CD40 ligand (CD154) stimulation of macrophages to produce HIV-1-suppressive beta-chemokines

Abstract

beta-chemokines play an important role in the development of immunologic reactions. Macrophages are major beta-chemokine-producing cells during T-cell directed, delayed-type hypersensitivity reactions in tissues, and have been reported to be important producers of beta-chemokines in the lymph nodes of HIV-1-infected individuals. However, the physiological signals responsible for inducing macrophages to produce beta-chemokines have not been established. Two soluble T cell products, interferon-gamma and granulocyte-macrophage colony stimulating factor, were added to cultured macrophages, but failed to stimulate the production of macrophage inflammatory protein-1alpha and -1beta; regulated upon activation, normal T cell expressed and secreted (RANTES); or monocyte chemoattractant protein-1. Instead, direct cell-cell contact between macrophages and cells engineered to express CD40L (also known as CD154) resulted in the production of large amounts of macrophage inflammatory protein-1alpha and -1beta, and RANTES (all ligands for CCR5), and monocyte chemoattractant protein-1 (a ligand for CCR2). Supernatants from CD40L-stimulated macrophages protected CD4(+) T cells from infection by a nonsyncytium-inducing strain of HIV-1 (which uses CCR5 as a coreceptor). These results have implications for granulomatous diseases, and conditions such as atherosclerosis and multiple sclerosis, where CD40L-bearing cells have been found in the macrophage-rich lesions where beta-chemokines are being produced. Overall, these findings define a pathway linking the specific recognition of antigen by T cells to the production of beta-chemokines by macrophages. This pathway may play a role in anti-HIV-1 immunity and the development of immunologic reactions or lesions.

Figure 1

CD40L induction of β-chemokine production by macrophages. (A) Flow cytometry measurement of CD40L surface expression. Isotype control staining of CD40L-293 cells with PE-conjugated anti-CD8 control mAb (thin solid line). Expression of human CD40L on control 293 cells (thin broken line) and CD40L-293 cells (thick solid line) assessed by using PE-conjugated anti-CD40L mAb 24–31. (B) β-chemokine production by macrophages. MDM were cultured in medium alone, with added IFN-γ or GM-CSF, or with added control 293 cells or CD40L-293 cells. LPS was used as a positive control. The mean chemokine concentrations (picogram per milliliter) of the supernatants from quadruplicate wells 24 hr later are shown (±SD). □, MIP-1α; ▨, MIP-1β; ▪, RANTES. (C) Abrogation of CD40L stimulation by anti-CD40 or anti-CD40L mAbs. In an experiment similar to B, neutralizing mAbs were added at the initiation of the CD40L-293 cell-MDM cocultures. Anti-CD40 mAb blocked >99% and anti-CD40L mAb blocked 94% of the β-chemokine release. (D) Stimulation of macrophages by CD40L-bearing plasma membranes. Acellular preparations of membranes from control 293 cells, 293 cells expressing a nonfunctional mutant of human CD40L (T147N), human CD40L-293 cells (CD40L), and murine CD40L-293 cells (mCD40L) were added to MDM in an experiment similar to B.

Figure 2

Inhibition of NSI HIV-1 replication in CD4⁺ T cells by CD40L-stimulated macrophage supernatants. CD4⁺ T cells (depleted of CD8⁺ T cells) were stimulated by PHA and IL-2, infected with the NSI isolate, (HIV-1_SF162), and cultured for 14 days in either R10 medium or a 1:20 dilution of MDM supernatants. Virus production was measured by ELISA for HIV-1 p24 antigen. To demonstrate that the β-chemokines in the CD40L-stimulated MDM supernatant accounted for the HIV-1 suppressive activity, the supernatant was pretreated with a mixture of neutralizing antibodies against MIP-1α, MIP-1β, and RANTES, which abrogated its effectiveness.

Figure 3

CD40L stimulation of MCP-1 production by MDM. Culture conditions were identical to that in Fig. 1B.

Figure 4

CD40L-macrophage pathway for the generation of β-chemokines. Stimulation of the T cell receptor (TCR) by peptidic antigen presented by major histocompatibility complex (MHC) molecules (Signal 1) is sufficient to stimulate a subset of T cells to rapidly express CD40L (42). Macrophages bearing CD40 respond to cell–cell contact with such T cells by producing β-chemokines within 3 hr. The resulting β-chemokines may then act to attract additional lymphocytes and monocyte/macrophages to the site of the developing immune reaction or block the infection of nearby CD4⁺ T cells by NSI strains of HIV-1.