Ficolins are soluble pattern recognition molecules that bind carbohydrate structures on the surface of microorganisms. Three ficolins have been identified in man: ficolin-1, ficolin-2, and ficolin-3. Ficolin-1 is derived from the FCN1 gene on chromosome 9 and is synthesized in monocytes and type II alveolar epithelial cells. Ficolin-1 has been shown to be present in secretory granules of human neutrophils, but which subset of the neutrophils secretory granules harbors ficolin-1 is not known. In order to determine the exact subcellular localization of ficolin-1 in neutrophils, recombinant ficolin-1 was expressed in Chinese hamster ovary cells and used for generation of polyclonal antibodies. This allowed detection of ficolin-1 in subcellular fractions of human neutrophils by ELISA and western blotting, and by immunohistochemistry. Real time PCR examination of normal human bone marrow showed FCN1 gene expression largely in myelocytes, metamyelocytes, and band cells with a profile quite similar to that of gelatinase. In accordance with this, immunohistochemistry and subcellular fractionation demonstrated that ficolin-1 is primarily localized in gelatinase granules, but also in highly exocytosable gelatinase poor granules, not previously described. Ficolin-1 is released from neutrophil granules by stimulation with fMLP or PMA, and a significant part becomes associated with the surface membrane of the cells and can be detected by flow cytometry. Our studies show that neutrophils are a major source of ficolin-1, which can be readily released to the surroundings by stimulation. Figure. Left: Distribution profile of ficolin-1 in unstimulated (control) neutrophils and neutrophils stimulated with fMLP or PMA. Right: Distribution profile of ficolin-1 and granule marker proteins in subcellular fractions of unstimulated human neutrophils. Figure. Left: Distribution profile of ficolin-1 in unstimulated (control) neutrophils and neutrophils stimulated with fMLP or PMA. Right: Distribution profile of ficolin-1 and granule marker proteins in subcellular fractions of unstimulated human neutrophils.
3775 Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Figure 1. mRNA expression profiles for OLFM4 and LCN2 in populations enriched in myeloblasts/promyelocytes (MB/PM), myelocytes/metamyelocytes (MY/MM), banded cells/segmented cells (BC) and peripheral blood neutrophils (pb-PMN) normalized to ACTB. Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not. Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Figure 2. Immunohistochemistry of OLFM4 in neutrophils. Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist. Disclosures: No relevant conflicts of interest to declare.
3784 Alpha-1-antitrypsin (A1AT) is an important inhibitor of the neutrophil serine proteases elastase, cathepsin G, and proteinase 3. A1AT is produced mainly by the liver and secreted to plasma. A1AT deficiency caused by the PiZZ mutation in the A1AT gene leads to accumulation of mutated A1AT in the liver which may induce liver cell necrosis and necessitate liver transplantation. In a recently performed profiling of mRNA expression during terminal neutrophil differentiation in the bone marrow, we found that A1AT mRNA increases from the promyelocyte stage and up, indicating that A1AT is a constituent of all neutrophil granules. We examined the localization and production of A1AT in healthy donor neutrophils and investigated whether the structure or function of neutrophils is affected in individuals with A1AT deficiency. RT- PCR for A1AT performed on neutrophil precursors isolated from normal human bone marrow showed that the mRNA level is highly upregulated as the cells mature in the bone marrow and even increases further as the cells enter the blood stream. Biosynthesis studies revealed that A1AT is produced by all stages of neutrophil maturation in the bone marrow and is efficiently retained in the cells as judged by pulse chase studies. Neutrophils from circulating blood also produce A1AT but this is not retained in the cells. Stimulation of neutrophils from peripheral blood with G-CSF during 24 hours resulted in a 20 fold increase in A1AT biosynthesis which was largely released to the medium. Subcellullar fractionation of blood neutrophils on a 4-layer Percoll density gradient revealed 3 forms of A1AT. A doublet band at 37 and 44 kD both with immunoreactivity against A1AT was observed in fractions corresponding to azurophil granules (cf biosynthesis of this form in promyelocytes). A band with mw of 52 kD, corresponding to the form present in blood plasma, was observed in fractions that contain NGAL, a marker of specific granules and in fractions that contain gelatinase. The 52 kD band was also observed in fractions containing albumin as expected, since secretory vesicles contain plasma proteins. The localization of A1at in neutrophil granules was further confirmed by exocytosis studies. Neutrophils were stimulated with PMA which mobilizes secretory vesicles and gelatinase granules efficiently and approximately 50% of specific granules. Only the 52 mw form of A1AT was released from cells during stimulation while none of the 37/44 double band was released. This is in agreement with localization of this double band in azurophil granules and with localization of the 52 kD form in specific granules, gelatinase granules and secretory vesicles. In addition, a high molecular weight form of A1AT was observed at 76 kD corresponding to the mw of A1AT complexed with neutrophil elastase. We isolated neutrophils from patients with the ZZ genotype of A1AT deficiency which had either been liver transplanted or lung transplanted. The neutrophils were examined by electron microscopy for detection of structural abnormalities and by exocytosis studies for detection of functional abnormalities. Electron micrographs did not reveal any abnormality in neutrophil structure and in none of the neutrophils examined (from 6 patients) did we observe abnormal granules akin to the intracellular accumulation of A1AT in liver cells from patients. We did, however, observe reduction in the total intracellular amount of A1AT in neutrophils from patients that had been lung transplanted but not in neutrophils from liver transplanted patients. This most likely reflects that secretory vesicles of neutrophils from lung transplanted will not contain A1AT as this is still severely deficient in plasma from lung transplanted patients, while liver transplanted patients will have normal levels of A1AT in plasma and hence take up normal amounts into their secretory vesicles. Release of granule proteins in response to stimulation by fMLP or PMA did not reveal any functional abnormality in neutrophils from A1AT deficient patients. Based on these studies we conclude that the A1AT deficiency does not inflict functional or structural abnormalities on neutrophils, and suggest that A1AT generated and released from neutrophils may contribute to anti-protease defense in tissues. Disclosures: No relevant conflicts of interest to declare.
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