Modulation of K+ currents in monocytes by VCAM-1 and E-selectin on activated human endothelium

Margaret Colden-Stanfield1 and Elaine K. Gallin2

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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 beta 1-integrins on the surface of monocytes induce immediate-early genes coding for inflammatory mediators, interleukin-1beta (IL-1beta ), tumor necrosis factor-alpha (TNF-alpha ), and interleukin-8 (IL-8), through a protein tyrosine kinase-mediated pathway (28, 48). A similar increase in the production of TNF-alpha also was noted after the direct engagement of beta 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 alpha -subunit ubiquitously expressed by all leukocytes, or the beta -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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 MOmega . These currents were compared with macroscopic current recordings obtained from THP-1 monocytes that were bound to polystyrene for 1-5 h. The pipette contained (in mM) 145 KCl, 0.1 EGTA, 1 CaCl2 (26.5 nM free Ca2+), and 10 HEPES, with pH adjusted to 7.2 with KOH; the Ringer bath solution contained (in mM) 137 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5.5 glucose, 10 HEPES, and 0.01% BSA, with pH adjusted to 7.3 with NaOH. Free Ca2+ concentration of the KCl pipette solution was determined by using fura 2 free acid (1.25 µM) and a calibration curve constructed with external CaCl2 standards (Molecular Probes, Eugene, OR) ranging from 0 to 40 µM. 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. RMP was measured in current-clamp mode [current (I) = 0] immediately after attainment of the whole cell configuration. The voltage-clamp pulse protocols were computer driven by use of pCLAMP software (Axon Instruments) and 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 (Vh) of -40 mV and stepped in 20-mV increments from -160 mV to +120 mV with each pulse lasting 450 ms at an interval of 1 s. The Vh of -40 mV was chosen because it was near the RMP for THP-1 cells expressing Iir. This Vh and the short interval between pulses resulted in partial inactivation (an ~35% reduction in maximal conductance) of Idr. However, this partial inactivation did not affect comparisons of the current profiles from THP-1 monocytes adherent to each substrate, since comparisons were made using identical Vh and pulse protocols. 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.

Immobilization 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
% adherence = <FR><NU>(test wells − HUVECs alone)</NU><DE>(total THP-1 cells − HUVECs alone)</DE></FR> × 100

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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Abstract
Introduction
Methods
Results
Discussion
References

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 -20.0 ± 3.6 mV (n = 12). The RMP remained unchanged for up to 5 h when THP-1 monocytes were incubated on polystyrene. The mean RMP of the pooled data from all time points was -24.3 ± 1.7 mV (n = 42). Under voltage-clamp conditions, step hyperpolarizations in 20-mV increments to -160 mV induced small time-independent currents in most of the cells (Table 1, Fig. 1A). In 14% of THP-1 monocytes (Table 1) bound to polystyrene, an inward current resembling the Iir (Fig. 1B) previously described in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages (14) and other monocytes (19) was observed. Iir showed a time-dependent inactivation for voltage steps more negative than -120 mV (Fig. 1B). Under our experimental conditions, Iir in THP-1 monocytes bound to polystyrene were smaller in amplitude (53.8 ± 7.6 pA at -100 mV, n = 7) than the Iir (~400 pA at -100 mV) observed by DeCoursey et al. (14) in PMA-differentiated THP-1 macrophages bound to glass.

                              
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Table 1.   Ionic current expression in THP-1 monocytes during adherence to various substrates


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Fig. 1.   Representative family of whole cell currents in 86% (A) or 14% (B) of THP-1 monocytes bound to polystyrene for 5 h. Cells were bathed in Ringer saline solution, and recording pipette was filled with high-KCl solution-pCa 8 (reversal potentials for K+ and Cl- = -86 and 0 mV, respectively). Voltage was stepped to between -160 and -40 mV in 20-mV increments from a holding potential of -40 mV. C: current-voltage (I-V) relationships of peak inward currents recorded in A (open circle ) and B (bullet ) measured during first 50 ms of 450-ms voltage pulse. Vm, membrane potential.

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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Fig. 2.   Family of whole cell currents recorded in THP-1 monocytes bound to polystyrene for 5 h before (A) and after (B) 30 nM exogenous charybdotoxin. Voltage was stepped to between -160 and +80 mV in 20-mV increments from a holding potential of -40 mV. C: I-V relationships before (square ) and after (black-square) exposure to charybdotoxin measured during last 50 ms of 450-ms voltage pulse. Recording conditions as described in Fig. 1 legend.


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Fig. 3.   Inhibition of anion conductance by SITS in THP-1 monocytes bound to polystyrene for 4 h. Family of whole cell currents is shown under control conditions in absence of delayed rectifier K+ currents (A) and 10 min after addition of 1 mM SITS to bath solution (B). Voltage was stepped to between -160 and +120 mV in 20-mV increments from a holding potential of -40 mV. C: I-V relationships before (triangle ) and after (black-triangle) exposure to SITS measured during last 50 ms of pulse. Recording conditions as described in Fig. 1 legend.

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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Fig. 4.   A: effect of lipopolysaccharide (LPS, 3 µg/ml) treatment of human umbilical vein endothelial cell (HUVEC) monolayers on time course of surface expression of E-selectin (black-square), intercellular adhesion molecule 1 (ICAM-1, bullet ), and vascular cell adhesion molecule 1 (VCAM-1, black-triangle). Each point is mean ± SE of quadruplicate wells in an experiment representative of 3 separate experiments. Open symbols, antigen expression on untreated HUVECs for that adhesion molecule. OD, optical density. B: THP-1 monocyte adherence to untreated (naive) and LPS-treated (LPS) HUVEC monolayers in absence or presence of saturating concentrations of anti-ICAM-1, anti-E-selectin, or anti-VCAM-1. Values are means ± SE of 5 replicates in an experiment representative of 3 separate experiments. Cont, control. * Significantly different from LPS-treated group in absence of antibody (P < 0.05).

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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Table 2.   Magnitude of THP-1 monocyte ionic conductances during adherence to different substrates

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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Fig. 5.   Family of whole cell currents recorded in THP-1 monocytes adherent to LPS-activated HUVECs (A) or polystyrene (B) for 5 h. Conductance-voltage relationships obtained from THP-1 monocytes adherent to LPS-activated HUVECs (C) and polystyrene (D) for 1 (triangle ), 3 (open circle ), or 5 h (black-square). Current amplitude for all conductance-voltage relationships was measured during last 50 ms of each voltage step and then normalized to membrane capacitance. Each point is mean ± SE of normalized current amplitudes from 5-18 cells. Recording conditions as described in Fig. 1 legend.

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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Fig. 6.   Whole cell recordings obtained from THP-1 monocytes adherent to LPS-activated endothelial cells for 5 h before (A) and after (B) exposure to externally applied 1 mM BaCl2. C: I-V relationships before (open circle ) and after (bullet ) exposure to BaCl2 measured during peak of inward current.

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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Table 3.   Mechanisms underlying induction of Iir in THP-1 monocytes

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.

To determine whether cycloheximide and herbimycin A were acting directly on the THP-1 monocytes, rather than on HUVECs, to inhibit the signaling events, identical experiments were performed with fixed LPS-treated HUVEC monolayers. THP-1 monocytes were pretreated with cycloheximide (100 µM) or herbimycin A (10 µM) for 1 h at 37°C before coincubation with fixed LPS-treated HUVEC monolayers. Both agents prevented the induction of the Iir in THP-1 monocytes [2.2 ± 0.3 (n = 16) and 2.7 ± 0.3 pA/pF (n = 18) for cycloheximide and herbimycin A, respectively] adherent to fixed LPS-treated HUVECs, indicating that the agents were inhibiting the signaling events in the 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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Fig. 7.   Effect of VCAM-1 engagement on THP-1 ionic conductances. A: family of whole cell currents recorded in THP-1 monocytes adherent to immobilized VCAM-1 for 5 h. B: conductance-voltage relationships of mean normalized current (n = 9-11) obtained from THP-1 monocytes adherent to VCAM-1 for 1 (square ), 3 (bullet ), or 5 (triangle ) h. C: conductance-voltage relationships of mean normalized current (n = 18-27) from THP-1 monocytes adherent to LPS-activated endothelial cells (black-square) and immobilized VCAM-1 (square ) for 5 h. Recording conditions as described in Fig. 1 legend.

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).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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-1beta , TNF-alpha , 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-1beta and tissue factor induced in THP-1 cells adherent to extracellular matrix proteins or ligation of their beta 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 Cl- current, and an H+ current. Two additional K+ currents are expressed in THP-1 cells after PMA-induced differentiation to more mature macrophages (14).

In 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 for >= 2-4 h. The increased expression of Iir in J774 cells was associated with an increase in RMP (35). Unlike J774 cells, only 14% of THP-1 monocytes that are adherent to polystyrene express Iir. However, adherence to LPS-stimulated endothelium or VCAM-1 activates signaling pathways leading to the expression of Iir within 2-4 h in 70-80% of the THP-1 monocytes. The magnitude of the Iir in the THP-1 cells expressing Iir after adherence to these substrates was comparable to the Iir reported in PMA-differentiated THP-1 cells (14). The fact that pretreating LPS-treated HUVECs with anti-VCAM-1 eliminated the enhanced Iir expression suggests that VCAM-1 is responsible for the induction of Iir in the THP-1 cells adherent to the activated endothelium. It also argues against the possibility that activation of the THP-1 cells occurred by exposure to traces of residual LPS remaining in HUVEC cultures after two washes. The lack of Iir expression in THP-1 cells incubated on polystyrene dishes previously exposed to LPS and washed to remove LPS provides additional evidence that LPS contamination does not mediate the induction of Iir in THP-1 cells under our experimental conditions.

In 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 (-50 to -40 mV vs. -60 to -80 mV), and Idr exhibits voltage-dependent inactivation. For example, increases in extracellular K+, which will depolarize cells, increase current flow through inwardly rectifying K+ channels but are likely to close (inactivate) delayed rectifier K+ channels. Studies in primary macrophages and in the murine macrophage cell line J774 indicate that monocytes that express inwardly rectifying K+ conductances generally have more negative RMP than cells that do not express Iir (20, 21, 35). The depolarized RMP (-24 mV) in THP-1 monocytes bound to polystyrene correlates well with the finding that the majority of these cells lack Iir. In 9 of 49 THP-1 monocytes adherent to activated endothelial cells in which the Iir was the predominant current, RMP ranged from -54 to -73 mV.

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-1beta (39, 45). Engagement of beta 1-integrins stimulates the translation of IL-1beta . Thus, by modifying intracellular K+, it is possible that the expression of Iir is important in facilitating the posttranslation processing and release of IL-1beta .

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.

    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.

    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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