Substance P up-regulates macrophage inflammatory protein-1beta expression in human T lymphocytes

Abstract

Substance P (SP) is an important modulator of neuroimmunoregulation. We have demonstrated that human T lymphocytes express SP and neurokinin-1 receptor (NK-1R), a primary SP receptor. In the present study, we investigated whether SP stimulates synthesis of macrophage inflammatory protein-1beta (MIP-1beta) in human T lymphocytes. SP significantly enhanced MIP-1beta expression at both the mRNA and protein level in a human T cell line (Jurkat) containing the SP receptor gene (J-SPR) as determined by real-time PCR and ELISA assays. SP-induced MIP-1beta expression is abrogated by the specific NK-1R antagonist (CP-96,345). The supernatants from SP-stimulated J-SPR T cell cultures enhanced T lymphocyte chemotaxis in vitro, indicating functional activity of SP-induced MIP-1beta. In addition, SP augmented secretion of MIP-1beta from primary cultures of peripheral blood lymphocytes (PBL) isolated from some of the donors. This donor variability was due to differential expression of the primary SP receptor (NK-1R) on PBL from different donors. PBL from two of seven donors that did not respond to SP stimulation had undetectable NK-1R expression. Our mechanistic studies showed that SP activated NF-kappaB promoter-directed luciferase activity, which may be responsible for its effect on MIP-1beta expression in human T cells. Our data provide a potential mechanism by which SP selectively influences cellular immune responses such as beta-chemokine expression in human T lymphocytes through NK-1R, which may have an important in vivo implication in inflammatory diseases.

Fig. 1

Effect of SP on the MIP-1β expression in J-SPR T cells. (A) J-SPR T cells were incubated with SP (10⁻¹¹ to 10⁻⁷ M) for 3 h and total RNA was isolated and subjected to RT-PCR, electrophoresis (insert), and real-time PCR to quantify MIP-1β mRNA. The data are expressed as mean ± S.D. of triplicate cultures of MIP-1β mRNA copy number per 10⁶ copies of GAPDH mRNA. (B) Effect of SP treatment on MIP-1β production in J-SPR T cells. MIP-1β protein was assayed using ELISA. The data shown are presented as mean ± S.D. of triplicate cultures and are representative of three independent experiments.

Fig. 2

Time-course of SP on the MIP-1β expression in J-SPR T cells. J-SPR T cells were incubated with (+) or without (−) SP (10⁻⁷ M), and total RNA was isolated from the cells after the indicated amount of time (h). (A) Real-time PCR: MIP-1β mRNA levels were quantified by real-time PCR using the same RNA samples as indicated, and the data are expressed as mean ± S.D. of MIP-1β mRNA copy number per 10⁶ copies of GAPDH mRNA. (B) MIP-1β protein was assayed using ELISA. The data shown are presented as mean ± S.D. of triplicate cultures and are representative of three independent experiments.

Fig. 3

Effect of SP antagonist, CP-96,345, on SP-induced MIP-1β expression in J-SPR T cells. (A) J-SPR T cells were incubated with SP (10⁻⁷ M), SP (10⁻⁷ M) plus CP-96,345 (10⁻⁶ M), CP–96,345 (10⁻⁶ M) and without compounds (as control) for 3 h. Cellular RNA was isolated and subjected to RT-PCR, electrophoresis (insert), and real-time RT-PCR to quantify MIP-1β mRNA. The data are expressed as mean ± S.D. of triplicate cultures and are representative of three independent experiments of MIP-1β mRNA copy number per 10⁶ copies of GAPDH mRNA. (B) MIP-1β protein was assayed using ELISA. The data shown are presented as mean ± S.D. of triplicate cultures and are representative of three independent experiments.

Fig. 4

Culture supernatants from SP-stimulated J-SPR T cells induce lymphocyte chemotaxis. Culture supernatants from SP stimulated J-SPR T cell cultures caused a significant increase of T lymphocyte migration compared with unstimulated controls. The culture supernatants from SP and/or CP-96,345-treated J-SPR T cell cultures had no effect on migration of T lymphocytes compared with that of unstimulated J-SPR T cell culture supernatants. The data shown are mean ± S.D. of three independent experiment (*P < 0.0001, SP-treated vs. untreated cells).

Fig. 5

Effect of SP on the MIP-1β production in PBL. PBL isolated from seven healthy donors (D1–D7) were pre-stimulated with PHA for 72 h. The cells were incubated with or without SP (10⁻⁷ M) for 12 h. Total RNA was extracted from PBL isolated from different donors and nested-RT-PCR was performed to amplify NK-1R mRNA (395 bp) (insert). Cell-free supernatants were collected and MIP-1β protein was assayed using ELISA. The data shown are mean ± S.D. of MIP-1β in triplicate cultures. Plus (+) and minus (−) signs indicate presence or absence of SP.

Fig. 6

Effect of SP on the activation of NF-κB in J-SPR T cells. J-SPR T cells were transfected with pNF-κB-luc using Fugene 6 Transfection Reagent (Fugene 6). The ratio of Fugene 6/plasmid was 6:1 (μl/μg). Twenty-four hours after transient transfection, the cells were treated with SP and/or CP-96,345 at different concentrations as indicated for 6 h. The effect of SP on activation of NF-κB in these transfected cells was determined by NF-κB-driven luciferase activity. Luciferase activity in the cell-free lysate was quantitated using a luciferase assay system (Promega) and a luminometor. CP: CP-96,345 (10⁻⁶ M), SP + CP: SP(10⁻⁷ M) plus CP-96,345 (10⁻⁶ M). The data are presented as relative light unit (RLU). The data shown are mean ± S.D. of three independent experiments.