c lymph nodes mounted on glass slides. Slides were rotated at 112 rpm (Labline Instrument, Melrose Park, IL) at 4°C for 30 min, and nonadherent cells were removed by gentle washing in cold PBS. Slides were fixed vertically in 3% glutaraldehyde/PBS for 1 h, permeabilized in 70% ethanol, and stained with 0.5% toluidine/absolute ethanol. A total of 300–500 HEV was examined by light microscopy; each sample was assayed in triplicate.
Ultrastructural immunolocalization of L-selectin
Localization of L-selectin on human PBL was determined by immunogold transmission electron microscopy 8 . Following incubation with 10 µl of goat serum (diluted 1/2) to block potential Fc receptor sites, PBL were labeled at 4°C with primary mAb (30 µg/ml of DREG-56 or isotype-matched negative control Ab), stained with 20 nm colloidal gold-conjugated secondary mAb, fixed in 3% glutaraldehyde/1% tannic acid (to stabilize cytoskeletal elements including microfilaments) 29 in 0.1 M phosphate buffer (pH 7.4), and processed for transmission electron microscopy by the Roswell Park Cell Analysis facility. Sections were viewed in an Elmiskop 101 electron microscope (Siemens, Iselin, NJ). Blind analysis of triplicate specimens was performed by counting gold particles associated with distinct surface structures (microvilli or cell body) on a total of 100 cells in each specimen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate whether L-selectin interacts with the lymphocyte
cytoskeletal framework, a detergent extraction-based analysis was
performed. Human PBL or Daudi B cells (Fig. 1
) were extracted by a protocol that
differentially removes most soluble proteins and phospholipids, leaving
the cortical cytoskeleton intact 28 . Plasma membrane proteins that
are not associated with the cytoskeleton are extracted with other
soluble proteins, whereas proteins that are anchored to the structural
cytoskeletal matrix are resistant to detergent extraction. To detect
L-selectin in the detergent-insoluble (cytoskeletal) fraction, cells
were stained with an FITC-labeled mAb, anti-Leu 8, which recognizes
an epitope in the N-terminal lectin domain of L-selectin, followed by
FITC-conjugated goat F(ab')2 anti-mouse IgG
cross-linking polyclonal Ab. Fluorescence was visualized by confocal
laser scanning fluorescence microscopy. Since the Daudi B cell line
constitutively expresses only low levels of L-selectin, it was
necessary to preincubate these cells with IFN- (500 IU/ml, 12
h) to up-regulate the synthesis and surface expression of this adhesion
molecule 9, 20, 21 .
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, left panels) exhibited punctate L-selectin surface staining
as described previously 21, 30 . When cells were extracted with 0.5%
Triton X-100 in CSB before staining with FITC-labeled Abs (Fig. 1, middle panels), L-selectin could not be detected in the
detergent-insoluble fraction, indicating that constitutive L-selectin
interactions with the structural cytoskeleton are either weak or
nonexistent in resting cells. In sharp contrast, when lymphocytes were
incubated with a saturating concentration of anti-Leu 8 mAb and
cross-linked by FITC-labeled goat F(ab')2 anti-mouse
polyclonal IgG before detergent extraction (Fig. 1, right
panels), significant association of L-selectin was observed with
the detergent-insoluble matrix underlying the plasma membrane. The
characteristic punctate surface staining pattern of L-selectin was
maintained in detergent-extracted cells, although the fluorescence
intensity was partially reduced, suggesting that a subset of surface
L-selectin molecules remains detergent soluble after Ab cross-linking.
Similar results were obtained using L-selectin-specific mAb directed
against independent epitopes in the amino-terminal lectin domain
22, 23, 24 (i.e., DREG-56, TQ1) as well as in extraction buffer
containing another nonionic detergent, Nonidet P-40 (data not shown).
Cross-linking at either 4 or 37°C of the functionally unrelated
ß2 integrin transmembrane protein, LFA-1, by mAb TS1/22
did not induce any cytoskeletal association of L-selectin (data not
shown).
L-selectin cross-linking is likely to occur during engagement by its
natural complex carbohydrate ligands on endothelial cell surfaces 1, 3 . To determine the extent of cross-linking necessary to stimulate
L-selectin interactions with the cytoskeleton, human PBL were bound
with a saturating amount of FITC-conjugated L-selectin-specific mAb
alone or followed by goat F(ab')2 anti-mouse IgG
cross-linking Ab and then subjected to detergent extraction.
Quantification of the percentage of cells expressing
detergent-resistant L-selectin was performed by flow cytometric
analysis. The results (Fig. 2A) confirm the confocal
microscopy data, demonstrating that constitutive association of
L-selectin could not be detected in the detergent-insoluble
cytoskeletal fraction in cells extracted before Ab ligation.
Significant redistribution of L-selectin to the detergent-insoluble
cytoskeleton occurred only following engagement by the combination of
anti-L-selectin mAb and goat F(ab')2 anti-mouse IgG
cross-linking Ab, whereas L-selectin-cytoskeletal
interactions were not detected following ligation by
anti-L-selectin mAb alone (Fig. 2A). Moreover, a goat
F(ab') anti-mouse Ig secondary Ab failed to induce L-selectin
association with the detergent-insoluble cytoskeleton (not shown).
These data suggest that a threshold level of L-selectin cross-linking
is required to initiate its association with the lymphocyte
cytoskeletal framework.
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B), reaching approximately
half-maximal levels within the shortest time frame that could be
measured (5 s). Moreover, stable L-selectin-cytoskeletal interactions
were maintained at a maximal level over a 2-h period in the presence of
cross-linking Ab (not shown). The rapidity of this response is
consistent with the short period in which lymphocytes interact
with HEV in an L-selectin-dependent manner in vivo 1, 2 .
To determine whether a physiologic ligand of L-selectin could also
stimulate cytoskeletal interactions, human PBL (comprised of 61.6%
L-selectin+ cells) were incubated with GlyCAM-1 purified
from mouse serum 31 , extracted with detergent, and then stained with
FITC-labeled L-selectin mAb (anti-Leu 8). In control experiments,
GLYCAM-1 did not block anti-Leu 8 mAb binding (not shown). Prior
studies by Hwang et al. 32 have demonstrated that human lymphocytes
are functionally responsive to murine GlyCAM-1. The data (Fig. 3) indicate that GlyCAM-1 acts in a
dose-dependent manner to initiate stable L-selectin interactions with
the detergent-insoluble fraction, with maximal effects detected at 8
µg/ml. Under these conditions, nearly all cells that were initially
L-selectin+ retained L-selectin in the detergent-insoluble
fraction. Pretreatment of cells with the L-selectin function-blocking
mAb, DREG-56, specifically inhibited GlyCAM-1 effects on
L-selectin-cytoskeletal associations detected with FITC-labeled
anti-Leu 8 mAb. These results cannot be explained by competitive
inhibition between DREG-56 and anti-Leu 8 mAb, since these mAb
recognize distinct epitopes within the L-selectin lectin domain and
thus are not cross-blocking Ab 22, 23, 24 (S. S. Evans, unpublished
observations). Notably, GlyCAM-1 at a concentration of 2 µg/ml, which
approximates the systemic level found in serum (i.e., 
1–2 µg/ml)
3, 31 , did not stimulate L-selectin interactions with the
cytoskeletal matrix, suggesting that the concentration of GlyCAM-1 in
the general circulation is below the level required to trigger
L-selectin-cytoskeletal interactions.
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The COOH-terminal 11-amino acid region of the L-selectin
cytoplasmic domain contains a binding site for -actinin as well as
potential sites of serine and tyrosine phosphorylation 7, 33, 49 .
Therefore, it was of interest to determine whether the cytoplasmic
domain of L-selectin is necessary for L-selectin interactions with the
detergent-insoluble cytoskeleton. Murine pre-B 300.19 cells transfected
either with native human L-selectin or a deletion mutant lacking the
COOH-terminal 11 amino acids of the cytoplasmic tail (Lcyto)
expressed similar levels of surface L-selectin (Fig. 4A), as reported previously
for these cell lines 16, 18 . Moreover, analysis of
L-selectin-specific binding of 300.19 cells to lymph node HEV confirmed
previous observations 16 that deletion of the cytoplasmic tail
significantly impairs adhesion (i.e., 4.23 ± 0.23
300.19/L-selectin cells bound/HEV compared with 1.04 ± 0.10
300.19/Lcyto cells bound/HEV; data represent the mean ± SD
of triplicate samples).
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cyto transfectants
when cells were extracted with detergent before Ab cross-linking (Fig. 4B), consistent with the results obtained in PBL or Daudi B
cells (Figs. 1 and 2). Thus, despite the constitutive interactions
between L-selectin and -actinin that have been documented in
300.19/L-selectin transfectants 18 , constitutive interactions between
L-selectin and the structural cytoskeletal framework could not be
demonstrated in these cells. However, when
300.19/L-selectin-transfectants were bound with anti-L-selectin mAb
and cross-linking Ab before detergent extraction, a marked increase in
the amount of detergent-resistant L-selectin was detected (Fig. 4B). In sharp contrast, Ab cross-linking failed to induce
L-selectin-cytoskeletal interactions in 300.19/Lcyto transfectants.
These data demonstrate an absolute requirement for the COOH-terminal 11
amino acids of the L-selectin cytoplasmic tail to support inducible
association with the cytoskeletal matrix. L-selectin adhesion and L-selectin-cytoskeletal interactions are differentially dependent on de novo polymerization of cytoskeletal elements and kinase activity
The results shown in Fig. 5 and in
previous reports 7, 15, 16, 17 indicate that L-selectin adhesion to
endothelial ligands under shear is dependent on both reorganization of
the structural cytoskeleton and kinase activity. Specifically,
L-selectin adhesion is abrogated by cytochalasins B or D (Fig. 5, A and B) 15, 16, 17 , fungal metabolites that
disrupt cytoskeletal organization by inhibiting the rate of actin
filament polymerization without affecting net depolymerization 34 .
The data shown in Fig. 5B and in a recent report by Haribabu
et al. 7 indicate that L-selectin adhesion is also suppressed, to
variable extents, by 1) colchicine and nocodozole, inhibitors of
microtubule polymerization; 2) staurosporine 7 , a broad inhibitor of
both serine/threonine protein kinases (e.g., PKC, PKA, and PKG) and
tyrosine kinases; 3) calphostin C, a specific inhibitor of PKC; and 4)
genistein, a tyrosine kinase-specific inhibitor. Equivalent
concentrations of these respective kinase inhibitors have been shown in
lymphocytes to block PKC- and tyrosine kinase-dependent L-selectin
shedding 20 , PKC- and tyrosine kinase-dependent homotypic aggregation
responses 35 , and tyrosine-kinase-dependent activation of
IFN-responsive gene expression 21 .
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A).
Consistent with the reported mechanism of action of cytochalasin B,
this inhibitor did not appear to significantly depolymerize existing
actin filaments in PBL at the concentrations used (i.e., 20 µM),
since the topography of actin-rich surface microvilli remained
unaltered, as assessed by scanning (not shown) and transmission
electron microscopy (Fig. 6B). Moreover, cytochalasin B did
not affect the restricted localization of L-selectin on lymphocyte
microvilli, as determined by ultrastructural immunogold labeling of
L-selectin (Fig. 6B). L-selectin/cytoskeletal associations
were also not prevented by colchicine or nocodozole (Fig. 6A). Thus, de novo polymerization of actin filaments and
microtubules is not required to support linkages between L-selectin and
the cytoskeleton, indicating that L-selectin ligation triggers stable
interactions with the preformed cytoskeletal network. Additionally,
inhibitors of serine/threonine and tyrosine kinase activity
(staurosporine, calphostin C, and genistein) had no effect on
L-selectin association with the cytoskeletal matrix (Fig. 6A), suggesting that this process is not dependent on PKC,
PKA, PKG, or tyrosine kinase activity. Taken together, these studies
indicate that distinct molecular events are required to initiate
L-selectin attachment to the cytoskeleton and to support cell-to-cell
adhesion.
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We have recently shown that direct exposure of lymphocytes to fever-range temperatures increases L-selectin-dependent adhesion of human or murine lymphocytes to lymph node HEV in vitro and enhances L-selectin-mediated lymphocyte trafficking to lymph nodes and Peyer’s patches in vivo 8, 9 . Because L-selectin surface density, distribution on microvilli, and lectin activity are unaffected by hyperthermia 8 , we hypothesize that the avidity of pre-existing L-selectin molecules is enhanced by thermal stimuli.
To investigate whether cytoskeletal interactions contribute to the
observed changes in L-selectin avidity in response to hyperthermia,
human PBL and 300.19/L-selectin transfectants (wild-type and Lcyto)
were cultured for 12 h at either 37 or 40°C, and adhesion to HEV
and L-selectin-cytoskeletal associations were evaluated. Direct
exposure of PBL or 300.19/L-selectin (wild-type) transfectants to
hyperthermia resulted in an approximately 1.6- to 1.7-fold increase in
L-selectin-dependent adhesion to lymph node HEV compared with cells
maintained at 37°C (Fig. 7A) as
previously described 8, 9 . In addition, analysis of
L-selectin-cytoskeletal interactions following heat treatment
demonstrated that exposure of PBL or 300.19/L-selectin transfectants to
hyperthermia caused a dramatic increase in the level of association of
L-selectin with the detergent-insoluble cytoskeletal matrix (Fig. 7B). In contrast, no increase in adhesion of the L-selectin
cytoplasmic-deletion mutant (Lcyto) was observed in response to
hyperthermia (Fig. 7A), and hyperthermia failed to induce
L-selectin-cytoskeletal interactions in the Lcyto deletion mutant
(Fig. 7B). Thus, the increase in L-selectin activity in
response to hyperthermia was associated with attachment of L-selectin
to the cytoskeleton through a mechanism requiring the L-selectin
cytoplasmic domain. Moreover, the effect of hyperthermia on
L-selectin-cytoskeletal interactions was specific, since fever-range
temperatures did not induce the association of LFA-1, ß1
integrins, or ICAM-1 with the detergent-insoluble cytoskeletal matrix
of PBL (Fig. 7C). Taken together, this independent evidence
of a direct correlation between L-selectin-mediated cell adhesion and
cytoskeletal interactions in PBL and L-selectin transfectants strongly
suggests that physical interactions between L-selectin and the cortical
actin-based cytoskeleton positively regulate L-selectin avidity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies identify a dynamic and previously unrecognized transition
step in L-selectin/cytoskeletal interactions. L-selectin was recently
shown to constitutively associate with the actin binding protein,
-actinin, in detergent-soluble cellular extracts 18 .
However, the present study indicates that this constitutive
association between L-selectin and -actinin is not sufficient for
attachment of L-selectin to the detergent-insoluble actin
cytoskeleton. Rather, a second event, triggered by ligand or
hyperthermia, is required to induce interactions between L-selectin and
the cytoskeletal matrix. The failure of a truncated form of L-selectin
lacking the COOH-terminal 11 amino acids to associate with the
detergent-insoluble cytoskeleton in response to Ab cross-linking or
hyperthermia establishes a necessary role for the cytoplasmic domain in
this process. These data together with previous evidence that the
COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain bind
directly to the rod domain of -actinin 18, 19 strongly implicate
-actinin in providing a linkage between L-selectin and the
actin-based detergent-insoluble membrane cytoskeleton.
The present findings support and extend the notion 16, 18 that
cytoskeletal interactions regulate the avidity of L-selectin for
endothelial ligands. We hypothesize that the constitutive association
of L-selectin with -actinin allows leukocytes to circulate in a
state of functional readiness. During the initial contact of leukocytes
with vascular endothelium, -actinin may serve to rapidly link the
L-selectin cytoplasmic tail to the membrane cytoskeletal matrix,
thereby altering the conformation of L-selectin and its avidity for
endothelial ligands. Stable interactions may be further facilitated by
additional actin binding proteins such as vinculin and talin, which
have been shown biochemically to interact with L-selectin in an
-actinin-dependent manner 18 . The recent finding by Kahn et al.
36 that the L-selectin cytoplasmic tail also interacts with
calmodulin, a known regulator of cytoskeletal architecture 37, 38 ,
raises the possibility that L-selectin-cytoskeletal interactions and
cell adhesion are controlled by the coordinate actions of calmodulin
and -actinin.
The requirement for extensive L-selectin cross-linking by
polyclonal F(ab')2 secondary Abs to stimulate L-selectin
redistribution to the detergent-insoluble matrix suggests that a
threshold level of receptor clustering is required under physiological
conditions to initiate L-selectin interactions with the filamentous
cytoskeleton. In support of this notion, GlyCAM-1, with its predicted
multivalent mucin-like structure, was shown to induce
L-selectin-cytoskeletal associations in a dose-dependent manner without
requirement for additional cross-linking. Thus, during lymphocyte
trafficking, L-selectin cross-linking and its subsequent association
with the cytoskeleton are predicted to occur in direct response to
interactions between L-selectin, clustered on microvillous surface
membranes 1, 8, 17 , and high local concentrations of
O-linked mucin domains on either membrane-bound or secreted
endothelial L-selectin ligands within HEV (i.e., GlyCAM-1, CD34, and
MAdCAM-1) 1, 2, 3 . Because the cytoskeleton is comprised of both rigid
and elastic fibers, anchoring of L-selectin to the structural
cytoskeleton may strengthen the ability of this adhesion molecule to
withstand high tensile forces characteristic of hemodynamic flow in
blood vessels. A similar role has recently been proposed for
-actinin-mediated linkages between the actin-based contractile
cytoskeleton and the cytoplasmic domains of ß1 and
ß2 integrins, ICAM-1, ICAM-2, and E-selectin 11, 12, 13, 14, 39, 40, 41 . It remains to be determined whether stable associations
between L-selectin and the cytoskeletal matrix are maintained following
ligand engagement or if the transient nature of L-selectin adhesion
results in its dissociation from cytoskeletal elements.
Hyperthermia has been shown to induce significant alterations in the structure of the filamentous cytoskeleton that may explain its stimulatory effects on L-selectin-cytoskeletal interactions and adhesion 42 . In lymphocytes, fever-range hyperthermia stimulates reorganization of the actin-binding cytoskeletal protein spectrin and its colocalization with heat shock proteins 43, 44 , molecules that are known to control protein folding and conformation, signal transduction, and the organization of the actin-based cytoskeleton 45, 46, 47 . Data demonstrating that temperatures within the fever range stimulate L-selectin-cytoskeletal interactions and L-selectin-mediated adhesion in lymphocytes (described in this report and in Refs. 8 and 9) are consistent with the proposed role of fever as a positive activator of lymphocyte immune function 48 . During a febrile response, stable associations between L-selectin and the structural cytoskeleton may increase the potential of circulating lymphocytes to adhere to HEV, thereby amplifying the recruitment of lymphocytes to tissues. It is of note that hyperthermia elicits highly selective effects on the association of various lymphocyte adhesion molecules with the detergent-insoluble cytoskeletal matrix. Specifically, stable interactions between L-selectin and the membrane cytoskeleton are strongly induced in response to hyperthermia, whereas the ß2 integrin LFA-1, ß1 integrins, and ICAM-1 all fail to associate with the structural cytoskeleton in heat-treated cells. These results closely parallel observations that fever-range hyperthermia selectively enhances L-selectin-mediated adhesion in lymphocytes but does not affect LFA-1 binding to ICAM-1 8 or ß1 integrin-mediated adhesion to extracellular matrix proteins (S. S. Evans and E. A. Repasky, unpublished observations). Taken together, these observations strongly suggest that the inducible, regulated attachment of L-selectin to the cytoskeleton is mechanistically related to the enhanced adhesion observed in heat-treated cells.
The emerging data indicate that complex intracellular signaling events are required to support L-selectin-mediated lymphocyte adhesion to HEV that are distinct from the mechanisms that initiate L-selectin attachment with the structural cytoskeleton. In this regard, specific inhibitors of various molecular processes, including de novo polymerization of actin and tubulin (cytochalasins B and D, colchicine, nocodozole), serine/threonine kinase activity (staurosporine, calphostin C), and tyrosine kinase activity (staurosporine, genistein), significantly impair L-selectin adhesion (this report and Refs. 7 and 15–17), although these inhibitors do not prevent the formation of stable interactions between L-selectin and the membrane cytoskeleton. In light of these data, it is tempting to speculate that L-selectin adhesion, subsequent to L-selectin linkage with the cytoskeletal matrix, is controlled by phosphorylation events involving signal transduction molecules with known relationships to L-selectin, such as mitogen-activated protein kinase, Ras, Rac2, JNK, and p56lck 33, 49, 50, 51 . Adhesion may also be regulated as a consequence of direct phosphorylation of serine or tyrosine residues within the L-selectin cytoplasmic domain, as shown to occur in response to multiple stimuli, including chemokines, L-selectin ligation by mAb, or PKC activation by phorbol esters 7, 33 .
Our findings parallel those reported by Yoshida et al. 41 for E-selectin in which association of E-selectin with the detergent-insoluble actin cytoskeleton of cytokine-activated endothelium could be induced through engagement of its lectin domain by natural ligand or by cross-linking of anti-E-selectin mAb by secondary Abs, but not by mAb to E-selectin alone. Attachment of E-selectin to the actin cytoskeleton is associated with greatly increased resistance to mechanical stress, which is abolished by treatment of cells with cytochalasin D 41 . Similarly, L-selectin-mediated adhesion under shear is highly sensitive to cytochalasin B (shown in this report and in Refs. 15–17). The fact that cytochalasin B blocks L-selectin-mediated adhesion without preventing its inducible association with the cytoskeletal matrix is interpreted to indicate that continual cytoskeletal remodeling, involving actin polymerization, is necessary to support L-selectin-mediated adhesion through a process that is independent of L-selectin linkage to the preformed filamentous cytoskeleton. While the molecular basis for the requirement of actin polymerization to support adhesion is not known, it is possible that polymerization of the filamentous cytoskeleton underlying the plasma membrane contributes to the physical extension of microvilli thought to be necessary to reduce the mechanical force imposed on L-selectin-mediated adhesive bonds by blood flow 52 . In support of this idea, cytochalasins B and D reportedly alter the structure of microvilli in cell types that are highly sensitive to these agents, such as neutrophils and pre-B cell lines 17, 53 . Although cytochalasin B does not affect the overall morphology of microvilli in human PBL or the restricted localization of L-selectin on lymphocyte microvilli, subtle changes in the dynamics of actin polymerization within microvilli might explain the observed suppression of L-selectin-mediated adhesion of lymphocytes to HEV in the presence of this inhibitor.
A critical distinction between L-selectin and E-selectin is that the cytoplasmic tail of E-selectin is not required to initiate adhesion 19, 41 . Thus, inducible interactions between E-selectin and the actin-based cytoskeleton are speculated to participate in the complex process of leukocyte extravasation at a level subsequent to the initial contact between leukocytes and endothelium. It remains to be determined whether intracellular signals transmitted through high affinity interactions of L-selectin with the lymphocyte cytoskeleton also influence the function of downstream events required for transendothelial migration. Interestingly, L-selectin ligation by mAb or GlyCAM-1, shown in this report to promote L-selectin-cytoskeletal interactions, also activates ß2 and ß1 integrin binding activity in human neutrophils, naive T cells, and the CD56bright NK cell subset 22, 32, 54, 55 . Our studies demonstrating a dynamic relationship between L-selectin and the cortical cytoskeletal matrix provide a framework for future investigation of the molecular mechanisms and biochemical consequences of these interactions during lymphocyte-endothelial adhesion.
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2 Address correspondence and reprint requests to Dr. S. S. Evans, Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263. E-mail address:
3 Abbreviations used in this paper: HEV, high endothelial venule; GlyCAM-1, glycosylation-dependent cell adhesion molecule-1; MAdCAM-1, mucosal addressin cell adhesion molecule-1; CSB, cytoskeletal stabilizing buffer; PK, protein kinase.
Received for publication August 25, 1998. Accepted for publication December 10, 1998.
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