Thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells

Abstract

Compelling evidence suggests that the epithelial cell-derived cytokine thymic stromal lymphopoietin (TSLP) may initiate asthma or atopic dermatitis through a dendritic cell-mediated T helper (Th)2 response. Here, we describe how TSLP might initiate and aggravate allergic inflammation in the absence of T lymphocytes and immunoglobulin E antibodies via the innate immune system. We show that TSLP, synergistically with interleukin 1 and tumor necrosis factor, stimulates the production of high levels of Th2 cytokines by human mast cells (MCs). We next report that TSLP is released by primary epithelial cells in response to certain microbial products, physical injury, or inflammatory cytokines. Direct epithelial cell-mediated, TSLP-dependent activation of MCs may play a central role in "intrinsic" forms of atopic diseases and explain the aggravating role of infection and scratching in these diseases.

Figure 1.

Human MCs expressed functional receptor for TSLP. (A) TSLP-R and IL-7Rα chain expression was determined at mRNA on MCs and peripheral blood T cells (mean ± SEM of eight experiments on different MC lines) and protein levels. (B) Tissue sections from the bronchial mucosa of asthmatic patients were stained with TSLP-R mAb using an HRP system (brown) and Astra Blue (blue) for the identification of MCs. (C) MCs were stimulated with TSLP alone or together with different inflammatory cytokines. The 24-h culture supernatants were tested for their content in IL-5. One representative of three experiments is shown; mean ± SD of triplicates. (D) MCs were stimulated with varying concentrations of TSLP in the presence of 10 ng/ml IL-1 and 25 ng/ml TNF. One representative of three experiments is shown; mean ± SD of triplicates. (E) Time course of cytokine production by MCs stimulated with 10 ng/ml IL-1β/TNF and TSLP. Mean ± SEM (n = 5). (F and G) MCs were stimulated in the presence of neutralizing mAb to TSLP (F) or TSLP-R (G) and isotype control IgG (each at 10 μg/ml). One representative of three experiments is shown; mean ± SD of triplicates.

Figure 2.

TSLP-stimulated secretion of cytokines and chemokines by MCs. Cytokine (A) and chemokine (B) secretion by MCs (10⁵ cells/ml) stimulated for 24 h with 10 ng/ml IL-1β/TNF or/and TSLP was assessed by ELISA. Mean ± SEM (n = 11).

Figure 3.

Induction of TSLP production by primary human airway epithelial cells. (A) SAECs were stimulated as indicated, and the 48-h culture supernatants were tested for their content in TSLP by ELISA. Mean ± SEM (n = 5). (B) Expression of the indicated TLR mRNA in SAECs was determined by real-time PCR. (C) BAF cells (10⁴ cells/well) expressing the human TSLP-R and IL-7Rα chains were cultured in the presence of SAEC supernatants and in the presence or absence of neutralizing anti-TSLP mAb, and their proliferation was assessed after 3 d. One representative of three experiments is shown; mean ± SD of triplicates. (D–F) MCs were cultured in the presence or absence of supernatants of SAECs (50% vol/vol) described in A that were obtained upon stimulation with IL-1α/TNF (D), PGN (E), or polyI:C (F). IL-13 and IL-5 (not depicted) were measured after 24 h of MC stimulation. Mean ± SEM of four to five experiments.

Figure 4.

MC activation by skin-derived TSLP. (A) MCs were cultured with or without lesional or nonlesional skin fragments from AD patients in the presence of IL-1β/TNF with or without neutralizing mAb to TSLP. IL-13 and IL-5 (not depicted) were measured in the supernatants after 24 h of culture. (B) TSLP mRNA was assessed in the lesional and nonlesional skin of AD patients by real-time PCR. (C) Skin explants from nonallergic patients undergoing plastic surgery were minced and cultured for 24 h. Their cell-free culture supernatants (50% vol/vol) were used to stimulate MCs in the presence of IL-1β/TNF with or without mAb to TSLP and TSLP-R or isotype control. IL-13 was measured after 24 h of culture. One representative of three experiments is shown; mean ± SD of triplicates. (D) TSLP mRNA was assessed on freshly isolated or cultured for 24-h skin explants. (E) TSLP protein was measured in the supernatant fluids of these cultures. One representative of three experiments is shown; mean ± SD of triplicates.