Defensin-rich dense granules of human neutrophils

Abstract

Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta- glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

ProHNPs are the principal α‐defensins of human plasma

“…This is in agreement with the differences in routing of proHNP and mature HNP in neutrophil precursors which secrete most proHNP into plasma, while effectively storing HNP in azurophil granules [3,38]. In addition, azurophil granules largely fuse with phagosomes and only undergo a very low degree of exocytosis (<5%) upon stimulation [1,8,30]. The high BM/PM plasma ratio of proHNP substantiated BM cells as the major source of plasma proHNP.…”

“…Their granules contain proteins which are crucial for their capacity to kill microorganisms. Neutrophil a-defensins, also known as human neutrophil peptides 1-3 (HNP-1-3), are the most abundant of all neutrophil granule proteins, accounting for 5-7% of total cell protein [1]. HNPs belong to a family of small, cationic peptides with broad antimicrobial activities [2].…”

CXC chemokines and antimicrobial peptides in rhinovirus‐induced experimental asthma exacerbations

“…Antimicrobial peptides of the defensin or cathelicidin families comprise a significant part of the neutrophilic armamentarium against these pathogens [6]. The a-defensins (HNP 1-3) are stored in primary (azurophil) neutrophil granules and constitute 30-50% of the total protein of these organelles [9]. It has been hypothesized that human rhinovirus infections should increase levels of a-defensins in the airways [10], as they lead to marked neutrophil infiltration and degranulation in the airways [11] which are associated with clinical severity of virus-induced asthma [5,12].…”

The human neutrophil. Function and dysfunction

“…The peptides, 33-34 amino acids residues in length, are rich in cysteine and arginine. The defensins are localized in a high density subset of azurophil granules (94), where they constitute approximately 50% of the total amount of protein. The mechanism of action of these peptides has not been identified, but the antibiotic spectrum is very broad.…”