1 Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 30310; and 2 Women's Health Initiative, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203
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ABSTRACT |
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Resting membrane potential (RMP) and whole cell currents were
recorded in human THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cells (HUVECs),
lipopolysaccharide (LPS)-treated HUVECs, immobilized
E-selectin, or vascular cell adhesion molecule 1 (VCAM-1)
using the patch-clamp technique. RMP after 5 h on polystyrene was
24.3 ± 1.7 mV (n = 42) with delayed rectifier K+
(Idr) and
Cl
currents
(ICl) present
in >75% of the cells. Inwardly rectifying K+ currents
(Iir) were
present in only 14% of THP-1 cells. Adherence to unstimulated HUVECs
or E-selectin for 5 h had no effect on Iir or
ICl but decreased
Idr. Five hours
after adherence to LPS-treated HUVECs, outward currents were unchanged,
but Iir was
present in 81% of THP-1 cells. A twofold increase in
Iir and a
hyperpolarization (
41.3 ± 3.7 mV,
n = 16) were abolished by pretreatment
of THP-1 cells with cycloheximide, a protein synthesis inhibitor, or
herbimycin A, a tyrosine kinase inhibitor, or by pretreatment of the
LPS-treated HUVECs with anti-VCAM-1. Only a brief (15-min) interaction
between THP-1 cells and LPS-treated HUVECs was required to
induce Iir expression 5 h later. THP-1 cells adherent to VCAM-1 exhibited similar
conductances to cells adherent to LPS-treated HUVECs. Thus engagement
of specific integrins results in selective modulation of different
K+ conductances.
monocytic leukemia; integrin; adhesion molecules; ion channels; signaling
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INTRODUCTION |
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ADHESION OF MONOCYTES to extracellular matrix molecules
or other cells not only mediates cell rolling and diapedesis, it also triggers intracellular signaling pathways, resulting in
monocyte/macrophage activation (42). These interactions, mediated by
the engagement of several different families of integrins, induce the
expression of a number of genes associated with the inflammatory
response. Binding and cross-linking of
1-integrins on the surface of
monocytes induce immediate-early genes coding for inflammatory
mediators, interleukin-1
(IL-1
), tumor necrosis factor-
(TNF-
), and interleukin-8 (IL-8), through a protein
tyrosine kinase-mediated pathway (28, 48). A similar increase in the
production of TNF-
also was noted after the direct engagement of
2-integrin, CD11b/CD18, in
human monocytes (18).
Changes in ionic conductances and/or homeostasis after
integrin-ligand interactions have been reported in various cell types. Integrin-ligand interactions enhance
K+ conductances, leading to
membrane hyperpolarization in neuroblastoma and erythroleukemia cells
after interaction with fibronectin (2, 5). Other studies have linked
increases in intracellular Ca2+
levels to integrin-ligand signaling cascades. In the nervous system,
outgrowth from cerebellar neurons (46) and pheochromocytoma cells (15)
is triggered by Ca2+ influx
induced by engagement of N-cadherin, neural cell adhesion molecule, and
L1. Cross-linking of CD18, the integrin -subunit ubiquitously
expressed by all leukocytes, or the
-subunit CD11b/CD18 increases
intracellular Ca2+ in human
monocytic leukemia THP-1 cells (1).
THP-1 monocytes closely resemble monocyte-derived macrophages in their functional characteristics, surface receptors, and ionic conductances (3, 44). Similar to primary monocytes, THP-1 cells display a variety of adhesion molecules on their surface that mediate their interactions with extracellular matrix molecules and other cells. Thus they provide a well-characterized, easily accessible model to examine the effects of integrin-ligand interactions on the membrane conductances and resting membrane potential (RMP) changes of monocytes. Furthermore, THP-1 cells exhibit many of the K+ conductances present in primary monocytes and macrophages (14, 25). One of these conductances, an inwardly rectifying K+ conductance, has been reported in primary human and murine macrophages (20), and when it is present, it sets the RMP to more negative levels. The inwardly rectifying K+ conductance also is present in the J774 murine monocytic cell line, where it increases after adherence (21, 35). After THP-1 cells are treated with phorbol esters to induce differentiation into macrophage-like cells, inwardly rectifying K+ currents (Iir), absent in undifferentiated THP-1 cells, are expressed (14, 25).
The current study examines the electrophysiology of undifferentiated THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cell (HUVEC) monolayers, lipopolysaccharide (LPS)-treated HUVEC monolayers expressing E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), or immobilized, purified adhesion molecules. We demonstrate that two K+ conductances, a delayed rectifier K+ conductance and an inwardly rectifying K+ conductance, are differentially modulated by adherence to distinct substrates. Interaction of undifferentiated THP-1 monocytes with immobilized soluble E-selectin decreases the delayed rectifier K+ currents (Idr) without affecting Iir expression, whereas interaction with immobilized soluble VCAM-1 decreases the amplitude of Idr and increases the size and the number of cells expressing Iir.
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METHODS |
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Cell Cultures
Undifferentiated THP-1 cells (American Type Culture Collection, Rockville, MD) were cultured in suspension with RPMI culture medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (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, 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 HUVECs (American Type Culture Collection) was maintained in MCDB107 medium (American Biorganics, Niagara Falls, NY) supplemented with 10% fetal bovine serum, 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 HUVECs in passages 17-19 and at 1-2 days postconfluency. Some HUVEC monolayers were pretreated with LPS (3 µg/ml) at 37°C in a 5% CO2-humidified incubator to increase the surface expression of E-selectin, ICAM-1, and VCAM-1. To eliminate the possibility of LPS activation of the THP-1 monocytes, LPS-treated HUVECs were washed twice with complete RPMI before the THP-1 cells were added to the cultures.
Patch-Clamp Recording of Ionic Currents
Macroscopic whole cell patch-clamp recordings (22) were obtained from THP-1 cells that were coincubated with unstimulated HUVEC monolayers, LPS-treated HUVEC monolayers, or immobilized purified adhesion molecules using patch electrodes (model BF100-50-10, Sutter Instruments) with resistances of 4-7 MImmobilization of Soluble Adhesion Molecules
A 50-µl aliquot of recombinant soluble ICAM-1 (R & D Systems, Minneapolis, MN), E-selectin (formerly known as ELAM-1), or VCAM-1 (50 µg/ml; the latter both generous gifts from Dr. Roy Lobb, Biogen, Cambridge, MA) (29) in a binding buffer (15 mM NaHCO3-35 mM Na2CO3, pH 9.2) was placed in the middle of 35-mm polystyrene bacteriologic dishes (Falcon 1008, Fisher Scientific, Norcross, GA) for coating overnight at 4°C. Nonspecific binding was blocked with PBS-1% BSA for 1 h at 4°C, and the dish was washed once with complete RPMI before THP-1 cells (1 × 106 cells/ml) in complete RPMI were added for incubation at 37°C for 1-5 h. Nonadherent THP-1 monocytes were removed by washing the dish with Ringer bath solution twice before macroscopic currents were obtained from remaining adherent THP-1 monocytes.Surface Antigen Detection of Adhesion Molecules
HUVECs (104 cells/well) were seeded in collagen-coated 96-well plates 24 h before confluent monolayers were incubated with complete MCDB107 medium or LPS (3 µg/ml) in complete MCDB107 medium for various times at 37°C. The time course of surface antigen expression of endothelial adhesion molecules was assessed as previously described with a colorimetric ELISA (8) with use of mouse monoclonal antibodies raised against E-selectin, ICAM-1, and VCAM-1 (Becton-Dickinson, San Jose, CA).Adhesion Assay
To confirm the functional manifestation of upregulated adhesion molecules produced by LPS treatment of HUVEC monolayers, THP-1 monocyte adherence was assessed by a fluorescence-based microplate adhesion assay. 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded THP-1 monocytes (105 cells/well) suspended in complete MCDB107 medium were added to the LPS-treated HUVEC monolayers for 30 min at 37°C in a 5% CO2-humidified incubator. Unbound THP-1 cells were aspirated, and HUVEC monolayers were washed twice before the remaining fluorescence (i.e., adherent THP-1 cells) was read on a Cytofluor II fluorescence plate reader with use of fluorescein optics (485 nm excitation/530 nm emission). The percentage of THP-1 monocyte adherence was calculated with the measured fluorescence as follows
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Adherence assays also were performed to determine the contribution of ICAM-1, E-selectin, and VCAM-1 to THP-1 monocyte adherence to naive or LPS-treated HUVEC monolayers. Naive or LPS-treated HUVEC monolayers were pretreated with anti-ICAM-1, anti-E-selectin, or anti-VCAM-1 (2 µg/ml) for 30 min at 37°C before the adhesion assay was performed in the presence of the same monoclonal antibody.
Data Analysis
Values are means ± SE. Current amplitude normalized by Cm (pA/pF) and RMP were compared using the unpaired Student's t-test. The occurrence of specific currents was compared between THP-1 monocytes bound to polystyrene, adherent to endothelial cells, or immobilized adhesion molecules by means of the z-test. P > 0.05 was not considered significant. ![]() |
RESULTS |
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Electrophysiological Properties of THP-1 Monocytes Bound to Polystyrene
Whole cell recordings were obtained from undifferentiated THP-1 monocytes bound to polystyrene dishes for various periods of time. Under current-clamp conditions (I = 0), RMP recorded after 1 h of incubation on polystyrene was
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Outward currents in response to depolarizing voltage steps were larger
and more complex than the currents in response to hyperpolarizing voltage steps. In 76% of cells (37 of 49; Table 1) an inactivating outward current was activated at potentials more depolarized than 40 mV (Fig.
2A). A
component of the outward current was similar to the delayed rectifier
K+ conductance previously
described in THP-1 cells and other types of monocytes (19) and was
inhibited by 30 nM charybdotoxin (Fig. 2,
B and
C) or 100 µM 4-aminopyridine
(current profile identical to Fig.
2B). An additional current unmasked
by pharmacological blockade of
Idr (Fig.
2B) or step depolarizations greater
than +100 mV was present in 94% of the cells. This outward current was
most easily characterized in the 24% of the THP-1 monocytes without
Idr (Fig.
3A). It
was noninactivating, blocked by exogenous application of 1 mM SITS
(Fig. 3B), and reversed near 0 mV
(Fig. 3C), suggesting that
Cl
carried the current,
since the equilibrium potential for
Cl
under these experimental
conditions was 0 mV. Similar blockade was obtained with 200 µM DIDS
(current profile identical to Fig. 3B). No attempt was made to
pharmacologically characterize the small outward current (~50 pA at
+80 mV) remaining after SITS or DIDS blockade, but it is likely that
this third outward current represents the cation current previously
described in THP-1 monocytes (25). Neither the outward nor inward
currents (normalized to Cm) recorded
from undifferentiated THP-1 cells significantly changed when the cells
were bound to polystyrene for 1, 3, or 5 h (see Fig.
5D).
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Effect of Adherence to Unstimulated Endothelial Cells
Macroscopic currents were recorded from THP-1 monocytes that were incubated with unstimulated HUVEC monolayers for 5 h. Under normal conditions the endothelial cell monolayer lining blood vessels is nonthrombogenic and does not act as an adhesive surface for leukocytes. As illustrated in Fig. 4A, HUVECs constitutively express ICAM-1 (open circle) on their surface, but VCAM-1 (open triangle) and E-selectin (open square) are absent. Lack of VCAM-1 and E-selectin expression results in low THP-1 monocyte adherence to unstimulated HUVEC monolayers (Fig. 4B, open bar).
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RMP in THP-1 cells adherent to unstimulated HUVEC monolayers (basal HUVECs in Table 1) was not significantly different from that in THP-1 cells bound to polystyrene. As we observed in THP-1 monocytes bound to polystyrene, 5 h after incubation with basal HUVEC monolayers the majority of THP-1 cells (94%) possessed little or no Iir (Table 1). A comparison of the normalized mean inward current measured in THP-1 monocytes adherent to basal HUVECs to current in THP-1 cells bound to polystyrene reveals no difference in magnitude (Table 2). Similarly, ICl were not different. However, Idr were significantly smaller in THP-1 cells on basal HUVEC monolayers than in THP-1 monocytes bound to polystyrene.
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Effect of Adherence to LPS-Activated Endothelial Cells
To determine whether the engagement of integrins with specific adhesion molecules on activated endothelial cells modifies the ionic currents expressed by THP-1 cells, macroscopic currents were recorded from THP-1 cells that were coincubated for various times with HUVEC monolayers pretreated with LPS (3 µg/ml). ELISA demonstrates that LPS treatment of HUVEC monolayers for 8 h resulted in a 344% increase in surface expression of ICAM-1, which remained elevated for the next 64 h (Fig. 4A). LPS treatment also induced the expression of E-selectin and VCAM-1, which are absent from the surface of unstimulated HUVECs, both of which remained elevated for at least 72 h. Twenty-four hours after LPS treatment of HUVEC monolayers, when all three of these adhesion molecules were upregulated, the percentage of THP-1 cells adhering to LPS-treated HUVEC monolayers was increased fivefold over the percentage of THP-1 cells adhering to unstimulated HUVEC monolayers (Fig. 4B). Antibody-blocking experiments (see METHODS for procedure) revealed that although basal adherence was not affected by antibody against any of the three adhesion molecules, LPS-induced THP-1 monocyte adherence was inhibited by 25.3 ± 2.5% (n = 3) in the presence of anti-VCAM-1 (Fig. 4B). In contrast, LPS-induced THP-1 monocyte adherence was not inhibited by anti-E-selectin or anti-ICAM-1.For patch-clamp studies, HUVEC monolayers were treated with a
saturating concentration of LPS (3 µg/ml) for 17-24 h before the
monolayers were washed and THP-1 monocytes were added to the monolayers. Under current-clamp conditions
(I = 0), RMP of THP-1 monocytes
adherent to LPS-treated HUVEC monolayers for 1 h was not significantly
different from RMP of THP-1 cells bound to polystyrene for 1 h:
27 ± 4.0 mV (n = 10) vs.
20.0 ± 3.6 mV (n = 12). The magnitude of the inward currents in response to hyperpolarizing voltage
steps in THP-1 cells adherent to LPS-treated HUVECs for 1 h was also
similar to inward currents observed in THP-1 cells adherent to
polystyrene (cf. Fig. 5,
C and
D, triangles). Furthermore, outward
currents measured at 0 mV
(Idr) and +120
mV (predominantly ICl) were
similar in THP-1 monocytes adherent to activated endothelium and in
those bound to polystyrene for 1 h.
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After 5 h of adherence to monolayers of LPS-treated HUVECs, RMP of THP-1 cells was hyperpolarized by ~20 mV compared with RMP in THP-1 cells bound to polystyrene or in THP-1 cells adherent to unstimulated HUVECs (Table 1). In addition, Iir were now present in 81% of the cells, and the normalized Iir amplitude in response to inward voltage steps was greater than that for THP-1 monocytes bound to polystyrene (Tables 1 and 2, cf. Fig. 5, A and B). Interestingly, although the percentage of cells expressing Idr significantly increased by 17%, the proportion of cells expressing ICl decreased by the same percentage (Table 1). However, the magnitude of these currents in THP-1 cells adherent to LPS-treated HUVECs compared with THP-1 cells bound to polystyrene was similar (Table 2). The conductance-voltage relationships shown in Fig. 5C illustrate the time-dependent development of inward and outward currents (normalized to total Cm) in THP-1 cells after adherence to LPS-treated HUVEC monolayers. In contrast, there were no time-dependent changes in inward or outward currents in THP-1 monocytes bound to polystyrene (Fig. 5D).
To eliminate the possibility that any remaining LPS after the wash procedure of LPS-treated HUVECs caused the increased Iir expression, we recorded macroscopic currents from THP-1 cells incubated on polystyrene that had been exposed to the same concentration of LPS used to treat HUVECs and washed twice with complete (endotoxin-free) RPMI before THP-1 cells were added. There was no enhanced normalized Iir in THP-1 monocytes after 5 h of incubation on polystyrene that had been previously exposed and washed to remove LPS (1.9 ± 0.4 pA/pF, n = 8).
Whole cell currents were recorded in THP-1 monocytes adherent to
LPS-treated HUVEC monolayers before and after exposure to BaCl2 or CsCl, two
well-established blockers of inwardly rectifying K+ channels (10, 21). In the
absence of blocker, inward currents were activated at steps negative to
80 mV and exhibited voltage-dependent inactivation at potentials
more negative to
120 mV (Fig.
6A). Exogenous application of Ba2+ (1 mM) completely blocked the inward current (Fig. 6,
B and
C) and depolarized the THP-1
monocytes by ~30 mV. In separate experiments, addition of
Cs+ (10 mM) to the bath solution
also blocked Iir
(data not shown).
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Signaling Mechanisms Underlying Induction of Iir
THP-1 currents induced by acute adherence to LPS-activated endothelial cells. It is possible that the initial contact between THP-1 cells and activated HUVECs triggers a signaling cascade that subsequently leads to increased Iir expression. To examine this possibility, macroscopic currents were recorded from THP-1 monocytes allowed to adhere to LPS-treated HUVEC monolayers for 15 min, removed from the monolayer, and incubated on polystyrene for the remaining 4-5 h. Table 3 illustrates that THP-1 cells treated in this manner expressed the same magnitude of Iir, having only been briefly exposed to LPS-treated HUVECs. In addition, the percentage of cells (82%, 9 of 11 cells) expressing Iir was identical to the percentage of cells expressing the same current in THP-1 cells adherent to LPS-treated HUVECs for the entire 5-h period. Thus continuous exposure of THP-1 cells to LPS-treated HUVECs is not necessary to induce Iir expression.
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Requirement of protein synthesis and tyrosine phosphorylation. The protein synthesis inhibitor cylcoheximide was used to determine whether de novo protein synthesis was required for the increase in Iir in THP-1 cells 4-5 h after interaction with LPS-treated HUVECs. THP-1 cells were pretreated with cylcoheximide (100 µM) for 1 h before they were incubated with LPS-treated HUVECs in the presence of cylcoheximide for 4-5 h. The development of Iir was completely abolished by cylcoheximide treatment, whereas the magnitude of the Idr and ICl in the cylcoheximide-treated THP-1 cells was similar to that in untreated THP-1 cells on activated endothelium (Table 3). Similar results were observed when THP-1 monocytes were pretreated with 10 µM herbimycin A, a tyrosine kinase inhibitor (Table 3). Therefore, the signaling events that induced expression of Iir after THP-1 monocyte adherence to LPS-treated HUVECs probably involve stimulation of de novo protein synthesis and tyrosine phosphorylation in THP-1 monocytes.
THP-1 currents during adherence to fixed LPS-activated endothelial
cells.
To test the possibility that an endothelium-derived factor(s) released
by HUVECs after their interaction with THP-1 monocytes results in the
changes in THP-1 current profile, macroscopic currents were recorded
from THP-1 monocytes incubated for 4 h with LPS-treated HUVEC
monolayers that had been fixed in paraformaldehyde. Prior fixation of
LPS-treated HUVECs did not prevent the expression of
Iir in THP-1
monocytes (18 of 25 cells, 72%), nor did it significantly affect the
shift in RMP that occurred in THP-1 cells 4 h after interaction with
LPS-treated HUVECs (2.2 ± 2.9 mV,
n = 22). The peak amplitude of the
Iir was not
significantly different from the normalized
Iir in THP-1
monocytes adherent to unfixed LPS-treated HUVECs (6.5 ± 0.9 pA/pF,
n = 18 at
160 mV), indicating
that the activated endothelium is not releasing factors to induce the
Iir in THP-1
monocytes.
THP-1 Currents During Adherence to Immobilized Purified Adhesion Molecules
To elucidate the role of adhesion molecules, macroscopic currents were recorded from THP-1 cells incubated on immobilized purified E-selectin or VCAM-1 for 5 h (see Immobilization of Soluble Adhesion Molecules). Similar experiments were attempted with ICAM-1, but THP-1 monocytes did not adhere to immobilized purified ICAM-1. ICAM-1 does not support monocyte adherence to endothelium (40). The presence of ICAM-1 on the surface of the dish was confirmed by positive binding of the human promyelocytic leukemia cell line HL-60 differentiated with DMSO to neutrophil-like cells (32).In contrast to our results with ICAM-1, THP-1 cells began to adhere to
immobilized E-selectin and VCAM-1 within minutes. Engagement of THP-1
monocytes to E-selectin for 5 h produced a current profile during step
hyperpolarizations similar to THP-1 cells bound to polystyrene (cf.
Fig. 5B, Tables 1 and 2). That is,
82% of the cells did not express
Iir and possessed
a mean RMP of 25 mV for the entire group. In contrast, the
normalized Idr
during depolarization were significantly smaller in THP-1 cells
adherent to E-selectin than in THP-1 cells adherent to polystyrene or
LPS-treated HUVECs. Normalized
ICl were similar
in magnitude to THP-1 cells bound to polystyrene but smaller than THP-1
cells on LPS-treated HUVECs. These data indicate that engagement of
THP-1 monocytes with E-selectin modifies the amplitude of
Idr but does not
increase Iir.
A firmer adherence was observed when THP-1 monocytes were incubated with immobilized VCAM-1 than when they were incubated with E-selectin. When the THP-1 cells were incubated on VCAM-1 for 1 h, depolarizing voltage steps produced similar outward currents observed previously with THP-1 monocytes adherent to the other substrates. Iir were absent in response to step hyperpolarizations. Longer incubation periods on immobilized VCAM-1 produced an Iir profile identical to that observed in THP-1 cells adherent to LPS-treated HUVECs (cf. Figs. 7 and 5, A and C). Ba2+-sensitive Iir were activated in 73% of the THP-1 cells tested (Table 1) and increased in magnitude over time (Fig. 7B, Table 2). Furthermore, as observed in THP-1 monocytes adherent to LPS-treated HUVECs for 5 h, THP-1 cells adherent to VCAM-1 had more hyperpolarized RMP than THP-1 cells adherent to polystyrene (Table 1). In contrast, the Idr and ICl in THP-1 cells adherent to VCAM-1 were much smaller in magnitude than those recorded in THP-1 cells adherent to LPS-treated HUVECs (Fig. 7C, Table 2), whereas only Idr were smaller than those recorded in THP-1 cells bound to polystyrene (Table 2).
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The finding that the
Iir was identical
in THP-1 monocytes adherent to LPS-treated HUVECs and VCAM-1 suggests
that VCAM-1 may be the adhesion molecule responsible for the enhanced
expression of Iir
in THP-1 monocytes adherent to LPS-treated HUVECs. To examine this possibility, we recorded whole cell currents in THP-1 monocytes adherent to LPS-treated HUVECs that had been pretreated with a saturating concentration of monoclonal antibody raised against VCAM-1.
The presence of anti-VCAM-1 completely blocked the expression of
Iir in 87% of
THP-1 monocytes adherent to activated endothelium, with
Iir in the
remaining THP-1 monocytes similar in magnitude to that observed on
polystyrene (Table 3). The increase in RMP observed on activated
endothelium (41 mV) was not observed when anti-VCAM-1 was
present (
22.0 ± 3.3 mV, n = 8).
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DISCUSSION |
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The acute monocytic leukemia cell line THP-1 provides a relatively
uniform population of monocytic cells that can be differentiated into
mature macrophage-like cells, and therefore they have been used
extensively to study gene expression during monocytic differentiation (3). Induction of immediate-early response genes that code for IL-1,
TNF-
, IL-8, and tissue factor are associated with monocytic
differentiation (3). Engagement of integrin receptors regulates the
levels of cytokine messages by triggering a cascade of signaling events
that involve tyrosine phosphorylation. Tyrosine kinase inhibitors block
increased levels of mRNAs for IL-1
and tissue factor induced in
THP-1 cells adherent to extracellular matrix proteins or ligation of
their
1-integrins (29, 34). Integrin-mediated adhesion also has been shown to activate other signaling pathways that involve ionic permeability changes (1, 2, 6).
In this report, we evaluate the role of monocyte-endothelial cell
interactions and, in particular, integrin-ligand interactions in
modifying the electrophysiological properties of THP-1 monocytes.
General Electrophysiological Properties of THP-1 Cells
A recent series of papers has described the electrophysiological characteristics of undifferentiated and PMA-differentiated THP-1 cells (13, 14, 25). Five different ionic conductances, many of which were previously described in primary macrophages, were noted in undifferentiated THP-1 monocytes bound to glass by use of the patch-clamp technique (25). These included two K+ currents, a delayed rectifier K+ and a small-conductance Ca2+-sensitive K+ current, as well as a cation current, a ClIn comparing the currents reported here with those previously described by Kim et al. (25), two facts should be noted. First, our recording conditions were designed to buffer intracellular Ca2+ concentration at 26.5 nM. Thus neither the small-conductance Ca2+-activated K+ channels present in undifferentiated THP-1 cells (25) nor the large-conductance Ca2+-activated K+ channels (14) in PMA-differentiated cells would be activated during our recordings. Second, the lack of H+ gradients between the electrode and the bath solutions, together with the voltage ranges examined, makes it unlikely that H+ currents observed by DeCoursey and Cherny (13) would be activated under our recording conditions. When these different recording conditions are taken into account, it is not surprising that the three main currents reported in our recordings were the Idr, the Iir, and the ICl that were previously described in THP-1 cells. The properties of each of these currents, in terms of their voltage activation and/or inactivation and the action of pharmacological blockers, such as charybdotoxin, Ba2+, and DIDS, confirmed that these ionic currents were similar to those previously described in THP-1 cells and other macrophages.
Adherence to Specific Substrates Selectively Modulates Expression of Idr and Iir
Changes in ionic currents after the adherence of macrophages were first noted in J774 cells after adherence to polystyrene (21). In those cells, Iir were absent in cells allowed to adhere for only 1 h but were present in cells adherent to polystyrene forIn contrast, adherence to E-selectin or unstimulated endothelial cells had no effect on the expression or magnitude of Iir. This pattern was very different from the effect of adherence on Idr. Idr, present in 76% of THP-1 monocytes on polystyrene, were expressed in the majority of adherent THP-1 cells regardless of the surfaces to which they adhered. However, Idr magnitude was significantly decreased when cells were plated on E-selectin, VCAM-1, or basal HUVECs. Surprisingly, Idr was not decreased after adherence to stimulated HUVECs, suggesting that an unknown factor/ligand associated with LPS-stimulated HUVECs prevented the decreased Idr density induced by the VCAM-1 and E-selectin expressed on stimulated endothelium. Other studies on THP-1 cells have shown that adherence to different substrates selectively induces the expression of different inflammatory response genes (17). Our results indicate that this selectivity is also present with regard to effects on K+ conductances expressed.
Signaling Pathways Involved in Iir Expression
Iir expression is associated with differentiation of several cell types, including mast cells (33), cardiomyocytes (31), skeletal muscle (41), neuroblastoma cells (23), and monocytes (14, 21, 35), with obligate de novo protein synthesis occurring. McKinney and Gallin (35) showed that upregulation of Iir in J774 cells 2-8 h after adherence to polystyrene was inhibited by the protein synthesis inhibitor cylcoheximide. Our studies similarly show that the inhibition of protein synthesis blocks the upregulation of Iir in THP-1 cells adherent to stimulated endothelium.The signals propagated by adhesion molecule interaction with integrin receptors result in the triggering of a number of familiar signaling pathways, including activation of protein tyrosine kinases (37, 38). Ca2+ channel activation and subsequent neurite outgrowth activated by engagement of rat cerebellar neurons with cell adhesion molecules, NCAM, N-cadherin, or L1 are inhibited by genistein, a tyrosine kinase inhibitor (47). The engagement of VLA-4 on T lymphocytes activates protein tyrosine kinase and is believed to be an early and obligatory event in the activation and proliferation of T cells (26, 37). Our results with use of the tyrosine kinase inhibitor herbimycin A suggest that the induction of Iir in THP-1 monocytes adherent to activated endothelial cells also involves tyrosine phosphorylation.
Physiological Relevance of Changes in K+ Currents
K+ channels serve to help set the membrane potential in several different cell types (11, 35) and to allow K+ to move into or out of the cell (depending on the electrochemical gradient). Both effects may have physiological implications for the cell. Although Idr and Iir probably contribute to the RMP of THP-1 cells, their relative contributions will vary, because the voltage range of action of the delayed rectifier K+ conductance in THP-1 monocytes differs from that of the inwardly rectifying K+ conductance (By increasing the driving force for Ca2+, hyperpolarization of membrane potential enhances the magnitude of Ca2+ entry into cells that possess non-voltage-gated Ca2+ influx pathways (43). Human T lymphocyte activation is associated with an increase in intracellular Ca2+ concentration that is regulated by membrane hyperpolarization produced via K+ efflux through K+ channels (27). Macrophages exhibit a non-voltage-gated Ca2+ influx pathway (30). In support of this hypothesis, preliminary evidence from our laboratory indicates that thapsigargin-induced Ca2+ entry is enhanced when THP-1 cells are adherent to VCAM-1 (9).
In addition to setting the RMP of cells to potentials near the
equilibrium potential for K+ (if
other ionic permeabilities are minimal),
K+ conductances also modulate
intracellular and extracellular K+
concentrations. For example, the inwardly rectifying
K+ conductance in glial cells
helps regulate extracellular K+
levels in the nervous system. In retinal glial cells,
Iir density is
highest in the end foot region. In those cells,
Iir
sublocalization is important in the spatial buffering and siphoning off
of extracellular K+ from focal
regions of activity (36).
Iir may play a
similar role in the tissue macrophages at sites of chronic inflammation if K+ levels rise in focal
restricted areas that do not result in changes in the cell's membrane
potential (19).
Iir also may be
important in modifying intracellular
K+. Studies have demonstrated that
agents that deplete intracellular K+ stimulate the posttranslational
processing and release of the inflammatory mediator IL-1 (39, 45).
Engagement of
1-integrins stimulates the translation of IL-1
. Thus, by modifying intracellular K+, it is possible that the
expression of Iir
is important in facilitating the posttranslation processing and release
of IL-1
.
Monocyte adhesion to endothelial cells is a crucial, early event for atherogenesis and inflammation (5) and involves induction of E-selectin and VCAM-1 (7, 12). Expression of VCAM-1 but not E-selectin is maintained at sites of inflammation such as early foam cell lesions leading to atherosclerotic plaque formation (12, 16). Long-lived macrophages accumulate at sites of chronic inflammation and contribute to the inflammatory response (24, 40). Our results suggest that monocytes that accumulate at those sites are likely to have higher levels of inwardly rectifying K+ channels in their cell membranes than circulating blood monocytes. Because THP-1 monocytes are readily available and relatively homogeneous, they provide an excellent model to use in future studies designed to dissect the pathways controlling inwardly rectifying K+ channel expression and determine its relevance to macrophage activation and inflammation.
In summary, we have demonstrated that the adherence of THP-1 monocytes to LPS-treated HUVECs or VCAM-1 induces the expression of inwardly rectifying K+ channels in the cell surface. Adherence to unstimulated HUVECs or immobilized E-selectin has no effect on Iir expression but decreases the Idr amplitude. Thus the expression of Idr and Iir in THP-1 monocytes is differentially modified by interaction with adhesion molecules.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Gordon Leitch and Pamela Gunter-Smith for critical evaluation of the manuscript and Dr. Mary Scanlon for confirming the free intracellular Ca2+ concentration of our pipette solution using the Digital Imaging System at Morehouse School of Medicine. We acknowledge the excellent technical assistance of Esther Carlisle-Doele and Roberta Hawkins.
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FOOTNOTES |
---|
This work was supported by National Institute of General Medical Sciences Grant GM-08248-10S1, a grant from the American Heart Association-Georgia Affiliate, and in part by National Institutes of Health Grant RR-03034.
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. §1734 solely to indicate this fact.
Address for reprint requests: M. Colden-Stanfield, Morehouse School of Medicine, Dept. of Physiology, Rm. 1311, 720 Westview Dr. SW, Atlanta, GA 30310.
Received 4 February 1998; accepted in final form 9 April 1998.
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