The Src kinase Lyn is required for CCR5 signaling in response to MIP-1beta and R5 HIV-1 gp120 in human macrophages

Abstract

CCR5 is a receptor for several beta chemokines and the entry coreceptor used by macrophage-tropic (R5) strains of HIV-1. In addition to supporting viral entry, CCR5 ligation by the HIV-1 envelope glycoprotein 120 (gp120) can activate intracellular signals in macrophages and trigger inflammatory mediator release. Using a combination of in vitro kinase assay, Western blotting for phospho-specific proteins, pharmacologic inhibition, CCR5 knockout (CCR5Delta32) cells, and kinase-specific blocking peptide, we show for the first time that signaling through CCR5 in primary human macrophages is linked to the Src kinase Lyn. Stimulation of human monocyte-derived macrophages with either HIV-1 gp120 or MIP-1beta results in the CCR5-mediated activation of Lyn and the concomitant Lyn-dependent activation of the mitogen-activated protein (MAP) kinase ERK-1/2. Furthermore, activation of the CCR5/Lyn/ERK-1/2 pathway is responsible for gp120-triggered production of TNF-alpha by macrophages, which is believed to contribute to HIV-1 pathogenesis. Thus, Lyn kinase may play an important role both in normal CCR5 function in macrophages and in AIDS pathogenesis in syndromes such as AIDS dementia where HIV-1 gp120 contributes to inappropriate macrophage activation, mediator production, and secondary injury.

Figure 1.

R5 gp120 and MIP-1β activate ERK-1/2 in human macrophages. MDMs were stimulated with (A) R5 gp120 (40 nM) or (B) MIP-1β (25 nM). Cell lysates were harvested at the indicated time points and subjected to Western blot with phospho-specific ERK-1/2 (P-ERK) antibodies. Membranes were stripped and reblotted with antibodies to determine total ERK-1/2 protein (T-ERK). ΔHI indicates protein subjected to heat denaturation.

Figure 2.

Lyn kinase activity is up-regulated by gp120 and MIP-1β in macrophages. MDMs were stimulated with (A,C) R5 gp120 (40 nM) or (B) MIP-1β (25 nM). At the indicated time points cell lysates were immunoprecipitated with antibodies specific for Lyn or Hck kinases and subjected to in vitro kinase assay (KA; top rows). In parallel, retained cell lysates (25 μg) were subjected to Western blotting for total Lyn or Hck protein (WB; bottom rows).

Figure 3.

Lyn kinase activation is CCR5-dependent. MDMs were harvested from donors homozygous for either the CCR5 wild-type (WT) or Δ32 deletion (CCR5Δ32) alleles, pretreated for 1 hour with or without the Src inhibitor PP2 (10 μM), and stimulated for 1 minute with R5 gp120 (40 nM) or MIP-1β (25 nM). Cell lysates were then either immunoprecipitated using anti-Lyn antibodies followed by in vitro kinase assay (KA; top row), or analyzed for total Lyn protein by Western blot (WB; bottom row).

Figure 4.

CCR5 mediates ERK-1/2 phosphorylation. (A) MDMs from wild-type or CCR5Δ32 homozygous donors were stimulated for 15 minutes with R5 gp120 (40 nM) or MIP-1β (25 nM). Cell lysates were then subjected to Western blot using antibodies specific for phosphorylated (P-ERK) or total ERK-1/2 protein (T-ERK). (B) MDMs were pretreated for 1 hour with the CCR5 antagonist M657 (1 μM), stimulated in the continued presence of the inhibitor with R5 gp120 or MIP-1β, and analyzed by Western blot for phosphorylated or total forms of ERK-1/2.

Figure 5.

ERK-1/2 activation through CCR5 is sensitive to Src inhibition. MDMs were pretreated for 1 hour with the Src kinase inhibitor PP2 (10 μM) or the negative control PP3 (10 μM), then stimulated for 15 minutes in the continued presence of the inhibitor with (A) R5 gp120 (40 nM) or (B) MIP-1β (25 nM). Cell lysates were subjected to ERK-1/2 Western blot using phospho-specific (P-ERK) or total ERK-1/2 antibodies (T-ERK).

Figure 6.

Lyn inhibition blocks CCR5-mediated ERK-1/2 activation. MDMs were pretreated for 2 hours with the Lyn kinase peptide inhibitor KRX-123.302 (0.1 μM) or the negative control peptide KRX-107.110 (0.1 μM). Cultures were then stimulated for 15 minutes with (A) MIP-1β (25 nM) or (B) R5 gp120, in the continued presence or absence of peptides. Cell lysates were subjected to ERK-1/2 Western blot using phospho-specific antibodies (P-ERK). Membranes were then stripped and reblotted with total ERK-1/2 antibodies (T-ERK).

Figure 7.

The gp120-triggered macrophage TNF-α production depends on Src, ERK-1/2 and CCR5. (A) MDMs from donors homozygous for CCR5 wild-type or Δ32 alleles were maintained in 96-well plates, stimulated with R5 gp120 (40 nM), or LPS (10 ng/mL), and TNF-α levels in supernatant measured 16 hours later by ELISA. (B-D) MDMs from CCR5 wild-type donors were pretreated for 1 hour with the Src inhibitor PP2 or control compound PP3 (each at 10 μM), the ERK-1/2 inhibitors PD98059 or U0196 (each at 10 μM), or the Lyn-specific inhibitory peptide KRX-123.302 (at either 0.1 or 1.0 μM). They were then exposed to R5 gp120 in the continued presence of the inhibitor, and TNF-α levels in supernatant were measured 16 hours later.