Tachykinin receptor modulation of cyclooxygenase-2 expression in human polymorphonuclear leucocytes

Abstract

Background and purpose: We investigated the ability of natural and synthetic selective NK receptors agonists and antagonists to modulate cyclooxygenase-2 (COX-2) expression in human polymorphonuclear leucocytes (PMNs).

Experimental approach: The presence of all three tachykinin in PMNs was assessed by Western blot and PCR techniques. Natural and synthetic ligands selective for the tachykinin receptors were used to modulate COX-2 protein (measured with Western blotting) and activity [as prostaglandin E(2) (PGE(2)) output]. Effects of substance P (SP) on phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB) activation were studied to analyse the signalling pathway involved in COX-2 up-regulation mediated by SP.

Key results: Stimulation of NK receptors with the natural ligands SP, neurokinin A (NKA) and neurokinin B, in the pmol.L(-1)-micromol.L(-1) concentration range, modulated COX-2 expression and PGE(2) release in a concentration- and time-dependent manner. Experiments with synthetic selective agonists [Sar(9), Met(O(2))(11)]SP, [beta-Ala(8)] NKA(4-10), senktide or selective antagonists L703,606, SR48,968 or SR142801, confirmed that COX-2 up-regulation was mediated by NK receptors. We found that mainly p38, p42 and p46 MAPKs were phosphorylated by SP and SB202190, PD98059 and SP600125, which are selective inhibitors of these kinases, blocked SP-induced COX-2 expression. SP also induced nuclear translocation of NF-kappaB concentration-dependently, with a maximum effect at 1 nmol.L(-1).

Conclusions and implications: Human PMNs possess functional NK(1), NK(2) and NK(3) receptors, which mediate the induction of COX-2 expression and NF-kappaB activation by SP.

Figure 1

Identification of NK receptors on PMNs. (A) Agarose gel showing products of RT-PCR assay of PMNs isolated from six healthy volunteers. Single transcripts corresponding to the sizes predicted for NK₁, NK₂ and NK₃ receptors were detected. β-actin was used as a housekeeping gene. (B) Western blot analysis of NK₁, NK₂ and NK₃ receptors in PMNs isolated from six healthy volunteers. β-Actin was used to confirm the homogeneity of the protein loaded. (C) Quantitative evaluation of NK receptors expression by densitometry. Results represent the means ± SE of the analysis performed in six healthy volunteers, expressed as integrated optical density (IOD), for each of the NK receptors.

Figure 2

Time course and concentration dependency of COX-2 protein induction by SP. (A) PMNs were stimulated with SP (1 nmol·L⁻¹) from time 0 to 8 h. At the times indicated, cells were processed for Western blot analysis as described in the Methods section and analysed by densitometry. Viability of the cells at each hour is shown as % cell death (right hand axis). (B) Immunoblot of one experiment representing modulation of COX-2 protein expression by SP (1 nmol·L⁻¹). In the same experimental conditions COX-1 and iNOS were not affected by SP; β-actin was used to confirm the homogeneity of the protein loaded. (C) PMNs were incubated with SP or NKA or NKB (1 pmol·L⁻¹–1 µmol·L⁻¹) for 5 h, and cells were subjected to Western blot analysis as described in the Methods section and analysed by densitometry. Densitometric results, normalized to β-actin, are expressed as fold increases over the control value (=1), which was subtracted from all experimental values (means ± SE of six separate experiments). Data were analysed by one-way analysis of variance (anova) and the Student-Newman-Keuls test (*, ^#, ^§P < 0.05 vs. control).

Figure 3

Time course and concentration dependency of PGE₂ release induced by SP. PMNs were stimulated with SP (1 nmol·L⁻¹) from time 0 to 8 h (A) or with SP (1 pmol·L⁻¹–1 µmol·L⁻¹) for 5 h (B) and analysed for PGE₂ release as described in the Methods section. The results are expressed as means ± SE of six independent experiments performed in duplicate. Data were analysed by one-way analysis of variance (anova) and the Student-Newman-Keuls test. *P < 0.05 versus basal level of PGE₂, which was 40 pg·mL⁻¹ for panel (A) and 300 pg·mL⁻¹ for panel (B).

Figure 4

Effect of SP on phosphorylation of p38, p42/44 and p46/55 MAPKs in PMNs. (A) Cells were incubated with SP (1 nmol·L⁻¹) from 0 to 45 min, and processed at the time indicated for Western blot analysis of the phosphorylated forms (ph-) of p38, p42/44 or p46/55 as described in the Methods section. Immunoblot of one experiment representative of the six performed. β-actin was used to confirm the homogeneity of the protein loaded. (B) PMNs were pre-incubated for 60 min with the p38 MAPK inhibitor SB202190 (50 µmol·L⁻¹) or the p42/44 MAPK inhibitor PD98059 (50 µmol·L⁻¹) or the p46/55 MAPK inhibitor SP600125 (20 µmol·L⁻¹) followed by the addition of SP (1 nmol·L⁻¹) for 5 h. Cells were then processed for Western blot analysis as described in the Methods section. Densitometric results, normalized to β-actin, are expressed in densitometric units (O.D.) with control = 1 (means ± SE of six separate experiments). Data were analysed by one-way analysis of variance (anova) and the Student-Newman-Keuls test. *P < 0.05 versus control; **P < 0.01, versus SP.

Figure 5

Effect of SP on NF-κB activation. (A) Immunoblot of one experiment representing NF-κB translocation from cytosol to nucleus in PMNs stimulated with SP (1 nmol·L⁻¹). (B) Densitometric analysis of Western blot performed to evaluate NF-κB translocation is expressed as fold increase over the control value (=1), which was subtracted from all experimental values (means ± SE of six separate experiments). Data were analysed by one-way analysis of variance (anova) and the Student-Newman-Keuls test. *P < 0.05 versus untreated PMNs.

Figure 6

Effect of tachykinin receptor antagonists on COX-2 expression and NF-κB activation. (A) PMNs were stimulated with SP or [Sar⁹, Met(O₂)¹¹]SP or [β-Ala⁸] NKA(4-10) or senktide 1 pmol·L⁻¹–1 µmol·L⁻¹ for 5 h and processed for Western blot analysis as described in the Methods section. Dentitometric results, normalized to β-actin, are expressed as fold increase over the control value (=1), which was subtracted from all experimental values (means ± SE of six separate experiments). Data were analysed by one-way analysis of variance (anova) and the Student-Newman-Keuls test (*, ^§, ^#, ^@, P < 0.05 vs. control). (B) PMNs were stimulated with 1 nmol·L⁻¹ SP or with SP plus L703,606 (1 nmol·L⁻¹) or SP plus SR48,968 (1 nmol·L⁻¹) or SP plus SR142801 (1 nmol·L⁻¹) or SP plus all antagonists (all 1 nmol·L⁻¹) for 5 h and processed for Western blot analysis as described in the Methods section. Densitometric results, normalized to β-actin, are expressed as fold increase over the control value (=1), which was subtracted from all experimental values (means ± SE of six separate experiments). Data were analysed by one-way analysis of variance (anova) and the Student-Newman-Keuls test (*P < 0.05 vs. SP; **P < 0.01 vs. SP).

Figure 7

Effect of ‘chronic’ treatment with SP on COX-2 expression in PMNs. Cells were incubated with SP (1 nmol·L⁻¹), washed twice every 2 h and SP was re-added, for up to 6 h. Every 2 h, cells were processed for Western blot or PGE₂ release analysis as described in the Methods section. Densitometric results, normalized to β-actin, are expressed as fold increase over the control value (=1), which was subtracted from all experimental values (means ± SE of six separate experiments). Data were analysed by one-way analysis of variance (anova) and the Student-Newman-Keuls test. *P < 0.05 versus basal expression of COX-2; ^#P < 0.05 versus basal release of PGE₂.