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Biomedical subjects

N Borregaard

Publications and source records attributed to N Borregaard.

At least 73 records · Page 4Linked to original sources
PubMed1996

Current concepts about neutrophil granule physiology.

During the past decade it has become clear that neutrophil granules are not simple bags of proteolytic and microbicidal substances. Their membranes contain proteins of paramount importance for the neutrophil to carry out its function as the body's primary mobile phagocyte. It has recently become evident that the heterogeneity of neutrophil granules is much wider than previously thought and that a highly mobilizeable organelle, termed the secretory vesicle, may play a key role in modulating the neutrophil's surface protein profile in response to chemotactic factors. Clearly correct targeting of both membrane and matrix proteins into these different mobilizeable organelles is important for optimal function of the neutrophil. The expanding knowledge of protein targeting into granules and of control of their subsequent mobilization under the specialized conditions of rolling, adhesion, diapedesis, and phagocytosis is the subject of this review.

Biomarkers↗
PubMed1996

Apoptosis following interleukin-2 withdrawal from T cells: evidence for a regulatory role of CD18 (beta 2-integrin) molecules.

Following a successful immune response against invading microorganisms, the majority of activated T cells is eliminated, while a minor fraction survives as memory T cells. A decline in T lymphocyte growth factors such as interleukin-2 (IL-2) appears to play a role in the elimination of previously activated T cells. Thus, removal of IL-2 from proliferating T cells not only induces growth arrest, but triggers a massive cell death due to apoptosis. While the apoptotic response involves a series of well-described events, it remains less clear how apoptosis is regulated following IL-2 withdrawal. Here, we provide evidence that CD18 molecules (beta 2-integrins) play a regulatory role in the apoptotic response following removal of IL-2 from previously activated, antigen specific CD4+ T cell lines. Thus, CD18 mAb inhibited the apoptotic response to IL-2 deprivation, whereas mAb against other adhesion molecules (CD28, CD29, CD49d, CD80, CD86) did not. Secondly, IL-2 withdrawal resulted in a retarded apoptotic response in LFA-1 (CD11a/CD18) negative T cells obtained from a leukocyte adhesion deficiency (LAD) patient, as compared to LFA-1 positive T cell lines. Thirdly, co-culture of LFA-1 positive- and negative-T cells at different ratios induced apoptotic responses that were higher than expected, had the two lymphocyte populations not been interacting and significantly higher than that seen in pure LFA-1 negative T cells. Supernatants from LFA-1 positive T cell cultures undergoing apoptosis did not induce an enhanced apoptotic responses in LFA-1 negative T cells, and, reversely, culture supernatants from LFA-1 negative T cells did not rescue LFA-1 positive cells from undergoing apoptosis. The apoptotic response was partly blocked by IL-15, a newly identified T cell growth factor. Taken together, these findings suggest that CD18 molecules (beta 2-integrins) play a regulatory role in the apoptotic response following cytokine withdrawal, and that the regulation is mediated, at least partly, through T-T cell interactions. Thus, apoptotic death following IL-2 deprivation appears to be under "social" control by surrounding T cells.

Apoptosis↗
PubMed1996

Induction of NGAL synthesis in epithelial cells of human colorectal neoplasia and inflammatory bowel diseases.

In inflammatory and neoplastic disorders of the colon a defect barrier function of the mucosa may result in absorption of bacterial products from the intestinal lumen. These products may further recruit inflammatory cells and thus augment the inflammatory response. A novel lipocalin in neutrophils, neutrophil gelatinase associated lipocalin (NGAL), with the ability to bind bacterial formylpeptides, has been described and therefore it is of interest to investigate the expression of this protein in diseases of the colon. Expression of NGAL was investigated by immunohistochemistry and by mRNA in situ hybridisation in normal colon and in neoplastic and inflammatory colorectal diseases. A very high expression of NGAL was seen in colonic epithelium in areas of inflammation, both in non-malignant epithelium (diverticulitis, inflammatory bowel disease, and appendicitis) as well as in premalignant and malignant neoplastic lesions of the colon. In adenocarcinoma, the NGAL expression was especially abundant in the transitional mucosa and in the superficial ulcerated area. On the other hand, no NGAL expression could be detected in lymph node metastases from these adenocarcinomas. A weak expression of NGAL in some epithelial cells was only occasionally seen in normal colon. In conclusion, NGAL synthesis is induced in epithelial cells in inflammatory and neoplastic, colorectal diseases. NGAL may serve an important anti-inflammatory function as a scavenger of bacterial products.

Acute-Phase Proteins↗
PubMed1996

Human neutrophil gelatinase and associated lipocalin in adult and localized juvenile periodontitis.

In search of direct in vivo evidence of matrix metalloproteinases (MMPs) in periodontal tissue destruction, we studied the presence and localization of MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL) in adult periodontitis (AP) and localized juvenile periodontitis (LJP) gingival tissue specimens by immunohistochemistry, and the activities of gelatinases by Western blot, enzymography, and activity measurements, using radioactive gelatin as substrate in gingival crevicular fluid (GCF) and saliva. In gingival tissue obtained from AP and LJP patients, polymorphonuclear leukocyte (PMN) 92-kDa MMP-9 and NGAL were seen in the connective tissue, but both the sulcular and the oral epithelia were consistently negative. Whereas PMNs located in the gingival blood vessels showed strictly cytoplasmic MMP-9 and NGAL immunoreactivities, in the case of PMN extravasation the staining reactions extended extracellularly. Gelatinase activities consisting mainly of 92-kDa gelatinase were increased in AP GCF relative to LJP GCF and periodontally healthy control GCF. Western blot with specific anti-NGAL antibodies revealed the presence of 25-kDa NGAL and its high-molecular-weight forms in AP and LJP GCF and saliva and in culture medium of oral keratinocytes, but not in gingival fibroblast culture medium. We conclude that extravasated degranulating PMNs are the major source of MMP-9 and NGAL in periodontitis gingiva, GCF, and saliva.

Acute-Phase Proteins↗
PubMed1996

Granules and secretory vesicles in human neonatal neutrophils.

Neonates have an increased susceptibility to bacterial infections that may be due to defective adherence and migration of neonatal neutrophils (NN). Because receptors of relevance for these activities are located mainly in intracellular granules and secretory vesicles that have only recently been characterized in adult neutrophils (AN), we investigated whether the same structures are present in NN and to what extent they are mobilized in response to chemotactic and inflammatory mediators. Subcellular fractionation of NN on a three-layer Percoll density gradient revealed that secretory vesicles, identified by latent alkaline phosphatase and albumin, are present in NN. We also demonstrated the presence of gelatinase granules distinct from specific granules, although the content of their respective markers, gelatinase and lactoferrin, was markedly reduced. The low content of lactoferrin may explain an observed lower isopycnic density of specific granules in NN. Mobilization of granules by a variety of stimuli was slightly higher in NN compared with AN, whereas mobilization of secretory vesicles was normal. This shows that NN contain both secretory vesicles and all subsets of granules identified in AN, and that these are readily mobilized, although a marked structural difference exists between peroxidase-negative granules of NN and AN that may reflect differences during myelopoiesis.

Alkaline Phosphatase↗
PubMed1995

Subcellular distribution of docking/fusion proteins in neutrophils, secretory cells with multiple exocytic compartments.

Neutrophils contain at least four distinct types of secretory organelles, which undergo exocytosis during infection and inflammation. The signaling pathways leading to secretion of individual granules and their kinetics of exocytosis vary greatly, causing temporal and regional differences in docking and fusion with the plasma membrane. As a step toward understanding the processes underlying differential granular secretion in neutrophils, we assessed the presence and distribution of a number of proteins reported to be involved in vesicular docking and/or fusion in other systems. Specific Abs were used for immunoblotting of cells fractionated by density gradients and free-flow electrophoresis, and for localization by confocal immunofluorescence and electron microscopy. Syntaxin 1, VAMP (vesicle-associated membrane protein)-1, synaptosome-associated protein-25 (SNAP-25), synaptophysin, and cellubrevin were not detectable in human neutrophils. In contrast, syntaxin 4, VAMP-2, and the 39-kDa isoform of secretory carrier membrane protein (SCAMP) were present. SCAMP was found mainly in secondary and tertiary granules and in a fraction containing secretory vesicles, but was virtually absent from the primary (lysosomal) granules. This profile is consistent with the proposed "post-Golgi" distribution of SCAMP. VAMP-2 was largely absent from primary and secondary granules, but concentrated in tertiary granules and secretory vesicles. This pattern of distribution parallels the increasing sensitivity of these exocytic compartments to intracellular free calcium. Accordingly, ionomycin induced translocation of VAMP-2 toward the plasma membrane. Syntaxin 4 was found almost exclusively in the plasma membrane, and it accumulated in lamellipodia of migrating cells. This regional accumulation may contribute to localized secretion into the phagosomal lumen.

Cells, Cultured↗
PubMed1995

An ELISA for grancalcin, a novel cytosolic calcium-binding protein present in leukocytes.

Grancalcin is a newly discovered cytosolic calcium-binding protein, belonging to the group of EF-hand proteins. Grancalcin is specifically associated with cells originating in the bone marrow. Grancalcin binds reversibly to secretory vesicles and plasma membranes in human neutrophils and might therefore play a role in the regulation of vesicle/granule exocytosis. We describe here the production of recombinant grancalcin, the generation of antibodies to the protein and the development of a specific, accurate and sensitive ELISA for the detection of human leukocytic grancalcin. This ELISA may be useful for monitoring leukocyte infiltration into tissues.

Antibody Specificity↗
PubMed1995

hCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules.

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-39/CAP-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.

Amino Acid Sequence↗
PubMed1995

Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils.

The extent of mobilization of four different intracellular compartments was measured during in vivo exudation of neutrophils into skin chambers and compared with resting neutrophils obtained from blood. Exudation of neutrophils induced increased surface expression of alkaline phosphatase, complement receptor 1, and Mac-1, and a complete loss of L-selectin. The increase in the content of surface molecules in the plasma membrane is in accordance with complete mobilization of secretory vesicles. Granule matrix proteins were secreted into the chamber fluid by the exudated neutrophils and the exocytosed proteins were recovered in the skin chamber fluid. Release of gelatinase from gelatinase granules was 38.1%, lactoferrin release from specific granules was 21.9%, and myeloperoxidase release from azurophil granules was 7.0%, clearly illustrating a hierarchy in mobilization among granules. When exudate neutrophils were stimulated with FMLP, additional mobilization of granules was observed and the rank order regarding release was preserved. This is the first report to evaluate the mobilization of secretory vesicles during in vivo exudation of human neutrophils. It is shown that secretory vesicles are regulated exocytotic vesicles that are fully mobilized during in vivo exudation. Once exocytosed, secretory vesicles are not re-formed within a period of 6 h.

Alkaline Phosphatase↗
PubMed1995

Biosynthesis of granule proteins in normal human bone marrow cells. Gelatinase is a marker of terminal neutrophil differentiation.

Differentiation and maturation of myeloid cells is characterized by the sequential acquisition of two distinct cytoplasmic granule subsets, azurophil granules and specific granules. We recently showed the existence of a third granule subset, gelatinase granules. To investigate whether appearance of gelatinase granules marks a further step in maturation of myeloid cells beyond the appearance of specific granules, we sorted normal human bone marrow cells into one of three groups according to maturity by centrifugation on Percoll density gradients. The biosynthesis of myeloperoxidase (MPO) (an azurophil granule marker), lactoferrin and neutrophil gelatinase-associated lipocalin NGAL (specific granules markers) and gelatinase was then studied in each of these groups. We found that gelatinase was synthesized mainly in the group containing band cells and segmented cells. This contrasted with lactoferrin and NGAL, which were synthesized almost exclusively in the group containing myelocytes and metamyelocytes, and with MPO, which was mainly synthesized in the group containing myeloblasts and promyelocytes. Immunocytochemistry was in full agreement with the biosynthesis data, and showed that gelatinase appears in band cells, whereas NGAL and lactoferrin both appear in myelocytes. Thus, acquisition of gelatinase granules marks a step in neutrophil differentiation beyond the appearance of specific granules.

Animals↗
PubMed1995

The exocytotic fusion pore of small granules has a conductance similar to an ion channel.

We measured capacitance changes in cell attached patches of human neutrophils using a high frequency lock-in method. With this technique the noise level is reduced to 0.025 fF such that capacitance steps of 0.1 fF are clearly detected corresponding to exo- and endocytosis of single 60 nm vesicles. It is thus possible to detect almost all known exocytotic and endocytotic processes including exocytosis of small neurotransmitter containing vesicles in most cell types as well as endocytosis of coated and uncoated pits. In neutrophils we demonstrate a stepwise capacitance decrease generated by 60-165 nm vesicles as expected for endocytosis of coated and non-coated pits. Following ionomycin stimulation a stepwise capacitance increase is observed consisting of 0.1-5 fF steps corresponding to the different granule types of human neutrophils from secretory vesicles to azurophil granules. The opening of individual fusion pores is resolved during exocytosis of 200 nm vesicles. The initial conductance has a mean value of 150 pS and can be as low as 35 pS which is similar to the conductance of many ion channels suggesting that the initial fusion pore is formed by a protein complex.

Cytoplasmic Granules↗
PubMed1995

Identification and characterization of gelatinases/type IV collagenases in jaw cysts.

The molecular mechanisms of jaw cyst expansion probably involve interactions of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs). In this study, molecular species of gelatinases present in neutral salt extracts of cyst walls and cyst fluids were characterized by functional activity measurements (type I gelatin and alpha-casein zymography) and immunologically (Western-blotting). The effects of various protein thiol-group or cysteine-switch reactants involved in the activation of collagenases were studied on cyst gelatinases and a gelatinases purified from human gingival fibroblasts (72 kD MMP-2), gingival keratinocytes (92 kD MMP-9) and polymorphonuclear neutrophilic leukocytes (92 kD MMP-9). Western-blotting revealed the presence of both 92 kD (MMP-9) and 72 kD (MMP-2) gelatinases in cyst wall extracts and cyst fluids. Western-blot studies further suggested that jaw cyst gelatinases were only in part complexed with and thus inhibited by TIMP-1 or TIMP-2, suggesting that both MMP-9 and MMP-2 may participate in cyst expansion. MMP-2 was also partially fragmented to a 68 kD form and additional lower molecular weight proteinases (< 60 kD) were detected by alpha-casein zymography and by Western-blotting, suggesting proteolytic fragmentation. MMP-9 was at least partially activated by all protein-thiol group reactants and rather resistant to oxidative inhibition by hypochlorite (NaOCl); in contrast, MMP-2 was activated by APMA but not at all by gold thioglucose (GTG) and was clearly inactivated by hypochlorite (NaOCl).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Lysozyme in human neutrophils and plasma. A parameter of myelopoietic activity.

Lysozyme was found to be present in all three types of human neutrophil granules (azurophil-, specific- and gelatinase granules) as determined by subcellular fractionation, employing a three-layer Percoll gradient and measurement of lysozyme by a novel ELISA. The content of lysozyme was also measured in plasma. In contrast to other neutrophil granule proteins (lactoferrin, NGAL, and gelatinase), plasma lysozyme was unaffected by increase in the number of circulating neutrophils induced by intravenous administration of methylprednisolone to healthy individuals. Also in contrast to lactoferrin, NGAL, and gelatinase, plasma lysozyme was found to rise 1 week prior to detectable increases in the number of circulating neutrophils in patients undergoing allogeneic bone marrow transplantation. We conclude that plasma lysozyme is a parameter of myelopoietic activity and may be useful as a marker for bone marrow repopulation after transplantation.

Bone Marrow Cells↗
PubMed1995

Purification of lysozyme from human neutrophils, and development of an ELISA for quantification in cells and plasma.

Lysozyme was purified from exocytosed granule material from PMA-stimulated human neutrophils by polyethyleneglycol precipitation, cation exchange chromatography and molecular sieve chromatography. Rabbit antibodies were biotinylated and affinity purified on a lysozyme column, for subsequent development of a novel ELISA. This ELISA for lysozyme is sensitive and accurate, and applicable to determination of lysozyme in neutrophils and plasma.

Animals↗