Background:  HIV infection is initiated by binding of the viral envelope glycoprotein gp120 first to the principal receptor CD4, followed by interactions with the co-receptor CCR5 or CXCR4. Both CCR5 and CXCR4 are expressed in macrophages, which are important targets for HIV-1 in vivo and play a central role in neuropathogenesis of AIDS. Besides mediating infection, HIV-1 gp120 is a functional ligand for CCR5 and CXCR4 on macrophages. Our group showed that gp120 stimulation of CCR5 on primary monocyte-derived macrophages (MDM) stimulates multiple protein kinases, including focal adhesion-related kinase Pyk2, phosphatidylinositol-3 kinase (PI3K), and the Src kinase Lyn. How these kinases regulate macrophage function after gp120/CCR5 stimulation is not well defined.

Methods:  MDM were stimulated with recombinant gp120 from a CCR5-utilizing HIV strain or the natural ligand MIP-1b. Chemotaxis was assayed by the ability to migrate across a Transwell membrane. Specific inhibitors were used to define the role of particular signaling molecules in macrophage chemotaxis.

Results:  Stimulation of CCR5 with gp120 or MIP-1b induced chemotaxis of MDM in a concentration-dependent manner. Both gp120- and MIP-1b-stimulated chemotaxis was blocked by the CCR5 antagonist M657, confirming that the response is mediated via binding to CCR5. Chemotaxis was also inhibited by pertussis toxin, suggesting that it requires coupling of G_ia protein. Furthermore, CCR5-mediated chemotactic response was attenuated by inhibitors for Pyk2 (AG17 and dantrolene) and PI3K (wortmannin and LY294002), implicating their involvement in the chemotactic response. CCR5-stimulated MDM chemotaxis was also abrogated by the Src inhibitor PP2, and by the Lyn-specific peptide inhibitor KRX123.302.

Conclusions:  Chemotaxis of immune cells plays an important role in inflammatory responses to infection. Our results indicated that macrophage chemotaxis in response to either gp120 or MIP-1β requires interaction with CCR5, coupling of G_iα protein, and activation of multiple protein kinases, including Pyk2, PI3k, and Lyn. HIV-1-associated dementia (HAD) is characterized by massive infiltration of macrophages to the brain where both infected and uninfected macrophages are activated. Chemokines or gp120-stimulated chemotaxis could induce recruitment of activated macrophages to sites of viral replication, thereby promoting the propagation of virus as well as further activation. This may contribute to the neuropathogenesis of HAD.