No abstract

No abstract

Granules are essential for the ability of neutrophils to fulfill their role in innate immunity. Granule membranes contain proteins that react to environmental cues directing neutrophils to sites of infection and initiate generation of bactericidal oxygen species. Granules are densely packed with proteins that contribute to microbial killing when liberated to the phagosome or extracellularly. Granules are, however, highly heterogeneous and are traditionally subdivided into azurophil granules, specific granules, and gelatinase granules in addition to secretory vesicles. This review will address issues pertinent to formation of granules, which is a process intimately connected to maturation of neutrophils from their precursors in the bone marrow. We further discuss possible mechanisms by which decisions are made regarding sorting of proteins to constitutive secretion or storage in granules and how degranulation of granule subsets is regulated.

Synthesis of the antimicrobial protein neutrophil gelatinase-associated lipocalin (NGAL) increases dramatically in bronchial epithelial cells and alveolear type II pneumocytes during lung inflammation. IL-1β induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-α, IL-6, and LPS had no effect. Similar IL-1β selectivity was demonstrated in primary bronchial epithelial cells and epidermal keratinocytes and for an NGAL promoter fragment transfected into A549 cells. By deletion and substitution analysis of the NGAL promoter, a 40-bp region containing an NF-κB consensus site was found to control the IL-1β-specific up-regulation. Involvement of the NF-κB site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-κB pathway, and by EMSA. TNF-α activation of NF-κB, in contrast, did not increase NGAL synthesis, even though induced binding of NF-κB to the NGAL promoter was observed in vitro. IL-1β specificity was not contained within the NF-κB site of the NGAL promoter, as determined by exchanging the NGAL promoter′s NF-κB-binding sequence with that of the IL-8 promoter or with the NF-κB consensus sequence and by testing the NF-κB-binding sequence of the NGAL promoter against the heterologous SV40 promoter. Selectivity for the IL-1 pathway was substantiated by demonstrating that NGAL promoter activity could be induced by LPS stimulation of A549 cells transiently expressing Toll-like receptor 4, which use the same intracellular signaling pathway as the IL-1R. Together, this demonstrates a selective up-regulation of NGAL by the IL-1 pathway.

No abstract

In addition to acting as a physical barrier against microorganisms, the skin produces antimicrobial peptides and proteins. After wounding, growth factors are produced to stimulate the regeneration of tissue. The growth factor response ceases after regeneration of the tissue, when the physical barrier protecting against microbial infections is re-established. We found that the growth factors important in wound healing, insulin-like growth factor I and TGF-α, induce the expression of the antimicrobial peptides/polypeptides human cationic antimicrobial protein hCAP-18/LL-37, human β-defensin 3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor in human keratinocytes. Both an individual and a synergistic effect of these growth factors were observed. These findings offer an explanation for the expression of these peptides/polypeptides in the skin disease psoriasis and in wound healing and define a host defense role for growth factors in wound healing.

To characterize the transcriptional program that governs terminal granulocytic differentiation in vivo, we performed comprehensive microarray analyses of human bone marrow populations highly enriched in promyelocytes (PMs), myelocytes/ metamyelocytes (MYs), and neutrophils (bm-PMNs). These analyses identified 11 310 genes involved in differentiation, of which 6700 were differentially regulated, including previously unidentified effector proteins and surface receptors of neutrophils. Differentiation of PMs toward MYs was accompanied by a marked decline of proliferative and general cellular activity as defined by down-regulation of E2 promoter binding factor (E2F) target genes; cyclin dependent kinases 2, 4, and 6; and various metabolic, proteasomal, and mitochondrial genes. Expression patterns of apoptosis genes indicated death control by the p53 pathway in PMs and by death receptor pathways in bm-PMNs. Effector proteins critical for host defense were expressed successively throughout granulocytic differentiation, whereas receptors and receptor ligands essential for the activation of the host defense program were terminally up-regulated in bm-PMNs. The up-regulation of ligandreceptor pairs, which are defined inducers as well as target genes of nuclear factor-B (NF-B), suggests a constitutive activation of NF-B in bm-PMNs by autocrine loops. Overall IntroductionPolymorphonuclear neutrophilic granulocytes (neutrophils/PMNs) constitute the most abundant population of white blood cells and are essential players in innate immune defense of mammalian hosts against microorganisms. Once neutrophils have migrated to sites of infection, they recognize microorganisms and their products to initiate a first line of defense using a number of distinct mechanisms. These defense mechanisms include phagocytosis, generation of reactive oxygen intermediates, and the release of antimicrobial granule proteins for killing and degradation of microorganisms. 1,2 Neutrophils are short-lived cells, which are continuously generated from hematopoietic stem cells (HSCs) in the bone marrow (BM) by a process called granulopoiesis. The hallmark of early granulopoiesis is the successive commitment of pluripotent HSCs via multipotent common myeloid progenitors (CMPs) and bipotent granulocyte-macrophage progenitors (GMPs) toward unipotent progenitors restricted to the granulocytic lineage. 3,4 Once the progenitors are committed to the granulocytic lineage, they initiate terminal granulopoiesis and differentiate into mature neutrophils. Terminal granulopoiesis gives rise to a series of morphologically distinct stages, which are readily identified by their characteristic nuclear shape and their content of granules. At the myeloblast/ promyelocyte (MB/PM) stages the cells still proliferate and generate primary granules with their constituting proteins. At the myelocyte/metamyelocyte (MC/MM) stages, cell proliferation and expression of primary granule proteins stop concomitantly with the successive generation of secondary and tertiary granules and the...

A 19 kDa protein was identified in specific granules of human neutrophils. A full-length cDNA clone was isolated from a human CML cDNA library, based on amino-acid sequences of isolated tryptic fragments. This clone includes the recently identified cDNA for FALL-391CAP-18. Aminoacid sequences of proteolytic fragments derived both from the conserved N-terminal cathelin-like region and the highly variable C-terminal region characteristic of this family of bactericidal, LPS binding proteins, were in complete agreement with the sequence deduced from the cDNA. Thus, the 19 kDa protein is hCAP-18, stored as a 'pro-peptide' in specific granules. Materials and methods Subcellular fractionationHuman neutrophils were isolated from freshly drawn blood and disrupted by nitrogen cavitation following treatment with 5 mM DFP (Aldrich) [6]. The post-nuclear supernatant (10 ml), was loaded on a 3-layer Percoll (Pharmacia) density gradient (gradient volume 27 ml) and fractionated [5]. Azurophil granules were identified by their content of myeloperoxidase [7], specific granules by lactoferrin [7], gelatinase granules by gelatinase [5,8], and plasma membranes by HLA, all assayed by ELISA [5,9,10]. Secretory vesicles were identified by their content of latent alkaline phosphatase [4], assayed as described [11] and by their content of albumin, assayed by ELISA [12].