TNFR1 links TNF exocytosis to TNF production in allergen-activated RBL-2H3 cells

Abstract

We previously reported that the maximal production of Tumor Necrosis Factor (TNF or TNFα) in antigen-activated RBL-2H3 cells (a tumor analog of mucosal mast cells) requires Munc13-4, a regulator of exocytic fusion. In this study, we investigated the involvement of various fusion catalysts in TNF production. We observed a strong correlation between the total TNF level and TNF exocytosis in RBL-2H3 cells. RT-qPCR shows that TNFR1 (TNF receptor 1) is the sole TNFR expressed in these cells, and that its transcription is upregulated upon allergen-mediated activation. Importantly, the addition of soluble TNFR1 inhibits antigen-elicited TNF production in a dosage-dependent fashion. Likewise, TNF production is diminished in the presence of TACE (TNFα Converting Enzyme) inhibitor KP-457, which prevents the generation of soluble TNF (sTNF). Together, these findings indicate that sTNF and TNFR1 function as autocrine agent and receptor respectively at the mast cell surface to boost TNF proliferation during allergic inflammation.

Keywords: Allergy; Autocrine signaling; Degranulation; Inflammation; Mast cell; Munc18.

Figure 1.. TNF exocytosis correlates with TNF level.

(A) TNP-BSA/IgE (anti-TNP)-triggered TNF exocytosis from Munc13–4^KO cells, stable Munc18a^KD cells, stable Munc18b^KD cells, or VAMP8^KD cells (via siRNA) was measured 1 h after stimulation, and the values were normalized against that of their respective control cells that were treated in the same manner. (B) Likewise, total TNF protein levels were calculated for activated KO or KD cells and normalized against that of the control cells. Averages and standard deviations from at least 4 biological repeats are presented. * p < 0.05; ** p < 0.01; *** p < 0.001

Figure 2.. TNFR1 is expressed and upregulated in activated RBL-2H3 cells.

RBL-2H3 cells sensitized with anti-TNP IgE or buffer were incubated with TNP-BSA for 1 h. (A) Mast cell degranulation was measured and expressed as the percentage of β-hexosaminidase in the supernatant. Meanwhile, lysates from activated (with IgE) or mock treated (with buffer) cells were subject to reverse transcription followed by either qPCR to quantify the relative amount of TNFR mRNA (B) or regular PCR to visually compare the expression of TNFR1 and TNFR2 on 2% agarose gel (lane 1 to 4). In lanes 5 and 6, one μL of H₂O and rat universal cDNA were used respectively as template.

Figure 3.. TNF feedback loop is mediated by TNFR1.

(A) RBL-2H3 cells sensitized with anti-TNP IgE were challenge with TNP-BSA along with specified amounts of sTNFR1 or buffer (PBS). At 1 h time point, total TNF protein level in all three conditions were measured using ELISA. The total TNF in activated cells subtracted by that in resting cells was set at 1 (lane 1) and used to calculate the relative values of TNF production in the presence of sTNFR1 (lanes 2 and 3). Averages and standard deviations from 3 biological repeats are presented. ** p < 0.01; *** p < 0.001. (B) Model of TNF autocrine loop. Mast cells elicited by allergens undergo calcium dependent exocytosis of full length, membrane-bound TNF (depicted in blue) that is prestored in secretory apparatuses such as MVB (1). Allergens also boost de novo synthesis of TNF, which is deposited into ER (omitted for simplicity) and subsequently delivered onto the plasma membrane via the classical secretory pathway (2). Upon TACE/Adam17 (depicted in red)-dependent cleavage, soluble TNF is released to the extracellular space, where it binds TNFR1 to promote additional TNF production. TNF proteins that have escaped proteolysis can be internalized and delivered to MVB for storage (3).

Figure 4.. TACE inhibitor KP-457 reduces TNF proliferation.

RBL-2H3 cells sensitized with anti-TNP IgE were challenge with TNP-BSA along with specified amounts of KP-457 or solvent (DMSO). At 1 h time point, β-hexosaminidase secretion (A), released TNF (recovered from the supernatant) (B) and total TNF protein level (C) were measured and the values were normalized against that of the solvent control (lane 2). Averages and standard deviations from at least 5 biological repeats are presented. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Figure 5.. Effect of MAPK inhibitors on exocytosis and TNF production.

Where indicated, MAP kinase inhibitors including (A) Doramapimod/BIRB 796 (IC₅₀ of 38 nM for p38α, 65 nM for p38β, 200 nM for p38γ, and 520 nM for p38δ), (B) AX-15836 (IC₅₀ of 8 nM for ERK5), (C) Ravoxertinib/GDC-0994 (IC₅₀ of 6.1 nM and 3.1 nM for ERK1 and ERK2 respectively), and (D) JNK-IN-8 (IC₅₀ of 4.7 nM, 18.7 nM, and 1 nM for JNK1, JNK2, and JNK3, respectively) were added to sensitized RBL-2H3 cells 30 min before activation by TNP-BSA. Total TNF, TNF secretion (% of total TNF in the supernatant), and β-hexosaminidase secretion (% of total β-hexosaminidase activity in the supernatant) were measured and normalized against that of the mock treatment (DMSO). Averages and standard deviations from 3 to 5 biological repeats are presented. * p < 0.05; ** p < 0.01