Highly glycosylated α1‐acid glycoprotein is synthesized in myelocytes, stored in secondary granules, and released by activated neutrophils

Acid glycoprotein (AGP) is an acute- phase protein produced by hepatocytes and secreted into plasma in response to infection/injury. We recently assessed the transcriptional program of terminal granulocytic differentiation by microar- ray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/ metamyelocytes (MYs), and BM neutrophils. These analyses demonstrated a transient, high mRNA ex- pression of genuine secondary/tertiary granule proteins and AGP in MYs. In agreement with this, immunocytochemistry revealed the presence of AGP protein and the secondary granule protein lactoferrin in cells from the MY stage and through- out granulocytic differentiation. Immunoelectron microscopy demonstrated the colocalization of AGP and lactoferrin in secondary granules of neutrophils. This finding was substantiated by the failure to detect AGP and lactoferrin in blood cells from a patient with secondary/tertiary (specific) granule deficiency. In addition, Western blot analysis of subcellular frac- tions isolated from neutrophils revealed that neutro- phil-derived AGP, localized in secondary granules, was abundant and highly glycosylated compared with endocytosed, plasma-derived AGP localized in secre- tory vesicles. Exocytosis studies further demon- strated a marked release of AGP and lactoferrin by activated neutrophils. Finally, induction of CCAAT/ enhancer-binding protein (C/EBP)- in a myeloid cell line was shown to increase AGP transcript levels, indicating that AGP expression in myeloid cells, like in hepatocytes, is partially regulated by members of the C/EBP family. Overall, these findings define AGP as a genuine secondary granule protein of neutro- phils. Hence, neutrophils, which constitute the first line of defense, are likely to serve as the primary local source of AGP at sites of infection or injury. J. Leu- koc. Biol. 78: 000-000; 2005.