Regulation of the receptor specificity and function of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) by dipeptidyl peptidase IV (CD26)-mediated cleavage

Abstract

CD26 is a leukocyte activation marker that possesses dipeptidyl peptidase IV activity but whose natural substrates and immunological functions have not been clearly defined. Several chemo-kines, including RANTES (regulated on activation, normal T cell expressed and secreted), have now been shown to be substrates for recombinant soluble human CD26. The truncated RANTES(3-68) lacked the ability of native RANTES(1-68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colony-stimulating factor. Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity. RANTES(3-68) showed a reduced activity, relative to that of RANTES(1-68), with cells expressing the recombinant CCR1 chemokine receptor, but retained the ability to stimulate CCR5 receptors and to inhibit the cytopathic effects of HIV-1. Our results indicate that CD26-mediated processing together with cell activation-induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.

Figure 1

RANTES cleavage products after digestion with sCD26. RANTES was incubated overnight with the indicated amounts of E⁺ or sCD26 and samples were subjected to ES-MS analysis. The peaks in the spectrum at masses (m) of 7,905 to 7,906 and 7,887 to 7,890 are tentatively identified as [M + K⁺]⁺ of RANTES with (7,904 daltons) and without (7,886 daltons) a molecule of H₂O, respectively; the labeled peaks at the left of the spectrum correspond to each of these molecular ions minus a Ser-Pro dipeptide (184 daltons).

Figure 2

Competitive inhibition of DPPIV by RANTES(1–68). Colorimetric DPPIV enzyme assay was performed using human placental DPPIV and the Gly-Pro-pNA substrate, in the presence or absence of the test competitors Ile-Pro-Ile, RANTES(1–68), or RANTES(3–68); the competitor concentration is indicated on the horizontal axis. Data are means ± SEM (n = 3), except for the highest concentration of RANTES(1–68) and RANTES(3–68), for which only one sample was assayed in order to conserve material. Similar results were obtained in a repeat experiment.

Figure 3

Reverse transcriptase– PCR analysis of chemokine receptor transcripts in monocytes cultured in the absence (M) or presence (M + M-CSF) of M-CSF. Total cellular RNA was subjected to reverse transcriptase– PCR analysis as described in Materials and Methods. Control reactions performed without reverse transcriptase were negative for each PCR product.

Figure 4

Effects of chemokines on [Ca²⁺]_i in monocytes cultured in the absence (M) or presence (M + M-CSF) of M-CSF. Fura-2–labeled cells were exposed (at the times indicated by arrowheads) to chemically synthesized RANTES variants (100 nM) or other indicated rh chemokines (30 nM; R & D Systems), and Ca²⁺ responses were measured. The final concentrations of chemokines in this and subsequent experiments were sufficient to induce a maximal increase in [Ca²⁺]_i in the responding cells, and further challenge with the same dose produced little or no detectable change in [Ca²⁺]_i. The duration (∼100 s) and amplitude (∼20– 30% of Fura-2 saturation) of Ca²⁺ responses were similar to those obtained for chemokines with human monocytes (36). Similar results were obtained in two additional experiments.

Figure 5

Desensitization of chemokine-induced Ca²⁺ responses by full-length or truncated RANTES. Fura-2–labeled cells were stimulated first with 100 nM RANTES(1–68) or RANTES(3–68), or were left unstimulated. After ∼150 s, the cells were challenged with the RANTES variants (100 nM) or other chemokines (30 nM) as indicated, and Ca²⁺ responses were measured.

Figure 6

Activity of full-length and truncated RANTES in cells expressing recombinant CCR5 or CCR1 receptors. The [Ca²⁺]_i was measured in HEK-293 cells expressing CCR5 (A and C) and HOS-CD4 cells expressing CCR1 (B and D). (A and B) Cells were stimulated with various concentrations of the two RANTES variants as indicated and maximal fluorescence values were calculated from the peaks of the Ca²⁺ response curves. (C and D) Homologous and heterologous desensitization of the responses induced by RANTES(1–68) and RANTES(3–68) was measured in transfectants as described in Fig. 5.

Figure 7

Effects of full-length and truncated RANTES on HIV-1–induced cytopathicity. (A) HOS-CD4.CCR5 cells were incubated with uninfected PM1 cells or PM1 cells chronically infected with MV3-HXB2 virus in the presence or absence of the indicated concentrations of RANTES variants. After 3 d, cell viability was measured by the XTT (2,3-bis[2-methoxy- h-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) method. Data are means of triplicate samples (SEM, <20% of mean). (B) Representative photomicrographs of HOS-CD4.CCR5 cells cultured with HIV-1–infected PM1 cells in the absence or presence of RANTES(1–68) or RANTES(3–68), as indicated.