The LD78beta isoform of MIP-1alpha is the most potent CCR5 agonist and HIV-1-inhibiting chemokine

Abstract

LD78alpha and LD78beta are 2 highly related nonallelic genes that code for different isoforms of the human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). Two molecular forms of natural LD78beta (7.778 and 7.793 kDa) were identified from conditioned media of stimulated peripheral blood mononuclear cells. Although LD78alpha and LD78beta only differ in 3 amino acids, both LD78beta variants were 100-fold more potent chemoattractants for mouse lymphocytes than was LD78alpha. On the contrary, LD78beta was only 2-fold more efficient than LD78alpha in chemoattracting human lymphocytes and monocytes. Using CC chemokine receptor-transfected cells, both molecular forms of LD78beta proved to be much more potent than LD78alpha in inducing an intracellular calcium rise through CCR5. Compared with LD78alpha and RANTES, this preferential binding of LD78beta to CCR5 resulted in a 10- to 50-fold higher potency in inhibiting infection of peripheral blood mononuclear cells by CCR5-using (R5) HIV-1 strains. To date, LD78beta is the most potent chemokine for inhibiting HIV-1 infection, and can be considered as a potentially important drug candidate for the treatment of infection with R5 HIV-1 strains.

Figure 1

Purification of PBMC-derived MIP-1α to homogeneity by RP-HPLC. (a) Prepurified MIP-1α immunoreactivity from conditioned medium of PBMCs was loaded on a C₈ RP-HPLC column. Proteins were eluted with an acetonitrile gradient (dashed line) and detected by measuring UV absorption at 220 nm (solid line). Fractions were tested for the presence of MIP-1α immunoreactivity (histogram) and ESb/MP cell chemotactic activity (solid line broken by circles). (b) Proteins eluted from the HPLC column were analyzed for purity by SDS-PAGE under reducing conditions and were stained with silver. Markers used are indicated in Methods.

Figure 2

Comparison of the chemotactic potencies of LD78α and the LD78β isoforms. The chemotactic activities of LD78α (filled circles), 7.778-kDa LD78β (filled diamonds), and 7.793-kDa LD78β (open diamonds) were determined in the Boyden microchamber assay, using ESb/MP lymphocytic cells, THP-1 monocytic cells, and PBMC CD3⁺ lymphocytes and monocytes. Results represent the mean chemotactic index ± SEM of 3 or more independent experiments (each performed in triplicate).

Figure 3

Diverging signaling capacities of LD78α, LD78β, and RANTES through CCR1 and CCR5. Intracellular calcium mobilization experiments were performed on CCR1- and CCR5-transfected HOS cells. Results represent the mean [Ca²⁺]_i rise ± SEM, obtained in 2 or more independent experiments. The limit for significant increases in [Ca²⁺]_i (20 nM) is indicated by the dashed line.

Figure 4

Inhibition of R5 HIV-1 infection by LD78β. PBMCs were protected from infection with the R5 BaL strain by the CC chemokines LD78α (filled circles), 7.778-Da LD78β (filled diamonds), 7.793-kDa LD78β (open diamonds), or RANTES (open circles). HIV-1 inhibition was evaluated by measuring viral p24 core antigen. Results at left show 1 representative experiment out of 2, whereas the results at right represent the mean ± SEM of 4 independent experiments.