CXCR2 specific endocytosis of immunomodulatory peptide LL-37 in human monocytes and formation of LL-37 positive large vesicles in differentiated monoosteophils

Immunomodulatory peptide cathelicidin/LL-37 induces human monocyte differentiation into a novel bone repair cell, the monoosteophil. We now demonstrate that LL-37 is endocytosed by monocytes over a period of 6 days producing large (10 × 2 μm), specialized LL-37 and integrin α3 positive vesicles. CXCR2, a membrane receptor previously associated with the binding of LL-37 to neutrophils, was co-endocytosed with LL-37 where both markers remained within the cytosol over a 16 h observation period. Endocytosis of LL-37 was mediated by a clathrin- and cavoelin/lipid raft-dependent pathway into early Rab5+ endosomes expressing APPL1 and EEA1. From 4 to 16 h, LL-37 vesicles co-localized with the Golgi, mitochondria, and to a lesser extent lysosomes and ER. By day 6, LL-37 was associated with large (>10 μm) vesicles, adjacent to Golgi, mitochondria, ER and lysosomes. LL-37 co-stained with integrin α3, tetraspanin CD9, GPI-linked CD59 and costimulatory molecule CD276 (B7-H3) in these vesicles. Continuous tracking of LL-37 with its associated vesicles over 6 days indicates that LL-37 is an extremely stable, membrane-associated peptide that plays a critical role in the differentiation of monocytes into monoosteophils.