Natural Killer Cells Recognize Pulmonary Epithelial Stress Molecules during Primary Graft Dysfunction

Abstract

<h3>Purpose</h3> Primary graft dysfunction (PGD) occurs in 1/3 of all lung transplants. It is defined by epithelial dysfunction and innate immune cell infiltration. Natural killer (NK) cells are innate lymphocytes that are activated by NKG2D receptor binding of specific stress molecules. We hypothesized that NK cells mediate PGD by recognizing stress molecules induced on lung epithelial cells and causing injury via direct cytotoxicity. <h3>Methods</h3> In an established experimental PGD model, left hilar clamp (HC) was compared to sham (S) surgery in C57BL/6 mice. Stress molecules (RAE-1, MULT1) and NK cell receptors were measured by flow cytometry median fluorescent intensity (MFI) on dissociated lung cells. Mice were given blocking anti-NKG2D or isotype control antibodies preceding HC. Human stress molecules (MICA, MICB, ULBP1, 3, and 2/5/6) were measured by flow cytometry on bronchial epithelial cells incubated for 4 hours in hypoxia (1% O₂) versus normoxia conditions. The Mann-Whitney U test was applied for pairwise comparisons. <h3>Results</h3> NK cells were increased as a percent of lymphocytes (p = 0.008) and by count (p = 0.04) in HC (n = 5) versus S (n = 5) lungs and infiltrated airways (Figure 1A). The NKG2D receptor was increased on NK cells following HC (p = 0.005). The stress molecules MULT1 (p = 0.0002) and RAE-1 (Figure 1B, p = 0.002) were increased on epithelial cells in HC compared to S lungs. NKG2D blockade (n = 9) resulted in less pulmonary edema following HC compared to isotype control antibody (Figure 1C, n = 9, p = 0.04). Hypoxic human bronchial epithelial cells (n = 6) had increased MICB (p = 0.008), ULBP-1 (p = 0.05), and ULBP-2,5,6 (Figure 1 D, p = 0.004) compared to normoxic cells (n = 6). <h3>Conclusion</h3> PGD induces NK cell stress ligands leading to NK cell recruitment that contributes to PGD. NKG2D blockade, under investigation in clinical trials of other diseases, resulted in decreased lung injury. The induction of human NKG2D stress molecules during hypoxia suggests that this NK cell receptor-ligand interaction may mediate PGD.