Researcher University of California Los Angeles Los Angeles, CA
Aim: Transplant recipients developing donor specific HLA antibodies (DSA) are at risk for acute and chronic antibody-mediated rejection (AMR). DSA contribute to the process of AMR by binding to HLA molecules on endothelial cells (EC) and eliciting EC proliferation and leukocyte recruitment. HLA molecules lack signal motifs and kinase activity in their short cytoplasmic tail. Previously we reported that HLA-I partnered with integrin b4 (ITGB4) to regulate HLA-I-stimulated cell proliferation and migration. We also showed that HLA I molecules engage in crosstalk with TLR4 signaling. We therefore postulated that TLR4 and ITGB4 regulate HLA-I-mediated EC activation via different signal pathways.
Methods: Gene silencing was determined with siRNA transfection. For signal complex formation, EC were stimulated with anti-HLA I mAb and cell lysates were immunoprecipitated with HLA-I Ab. Complex formation was detected by Western Blot, monocyte adhesion was assessed by CFSE labeling, measured by fluorescence microscopy, cell proliferation was measured by BrdU incorporation.
Results: Ligation of HLA-I molecules on EC with anti-HLA-I Ab induced complex formation with TLR4, ITGB4, or Piezo1. ITGB4 was found to associate with HLA-I, but not in complexes that included TLR4, MyD88, or Piezo1. Silencing TLR4, MyD88, or Piezo1 with siRNA inhibited HLA-I Ab-induced phosphorylation of NF-κB, ERK, JNK, and p38 MAP kinase, and blocked HLA-I–mediated P-selectin expression and monocyte adhesion to ECs. In contrast, ITGB4 knockdown did not affect these responses. However, ITGB4 siRNA abrogated HLA-I Ab-induced phosphorylation of S6K and S6RP, as well as EC proliferation.
Conclusion: Ligation of HLA-I molecules on the endothelial cell (EC) surface with anti-HLA-I Ab induced complex formation with TLR4, ITGB4, or Piezo1. ITGB4 was found to associate with HLA-I, but not in complexes that included TLR4, MyD88, or Piezo1. Silencing TLR4, MyD88, or Piezo1 with siRNA inhibited HLA-I Ab-induced phosphorylation of NF-κB, ERK, JNK, and p38 MAP kinase, and blocked HLA-I–mediated P-selectin expression and monocyte adhesion to ECs. In contrast, ITGB4 knockdown did not affect these responses. However, ITGB4 siRNA abrogated HLA-I Ab-induced phosphorylation of S6K and S6RP, as well as EC proliferation.
