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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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INTEGRINS ARE HETERODIMERS that are defined by an alpha -chain sharing a noncovalent link with a beta -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 beta 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 beta 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 beta 1 integrin-mediated adhesion and migration (28). The T cell K+ channels appear to be physically associated with beta 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 alpha vbeta 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).


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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 alpha 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 MOmega . 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.


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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).


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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.


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


                              
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Table 2.   Magnitude of THP-1 ionic conductances while adherent to different substrates

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.


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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).


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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).


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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.


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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.


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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 (open circle ) 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.


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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.


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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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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