Proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma alter tight junction structure and function in the rat parotid gland Par-C10 cell line

Abstract

Sjögren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-alpha and IFN-gamma decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I(sc)) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y(2) nucleotide receptor agonist. In contrast, TNF-alpha and IFN-gamma had no effect on agonist-induced increases in the intracellular calcium concentration [Ca(2+)](i) in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-alpha and IFN-gamma increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-alpha, but not IFN-gamma, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-gamma causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.

Fig. 1.

Tumor necrosis factor-α (TNF-α) and/or interferon-γ (IFN-γ) alone or in combination cause a time- (A) and dose-dependent (B) decrease in transepithelial resistance (TER) in Par-C10 cell monolayers. Par-C10 cells were cultured on permeable supports, as described in materials and methods. After reaching maximal TER, cells were exposed to TNF-α (10 ng/ml) and/or IFN-γ (10 ng/ml) for 18–48 h (A) or TNF-α and/or IFN-γ (0–100 ng/ml each) for 48 h (B). TER values were obtained with an epithelial volt-ohmmeter (EVOM) equipped with miniature dual chopstick electrodes. Following subtraction of medium resistance (120 Ω), tissue resistance was multiplied by the effective membrane area (1.13 cm²) and expressed as means ± SE of results from 10 independent experiments, where *P < 0.05 indicates a significant difference from control cells.

Fig. 2.

TNF-α and/or IFN-γ decrease agonist-induced transepithelial anion secretion in Par-C10 cell monolayers. Par-C10 cells were cultured on permeable supports, as described in materials and methods. After reaching maximal TER, cells were exposed to TNF-α (10 ng/ml) and/or IFN-γ (10 ng/ml) for 48 h. Permeable supports were placed in Ussing chambers and changes in short-circuit current (I_sc) were monitored in response to UTP (100 μM) applied to the apical compartment (A) or carbachol (100 μM) applied to the basolateral compartment (B). Values represent the maximum I_sc obtained and are expressed as means ± SE of results from 3 or more experiments, where *P < 0.05 indicates significant differences from control cells.

Fig. 3.

TNF-α and/or IFN-γ do not alter agonist-induced calcium signaling in Par-C10 cell monolayers. Par-C10 cells were cultured on permeable supports, as described in materials and methods. After reaching maximal TER, cells were exposed to TNF-α (10 ng/ml) and/or IFN-γ (10 ng/ml) for 48 h. UTP (100 μM) was applied to the apical compartment (A) or carbachol (100 μM) was applied to the basolateral compartment (B) and changes in [Ca²⁺]_i were monitored in Par-C10 cell monolayers, as described in materials and methods. Changes (Δ) in [Ca²⁺]_i were expressed by subtracting the basal [Ca²⁺]_i (before agonist addition) from the peak agonist-induced increase in [Ca²⁺]_i. Data are expressed as means ± SE of results from 3 experiments.

Fig. 4.

TNF-α and/or IFN-γ increase the paracellular permeability of normally impermeant proteins in Par-C10 cell monolayers. Par-C10 cells were cultured on permeable supports, as described in materials and methods. After reaching maximal TER, cells were exposed to TNF-α (10 ng/ml) and/or IFN-γ (10 ng/ml) for 48 h. Then, microperoxidase, peroxidase, or lactoperoxidase (42 μg/ml) was added to the basolateral compartment. After 2 h, peroxidase activity in 100 μl of medium from the apical compartment was detected with 100 μl of the liquid peroxidase substrate [2′2-Azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid] system for ELISA (Sigma). After 5 min, the reaction was stopped with 100 μl of 1% (wt/vol) SDS and the absorbance was measured at 405 nm. Data are expressed as means ± SE of results from 3 independent experiments, where *P < 0.05 indicates a significant difference from control cells.

Fig. 5.

TNF-α and IFN-γ alter Par-C10 cell morphology. Shown are transmission electron micrographs (TEMs) of Par-C10 cells grown on permeable supports for 48 h in the absence (A) or presence of TNF-α (10 ng/ml), IFN-γ (10 ng/ml; B and C), or TNF-α (10 ng/ml) and IFN-γ (10 ng/ml; D). Cells were processed for morphological analysis, as described in materials and methods. Magnification ×4,000.

Fig. 6.

TNF-α and IFN-γ alter tight junction (TJ) morphology in Par-C10 cell monolayers. TEM of freeze-fractured membrane replicas from untreated Par-C10 cells (A) or cells treated with TNF-α (10 ng/ml; B), IFN-γ (10 ng/ml; C), or TNF-α (10 ng/ml) and IFN-γ (10 ng/ml; D) was performed, as described in materials and methods. TJ strands are shown between black arrows (A–D). E: quantification of TJ depth and strand number. F: methods for quantifying TJ morphometry, as described previously (3). Main grid lines (black) perpendicular to the parallel strands were drawn at 3-cm intervals over the TJ area. The maximum depth (red arrow a) and the minimum depth (red arrow b) were measured for each compartment separated by the grid lines. Three additional grid lines (green) were drawn parallel to the main grid lines at 1-cm intervals in each compartment. The total number of strands was determined as the number of points where strands crossed the 3 grid lines (green). #1 cm. Data are expressed as means ± SE of results from 3 experiments, where *P < 0.05 indicates significant differences from control cells.

Fig. 7.

TNF-α and IFN-γ downregulate expression of claudin-1, but not other TJ proteins, in Par-C10 cell monolayers. A: lysates were prepared from Par-C10 cell monolayers treated with or without 10 ng/ml TNF-α and/or IFN-γ and claudin-1 and total ERK expression were detected by Western analysis, as described in materials and methods. Data are expressed as means ± SE of results from 3 or more experiments, where *P < 0.05 indicates significant differences from control cells. Results from a representative experiment are shown at the top of the figure. B: lysates were prepared from Par-C10 cell monolayers treated with or without 10 ng/ml TNF-α and/or IFN-γ and expression of ZO-1, occludin, JAM-1, and claudins-3, -4, and -10 was detected by Western blot analysis. Results from a representative experiment (n = 3) are shown.

Fig. 8.

TNF-α-mediated decreases in TER in Par-C10 cell monolayers are reversible. Par-C10 cell monolayers were treated with or without 10 ng/ml TNF-α and/or IFN-γ for 48 h and cells were washed and incubated in cytokine-free medium for 24 h. Then, TER was measured, as described for Fig. 1. Data are expressed as means ± SE of results from 3 or more experiments, where *P < 0.05 indicates a significant difference from cells treated without TNF-α removal.

Fig. 9.

TNF-α-mediated decreases in agonist-induced transepithelial anion secretion in Par-C10 cell monolayers are reversible. Par-C10 cell monolayers were treated with or without 10 ng/ml TNF-α and/or IFN-γ for 48 h and cells were washed and incubated in cytokine-free medium for 24 h. Then, changes in I_sc were determined in response to UTP (100 μM; A) or carbachol (100 μM; B), as described for Fig. 2. Data are expressed as means ± SE of results from 3 or more experiments, where *P < 0.05 indicates significant differences from cells treated without TNF-α removal.

Fig. 10.

TNF-α-mediated decreases in claudin-1 expression in Par-C10 cell monolayers are reversible. Par-C10 cell monolayers were treated with or without 10 ng/ml TNF-α and/or IFN-γ for 48 h and cells were washed and incubated in cytokine-free medium for 24 h. Claudin-1 expression in cell lysates was normalized to total ERK, as described for Fig. 7. Data are expressed as means ± SE of results from 3 or more experiments, where *P < 0.05 indicates a significant difference from cells treated without TNF-α removal. Results from a representative experiment are shown at the top.