Intractable diarrhoea of infancy caused by neutrophil specific granule deficiency and cured by stem cell transplantation.
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Cytoplasmic Granules↗
Biomedical subjects
N Borregaard
Publications and source records attributed to N Borregaard.
Patient education--a strategy for prevention of infections caused by permanent central venous catheters in patients with haematological malignancies: a randomized clinical trial.
A functioning tunnelled central venous catheter (CVC) is a crucial device for patients with haematological malignancies receiving high-dose intravenous chemotherapy. Despite the advantages, CVC infections are a major cause of sepsis and prolonged hospital stay. This study investigated the impact of patient education regarding provision of their own catheter care on the frequency of CVC-related infections (CRIs) and was conducted at a specialized haematological unit at the University Hospital of Copenhagen Rigshospitalet. From May to September 2002, 82 patients fitted with tunnelled double-lumen Hickman catheters were randomized consecutively. The intervention group (42 participants) received individualized training and supervision by a clinical nurse specialist, with the aim of becoming independently responsible for their own catheter care. The control group (40 participants) followed the standard CVC procedures carried out by nurses inside and outside the central hospital. A significant reduction in CRIs was found in the intervention group, with a >50% reduction in the incidence rate of CRIs. We conclude that systematic individualized, supervised patient education is able to reduce catheter-related infections.
Adolescent↗
Isolation of human cationic antimicrobial protein-18 from seminal plasma and its association with prostasomes.
BACKGROUND: Cathelicidins are a group of antibiotic peptides with broad antimicrobial activity. They are considered to be an essential part of the innate immune system. The only known human cathelicidin is the human cationic antimicrobial protein (hCAP-18), from which the antimicrobial peptide LL-37 is released. METHODS AND RESULTS: In the present study, we purified hCAP-18 from seminal plasma and confirmed its identity by N-terminal amino acid sequencing. Gel filtration of seminal plasma showed the presence of hCAP-18 in both a low and a high molecular weight peak. Fractions corresponding to the high molecular form of hCAP-18 also contained dipeptidyl peptidase IV (CD26), a prostasome marker. This finding suggested that hCAP-18 found in fractions corresponding to high molecular weight molecules, is prostasome-associated. Flow cytometry confirmed the association of hCAP-18 with prostasomes and indicated that the molecule is surface bound. Western blot showed the presence of intact hCAP-18 in sperm, prostasomes and ultracentrifuged seminal plasma. CONCLUSIONS: These findings suggest that hCAP-18 may have an important role in antimicrobial defence during human reproduction. The binding of hCAP-18 to prostasomes indicates that protasomes can serve as a reservoir of this precursor of the antibiotic peptide LL-37.
Antimicrobial Cationic Peptides↗
Structure of Ca(2+)-loaded human grancalcin.
Grancalcin is a cytosolic Ca(2+)-binding protein originally identified in human neutrophils. It belongs to a new class of EF-hand proteins, called PEF proteins, which contain five EF-hand motifs. At the N-terminus of grancalcin there is a approximately 50 residue-long segment rich in glycines and prolines. The fifth EF-hand, unpaired within the monomer, provides a means for dimerization through pairing with its counterpart in a second molecule. The structure of full-length grancalcin in the apo form and with one EF3 within the dimer occupied by a Ca(2+) ion have been determined. Although the N-terminal segment was present in the molecule, this part was disordered in the crystals. Here, the structure of a truncated form of grancalcin, which is lacking 52 N-terminal residues, in the presence and absence of Ca(2+) is presented. In the Ca(2+)-bound form the ions are found in the EF1 and EF3 hands. Binding of Ca(2+) to these two EF hands produces only minor conformational changes, mostly within the EF1 Ca(2+)-binding loop. This observation supports the hypothesis, formulated on the basis of the structure of a homologous protein ALG-2 which shows significant differences in the orientation of EF4 and EF5 compared with grancalcin, that calcium is a necessary factor but not sufficient alone for inducing a significant conformational change in PEF proteins.
Amino Acid Sequence↗
The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL.
Detection of matrix metalloproteinase (MMP) activities in the urine from patients with a variety of cancers has been closely correlated to disease status. Among these activities, the presence of a group of high molecular weight (HMW) MMPs independently serves as a multivariate predictor of the metastatic phenotype (). The identity of these HMW MMP activities has remained unknown despite their novelty and their potentially important applications in non-invasive cancer diagnosis and/or prognosis. Here, we report the identification of one of these HMW urinary MMPs of approximately 125-kDa as being a complex of gelatinase B (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL). Multiple biochemical approaches verified this identity. Analysis using substrate gel electrophoresis demonstrated that the 125-kDa urinary MMP activity co-migrates with purified human neutrophil MMP-9 x NGAL complex. The 125-kDa urinary MMP-9 x NGAL complex was recognized by a purified antibody against human NGAL as well as by a monospecific anti-human MMP-9 antibody. Furthermore, these same two antibodies were independently capable of specifically immunoprecipitating the 125-kDa urinary MMP activity in a dose-dependent manner. In addition, the complex of MMP-9 x NGAL could be reconstituted in vitro by mixing MMP-9 and NGAL in gelatinase buffers with pH values in the range of urine and in normal urine as well. Finally, the biochemical consequences of the NGAL and MMP-9 interaction were investigated both in vitro using recombinant human NGAL and MMP-9 and in cell culture by overexpressing NGAL in human breast carcinoma cells. Our data demonstrate that NGAL is capable of protecting MMP-9 from degradation in a dose-dependent manner and thereby preserving MMP-9 enzymatic activity. In summary, this study identifies the 125-kDa urinary gelatinase as being a complex of MMP-9 and NGAL and provides evidence that NGAL modulates MMP-9 activity by protecting it from degradation.
Acute-Phase Proteins↗
Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3.
Cathelicidins are a family of antimicrobial proteins found in the peroxidase-negative granules of neutrophils. The known biologic functions reside in the C-terminus, which must be cleaved from the holoprotein to become active. Bovine and porcine cathelicidins are cleaved by elastase from the azurophil granules to yield the active antimicrobial peptides. The aim of this study was to identify the physiological setting for cleavage of the only human cathelicidin, hCAP-18, to liberate the antibacterial and cytotoxic peptide LL-37 and to identify the protease responsible for this cleavage. Immunoelectron microscopy demonstrated that both hCAP-18 and azurophil granule proteins were present in the phagolysosome. Immunoblotting revealed no detectable cleavage of hCAP-18 in cells after phagocytosis. In contrast, hCAP-18 was cleaved to generate LL-37 in exocytosed material. Of the 3 known serine proteases from azurophil granules, proteinase 3 was solely responsible for cleavage of hCAP-18 after exocytosis. This is the first detailed study describing the generation of a human antimicrobial peptide from a promicrobicidal protein, and it demonstrates that the generation of active antimicrobial peptides from common proproteins occurs differently in related species. (Blood. 2001;97:3951-3959)
Antimicrobial Cationic Peptides↗
Profiling of gene expression in individual hematopoietic cells by global mRNA amplification and slot blot analysis.
The pattern of expressed genes defines the structure and functional status of cells. Currently, most methods used in gene expression studies depend on large numbers of cells. Thus, their application may be hampered by the heterogeneity of cell populations, and by the low numbers of cells obtainable from in vivo sources. Such drawbacks may be overcome by methods suitable for the profiling of gene expression at the single cell level. We studied whether polymerase chain reaction (PCR) products synthesized from individual cells by global amplification of messenger RNA (mRNA) were suitable as probes for gene expression analysis. For this purpose, cells were subjected to reverse transcription and PCR using sequence independent primers (SIP RT-PCR). The resultant cDNA products were radiolabeled and hybridized to cDNA clones arrayed on a nylon membrane by vacuum slot blotting (a method referred to as slot blot analysis). The SIP RT-PCR procedure was reproducible and allowed the detection of twofold changes in input RNA copies per cell (range: 80-10.000 copies of an in vitro transcribed poly(A)-tailed RNA/cell). Analysis of total RNA and amplified cDNA, obtained from neutrophil granulocytes and the promyelocytic HL-60 cell line, demonstrated comparable gene expression profiles as measured by Northern blot and slot blot analysis. Slot blot analysis of HL-60 cells indicated that individual cells from an apparently homogenous population have varying expression of specific transcripts, which all contribute to the mRNA phenotype of their population. Interestingly, the genes that were detected in some but not all individual HL-60 cells were those found to peak within 2 days of retinoic acid-induced granulocytic differentiation. This study demonstrates the potential of cDNA, synthesized from individual cells by global amplification of mRNA, as probes for cDNA arrays.
Blotting, Northern↗
Deficiency of the specific granule proteins, R-binder/transcobalamin I and lactoferrin, in plasma and saliva: a new disorder.
The mechanisms of hereditary deficiency of R binder, which originates in neutrophils and exocrine gland epithelium, are unknown and may be multiple. This led us to examine if defective R binder synthesis also involves proteins that colocalize with it in neutrophil-specific granules and exocrine epithelial cells and may be under common regulatory control. Stored plasma and saliva samples from five unrelated R binder-deficient patients and control subjects were assayed for R binder, lactoferrin, cationic antimicrobial protein-18, neutrophil gelatinase-associated lipocalin, gelatinase, lysozyme, and myeloperoxidase. One patient, patient A, had lactoferrin levels below the limits of detection in both plasma and saliva in addition to his R binder deficiency. Although his deficiency involved lactoferrin as well, he had no history of predisposition to infection. PCR amplification of his R binder gene promoter region and the beginning of the first exon revealed no DNA abnormalities. His son and the son of his equally deficient brother, both presumptive heterozygotes, had mild deficiency of both R binder and lactoferrin. The results show that R binder deficiency exists in at least two forms. One, presumably the less common of the two forms, is the new hereditary entity described here, which is characterized by deficiency of more than one specific granule protein in both plasma and saliva. Despite this more widely distributed absence of the proteins than is found in congenital specific granule deficiency, infection posed no clinical problem in the affected patient.
Female↗
Subcellular distribution and cytokine- and chemokine-regulated secretion of leukolysin/MT6-MMP/MMP-25 in neutrophils.
Leukolysin, originally isolated from human leukocytes, is the sixth member of the membrane-type matrix metalloproteinase (MT-MMP) subfamily with a potential glycosylphosphatidylinositol (GPI) anchor. To understand its biological functions, we screened subpopulations of leukocytes and localized the expression of leukolysin at the mRNA level to neutrophils. Polyclonal and mono-specific antisera raised against a synthetic peptide from its hinge region recognized a major protein species at 56 kDa and several minor forms between 38 and 45 kDa in neutrophil lysates. In resting neutrophils, leukolysin is distributed among specific granules ( approximately 10%), gelatinase granules ( approximately 40%), secretory vesicles ( approximately 30%), and the plasma membrane ( approximately 20%), a pattern distinct from that of neutrophil MMP-8 and MMP-9. Consistent with its membrane localization and its reported GPI anchor, leukolysin partitions into the detergent phase of Triton X-114 and can be released from intact resting neutrophils by glycosylphosphatidylinositol-specific phospholipase C. Phorbol myristate acetate stimulates neutrophils to discharge 100% of leukolysin from specific and gelatinase granules and approximately 50% from the secretory vesicles and plasma membrane, suggesting that leukolysin can be mobilized by physiological signals to the extracellular milieu as a soluble enzyme. Indeed, interleukin 8, a neutrophil chemoattractant, triggered a release of approximately 85% of cellular leukolysins by a process resistant to a mixture of proteinase inhibitors, including aprotinin, BB-94, pepstatin, and E64. Finally, purified recombinant leukolysin can degrade components of the extracellular matrix. These results not only establish leukolysin as the first neutrophil-specific MT-MMP but also implicate it as a cytokine/chemokine-regulated effector during innate immune responses or tissue injury.
Amino Acid Sequence↗
Biochemical characterization of the penta-EF-hand protein grancalcin and identification of L-plastin as a binding partner.
Grancalcin is a recently described Ca(2+)-binding protein especially abundant in human neutrophils. Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca(2+) although it has an unusual Ca(2+) coordination loop and one that does not bind Ca(2+) but is directly involved in homodimerization. We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated. This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca(2+) load. Flow dialysis showed that recombinant grancalcin binds two Ca(2+) per subunit with positive cooperativity and moderate affinity ([Ca(2+)](0.5) of 25 and 83 microm in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca(2+)-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca(2+) and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca(2+)-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice versa.
Calcium↗
Regulation of human neutrophil granule protein expression.
The function of the mature polymorphonuclear neutrophil is dependent on its granules, each with its characteristic content of proteins. The granule proteins are formed at different stages during maturation of neutrophils from myeloblasts to segmented cells. The regulation of granule protein expression is controlled by a number of transcription factors, many of which are also essential for commitment of multipotent hematopoietic stem cells to lineage-committed myeloid progenitor cells and for differentiation of these progenitor cells; among these, PU.1 and C/EBPalpha stand out as critical for all granule proteins whereas AML-1 is critical for primary granule protein expression and C/EBPepsilon for secondary and tertiary granule protein expression.
Blood Proteins↗
Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse.
Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa lipocalin originally purified from human neutrophils. It exists in monomeric and homo- and heterodimeric forms, the latter as a dimer with human neutrophil gelatinase. It is secreted from specific granules of activated human neutrophils. Homologous proteins have been identified in mouse (24p3/uterocalin) and rat (alpha(2)-microglobulin-related protein/neu-related lipocalin). Structural data have confirmed a typical lipocalin fold of NGAL with an eight-stranded beta-barrel, but with an unusually large cavity lined with more polar and positively charged amino acid residues than normally seen in lipocalins. Chemotactic formyl-peptides from bacteria have been proposed as ligands of NGAL, but binding experiments and the structure of NGAL do not support this hypothesis. Besides neutrophils, NGAL is expressed in most tissues normally exposed to microorganisms, and its synthesis is induced in epithelial cells during inflammation. This may indicate either a microbicidal activity of NGAL or a role in regulation of inflammation or cellular growth, putative functions yet to be demonstrated.
Acute-Phase Proteins↗
Crystal structure of human grancalcin, a member of the penta-EF-hand protein family.
Grancalcin is a Ca(2+)-binding protein expressed at high level in neutrophils. It belongs to the PEF family, proteins containing five EF-hand motifs and which are known to associate with membranes in Ca(2+)-dependent manner. Prototypic members of this family are Ca(2+)-binding domains of calpain. Our recent finding that grancalcin interacts with L-plastin, a protein known to have actin bundling activity, suggests that grancalcin may play a role in regulation of adherence and migration of neutrophils. The structure of human grancalcin has been determined at 1.9 A resolution in the absence of calcium (R-factor of 0.212 and R-free of 0.249) and at 2. 5 A resolution in the presence of calcium (R-factor of 0.226 and R-free of 0.281). The molecule is predominantly alpha-helical: it contains eight alpha-helices and only two short stretches of two-stranded beta-sheets between the loops of paired EF-hands. Grancalcin forms dimers through the association of the unpaired EF5 hands in a manner similar to that observed in calpain, confirming this mode of association as a paradigm for the PEF family. Only one Ca(2+) was found per dimer under crystallization conditions that included CaCl(2). This cation binds to EF3 in one molecule, while this site in the second molecule of the dimer is unoccupied. This unoccupied site shows higher mobility. The structure determined in the presence of calcium, although does not represent a fully Ca(2+)-loaded form, suggests that calcium induces rather small conformational rearrangements. Comparison with calpain suggests further that the relatively small magnitude of conformational changes invoked by calcium alone may be a characteristic feature of the PEF family. Moreover, the largest differences are localized to the EF1, thus supporting the notion that calcium signaling occurs through this portion of the molecule and that it may involve the N-terminal Gly/Pro rich segment. Electrostatic potential distribution shows significant differences between grancalcin and calpain domain VI demonstrating their distinct character.
Amino Acid Sequence↗
Ligand preference inferred from the structure of neutrophil gelatinase associated lipocalin.
Neutrophil gelatinase associated lipocalin (NGAL), a constituent of neutrophil granules, is a member of the lipocalin family of binding proteins. NGAL can also be highly induced in epithelial cells in both inflammatory and neoplastic colorectal disease. NGAL is proposed to mediate inflammatory responses by sequestering neutrophil chemoattractants, particularly N-formylated tripeptides and possibly leukotriene B(4) and platelet activating factor. The crystal structures of NGAL display a typical lipocalin fold, albeit with an unusually large and atypically polar binding site, or calyx. The fold of NGAL is most similar to the epididymal retinoic acid-binding protein, another lipocalin, though the overall architecture of the calyces are very different. The crystal structures also reveal either sulfate ions or an adventitiously copurified fatty acid bound in the binding site. Neither ligand is displaced by added N-formylated tripeptides. The size, shape, and character of the NGAL calyx, as well as the low relative affinity for N-formylated tripeptides, suggest that neither the copurified fatty acid nor any of the proposed ligands are likely to be the preferred ligand of this protein. Comparisons between the crystal structures and the recently reported solution structure of NGAL reveal significant differences, in terms of both the details of the structure and the overall flexibility of the fold.
Acute-Phase Proteins↗
Innate immunity: from plants to humans.
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Animals↗
The human cationic antimicrobial protein (hCAP-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa.
Innate immunity is important for the integrity of the host against potentially invasive pathogenic microorganisms in the environment. Antibiotic peptides with broad antimicrobial activity are part of the innate immune system. We investigated the presence of the cathelicidin, human cationic antimicrobial protein (hCAP-18), in the male reproductive system. We found strong expression of the hCAP-18 gene by in situ hybridization and hCAP-18 protein, as detected by immunohistochemistry, in the epithelium of the epididymis, but not in the testis. The highest expression in the epididymis was in the caudal part. Western blotting showed a doublet band, the upper part corresponding to the size of hCAP-18 in plasma and neutrophils. Using a specific enzyme-linked immunosorbent assay (ELISA), levels of 86.5 +/- 37.8 microg/ml (mean +/- standard deviation; range, 41.8 to 142.8 microg/ml; n = 10) were detected in seminal plasma from healthy donors, which is 70-fold higher than the level in blood plasma. Flow cytometry and immunocytochemistry revealed the presence of hCAP-18 on spermatozoa. ELISA measurement showed levels of 196 ng/10(6) spermatozoa, corresponding to 6.6 x 10(6) molecules of hCAP-18 per spermatozoon. Our results suggest a key role for hCAP-18 in the antibacterial integrity of the male reproductive system. The attachment of hCAP-18 to spermatozoa may implicate a role for hCAP-18 in conception.
Anti-Bacterial Agents↗
Subcellular fractionation of human neutrophils on Percoll density gradients.
Subcellular fractionation has been an important tool in the investigation of neutrophil structural organization including granule heterogeneity, composition and mobilization. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nitrogen cavitation as an efficient but gentle means of disrupting neutrophils and with Percoll as a density medium. This paper describes in detail the methodology of subcellular fractionation of nitrogen cavitated neutrophils on one-, two-, and three-layer Percoll density gradients. Appropriate marker proteins are presented for neutrophil organelles including azurophil, specific and gelatinase granules, in addition to secretory vesicles and plasma membranes. The dynamics of granule and secretory vesicle exocytosis is demonstrated by subcellular fractionation of resting and activated human neutrophils. Finally, the paper describes the applications of subcellular fractionation in the investigation of the localization of neutrophil constituents, in protein purification schemes and in the study of translocation of cytosolic proteins to isolated neutrophil organelles.
Biological Transport↗