Substance P inhibits natural killer cell cytotoxicity through the neurokinin-1 receptor

Abstract

SP is a potent neuroimmunomodulator that functions through ligating members of the neurokinin receptor family, one of which, NK1R, is widely expressed in immune cells. As in humans, circulating SP levels are increased in pathologic states associated with impairment of NK cell functions, such as depression and HIV infection, we hypothesized that SP has a direct, inhibitory effect upon NK cells. We have studied a clonal human NK cell line (YTS) as well as ex vivo human NK cells and have determined that truncated and full-length NK1R isoforms are expressed in and SP bound by ex vivo NK cells and the YTS NK cell line. Incubation of YTS cells with 10⁻⁶ M SP and ex vivo NK cells with 10⁻⁵ M SP inhibited cytotoxic ability by ∼20% and reduced degranulation. This inhibitory effect upon cytotoxicity was partially prevented by the NK1R antagonist CP96,345. The treatment of YTS or ex vivo NK cells with SP neither down-modulated NCR expression nor affected triggering receptor-induced NF-κB activation. Preincubation of YTS cells with SP, however, did abbreviate the typically prolonged intracellular calcium increase induced by target cell engagement and reduced triggering receptor-induced pERK. Thus, SP has the potential to regulate NK cell functions and acts downstream from neurokinin receptors to modulate NK cell activation signaling. This mechanism may contribute to impairment of NK cell function in certain disease states associated with increased circulating SP. Antagonism of this system may present an opportunity to augment NK cell function therapeutically in selected human diseases.

Figure 1.. Expression of NK1R in human NK cells.

(A) qPCR of NK1R mRNA expression in human NK cell lines. Total cellular RNA from different NK cell lines, NKL, NK92, NK3.3, or IL-2 expanded ex vivo NK cells (LAK-A and -B from two individual donors), was evaluated using specific primers for NK1R to demonstrate quantitative differences among RNA from each cell line. Data are representative of two experiments. NK1R isoform expression in (B) YTS cells and (C) ex vivo NK cells. Full-length and truncated NK1R mRNA were amplified from YTS cells by real-time RT-PCR and the number of transcripts presented/million copies of GAPDH. The truncated NK1R mRNA level was greater than that of full-length NK1R in YTS cells (n=10; *P=0.0148; paired t test) and ex vivo NK cells (n=3; P=0.0046; paired t test). Mean copies of full-length NK1R =186.5 in YTS (se=82.8) and =102.3 in ex vivo NK cells (se=51.2), whereas truncated NK1R =1009 in YTS (se=257.4) and =31,805 in ex vivo NK cells (se=2204).

Figure 2.. Detection of full length of NK1R in YTS cells.

(A) YTS cells, the THP-1 monocytic cell line, and the A3.01 lymphoblastic cell line were each dual-labeled for expression of NK1R using first, an antibody directed against the common N-terminus extracellular epitope, followed by permeabilization and incubation with antibody recognizing the intracellular C-terminus (present only in full-length NK1R). Visualization of the N-terminus-bound antibody used FITC-conjugated goat anti-mouse (green) and C-terminus using HPR-conjugated goat anti-rabbit, followed by Alexa Fluor 568 tyramide (red). Nuclei were counterstained with DAPI (blue) and cells imaged using fluorescence microscopy. Merged fluorescent images are shown in the bottom row. Data are representative of three independent experiments. Rabbit and mouse IgG used as primary antibodies were included as negative controls and demonstrated no detectable signal (data not shown). (B) Western blot analysis of NK1R in YTS, THP-1, and A3.01 cell lysates using the same N-terminus- and C-terminus-specific antibodies used for the immunofluorescence. Actin was included as a protein-loading control. Approximate band MWs are listed beside the arrows. Results are representative of three separate experiments.

Figure 3.. SP retention by NK cells.

(A) YTS cells (left) or J-SPR cells (right) were incubated with 0.3 μM SP-OG or 0.3 μM SA-OG or were preincubated with 100 μM unconjugated SP at 4°C for 30 min prior to addition of SP-OG (SP+SP-OG) or SA-OG (SP+SA-OG) and then evaluated by flow cytometry. Data are representative of three independent experiments. (B) PBMCs were treated with the same conditions and combinations as the YTS and J-SPR cells and then incubated with fluorophore-conjugated mAb to allow determination of individual immune cell populations. Lymphocytes or monocytes were selected by side- and forward-scatter and then evaluated for fluorescence intensities (left). NK cells (CD56⁺ CD3^–), monocytes (CD14⁺CD45^–), and CTL (CD8⁺CD3⁺) were gated and evaluated for OG fluorescence (right). Data are representative of six separate experiments performed using distinct, unrelated, healthy donors.

Figure 4.. Effect of SP on NK cell cytotoxicity.

(A) YTS cells were pretreated with media (●) or 10⁻⁶ M SP for 30 min (▴) and 72 h (gray ■) prior to being added to ⁵¹Cr-labeled 721.221 target cells to measure cytotoxicity. Each point represents the mean cytotoxicity ± sd of three individual experiments (each performed in triplicate). Decreases at 30 min and 72 h were significantly different where noted (*P≤0.02, two-tailed) when compared with media control for 72 h and 30 min time-points compared with media. YTS cell viability for both time-points was ≥90%, as determined by trypan blue exclusion. (B) Ex vivo NK cells were pretreated with media (black line) or 10⁻⁵ M SP (gray line) and 10⁻⁵ M SP with 10⁻⁵ M NK1R antagonist (SPA; dashed line) for 30 min, prior to being added to ⁵¹Cr-labeled 721.221 target cells to measure cytotoxicity. Points are representative of two individual experiments, each performed in triplicate. NK cell viability was ≥90%, as determined by trypan blue exclusion. (C) Dependence of inhibitory effect of SP on YTS cell cytotoxicity upon NK1R ligation. YTS cells were incubated with 10⁻⁶ M SP, SP + SPA, SPA alone, or media for 30 min at 37°C and then combined with ⁵¹Cr-labeled 721.221 target cells to measure cytotoxicity. Bars indicate the percent inhibition in cytotoxicity at a 10:1 E:T cell ratio compared with media control. The mean ± sd of six individual experiments is shown, and statistically significant differences from the media control are noted (**P<0.001; *P<0.05, two-tailed).

Figure 5.. Effect of SP on ex vivo NK cell granzyme B and IFN-γ release.

(A) Granzyme B and (B) IFN-γ measured by ELISA in the supernatant of 16 h cultures of 1 × 10⁵ ex vivo NK cells, 1 × 10⁴ 721.221 target cells, or coincubated ex vivo NK and 721.221 target cells. Prior to culture, NK cells were treated with media (black) or 10⁻⁵ M SP (gray) for 30 min. Values represent the average of two independent experiments, each performed in duplicate.

Figure 6.. Effect of SP on degranulation of NK cells conjugated with target cells.

PKH26-labeled YTS (A–D) and ex vivo NK cells (E–H) were used as effectors to form conjugates with CFSE-labeled 721.221 target (721) cells for 10 min (A and E), 30 min (B and F), and 4 h (C and G). Histograms (A–G) demonstrate fluorescence intensity of CD107a on unconjugated effector cells (gray-filled), unconjugated target cells (black-filled), SP-treated effector cells (dashed, black lines), control-treated effector cell/target cell conjugates (black lines), and SP-treated effector cell/target cell conjugates (dotted, gray lines). Conjugates were selected as PKH26⁺/CFSE⁺, whereas unconjugated NK cells were PKH26⁺/CFSE^–, and unconjugated target cells were PKH26^–/CFSE⁺. Bar graphs (D and H) demonstrate mean percentage of control for CD107a MFI in SP-treated effector cell/target cell conjugates at 10 min (open bars), 30 min (gray bars), and 4 h (black bars); n = 2.

Figure 7.. Effect of SP upon NK cell activation receptor expression.

YTS cells were incubated with 10⁻⁶ M SP (dotted lines) or 10⁻⁶ M SP + SPA (dashed lines) or media (solid lines) for 30 min (A) or 2 h (B) at 37°C and then incubated with FITC- or PE-conjugated mAb for the specified activation receptors and analyzed by flow cytometry. IgG FITC- or PE-conjugated, control mAb (gray, shaded regions) were used for background control. The data are representative of three separate experiments. Any slight differences noted between SP and control were not consistent across experimental repeats and time-points.

Figure 8.. Effect of SP on activation-induced NF-κB function in YTS cells.

YTS cells were pretreated with 10⁻⁶ M SP or media for 30 min at 37°C and added to plates coated with IgG or anti-CD28 or to which PMA/ionomycin was added and incubated for 15 min at 37°C. Cells were lysed and lysates evaluated for IκB (upper) Western blot. As a loading control, the blot was stripped and probed with an anti-actin pAb (lower). Densitometric quantitation of bands was performed, and the values normalized to IgG control are listed beneath each lane. The data are representative of three separate experiments.

Figure 9.. Effect of SP on the kinetic calcium response in YTS cells induced by 721.221 target cells.

(A) YTS cells incubated with 2 μM fura-2 AM for 30 min and the 334 and 380 nm fluorescence ratios quantified after addition of 721.221 cells, which were pretreated for 30 min with 10⁻⁷ M SP (gray) or media (black). (B) In separate experiments, YTS cells were pretreated for 30 min with media (gray ♦), 10⁻⁷ M SP (■), 10⁻⁷ M SP + SPA (▴), or SPA (×). The observed change in Ca²⁺ was recorded from 30 to 50 cells/experiment, and the traces shown are representative of two separate experiments. (C) YTS cells to which 10-7 M SP was added in the absence of 721.221 target cells. The arrow denotes the time at which the SP was added to the YTS cells under observation. (D) YTS cells pretreated with an inactive form of SPA (CP96,344 - to control for antagonist pretreatment) to which 10-7 M SP was added (black line). The addition of SP to the cells under observation is denoted by the arrow. YTS cells pretreated with SPA without addition of SP in the absence of target cells (gray line) demonstrate baseline Ca²⁺.

Figure 10.. Effect of SP upon activation-induced pERK.

YTS cells pretreated for 30 min at 37°C with 10⁻⁶ M SP or media were exposed to immobilized IgG or anti-CD28 for 15 min. As an activation control, YTS cells were treated for 15 min with PMA/ionomycin. Cells were lysed and lysates evaluated for pERK (upper) and total ERK (lower). Densitometric quantitation of bands was performed, and the values normalized to IgG control are listed beneath each lane. The data are representative of three separate experiments.