*Purpose: The role of intragraft macrophages (Mϕs) during antibody-mediated rejection (AMR) remains unclear. Anti-HLA Abs during AMR drives vascular inflammation as seen by monocyte recruitment and endothelial cell (EC) injury. Prior studies in our lab have indicated that monocyte and HLA I Ab-activated EC interactions skew monocytes to resolving M2-like phenotypes (CD68+CD206+CD163+). In this study, we validated the capacity of HLA I Ab-activated EC polarized Mϕs to a) efferocytose apoptotic ECs and b) phagocytose E.coli particles as in the setting of graft injury or infection, respectively. Moreover, we identified immune responses post-engulfment.

*Methods: Using a transwell in vitro co-culture system, human primary aortic ECs were stimulated with allele specific HLA I hIgG or HLA I F(ab’)₂ Ab portion, anti-CD105 Ab, TNF-a, hIgG isotype control, or left untreated. Third-party human monocytes added above activated ECs transmigrate to the bottom chamber and differentiate into Mϕs (5-days). Cytokine-polarized Mϕs (M1/M2) were generated as controls. EC apoptosis was induced using TNF-a and cycloheximide (16-hours) and was validated via caspase-3 activation. Apoptotic ECs and E. coli particles were labeled with pHRodo (pH sensitive dye; measured by FITC). Mϕs were co-cultured with labeled apoptotic ECs or E.coli particles for 24-hours. The phenotype of FITC ^Pos Mϕs was measured by flow cytometry. Cytokine secretion of culture supernatants from Mϕs treated with target cells and untreated Mϕs was measured via Luminex.

*Results: All transwell polarized Mϕs exhibited significantly higher levels of apoptotic EC efferocytosis (~20-40% FITC^Pos) and E.coli phagocytosis (~60-80% FITC^Pos) compared to cytokine-polarized Mϕs. Immune profiling results show that FITC^Pos Mϕs post-apoptotic EC efferocytosis increased expression of CD40, CR1, and CD71 compared to FITC^Neg Mϕs. Precisely, TNF-a activated EC polarized Mϕs significantly increased CD163 (*p=0.03) compared to untreated EC polarized Mϕs. Moreover, all transwell conditions exhibited an added increased in adhesive molecules (ICAM-1, CCR2, and PSGL-1) post-E.coli phagocytosis. Specifically, HLA I IgG Ab-activated EC polarized Mϕs significantly increased CR1 (*p=0.03) compared to untreated EC polarized Mϕs. Mϕ culture supernatants following apoptotic EC efferocytosis significantly increased FGF-2 while culture supernatants post-E.coli phagocytosis increased inflammatory cytokines (e.g. IL-6, IL-8, and IL-1b).

*Conclusions: Our studies indicate that endothelium polarized M2-like Mϕs exhibit high efferocytic and phagocytic capacities, and activate immune responses which are enhanced by TNF-a and HLA I Abs. This provides insight into Mϕ functions which may help mediate vascular repair, and clearance of pathogens following transplantation.

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