Clustering of very late antigen-4 integrins modulates
K+ currents to alter Ca2+-mediated monocyte
function
Margaret
Colden-Stanfield
Department of Physiology, Morehouse School of Medicine,
Atlanta, Georgia 30310
 |
ABSTRACT |
Endothelial
cell vascular cell adhesion molecule-1 (VCAM-1) activates adherent
monocytes by clustering their very late antigen-4 (VLA-4) receptors,
resulting in the modulation of the inwardly rectifying
(Iir) and delayed rectifying
(Idr) K+ currents, hyperpolarization
of the cells, and enhanced Ca2+ influx (Colden-Stanfield M
and Gallin EK. Am J Physiol Cell Physiol 275:
C267-C277, 1998; Colden-Stanfield M and Scanlon M. Am J
Physiol Cell Physiol 279: C488-C494, 2000). The present study
was undertaken to test the hypothesis that monoclonal antibodies
(MAbs) against VLA-4 (MAbVLA-4) mimic VCAM-1 to cluster VLA-4
integrins, which play a key role in signaling an increase in the
secretion of the proinflammatory cytokine interleukin-8 (IL-8). Whole
cell ionic currents and IL-8 secretion from THP-1 monocytes that were
incubated on polystyrene, VCAM-1-immobilized MAbVLA-4 or an
isotype-matched MAb against CD45 (MAbCD45) were measured. Clustering of
VLA-4 integrins with a cross-linked MAbVLA-4, but not a monovalent
MAbVLA-4, modulated the K+ currents in an identical manner
to incubation of cells on VCAM-1. Similarly, cross-linked MAbVLA-4 or
VCAM-1 augmented Ca2+-mediated IL-8 secretion from THP-1
monocytes and was completely abolished by exposure to CsCl, an
Iir blocker. Thus VLA-4 integrin clustering by
cross-linked MAbVLA-4 mimics VCAM-1/VLA-4 interactions sufficiently to
be associated with events leading to monocyte differentiation, enhanced
Ca2+-mediated macrophage function, and possibly
atherosclerotic plaque formation.
acute monocytic leukemia; vascular cell adhesion molecule-1; ion
channels; interleukin-8; cell signaling
 |
INTRODUCTION |
INTEGRINS ARE
HETERODIMERS that are defined by an
-chain sharing a
noncovalent link with a
-chain and function to promote cell-to-extracellular matrix interactions and cell-to-cell interactions (25). Very late antigen-4 (VLA-4) integrins, which belong
to the
1 subfamily of integrin adhesion receptors, can interact with
endothelial vascular adhesion molecule-1 (VCAM-1) to mediate the
extravasation of immune cells from blood vessels, particularly at sites
of inflammation (39). Apart from the important structural role VLA-4 and other integrins play in cell adhesion, it is now evident
that integrin engagement with ligand, accompanied by integrin clustering, leads to activation of important signal transduction cascades such as tyrosine phosphorylation, which regulate cell migration, differentiation, activation, and growth (25).
For example, binding of T lymphocytes with extracellular matrix
proteins through VLA-4 integrins rapidly stimulates tyrosine
phosphorylation of cellular proteins that are essential for subsequent
T cell proliferation (20, 30). Ligation of VLA-4 by
monoclonal antibody (MAb) on both human monocytes and the monocytic
THP-1 cell line promotes tissue factor expression, a process that
likely contributes to local microvascular thrombosis (37).
Binding and cross-linking of
1 integrins on the surface of monocytes
induces immediate early genes coding for inflammatory cytokines
(32, 43, 44).
Ionic current flow through ion channels has been implicated in the
signaling pathways of various functions of immune cells (6, 7,
17). Selective inhibition of K+ channels has proven
to be a potential target for immunosuppression in the treatment of
autoimmune disorders (4, 46, 51). However, only recently
has a functional link been documented between ion channel activity and
integrins in immune cells (28, 38). For example, the
opening of voltage-gated Kv1.3 channels in T lymphocytes activates
1
integrin-mediated adhesion and migration (28). The T cell
K+ channels appear to be physically associated with
1
integrins, as are G protein-activated inward rectifier K+
channels, and this interaction is required for normal lymphocyte function (28, 38).
The engagement of integrins has also been shown to alter ion channel
activity to affect function in other cell types. Engagement of
v
3
integrins, vitronectin receptors expressed on vascular smooth muscle
cells, causes arteriolar vasodilation, in part, by stimulating
K+ channel activity (41). Ca2+
influx via L-type Ca2+ channels is inhibited by arteriolar
smooth muscle cell contact with vitronectin or fibronectin
(50). Rat PC12 and cerebellar neuronal cell interaction
with cell adhesion molecules stimulates Ca2+ influx through
voltage-dependent Ca2+ channels. This is an important step
for neurite outgrowth (49). Activation of an inwardly
rectifying K+ current (Iir) in
neuroblastoma cells adherent to fibronectin induces a membrane
hyperpolarization, an obligate event for neuritogenesis (2,
5). Relatively little is known about the impact of VLA-4 integrin interaction with VCAM-1 on ion channel activation as it
relates to cellular function in monocytes/macrophages.
To begin to address this question, we demonstrated that the interaction
of THP-1 monocytes with activated endothelium or immobilized VCAM-1
modulates two K+ currents and hyperpolarizes these cells
(11), presumably by engaging and clustering VLA-4
integrins. The current study extends our early work by comparing the
expression of Iir and delayed rectifying
K+ currents (Idr) in THP-1
monocytes incubated on VCAM-1 with the expression of these
K+ currents in monocytes incubated on MAbs raised against
VLA-4 integrins (MAbVLA-4), the ligand for VCAM-1. It is our hypothesis that MAbVLA-4 mimics VCAM-1 to cluster VLA-4 integrins, which plays a
key role in signaling an increase in the secretion of the
proinflammatory cytokine interleukin-8 (IL-8).
 |
MATERIALS AND METHODS |
Chemicals.
Lipopolysaccharide (LPS), CsCl, and tetraethylammonium chloride (TEA;
Sigma Chemical, St. Louis, MO) were dissolved in PBS, divided into
aliquots, and stored as 100× stock solutions at
20°C. Thapsigargin
(TG; Sigma Chemical) was reconstituted in DMSO, and aliquots were
stored at
20°C. Recombinant charybdotoxin (ChTX; Alomone Labs, Jerusalem, Israel) was dissolved in distilled
water, divided into aliquots, and stored at
20°C. All drugs were
diluted immediately before use in experiments. The diluted ChTX
solution also contained 0.1% BSA.
Cell culture.
The undifferentiated THP-1 human monocyte cell line [American Type
Culture Collection (ATCC), Manassas, VA] (3, 48) was cultured in suspension with RPMI culture medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone, and 2 mM L-glutamine
(all from Life Technologies). Cells were passaged every 3-4 days
by centrifugation by removal of all media and resuspension in fresh media at 1-2 × 106 cells/ml. THP-1 cell cultures
were passaged and used only for 1 mo upon thawing from liquid nitrogen.
The established cell line of human umbilical vein endothelial cells
(ECs; ATCC) was maintained in MCDB110 medium (Sigma Chemical) supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, 100 µg/ml heparin, and 30 µg/ml H-Neurext (Upstate Biotechnology, Lake Placid, NY) on
collagen-coated tissue culture dishes. All experimental data were
obtained using ECs in their 17th to 19th passage and at 1-2 days
postconfluency. EC monolayers were activated by pretreating with LPS (3 µg/ml) at 37°C in a 5% CO2-humidified incubator for
24 h to increase the surface expression of E-selectin,
intracellular adhesion molecule-1, and VCAM-1. For the IL-8
experiments described below, LPS-activated ECs (LPS-ECs) were fixed
with 2% paraformaldehyde for 15 min before interaction with THP-1 monocytes.
Immobilization of soluble VCAM-1 or mouse MAbs.
A 10-µl aliquot of recombinant soluble VCAM-1 (50 µg/ml; a generous
gift from Dr. Roy Lobb of Biogen, Cambridge, MA) (31) or
mouse MAb against the
4 subunit of human VLA-4 (MAbVLA-4 IgG; 50 µg/ml; Pharmigen, San Diego, CA) or CD45 (MAbCD45 IgG; 50 µg/ml; Pharmigen) in a binding buffer (15 mM NaHCO3/35 mM
Na2CO3, pH 9.2) was placed in the middle of
35-mm polystyrene bacteriological dishes (Falcon 1008; Fisher
Scientific, Norcross, GA) for coating overnight at 4°C. Nonspecific
binding was blocked with PBS/1% BSA for 0.5 h at 4°C and washed
once with complete RPMI before THP-1 monocytes (1 × 106 cells/ml) in complete RPMI were added to uncoated
polystyrene (POLY), VCAM-1- or MAbVLA-4-coated dishes for incubation at
37°C for 5 h. To cluster VLA-4 integrins on the surface of THP-1
monocytes, cells were incubated with immobilized MAbVLA-4 that had been
previously cross-linked with goat anti-mouse IgG (Sigma) in PBS/1% BSA
for 0.5 h at 4°C. Immobilized CD45 was treated with goat
anti-mouse IgG in the same manner before incubation with THP-1
monocytes. Nonadherent THP-1 monocytes then were removed by washing
with Ringer saline solution once before macroscopic currents were
recorded from the remaining adherent THP-1 monocytes.
Confocal microscopy.
To examine VLA-4 integrin clustering, THP-1 monocytes in complete RPMI
were incubated on uncoated glass, LPS-ECs, VCAM-1, monovalent or
cross-linked MAbVLA-4, or cross-linked CD45 immobilized on Lab-Tek
eight-well chamber glass slides (Fisher Scientific) for 60 min in a 5%
CO2-humidified incubator before being fixed in 2%
paraformaldehyde for 15 min. After fixation, cells were exposed to
three 5-min washes with PBS/50 mM NH4Cl before incubating the cells with PBS/2%FBS for 10 min at room temperature to block nonspecific binding. The fixed cells were incubated in turn with MAbVLA-4 (1:250 dilution, Pharmigen) overnight at 4°C, with
biotinylated goat anti-mouse IgG (1:250 dilution, Jackson
ImmunoResearch, West Grove, PA), and with streptavidin-conjugated
Oregon Green 488 (1:250 dilution, Jackson ImmunoResearch) at room
temperature for 60 min. Each of these incubations was conducted with
three 5-min washes of PBS/1%BSA between steps. The scaffolding of the
chamber slide was removed, and the slide was mounted in 50% (vol/vol) glycerol in Tris-buffered saline containing 0.1 M N-propyl
gallate as an anti-bleaching agent.
Fluorescence was visualized on cells by setting the emission line on
the argon laser of a Multiprobe 2001 laser scanning confocal microscope
to 488 nm (Molecular Dynamics, Sunnyvale, CA). A Nikon Diaphot inverted
epifluorescence microscope was used to locate the cell surface before
collection of confocal images, which were stored, processed, and
analyzed on an Iris Indigo workstation (Silicon Graphics, Mountain
View, CA) running ImageSpace software (Molecular Dynamics).
Patch-clamp recording of ionic currents.
Macroscopic whole cell patch-clamp recordings (19) were
obtained from THP-1 monocytes that were incubated on immobilized VCAM-1
or MAbVLA-4 using patch electrodes (BF100-50-10, Sutter Instruments) with resistances of 4-8 M
. In control experiments, current recordings were obtained from THP-1 monocytes that were bound
to polystyrene or cross-linked immobilized MAbCD45 (MAbCD45x). The
pipette contained (in mM): 150 KCl, 0.1 EGTA, 1 CaCl2 (free Ca2+ concentration = 26.5 nM), and 10 HEPES, pH to 7.2 with KOH, and the Ringer bath solution contained (in mM): 150 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5.5 glucose, and 10 HEPES, and 0.01% BSA, pH adjusted to 7.3 with NaOH. All experiments
were performed on cells at 22-24°C with an Axopatch 200A
amplifier (Axon Instruments, Foster City, CA) after correction of
junction potentials.
Resting membrane potential (RMP) was measured in current-clamp mode
(I = 0) immediately after attainment of the whole cell configuration. While rapid equilibration of the pipette solution with
cytoplasm during the whole cell configuration (42) may reduce the precision of the RMP measurement, we and others have demonstrated this method to be an accurate estimation of RMP (8, 10, 40). The voltage-clamp pulse protocols were computer driven using pCLAMP software (Axon Instruments) and were delivered through the
head stage of the Axopatch 200A amplifier to elicit ionic currents. A
typical pulse protocol clamped the cell at a holding potential of
40
mV and stepped in 20-mV increments from
160 to +60 mV, with each
pulse lasting 450 ms at an interval of 1 s (11).
Records were low-pass filtered at 2 kHz, and the data were collected,
stored, and analyzed with the use of the pCLAMP software programs (Axon
Instruments). Total membrane capacitance (Cm)
was measured in the whole cell mode by integrating the capacity transient and was then compensated electronically. Whole cell membrane
conductance values were normalized to Cm to
yield a value for specific ionic conductances.
Measurement of IL-8 release.
IL-8 secretion from THP-1 monocytes adherent to POLY,
paraformaldehyde-fixed LPS-ECs, VCAM-1, or immobilized MAbs for 5 h was measured in culture supernatant using a microplate immunoassay (R&D Systems, Minneapolis, MN). Briefly, diluted culture supernatant (1:50) was incubated for 2.5 h at room temperature on microplate wells coated with a murine MAb against human IL-8 in the presence of a
polyclonal antibody against IL-8 conjugated to horseradish peroxidase.
After six washes to remove any unbound substances, a substrate solution
containing hydrogen peroxide and tetramethylbenzidine was added to each
well and allowed to incubate for 30 min at room temperature. Color
development was stopped with 2 N sulfuric acid before the optical
density of each well was read using a SpectraMax 250 plate reader
(Molecular Devices) set to 450 nm. Wavelength correction was set to 540 nm. IL-8 concentration for the samples was calculated by creating a
standard curve (four-parameter logistic curve fit) using IL-8 standards
ranging from 0 to 2,000 pg/ml.
Data analysis.
Current amplitudes normalized by membrane capacitance (pA/pF) and RMP
in THP-1 monocytes were compared with those parameters in THP-1
monocytes adherent to immobilized VCAM-1, MAbVLA-4, MAbVLA-4x, or
MAbCD45x using the unpaired Student's t-test. The
percentage of cells expressing each current, Iir
or Idr, in THP-1 monocytes bound to POLY was
compared with the percentage of cells expressing these currents in
THP-1 monocytes adherent to immobilized VCAM-1, MAbVLA-4, MAbVLA-4x, or
MAbCD45x using the z-test in the SigmaStat Statistical
Software program (Jandel Scientific, Cedar Grove, CA). Significance was
set at P
0.05. The amount of IL-8 produced and
released from THP-1 monocytes adherent to POLY, fixed LPS-ECs, VCAM-1,
or immobilized MAbs was compared using the Student's t-test with significance set at P
0.05.
 |
RESULTS |
Ionic current expression in THP-1 monocytes with clustered VLA-4
integrins.
Whole cell recordings obtained from THP-1 monocytes bound to POLY for
5 h revealed that, in the majority of cells (86%), step hyperpolarization did not elicit inward current (Fig.
1, A and C;
Table 1). In contrast, the majority of
THP-1 monocytes (67%) adherent to immobilized VCAM-1 for 5 h
expressed a Cs+-sensitive Iir during
step hyperpolarization negative to
80 mV (Fig. 1, B and
C; Table 1) as previously reported (11).
Normalized Iir in THP-1 monocytes adherent to
VCAM-1 was about twofold greater in magnitude than in the small
percentage of cells bound to POLY expressing Iir
(Table 2). Most cells adherent to POLY or
VCAM-1 elicited the Idr during step
depolarization that we have previously described (Fig. 1,
A-C; Table 1) (11). THP-1 monocytes
adherent to VCAM-1 possessed a Idr magnitude
that was one-half the magnitude that cells bound to POLY possessed
(Fig. 1, B and C; Table 2).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
A
family of digitized whole cell recordings in THP-1 monocytes adherent
to polystyrene (A) or immobilized vascular cell adhesion
molecule-1 (VCAM-1; B) for 5 h. A 450-ms pulse stepping
to 160 to +60 mV in 20-mV increments was delivered every 1 s
from a holding potential of 40 mV. Recording conditions in Figs. 1,
2, 5, and 6 were identical unless otherwise noted. C:
current-voltage (I/V) relationships of the normalized
current (pA/pF) measured during the last 50 ms of each voltage pulse
are illustrated for THP-1 monocytes adherent to polystyrene (POLY) or
VCAM-1. Each point is the mean ± SE of normalized current
amplitudes from 11-35 cells.
|
|
To determine whether we could mimic the VCAM-1-induced changes in
Iir and Idr, we recorded
whole cell currents from THP-1 monocytes after VLA-4 integrins were
directly engaged or occupied with immobilized monovalent MAbVLA-4 or
cross-linked MAbVLA-4 (MAbVLA-4x). Adherence to monovalent MAbVLA-4 for
5 h produced an increase in the percentage of THP-1 monocytes
expressing Iir (74%) but did not alter the
magnitude of Iir (Fig.
2, A and C; Tables
1 and 2). The expression of Idr was not affected
by adherence to monovalent MAbVLA-4 (Fig. 2, Tables 1 and 2).
Clustering of integrins has been shown to be necessary to optimally
stimulate several signal transduction mechanisms in cells and can be
accomplished by cross-linking anti-integrins (32, 37).
Therefore, we recorded whole cell currents in THP-1 monocytes adherent
to MAbVLA-4x for 5 h. As in THP-1 monocytes adherent to VCAM-1,
the percentage of cells expressing Iir was
increased to 67%, and the magnitude of Iir was
enhanced by twofold in THP-1 monocytes adherent to MAbVLA-4x compared
with monocytes bound to POLY (Fig. 2B; Tables 1 and 2).
Similarly, as we observed in cells adherent to VCAM-1, Idr was attenuated by twofold in THP-1 monocytes
adherent to MAbVLA-4x, as demonstrated by the current-voltage
(I-V) relationship (Fig. 2C vs. 1C;
Table 2). Different profiles of current activation (Fig. 2C)
were evident in THP-1 monocytes with only engaged VLA-4 integrins
(monovalent MAbVLA-4) compared with THP-1 monocytes with engaged and
clustered VLA-4 integrins (MAbVLA-4x). These data suggest that, while
monovalent MAbVLA-4 does not completely mimic the effects on these two
K+ currents that we observed with VCAM-1, MAbVLA-4x did
produce identical effects.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2.
Representative whole cell current recordings in THP-1 monocytes
adherent to immobilized monovalent monoclonal antibody to very late
antigen-4 (MAbVLA-4; A) or cross-linked MAbVLA-4 (MAbVLA-4x;
B) for 5 h. C: comparison of the
I-V relationships of the normalized current measured as
described in Fig. 1 legend in THP-1 monocytes adherent to MAbVLA-4 or
MAbVLA-4x. Each point is the mean ± SE of normalized current
amplitudes from 5-10 cells.
|
|
As a control, THP-1 monocytes were incubated on immobilized,
cross-linked MAb (isotype matched) raised against the leukocyte common
antigen present on all human leukocytes, CD45, to determine specificity
of the VLA-4-induced modulation of Iir and
Idr. THP-1 adherence to MAbCD45x did not alter
the percentage of cells expressing Iir or
Idr (Table 1) or the magnitude of either
K+ current (Table 2). To further test the specificity of
the VLA-4-induced modulation of Iir and
Idr, whole cell currents were recorded from THP-1 monocytes incubated on immobilized extracellular matrix proteins,
fibronectin or collagen. Fibronectin binds to a region on the VLA-4
integrin that is distinct from the binding site for VCAM-1
(15). Collagen does not bind to VLA-4 integrin but binds to VLA-2 integrin (32). Adherence of THP-1 monocytes to
either extracellular matrix proteins did not significantly affect
Iir or Idr expression
(data not shown). These data suggest that modulation of
Iir and Idr was due to
the engagement and clustering of VLA-4 integrins upon the specific
interaction with VCAM-1 adhesion molecules.
To confirm that clustering of VLA-4 integrins occurred on THP-1
monocytes under our experimental conditions, we performed immunocytochemical staining of VLA-4 integrins on THP-1 monocytes adherent to immobilized VCAM-1 or MAbVLA-4x for 5 h and visualized the fluorescence via confocal microscopy. To more closely mimic the in
vivo scenario of VLA-4-VCAM-1 interactions, we stained for VLA-4
integrins on THP-1 monocytes adherent to fixed LPS-EC monolayers and
compared the staining to VLA-4 localization on THP-1 monocytes bound to
glass or MAbCD45x for 5 h. Clustering of VLA-4 integrins was
evident on the surface of THP-1 monocytes adherent to LPS-ECs compared
with cells bound to glass (Fig. 3, A and B). Similarly, THP-1 monocytes
adherent to immobilized VCAM-1 or MAbVLA-4x exhibited a high degree of
clustering of VLA-4 on the cell surface (Fig. 3, D and
E). No significant staining was present when cells adherent
to VCAM-1 were stained with biotinylated goat anti-mouse IgG alone
(Fig. 3C) or on cells adherent to the binding,
isotype-matched MAbCD45x (data not shown).

View larger version (94K):
[in this window]
[in a new window]
|
Fig. 3.
Immunocytochemical staining of VLA-4 surface antigen on
THP-1 monocytes adherent to glass (A), fixed
lipopolysaccharide-treated human umbilical endothelial cells (LPS-ECs;
B), immobilized VCAM-1 (D), or MAbVLA-4x
(E) for 5 h. Arrows indicate clustering of VLA-4
integrins. C: low nonspecific staining on THP-1 monocytes
adherent to VCAM-1 in the presence of secondary antibody alone, with
corresponding transmitted light image (F). Bar in
E denotes 2 µm for all images.
|
|
Effect of clustered VLA-4 integrins on resting membrane potential
in THP-1 monocytes.
Previously, we demonstrated that adherence of THP-1 monocytes to
LPS-activated ECs and, specifically, VCAM-1, hyperpolarizes the cells
(11). To ascertain whether direct engagement of VLA-4 integrins, the counterreceptors on monocytes for VCAM-1, induces a
hyperpolarization, RMP was recorded under current-clamp conditions (I = 0) in THP-1 monocytes adherent to monovalent
MAbVLA-4 or MAbVLA-4x for 5 h and compared with RMP recorded in
THP-1 monocytes adherent to POLY, VCAM-1, or MAbCD45x. Our previous
observations were confirmed, in that RMP of THP-1 monocytes bound to
POLY was depolarized at
25 mV and adherence to VCAM-1 hyperpolarized
monocytes by ~15 mV (Fig. 4). While
adherence of cells to monovalent MAbVLA-4 did not alter RMP, a
significant increase in RMP to
40 mV was recorded in THP-1 monocytes
adherent to MAbVLA-4x for 5 h, as was observed in cells adherent
to VCAM-1 (Fig. 4). Hyperpolarization was not observed in THP-1
monocytes adherent to isotype-matched MAbCD45x (Fig. 4), fibronectin,
or collagen (data not shown).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of THP-1 monocyte adherence to various substrates
on resting membrane potential (RMP). RMP was measured as described in
MATERIALS AND METHODS in THP-1 monocytes adherent to POLY,
VCAM-1, MAbVLA-4, MAbVLA-4x, or MAbCD45x for 5 h. Each bar
represents the mean ± SE of at least 7 cells. * Significantly
different from control group (POLY). +Significantly
different from MAbVLA-4 and MAbCD45x groups.
|
|
In most cells the presence of an Iir sets RMP
close to the equilibrium potential for K+
(EK) in the range of
55 to
85 mV (14,
18, 26). In fact, there were several individual THP-1 monocytes
adherent to VCAM-1 or MAbVLA-4x possessing RMP of
55 mV or greater in
which Iir was the predominant current (Fig.
5, A and B). To
substantiate this observation, we examined electrophysiological data
from THP-1 monocytes with clustered VLA-4 integrins according to
whether their RMP was greater or less than
55 mV. We compared the
ratio of normalized Iir to normalized
Idr in THP-1 monocytes with RMP <
55 mV
to the ratio in THP-1 monocytes with RMP
55. In 29 cells that
possessed RMP <
55 mV (Fig. 5C, open bar), the ratio of the two voltage-gated K+ currents indicated that even
when Iir was threefold greater in magnitude than
Idr, it was not sufficient to set RMP higher
than a mean of
31 mV in these cells. In contrast, in 17 cells that possessed a mean RMP of
70 mV, Iir was
18.3-fold greater in magnitude than the Idr
present in these cells (Fig. 5C, solid bar). While the
magnitude of Idr was identical in cells with
high or low RMP, the magnitude of Iir was
2.9-fold greater in THP-1 monocytes with the more hyperpolarized RMP,
indicating that Iir must be the principal current in the cell for RMP to approach EK (
86
mV) under our recording conditions.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 5.
Contribution of inwardly rectifying K+ current
(Iir) and delayed rectifying K+
current (Idr) to setting RMP in THP-1 monocytes
with clustered VLA-4 integrins. A: representative family of
whole cell recordings in THP-1 monocytes in which
Iir was the predominant current. RMP was 67 mV
in this cell. B: corresponding I-V relationship
demonstrating a substantial Iir activated at
potentials negative to equilibrium potential for K+
(EK) ( 86 mV). C: comparison of the
ratio of normalized Iir to normalized
Idr in THP-1 monocytes with RMP < 55 mV
or RMP 55 mV. The magnitude of normalized
Iir and Idr was measured
at step voltage to 160 and 0 mV, respectively. * Significantly
different from RMP < 55 mV group. Each bar is the mean ± SE of 42 cells (RMP < 55 group) and 17 cells (RMP 55
group).
|
|
Although 37% of THP-1 monocytes with clustered VLA-4 integrins had RMP
approaching EK, most of the cells were more
depolarized. We further investigated the likelihood that additional
membrane permeability via Idr contributed to
setting RMP to
40 mV in THP-1 monocytes with clustered VLA-4
integrins. To examine this possibility, whole cell currents and RMP
were recorded in THP-1 monocytes adherent to MAbVLA-4x, which possessed
both Iir and Idr, in the
absence and presence of ChTX (30 nM) or TEA (30 mM), known blockers of Idr (11, 14). Figure
6 illustrates representative
Iir and Idr elicited in
THP-1 monocytes with clustered VLA-4 integrins. The addition of
exogenous ChTX inhibited Idr as we have
previously demonstrated (Fig. 6A) (11). Under
these recording conditions, any outward current between
40 and 0 mV
would be carried by K+ exiting THP-1 monocytes via
K+ channels. The I-V relationship (Fig.
6A, inset) indicates that ChTX predominantly
inhibited K+ flow through Idr while
having no effect on Iir. Residual
outward current observed in the presence of blocker at potentials more positive to 0 mV is most likely the chloride conductance we have previously described (11). Blockade of
Idr by ChTX in cells possessing
Iir hyperpolarized RMP from a mean RMP of
29
to
72 mV (Fig. 6B). Identical results were observed in
cells when Idr was blocked with TEA in the
presence of Iir (Fig. 6C,
inset). TEA had no effect on Iir in
THP-1 cells (Fig. 6C, inset). RMP was
hyperpolarized from a mean of
30 to
66 mV in THP-1 monocytes exposed to TEA (Fig. 6D). Furthermore, inhibition of
Idr in THP-1 monocytes lacking
Iir depolarized cells ~15 mV from the
threshold activation voltage for Idr (data not
shown). These data indicate that the presence of
Idr sets RMP near the threshold activation voltage for Idr in THP-1 monocytes with
clustered VLA-4 integrins and that Iir has to be
the predominant K+ current for THP-1 monocytes to maintain
a RMP approaching EK.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of blockade of Idr on
current profile and RMP in THP-1 monocytes with clustered VLA-4
integrins. A: family of whole cell currents recorded in
THP-1 monocytes adherent to MAbVLA-4x for 5 h before
(left) and after (right) a 10-min exposure to 30 nM exogenous charybdotoxin (ChTX). Inset: I-V
relationships before [ , control (cont)] and after
( ) exposure to ChTX. B: RMP of 4 individual
cells before and after addition of ChTX to the bath solution.
C: family of whole cell currents recorded in THP-1 monocytes
with clustered VLA-4 integrins before (left) and after
(right) a 10-min exposure to 30 mM exogenous
tetraethylammonium chloride (TEA). Inset: I-V
relationships before ( ) and after ( )
exposure to TEA. D: RMP of 4 individual cells before and
after addition of TEA to the bath solution.
|
|
Effect of clustered VLA-4 integrins on IL-8 production in THP-1
monocytes.
Internal Ca2+ increases due, in part, by extracellular
Ca2+ entry, have been shown to play a predominant role in
regulating monocyte/macrophage function (1, 20-23,
33). We have shown recently that the VCAM-1-induced hyperpolarization in THP-1 monocytes significantly increases the driving force for Ca2+ entry triggered by exposure to TG, a
Ca2+-ATPase inhibitor, to enhance peak intracellular
Ca2+ concentration (12). To directly
demonstrate the impact of clustering VLA-4 integrins on
Ca2+-mediated cellular function, we measured basal and TG
(100 nM)-stimulated IL-8 release from THP-1 monocytes adherent to POLY,
paraformaldehyde-fixed LPS-ECs, VCAM-1, or immobilized MAbs for 5 h. There was minimal IL-8 release from THP-1 monocytes incubated on
POLY (Fig. 7, CONT, open bar). Basal
release of IL-8 from THP-1 monocytes adherent to fixed LPS-ECs, VCAM-1,
and MAbVLA-4x was increased 6.8-, 7.4- and 9.5-fold, respectively,
suggesting that monocyte function was enhanced as a result of VLA-4
integrin clustering.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 7.
Interleukin-8 (IL-8) production and release from THP-1 monocytes
adherent to POLY, fixed LPS-ECs, VCAM-1, MAbVLA-4, or MAbVLA-4x for
5 h in the absence (open bars) or presence of thapsigargin (TG;
100 nM; solid bars). Each bar is the mean ± SE of supernatant
IL-8 of 3 independent experiments performed in duplicate.
* Significantly different from the corresponding control group.
+Significantly different from the POLY TG group.
|
|
IL-8 release from THP-1 monocytes was increased 18.2-fold during the
incubation period on POLY by the presence of TG (100 nM), which
promotes store-depleted activation of Ca2+ influx (Fig. 7,
TG, filled bar). Moreover, THP-1 monocytes adherent to fixed LPS-ECs or
MAbVLA-4x secreted 77% and 104% more IL-8 in response to TG,
respectively. THP-1 monocytes adherent to VCAM-1 and monovalent
MAbVLA-4 secreted 27% and 22% more TG-stimulated IL-8 release from
THP-1 cells, respectively, compared with cells bound to POLY. Basal and
TG-stimulated IL-8 release from THP-1 monocytes adherent to MAbCD45x or
the extracellular matrix proteins, fibronectin and collagen, were not
different from cells bound to POLY (data not shown). Fixed LPS-EC
monolayers alone did not secrete detectable levels of IL-8 in the
absence or presence of TG (data not shown), indicating that the
production of IL-8 was of monocyte origin.
To demonstrate the direct link between the induction of
Iir, hyperpolarization of RMP, and enhanced
Ca2+-dependent IL-8 production, we quantified basal and
TG-stimulated IL-8 release from THP-1 monocytes with clustered VLA-4
integrins in the presence of Cs+, a metal ion we have shown
previously to block the VCAM-1-induced Iir and
TG-stimulated Ca2+ response (11, 12). Basal
IL-8 release from THP-1 monocytes was not altered by exposure to CsCl
during the 5-h incubation period (Fig. 8,
Cont vs. Cont+Cs). In contrast, the 59% enhancement of TG-stimulated
IL-8 from THP-1 monocytes adherent to MAbVLA-4x was completely
abolished by blocking Iir (Fig. 8, TG vs.
TG+Cs). These data suggest that clustering of VLA-4 integrins on THP-1 monocytes appears to trigger signaling pathways, one of which is to
induce Iir, leading to properties that reflect
monocyte activation and/or differentiation to macrophages.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 8.
Basal (Cont) and stimulated IL-8 (TG) release from THP-1
monocytes adherent to POLY or MAbVLA4x for 5 h in the absence or
presence of CsCl (10 mM). Each bar is the mean ± SE of
supernatant IL-8 of 3 independent experiments performed in duplicate.
* Significantly different from the corresponding POLY group.
+Significantly different from the control group.
|
|
 |
DISCUSSION |
The functional significance of ionic channels, especially
K+ channels, in immune cells has been the subject of
intense study over the past 20 years. The gating of K+
channels in immune cells plays a significant role in determining resting membrane potential, cellular proliferation and migration, initiation of cell differentiation, and volume regulation (6, 7,
17). However, there is a paucity of studies that have attempted
to define the functional role of ion channel activity during the
physical interaction between monocytes and endothelial cells. In this
present study, we sought to determine whether the modulation of
K+ currents and resulting hyperpolarization we have
previously described (11) alters Ca2+-mediated
monocyte function. The results from the current study demonstrate that
clustering of VLA-4 integrins on THP-1 monocytes with
cross-linked MAbVLA-4 mimicked the effect on Iir
and Idr observed in THP-1 cells adherent to
purified VCAM-1 or activated endothelial cells. Furthermore, the
presence of Iir augmented IL-8 release from
THP-1 monocytes. IL-8 is chemotactic for neutrophils, endothelial
cells, lymphocytes, and vascular smooth muscle cells, all contributors
to atherosclerotic plaque formation (27, 47, 52).
Role of induction of Iir in monocyte differentiation.
Our data demonstrate that THP-1 monocytes with clustered VLA-4
integrins possess enhanced oxidative burst activity (9) and cytokine production, suggesting that the VLA-4-induced
Iir expression may constitute an early event
leading to monocyte differentiation. The increased expression of
K+ currents has been linked to cell differentiation in mast
cells (36), cardiomyocytes (35), skeletal
muscle (45), neuroblastoma cells (24), and
monocytes (14), some resulting from integrin/ligand interactions. Differentiation of THP-1 monocytes into macrophage-like cells with phorbol 12-myristate 13-acetate enhances the appearance and
magnitude of, presumably, the same Cs+-sensitive
Iir induced by clustered VLA-4 integrins
(14, 26). A role for an Iir has
also been implicated in the differentiation of the human monocytic cell
line U937 to macrophages (16). Differentiation of the
neuroblastoma/glioma NG108-15 cell line to neuronlike type I cells
induces an increase in Iir density that is
associated with neurite outgrowth (40).
Contribution of Iir and Idr to RMP.
Human monocytic leukemia THP-1 cells exhibit many of the K+
conductances present in normal monocytes and macrophages (11, 18), one of which, the Iir, when present
sets the RMP to more negative levels. However, in the current study,
the majority of THP-1 monocytes with clustered VLA-4 integrins also
expressed a Idr , and this K+
current contributed to RMP by driving RMP toward its threshold activation voltage of
45 mV. The relative magnitudes of these two
voltage-gated K+ currents determined whether RMP was near
the threshold activation voltage for Idr or
approached EK (
86 mV). As long as
Iir was the predominant current in THP-1
monocytes, RMP approached EK. Activated
microglia that function as brain macrophages in the central nervous
system possess Iir and
Idr and a mean RMP near
45 mV, the threshold
activation voltage for the Idr (8).
However, there is clearly a subset of activated microglia in which the Iir is the dominant current, bringing the
membrane potential to near
70 mV. Inhibition of this current by
Ba2+ depolarizes RMP near
45 mV. We observed similar
depolarization in THP-1 monocytes by blocking
Iir with Ba2+ or Cs+
(11). In the present study, RMP of THP-1 monocytes
possessing both voltage-gated K+ currents hyperpolarized
closer to EK following inhibition of K+ efflux via Idr with ChTX or TEA.
In THP-1 monocytes possessing Idr in the absence
of Iir, RMP was close to the threshold
activation voltage for Idr. Blockade of
Idr in cells that lack the
Iir depolarized RMP, suggesting that both
K+ currents function to sustain THP-1 monocytes with
clustered VLA-4 integrins at hyperpolarized RMP.
Physiological role of VLA-4-induced hyperpolarization.
Functionally, we have shown in this present study that clustering of
VLA-4 integrins on THP-1 monocytes with fixed LPS-ECs, VCAM-1, or
cross-linked MAbVLA-4 significantly increases Ca2+-mediated
IL-8 release over release from cells on POLY, and this effect is
completely abolished by exposure to Cs+, a heavy metal ion
known to block Iir. These data suggest that the
VLA-4-induced hyperpolarization possesses an important regulatory role
in Ca2+-mediated macrophage function. The progression of
atherosclerosis may even be impacted, given that cholesterol-loaded
THP-1 cells possess higher mRNA levels of IL-8, which correlate with
increased IL-8 mRNA in macrophage-rich areas of human atheromas
(44). Internal Ca2+ increases due, in part, to
extracellular Ca2+ entry, have been shown to play a
predominant role in regulating monocyte/macrophage function including
the initiation of phagocytosis (21), expression of
inducible nitric oxide synthase (22), stimulation of
oxidative burst (22, 23), superoxide anion production
(22), stimulated eicosanoid release (1), and
cytokine production (33). Hyperpolarization of membrane
potential produced by clustered VLA-4 integrins would likely enhance
store-depleted activation of Ca2+ influx known to be
present in monocytes or macrophages (13, 29, 34). We have
recently demonstrated that Ca2+ store depletion triggered
by the sarcoplasmic reticulum Ca2+-ATPase inhibitor, TG,
increased the peak intracellular Ca2+ concentration
significantly in THP-1 monocytes adherent to VCAM-1 compared with cells
adherent to glass (12). This VCAM-1-induced enhancement is
blocked by exposure to Cs+ or 50 mM extracellular
K+ and is mimicked in THP-1 monocytes bound to glass that
are hyperpolarized by valinomycin, a K+ ionophore.
In summary, MAb-mediated engaging and clustering of VLA-4 integrins
appear to mimic multivalent interactions of VCAM-1 with THP-1 monocytes
to modulate the expression and magnitude of two voltage-gated
K+ currents, Iir and
Idr. The induction of the
Iir hyperpolarizes THP-1 monocytes, increases
Ca2+ entry, and stimulates IL-8 release. This is the first
time that the Iir has been demonstrated to play
a role in the secretion of a proinflammatory cytokine when VLA-4
integrins on monocytes are clustered. This study suggests that these
events may also regulate monocyte differentiation, enhanced macrophage
function, and possibly atherosclerotic plaque formation.
 |
ACKNOWLEDGEMENTS |
I thank Drs. Pamela Gunter-Smith, Gordon Leitch, and David Mann for
their critical evaluation of this manuscript. I also acknowledge the
excellent technical assistance that Esther Carlisle Doele and Roberta
Hawkins provided.
 |
FOOTNOTES |
This work was supported by American Heart Association/GA Affiliate and,
in part, by National Institutes of Health National Center for Research
Resources Grant NIH/G13 RR-03034.
Address for reprint requests and other correspondence: M. Colden-Stanfield, Morehouse School of Medicine, Dept. of Physiology, Rm
349, 720 Westview Dr., SW, Atlanta, GA 30310-1495 (E-mail: stanfiel{at}msm.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 29, 2002;10.1152/ajpcell.00481.2001
Received 13 October 2001; accepted in final form 17 May 2002.
 |
REFERENCES |
1.
Aepfelbacher, M,
Ziegler-Heitbrock HW,
Lux I,
and
Weber PC.
Bacterial lipopolysaccharide up-regulates platelet-activating factor-stimulated Ca2+ mobilization and eicosanoid release in human Mono Mac 6 cells.
J Immunol
148:
2186-2193,
1992[Abstract/Free Full Text].
2.
Arcangeli, A,
Becchetti A,
Mannini A,
Mugnai G,
De Filippi P,
Tarone G,
Del Bene MR,
Barletta E,
Wanke E,
and
Olivotto M.
Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels.
J Cell Biol
122:
1131-1143,
1993[Abstract].
3.
Auwerx, J.
The human leukemia cell line, THP-1: a multifaceted model for the study of monocyte-macrophage differentiation.
Experientia
47:
22-31,
1991[ISI][Medline].
4.
Beeton, C,
Barbaria J,
Giraud P,
Devaux J,
Benoliel A,
Gola M,
Sabatier J,
Bernard D,
Crest M,
and
Beraud E.
Selective blocking of voltage-gated K+ channels improves experimental autoimmune encephalomyelitis and inhibits T cell activation.
J Immunol
166:
936-944,
2001[Abstract/Free Full Text].
5.
Bianchi, L,
Arcangel A,
Bartolini P,
Mugnai G,
Wanke E,
and
Olivotto M.
An inward rectifier K+ current modulates in neuroblastoma cells the tyrosine phosphorylation of the pp125FAK and associated proteins: role in neuritogenesis.
Biochem Biophys Res Commun
210:
823-829,
1995[ISI][Medline].
6.
Cahalan, M,
Wulff H,
and
Chandy K.
Molecular properties and physiological roles of ion channels in the immune system.
J Clin Immunol
21:
235-252,
2001[ISI][Medline].
7.
Chow, CW,
Demaurex N,
and
Grinstein S.
Ion transport and the function of phagocytic cells.
Curr Opin Hematol
2:
89-95,
1995[Medline].
8.
Chung, S,
Jung W,
and
Lee M.
Inward and outward rectifying potassium currents set membrane potentials in activated rat microglia.
Neurosci Lett
262:
121-124,
1999[ISI][Medline].
9.
Colden-Stanfield, M.
Clustering of VLA-4 integrins enhances oxidative burst activity in human THP-1 monocytes (Abstract).
FASEB J
14:
A271,
2000.
10.
Colden-Stanfield, M,
Cramer E,
and
Gallin E.
Comparison of apical and basal surfaces of confluent endothelial cells: patch-clamp and viral studies.
Am J Physiol Cell Physiol
263:
C573-C583,
1992[Abstract/Free Full Text].
11.
Colden-Stanfield, M,
and
Gallin EK.
Modulation of K+ currents in monocytes by VCAM-1 and E-selectin on activated human endothelium.
Am J Physiol Cell Physiol
275:
C267-C277,
1998[Abstract/Free Full Text].
12.
Colden-Stanfield, M,
and
Scanlon M.
VCAM-1-induced inwardly rectifying K+ current enhances Ca2+ entry in human THP-1 monocytes.
Am J Physiol Cell Physiol
279:
C488-C494,
2000[Abstract/Free Full Text].
13.
Davis, W.
Increased capacity for store regulated calcium influx in U937 cells differentiated by treatment with dibutyryl cAMP.
Cell Calcium
17:
345-353,
1995[ISI][Medline].
14.
DeCoursey, T,
Kim S,
Silver M,
and
Quandt F, III.
Ion channel expression in PMA-differentiated human THP-1 macrophages.
J Membr Biol
152:
141-157,
1996[ISI][Medline].
15.
Elices, MJ,
Osborn L,
Takada Y,
Crouse C,
Luhowskyj S,
Hemler ME,
and
Lobb RR.
VCAM-1 on activated endothelium interacts with the leukocyte intergrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site.
Cell
60:
577-584,
1990[ISI][Medline].
16.
Floto, RA,
Mahaut-Smith MP,
Allen JM,
and
Somasundaram B.
Differentiation of the human monocytic cell line U937 results in an upregulation of the calcium release-activated current, ICRAC.
J Physiol
495:
331-338,
1996[Abstract].
17.
Gallin, EK.
Ion channels in leukocytes.
Physiol Rev
71:
775-811,
1991[Free Full Text].
18.
Gallin, EK,
and
Livengood DR.
Inward rectification in mouse macrophages: evidence for a negative resistance region.
Am J Physiol Cell Physiol
241:
C9-C17,
1981[Abstract/Free Full Text].
19.
Hamill, OP.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
20.
Hess, SD,
Oortgiesen M,
and
Cahalan MD.
Calcium oscillations in human T and natural killer cells depend upon membrane potential and calcium influx.
J Immunol
150:
2620-2633,
1993[Abstract/Free Full Text].
21.
Hishikawa, T,
Cheung JY,
Yelamarty RV,
and
Knutson DW.
Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages.
J Cell Biol
115:
59-66,
1991[Abstract].
22.
Hotchkiss, RS,
Bowling WM,
Karl IE,
Osborne DF,
and
Flye MW.
Calcium antagonists inhibit oxidative burst and nitrite formation in lipopolysaccharide-stimulated rat peritoneal macrophages.
Shock
8:
170-178,
1997[ISI][Medline].
23.
Hoyal, CR,
Gozal E,
Zhou H,
Foldenauer K,
and
Forman HJ.
Modulation of the rat alveolar macrophage respiratory burst by hydroperoxides is calcium dependent.
Arch Biochem Biophys
326:
166-171,
1996[ISI][Medline].
24.
Hu, Q,
and
Shi YL.
Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma x glioma cells.
Pflügers Arch
433:
617-625,
1997[ISI][Medline].
25.
Juliano, RL.
Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members.
Annu Rev Pharmacol Toxicol
42:
283-323,
2002[ISI][Medline].
26.
Kim, SY,
Silver MR,
and
DeCoursey TE.
Ion channels in human THP-1 monocytes.
J Membr Biol
152:
117-130,
1996[ISI][Medline].
27.
Koch, AE.
Interleukin-8 as a macrophage-derived mediator of angiogenesis.
Science
258:
1798-1801,
1992[ISI][Medline].
28.
Levite, M,
Cahalon L,
Peretz A,
Hershkoviz R,
Sobko A,
Ariel A,
Desai R,
Attali B,
and
Lider O.
Extracellular K+ and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and
1 integrins.
J Exp Med
191:
1167-1176,
2000[Abstract/Free Full Text].
29.
Li, HF,
Shen AY,
Jan CR,
and
Wu SN.
Co-activation of nonselective cation channels by store depletion and oxidative stress in monocytic U937 cells.
Chin J Physiol
41:
113-119,
1998[ISI][Medline].
30.
Lin, CS,
Boltz RC,
Blake JT,
Nguyen M,
Talento A,
Fischer PA,
Springer MS,
Sigal NH,
Slaughter RS,
Garcia ML,
Kaczorowski GJ,
and
Koo GC.
Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation.
J Exp Med
177:
637-645,
1993[Abstract].
31.
Lobb, RR,
Chi-Rosso G,
Leone DR,
Rosa MD,
Bixler S,
Newman BM,
Luhowskyj S,
Benjamin CD,
Dougas IG,
Goelz SE,
Hession C,
and
Chow EP.
Expression and functional characterization of a soluble form of endothelial-leukocyte adhesion molecule 1.
J Immunol
147:
124-129,
1991[Abstract/Free Full Text].
32.
Longhurst, CM.
Integrin-mediated signal transduction.
Cell Mol Life Sci
54:
514-526,
1998[ISI][Medline].
33.
Mahe, Y,
Wakasugi H,
Scamps C,
Chouaib S,
and
Tursz T.
Role of calcium on interleukin-1 production by monocytes: its relevance during T cell proliferation.
Lymphokine Cytokine Res
19:
165-172,
1991.
34.
Malayev, A,
and
Nelson DJ.
Extracellular pH modulates the Ca2+ current activated by depletion of intracellular Ca2+ stores in human macrophages.
J Membr Biol
146:
101-111,
1995[ISI][Medline].
35.
Maltsev VA, Wobus AM, Rohwedel J, Bader M, and Hescheler J. Cardiomyocytes differentiated in vitro from embryonic stem cells
developmentally express cardiac-specific genes and ionic currents.
Circ Res 233-244, 1994.
36.
McCloskey, MA,
and
Qian YX.
Selective expression of potassium channels during mast cell differentiation.
J Biol Chem
269:
14813-14819,
1994[Abstract/Free Full Text].
37.
McGilvray, ID,
Lu Z,
Bitar R,
Dackiw APB,
Davreux CJ,
and
Rotstein OD.
VLA-4 integrin cross-linking on human monocytic THP-1 cells induces tissue factor expression by a mechanism involving mitogen-activated protein kinase.
J Biol Chem
272:
10287-10294,
1997[Abstract/Free Full Text].
38.
McPhee, J,
Dang Y,
Davidson N,
and
Lester H.
Evidence for a functional interaction between integrins and G protein-activated inward rectifier K+ channels.
J Biol Chem
273:
34696-34702,
1998[Abstract/Free Full Text].
39.
Needham, LA,
Van Dijk S,
Pigott R,
Edwards RM,
Shepherd M,
Hemingway I,
Jack L,
and
Clements JM.
Activation dependent and independent VLA-4 binding sites on vascular cell adhesion molecule-1.
Cell Adhes Commun
2:
87-99,
1994[ISI][Medline].
40.
Pancrazio, J,
Ma W,
Grant G,
Shaffer K,
Kao W,
Liu Q,
Manos P,
Barker J,
and
Stenger D.
A role for inwardly rectifying K+ channels in differentiation of NG108-15 neuroblastoma x glioma cells.
J Neurobiol
38:
466-474,
1999[ISI][Medline].
41.
Platts, SH.
Role of K+ channels in arteriolar vasodilation mediated by integrin interaction with RGD-containing peptide.
Am J Physiol Heart Circ Physiol
275:
H1449-H1454,
1998[Abstract/Free Full Text].
42.
Pusch, M,
and
Neher E.
Rates of diffusional exchange between small cells and a measuring patch pipette.
Pflügers Arch
411:
204-211,
1988[ISI][Medline].
43.
Rosales, C,
and
Juliano RL.
Integrin signaling to NF-
B in monocytic leukemia cells is blocked by activated oncogenes.
Cancer Res
56:
2302-2305,
1996[Abstract].
44.
Rus, HG,
Vlaicu R,
and
Niculescu F.
Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall.
Atherosclerosis
127:
263-271,
1996[ISI][Medline].
45.
Shin, KS,
Park J,
Kwon J,
Chung CH,
and
Kang M.
A possible role of inwardly rectifying K+ channels in chick myoblast differentiation.
Am J Physiol Cell Physiol
272:
C894-C900,
1997[Abstract/Free Full Text].
46.
Strauss, U,
Wissel K,
Jung S,
Wulff H,
Zhu J,
Rolfs A,
and
Mix E.
K+ channel-blocking alkoxypsoralens inhibit the immune response of encephalitogenic T line cells and lymphocytes from Lewis rats challenged for experimental autoimmune encephalomyelitis.
Immunopharmacology
48:
51-63,
2000[ISI][Medline].
47.
Terkeltaub, R,
Boisvert WA,
and
Curtiss LK.
Chemokines and atherosclerosis.
Curr Opin Lipidol
9:
397-405,
1998[ISI][Medline].
48.
Tsuchiya, S,
Yamabe M,
Yamaguchi Y,
Kobayashi Y,
Konno T,
and
Tada K.
Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).
Int J Cancer
26:
171-176,
1980[ISI][Medline].
49.
Williams, EJ,
Doherty P,
Turner G,
Reid RA,
Hemperly JJ,
and
Walsh FS.
Calcium influx into neurons can solely account for cell contact-dependent neurite outgrowth stimulated by transfected L1.
J Cell Biol
119:
883-892,
1992[Abstract].
50.
Wu, X,
Mogford JE,
Platts SH,
Davis GE,
Meininger GA,
and
Davis MJ.
Modulation of calcium current in arteriolar smooth muscle by
v
3 and
5
1 integrin ligands.
J Cell Biol
143:
241-252,
1998[Abstract/Free Full Text].
51.
Wulff, H,
Miller M,
Hansel W,
Grissmer S,
Cahalon M,
and
Chandy K.
Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant.
Proc Natl Acad Sci USA
97:
8151-8156,
2000[Abstract/Free Full Text].
52.
Yue, TL,
Mckenna PJ,
Gu JL,
and
Feurerstein GZ.
Interleukin-8 is chemotactic for vascular smooth muscle cells.
Eur J Pharmacol
240:
81-84,
1993[ISI][Medline].
Am J Physiol Cell Physiol 283(3):C990-C1000
0363-6143/02 $5.00
Copyright © 2002 the American Physiological Society