Fig. 3. The glycosaminoglycan-mediated breast cancer cell-induced APRIL secretion from neutrophils utilizes the TLR4 pathway.

To determine whether APRIL secretion involves the TLR4 pathway, potentially via PKR or TLR3, we took advantage of available biological/chemical activators and inhibitors. TLR4 and PKR were activated using ultra-pure lipopolysaccharide extracted from E. coli strain K12 (LPS-EK) and polyinosinic:polycytidylic acid (poly I:C), respectively. TLR4 was inhibited using a novel cyclohexene derivative, CLI-095. PKR and TLR3 were inhibited using 2-aminopurine (2-AP), and TLR3 was specifically inhibited using (R)-2-(3-Chloro-6-fluorobenzo[b]thiophene-2-carboxamido)-3-phenylpropanoic acid (C₁₈H₁₃ClFNO₃S). Neutrophils (2 × 10⁶) were pre-treated (or untreated) with inhibitors [CLI-095 (5 mM; Invivogen), 2-AP (5 mM; Sigma), or C₁₈H₁₃ClFNO₃S (10 μM; Calbiochem)] for 30 min at 37 °C prior to stimulation with T47D BCCs (5 × 10⁵), HS (10 μg/mL), LPS-EK (10 μg/mL; Invivogen) or poly I:C (25 μg/mL; Invivogen) for 5 min at 37 °C. Secreted neutrophil APRIL was measured by ELISA as described in Fig. legend 1. a TLR4 inhibition prevents BCC-, HS-, and LPS-EK-induced neutrophil APRIL secretion. Neutrophils were treated (or untreated) with the TLR4-specific inhibitor, CLI-095, prior to incubation with BCCs, HS or the TLR4-specific agonist, LPS-EK. b The PKR and TLR3 inhibitor, 2-AP, prevents BCC-, HS- and poly I:C-induced neutrophil APRIL secretion. Neutrophils were treated (or untreated) with 2-AP then incubated with BCCs, HS or poly I:C. c The TLR3-selective inhibitor, C₁₈H₁₃ClFNO₃S, does not prevent BCC- and poly I:C-induced neutrophil APRIL secretion. Neutrophils were treated (or untreated) with C₁₈H₁₃ClFNO₃S then incubated with BCCs or poly I:C. Data are means ± SD from three replicates of a representative experiment; n = 4. One-tailed Student’s t-test was used to determine statistical significance at p < 0.05