HIV-1 Nef mediates lymphocyte chemotaxis and activation by infected macrophages

Abstract

Infection of macrophage lineage cells is a feature of primate lentivirus replication, and several properties of primate lentiviruses seem to have evolved to promote the infection of macrophages. Here we demonstrate that the accessory gene product Nef induces the production of two CC-chemokines, macrophage inflammatory proteins 1alpha and 1beta, by HIV-1-infected macrophages. Adenovirus-mediated expression of Nef in primary macrophages was sufficient for chemokine induction. Supernatants from Nef-expressing macrophages induced both the chemotaxis and activation of resting T lymphocytes, permitting productive HIV-1 infection. These results indicate a role for Nef in lymphocyte recruitment and activation at sites of virus replication.

Fig. 1

HIV-1 Nef influences CC-chemokine production by HIV-1-infected macrophages. a, Chemokine and cytokine profiles in mock-infected macrophages (▲) and in macrophages infected with wild-type (◇) or ΔNef (■) HIV-1_ADA (1 μg virus, based on gag p24, was used to infection of 1 × 10⁶ cells). RT, reverse transcriptase; MCSF, monocyte colony-stimulating factor. b, Chemokine RNA levels in macrophages infected with wild-type (WT) HIV-1_ADA or HIV-1_ADA ΔNef or mock-infected at the peak of supernatant reverse transcriptase activity. M, molecular size markers.

Fig. 2

Nef expression is sufficient for induction of CC-chemokine expression. a, Monocyte-derived macrophages were infected with an adenovirus vector expressing green fluorescent protein (Adeno GFP, 1,000 plaque-forming units/cell). Cells were photographed under fluorescent (bottom row; absorbance 495 nm; emission, 520 nm) and brightfield (top row) illumination. Above photos, times after infection. Original magnification, ×400. Adeno Nef, macrophages infected with an adenovirus vector expressing HIV-1 Nef (control). b, HIV-1 Nef expression (right, western blot analysis) and chemokine RNA levels (left, RT–PCR) 16 h after infection in mock-infected macrophages and in macrophages infected with Adeno Nef or Adeno GFP virus. By limiting dilution analysis (wedges; doubling dilutions of substrate RNA), induction of MIP-1α and MIP-1β RNA in macrophages infected with Adeno Nef relative to those infected with Adeno GFP was 16-fold and 64-fold, respectively. c, In situ analysis of Nef and MIP-1α co-expression in primary macrophages 15 h after infection with an adenovirus vector expressing HIV-1 Nef. Rows represent two independent cultures. d, Chemokine production by macrophages infected with Adeno Nef (■) or Adeno GFP (◇) viruses or with an Adeno vector lacking a ‘foreign’ insert (○). e, Chemokine production by macrophages after stimulation with recombinant CD40 ligand (▲; 25 μg/ml) or with a control protein (Δ; IgG1; 25 μg/ml).

Fig. 3

Induction of lymphocyte chemotaxis by supernatants from HIV-1-infected and Nef-expressing macrophage cultures. a, Culture supernatants of macrophages infected with wild-type HIV-1_ADA or HIV-1_ADA ΔNef or mock-infected (top row) were collected at the peak of viral replication (10 d after infection). Supernatants of macrophages infected (inf. Mϕ supt.) with Adeno Nef or Adeno GFP (middle row) were collected at 18 h after infection and added to top (T) or bottom (B) or both chambers of microchemotaxis plates. The ability of these culture supernatants to promote chemotaxis of unstimulated peripheral blood lymphocytes was compared with the chemotactic activity of recombinant MIP-1α and MIP-1β proteins (bottom row). b, Antibodies against MIP-1α and MIP-1β block lymphocyte chemotactic activity of HIV-1 Nef-expressing macrophage culture supernatants (Adeno-Nef inf. Mϕ supt.). Chemotaxis was assessed in the presence (′) and absence of antibodies against MIP-1α (2.5 μg/ml) and MIP-1β (10 μg/ml). T, top chamber; B, bottom chamber.

Fig. 4

Induction of lymphocyte activation and ‘permissiveness’ to HIV-1 infection by Nef-expressing macrophage supernatants. a, Culture supernatants from macrophages infected with Adeno Nef or Adeno GFP or mock-infected were collected 24 h after infection. Chemokines were immunodepleted from a portion of these culture supernatants using MIP-1α and MIP-1β antibodies conjugated to protein A/G sepharose. Right, MIP-1α and MIP-1β levels (in pg/ml) in macrophage supernatants before and after immunodepletion. Resting peripheral blood lymphocytes were cultured in immunodepleted and non-immunodepleted macrophage supernatants for 3 d, then cell division was evaluated by ³H-thymidine incorporation. Lymphocytes activated with the mitogen phytohemagglutinin (PHA-P) served as a positive control. ND, not determined. b, Resting lymphocyte cultures were incubated for 3d with supernatants derived from Adeno Nef (◇), Adeno GFP (∎) or mock-infected (▲) macrophages cultures. Cells were then infected with HIV-1_NL4-3 and virus replication was monitored by measurement of reverse transcriptase activity in culture. ○, phytohemagglutinin.

Fig. 5

β chemokine expression associated with SIV-infected macrophages. a, An aggregate of macrophages in the brain of an animal with SIV encephalitis immunostained for CD14. b and c, The macrophages in the lesions in a contained abundant viral antigen (b) and viral nucleic acid (c). d and e, perivascular aggregates of cells in adjacent tissue sections expressed high levels of MIP-1α (d) and MIP-1β (e), with expression of RANTES limited primarily to vascular endothelium (f). g–I, Expression of MIP-1α (g) and MIP-1β (h) and RANTES (i was minimal or absent in normal brain from age and sex-matched control animals.