HIV-1 gp120 and chemokines activate ion channels in primary macrophages through CCR5 and CXCR4 stimulation

Abstract

HIV type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry into target cells. Here we show that the HIV-1 envelope gp120 (Env) activates multiple ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. Env from both CCR5-dependent JRFL (R5) and CXCR4-dependent IIIB (X4) HIV-1 opened calcium-activated potassium (K(Ca)), chloride, and calcium-permeant nonselective cation channels in macrophages. These signals were mediated by CCR5 and CXCR4 because macrophages lacking CCR5 failed to respond to JRFL and an inhibitor of CXCR4 blocked ion current activation by IIIB. MIP-1beta and SDF-1alpha, chemokine ligands for CCR5 and CXCR4, respectively, also activated K(Ca) and Cl(-) currents in macrophages, but nonselective cation channel activation was unique to gp120. Intracellular Ca(2+) levels were also elevated by gp120. The patterns of activation mediated by CCR5 and CXCR4 were qualitatively similar but quantitatively distinct, as R5 Env activated the K(Ca) current more frequently, elicited Cl(-) currents that were approximately 2-fold greater in amplitude, and elevated intracellular Ca(+2) to higher peak and steady-state levels. Env from R5 and X4 primary isolates evoked similar current responses as the corresponding prototype strains. Thus, the interaction of HIV-1 gp120 with CCR5 or CXCR4 evokes complex and distinct signaling responses in primary macrophages, and gp120-evoked signals differ from those activated by the coreceptors' chemokine ligands. Intracellular signaling responses of macrophages to HIV-1 may modulate postentry steps of infection and cell functions apart from infection.

Figure 1

Elevation of cytoplasmic calcium in MDM induced by HIV-1 gp120. Macrophages were loaded with fura-2 AM and were stimulated with gp120 (200 nM) from HIV-1 JRFL (A) or IIIB (B). Intracellular calcium in individual cells was calculated from F340/F380 ratios.

Figure 2

Activation of ionic currents in MDM by gp120 and by chemokines. Patch clamp recordings were carried out on MDM stimulated with gp120 (200 nM) from HIV-1 JRFL (A) and IIIB (B). Incubation of gp120 with anti-Env antiserum blocked current activation but did not prevent subsequent activation by non-neutralized gp120 (C and D). MDM were also stimulated with the chemokines (1 μg/ml) MIP-1β (E) and SDF-1α (F).

Figure 3

CCR5 and CXCR4 dependence of MDM ion current activation by gp120. Incubation of MDM from CCR5 wild-type donors (WT) with the CXCR4 inhibitor AMD3100 (5 μg/ml) blocked currents elicited by IIIB gp120 but did not inhibit responses to JRFL or MIP-1β (A and B). In MDM from donors homozygous for the CCR5 Δ32 allele, no current was evoked by JRFL gp120 whereas both IIIB and SDF-1α elicited ion currents (C and D).

Figure 4

Potassium and chloride currents evoked by gp120 in MDM. The ionic nature of currents elicited in MDM by gp120 from JRFL (A) and IIIB (B) was determined from the reversal potential (right) and with channel inhibitors (left). At peak activation, the outward JRFL current reversed direction at ≈−78 mV (A, top right) whereas the peak inward currents elicited by JRFL (A, bottom right) and IIIB (B, right) reversed at ≈5 mV (squares). In low Cl⁻ bath solution, the current voltage relationship of the inward current elicited by both Envs shifted to ≈+40 mV (circles). Charybdotoxin (100 nM) blocked the JRFL-evoked outward current, and NPPB (10 μM) blocked the inward currents activated by both Envs (A and B, left).

Figure 5

Potassium and chloride currents evoked by chemokines in MDM. The ionic nature of currents elicited by MIP-1β (A) and SDF-1α (B) was determined from the reversal potential in normal (squares) and low Cl⁻ (circles) bath solution and the effect of channel inhibitors as described in Fig. 4.

Figure 6

Activation of a nonselective cation channel in MDM by gp120 but not by MIP-1β or SDF-1α. Currents were recorded by using a pipette solution in which Cs⁺ replaced K⁺, aspartate was substituted for Cl⁻, and the cell was voltage-clamped at the calculated E_Cl of −45 mV. NPPB (10 μM) was added to block any residual Cl⁻ current. Cells were stimulated with JRFL gp120 (A), IIIB gp120 (B), MIP-1β (C), and SDF-1α (D). At the peak of gp120-activated currents, the bath solution was replaced with a low sodium solution (30 mM) as indicated by the bars (A and B). The reversal potentials for currents evoked by gp120 were determined in normal (squares) and low sodium (circles) bath solutions (lower panels of A and B).

Figure 7

Calcium-permeant nature of the nonselective cation channel activated by gp120. Currents were recorded by using a pipette solution containing 120 mM Cs-aspartate and 25 mM CsCl, and extracellular bath containing 100 mM CaCl₂ and no other cations. The membrane was voltage-clamped at the calculated E_Cl (−50 mV), and NPPB (10 μM) was added to the bath. An inward calcium current was elicited by gp120 from JRFL (A) and IIIB (B) but not by MIP-1β (C) or SDF-1α (D).