Capsaicinoids cause inflammation and epithelial cell death through activation of vanilloid receptors

Abstract

Capsaicinoids, found in less-than-lethal self-defense weapons, have been associated with respiratory failure and death in exposed animals and people. The studies described herein provide evidence for acute respiratory inflammation and damage to epithelial cells in experimental animals, and provide precise molecular mechanisms that mediate these effects using human bronchiolar and alveolar epithelial cells. Inhalation exposure of rats to pepper sprays (capsaicinoids) produced acute inflammation and damage to nasal, tracheal, bronchiolar, and alveolar cells in a dose-related manner. In vitro cytotoxicity assays demonstrated that cultured human lung cells (BEAS-2B and A549) were more susceptible to necrotic cell death than liver (HepG2) cells. Transcription of the human vanilloid receptor type-1, VR1 or TRPV1, was demonstrated by RT-PCR in all of these cells, and the relative transcript levels were correlated to cellular susceptibility. TRPV1 receptor activation was presumably responsible for cellular cytotoxicity, but prototypical functional antagonists of this receptor were cytotoxic themselves, and did not ameliorate capsaicinoid-induced damage. Conversely, the TRPV1 antagonist capsazepine, as well as calcium chelation by EGTA ablated cytokine (IL-6) production after capsaicin exposure. To address these seemingly contradictory results, recombinant human TRPV1 was cloned and overexpressed in BEAS-2B cells. These cells exhibited dramatically increased cellular susceptibility to capsaicinoids, measured using IL-6 production and cytotoxicity, and an apoptotic mechanism of cell death. Surprisingly, the cytotoxic effects of capsaicin in TRPV1 overexpressing cells were also not inhibited by TRPV1 antagonists or by treatments that modified extracellular calcium. Thus, capsaicin interacted with TRPV1 expressed by BEAS-2B and other airway epithelial cells to cause the calcium-dependent production of cytokines and, conversely, calcium-independent cell death. These results have demonstrated that capsaicinoids contained in pepper spray products produce airway inflammation and cause respiratory epithelial cell death. The mechanisms of these cellular responses to capsaicinoids appear to proceed via distinct cellular pathways, but both pathways are initiated by TRPV1.

FIG. 1

Photomicrographs of nasal (A and B), tracheal (C), alveolar (D and E), and terminal bronchiolar (F) tissues from rats exposed (30 min) by nose-only inhalation to an aerosolized pepper spray extract (1.0-1.2 mg/kg). L, epithelial loss; D, epithelial dysplasia; N, normal epithelium; M, macrophages; CI, mononuclear cell infiltrate; BH, bronchiolar hemorrhage; AH, alveolar hemorrhage; CM, cuboidal metaplasia. Figures represent characteristic lesions observed at a moderate to marked degree (scored 1-4) in a minimum of 50% of the treated animals following a 24-h recovery (n = 6).

FIG. 2

(A) Cytotoxicity of capsaicin to BEAS-2B (filled circles), A549 (open squares), and HepG2 (open triangles) cells. Data represent the mean cell viability and SD at a given concentration of capsaicin from three independent experiments. (B) Normalized densitometric traces for TRPV1 and β-actin transcripts, amplified by RT/PCR from total RNA isolated from BEAS-2B (solid line), A549 (dotted line), and HepG2 (dashed line) cells. The inset of panel B shows the agarose gel used to generate the densitometric traces (Std. = Molecular weight standards, B = BEAS-2B, A = A549, and H = HepG2) An asterisk (*) represents data points that were significantly greater (p < 0.025) than the values for both A549 and BEAS-2B cells.

FIG. 3

Flow cytometric analysis of FITC-Annexin V and propidium iodide staining of BEAS-2B cells treated with 0 (untreated control), 50, and 100 μM capsaicin for 24 h. Data are representative of a single experimental population of cells. However, the experiments were reproduced on three separate occasions to ensure consistent results. The percentage of cells exhibiting apoptotic and necrotic characteristics is shown within the figure.

FIG. 4

Induction and inhibition of IL-6 production by BEAS-2B cells treated with capsaicin (100 μM) or capsaicin (100 μM) plus various modulators of TRPV1 function for 24 h. Capsazepine was used at a concentration of 10 μM, EGTA at 100 μM, and CaCl₂ at 10 μM. Data represent the mean and SD of triplicate determinations for fold increases in IL-6 concentrations versus untreated cells (control values approximately 175 pg/ml). ^aStatistically significant difference (p < 0.025) versus untreated control cells. ^bStatistically significant difference (p < 0.025) from cells treated with capsaicin only.

FIG. 5

(A) Normalized densitometric traces for TRPV1, V5-fusion, and β-actin transcripts amplified by RT/PCR from total RNA isolated from BEAS-2B (dashed line) and TRPV1 overexpressing (solid line) cells. The inset represents the agarose gel used to generate the densitometric traces. (B) Cytotoxicity of capsaicin to BEAS-2B cells (circles) compared to TRPV1 overexpressing (squares) and Geneticin resistant control (squares) cells. Data represent the mean cell viability and SD at a given concentration of capsaicin for three independent experiments. For the Geneticin-resistant cells, the data represent the mean and SD from experiments using four different clones/cell lines. *Values significantly lower (p < 0.025) than the values obtained for both “normal” BEAS-2B and Geneticin-resistant (but not TRPV1 overexpressing) control cell lines.

FIG. 6

Induction of IL-6 production by “normal” BEAS-2B (solid bars) and TRPV1 overexpressing cells (dotted bars) treated with increasing concentrations of capsaicin for 24 h. Data represent the mean and SD of triplicate determinations for fold increases in IL-6 concentration versus untreated cells (control values for IL-6 were approximately 175 pg/ml and 850 pg/ml for BEAS-2B and TRPV1 overexpressing cells, respectively). *Statistically significant difference (p < 0.025) between treated and untreated control cells.

FIG. 7

Flow cytometric analysis of FITC-Annexin V and propidium iodide staining of TRPV1 overexpressing cells treated with 0 (left), 0.5 (middle), and 1.0 μM (right) capsaicin for 24 h. Data are representative of a single experimental population of cells. However, the experiments were reproduced on three separate occasions to ensure consistent results. The percentage of cells exhibiting apoptotic and necrotic characteristics is shown within the figure.