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H Sengeløv

Publications and source records attributed to H Sengeløv.

At least 19 recordsLinked to original sources
PubMed2003

A comparison of T-, B- and NK-cell reconstitution following conventional or nonmyeloablative conditioning and transplantation with bone marrow or peripheral blood stem cells from human leucocyte antigen identical sibling donors.

This retrospective study compares the reconstitution of T, B and NK cells in three groups of patients transplanted for haematological malignancies with grafts from their HLA-identical sibling donors. In all, 15 patients received PBSC after a nonmyeloablative conditioning regimen consisting of fludarabine and 200 cGy TBI, 13 patients received PBSC after myeloablative conditioning and 37 patients received BM after myeloablative conditioning. In the nonmyeloablative group, the NK cells normalised after 1 month, the CD8+ T cells normalised after 3 months, the CD4+ T cells reached near normal values after 9 months and the B cell values were reduced until 12 months after transplant. In the two myeloablative groups, recipients of PBSC had a significantly higher number of CD4+ T cells after 4 months (P=0.004) and after 12 months (P=0.001), than recipients of BM. We found no differences in the T cell reconstitution between the two PBSC groups. This was of interest as the recipients of nonmyeloablative conditioning were older (P<0.001) and had a higher occurrence of chronic GVHD (P<0.05) than the recipients of myeloablative conditioning. In contrast, the recipients of nonmyeloablative conditioning had a delayed B cell recovery when compared to the patients who received myeloablative conditioning (P=0.04).

Adolescent↗
PubMed2000

Solid-phase synthesis of chemotactic peptides using alpha-azido acids.

Four chemotactic peptides, For-Met-Xxx-Phe-OMe, with an alpha,alpha-disubstituted amino acid at position 2 have been synthesized by the azido acid method [Meldal M, Juliano MA, Jansson AM. 1997. Azido acids in a novel method of solid-phase peptide synthesis. Tetrahedron Lett. 38: 2531-2534] on solid-phase, and were tested for biological activity. Dipropylglycine in the central position (Xxx) was found to be as active as the natural chemotactic peptide for chemotactic activity toward human neutrophils. Higher yields were obtained than previously reported solution-phase syntheses of chemotactic peptides, and EEDQ was used successfully for the difficult solid-phase formylation of amino groups.

Amino Acid Sequence↗
PubMed1998

Azurophilic granules of human neutrophilic leukocytes are deficient in lysosome-associated membrane proteins but retain the mannose 6-phosphate recognition marker.

During granulocyte differentiation in the bone marrow (BM), neutrophilic leukocyte precursors synthesize large amounts of lysosomal enzymes. These enzymes are sequestered into azurophilic storage granules until used days later for digestion of phagocytized microorganisms after leukocyte emigration to inflamed tissues. This azurophil granule population has previously been defined as a primary lysosome, i.e., a membrane-bound organelle containing acid hydrolases that have not entered into a digestive event. In this study, azurophil granules were purified and shown to contain large amounts of mannose 6-phosphate-containing glycoproteins (Man 6-P GP) but little lysosome-associated membrane proteins (LAMP). In addition, the fine structural localization of Man 6-P GP and LAMP was investigated at various stages of maturation in human BM and blood. Man 6-P GP were present within the azurophilic granules at all stages of maturation and in typical multivesicular bodies (MVB) as well as in multilaminar compartments (MLC), identified by their content of concentric arrays of internal membranes. LAMP was absent in all identified granule populations, but was consistently found in the membranes of vesicles, MVB, and MLC. The latter compartment has not been previously described in this cell type. In conclusion, the azurophilic granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By current criteria, they therefore cannot be classified as lysosomes, but rather may have the functional characteristics of a regulated secretory granule. Rather, the true lysosomes of the resting neutrophil are probably the MVB and MLC. Finally, the typical "dense bodies" or mature lysosomes described in other cells are not present in resting neutrophils.

Antigens, CD↗
PubMed1998

Human neutrophils are devoid of the integral membrane protein caveolin.

The secretory vesicles of human neutrophils are rapidly mobilizable vesicles that contain several GPI-linked proteins, a characteristic feature of caveolae in other cells. To investigate whether secretory vesicles are structurally related to caveolae, we examined human neutrophils for the presence of caveolin, a major constituent of caveolae, by immunoblotting using monoclonal and polyclonal antibodies. Caveolin was not detected in lysates of human neutrophils nor in isolated plasma membrane/light membrane fractions in which secretory vesicles localize. In contrast, caveolin was readily detected in isolated membranes of adipose cells. We conclude that human neutrophils are devoid of caveolin and that secretory vesicles are not related to caveolae nor dependent on caveolae for mobilization.

Adipocytes↗
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

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

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↗
PubMed1995

Complement receptors in neutrophils.

Human neutrophils contain numerous intracellular granules and vesicles that are exocytosed in a hierarchic manner on stimulation of the neutrophil with inflammatory mediators. Secretory vesicles are mobilized completely to the plasma membrane when neutrophils are stimulated with inflammatory mediators in nanomolar concentrations. There is evidence that secretory vesicles contain at least four different complement receptors, namely, C1qR, CR1, CR3, and CR4. These complement receptors are all transported to the neutrophil surface in parallel by the mobilization of secretory vesicles. A fifth complement receptor, C5aR, is constitutively present on the neutrophil plasma membrane. Because secretory vesicles are mobilized by virtually all inflammatory mediators known to stimulate neutrophils, a prompt upregulation of complement receptors is ensured early in neutrophil activation. The integrins CR3 and CR4 are located both in secretory vesicles and granules, and this diverse subcellular localization may reflect their functional versatility. The residence of complement receptors in several intracellular compartments ensures a graded upregulation of these receptors in response to stimulation.

Humans↗
PubMed1994

Molecular cloning and expression of a cDNA encoding NGAL: a lipocalin expressed in human neutrophils.

NGAL, a protein recently isolated from human neutrophils, is a novel member of the lipocalins. NGAL binds a derivative of the bacterial chemotactic peptide formylmethionyl-leucyl-phenylalanine and may have important immunomodulatory functions. We here report the cloning of a cDNA for NGAL covering a 63 bp 5' untranslated region and the coding region of 591 bp. The cDNA encodes a protein of 197 amino acids, with a 19 amino acid leader sequence and a mature protein of 178 amino acids. Alignment of the cDNA sequence of NGAL to the rat analogue, alpha 2-microglobulin related protein, demonstrates a very high degree of conservation of this lipocalin. Northern blotting of a variety of tissues revealed that NGAL is mainly expressed in myeloid cells, where a signal of approximately 850 bp is observed. A faint signal was observed in fetal and adult human lung tissue. The molecular cloning of the NGAL cDNA allowed the recombinant production of NGAL in E. coli.

Acute-Phase Proteins↗
PubMed1994

Secretory vesicles are the intracellular reservoir of complement receptor 1 in human neutrophils.

The subcellular localization of complement receptor 1 (CR1) was investigated in human neutrophils. CR1 was located exclusively in the light membrane fractions containing secretory vesicles and plasma membranes in Percoll density gradients of unperturbed neutrophils. Separation of plasma membranes from secretory vesicles by high-voltage free-flow electrophoresis of the light membranes from the Percoll gradient revealed that more than 80% of the CR1 was located intracellularly in secretory vesicles. After weak stimulation of neutrophils, the increase in CR1 surface expression closely paralleled both surface increase of known secretory vesicle membrane Ags and the release of matrix proteins from secretory vesicles. More potent stimulation of the neutrophils did not enhance CR1 surface expression further, in agreement with the lack of CR1 in granules as demonstrated on Percoll gradient. CR3 (Mac-1), which has been shown to be located both in secretory vesicles and in neutrophil granules, was up-regulated in parallel with CR1 after weak stimuli, whereas a profound increase of CR3 was observed after more potent stimuli in accordance with granule mobilization. These results identify secretory vesicles as the reservoir of CR1, which translocates to the plasma membrane after weak stimulation, and underscore the functional significance of this recently identified organelle.

Cytoplasmic Granules↗
PubMed1994

Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils.

The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.

Cell Fractionation↗
PubMed1994

Isolation and characterization of gelatinase granules from human neutrophils.

We recently confirmed the existence of gelatinase granules as a subpopulation of peroxidase-negative granules by double-labeling immunogold electron microscopy on intact cells and by subcellular fractionation. Further characterization of gelatinase granules has been hampered by poor separation of specific and gelatinase granules on both two-layer Percoll gradients and sucrose gradients. We have developed a three-layer Percoll density gradient that allows separation of the different granules and vesicles from human neutrophils; in particular, it allows separation of specific and gelatinase granules. This allows us to characterize these two granule populations with regard to their content of membrane proteins, which become incorporated into the plasma membrane during exocytosis. We found that gelatinase granules, defined as peroxidase-negative granules containing gelatinase but lacking lactoferrin, contain 50% of total cell gelatinase, with the remaining residing in specific granules. Furthermore, we found that 20% to 25% of both the adhesion protein Mac-1 and the NADPH-oxidase component cytochrome b558 is localized in gelatinase granules. Although no qualitative difference was observed between specific granules and gelatinase granules with respect to cytochrome b558 and Mac-1, stimulation of the neutrophil with FMLP resulted in a selective mobilization of the least dense peroxidase-negative granules, ie, gelatinase granules, which, in concert with secretory vesicles, furnish the plasma membrane with Mac-1 and cytochrome b558. This shows that gelatinase granules are functionally important relative to specific granules in mediating early inflammatory responses.

Centrifugation, Density Gradient↗
PubMed1994

Identification of neutrophil gelatinase-associated lipocalin as a novel matrix protein of specific granules in human neutrophils.

Neutrophil gelatinase-associated lipocalin (NGAL) is a novel 25-kD protein of human neutrophils, that is in part covalently complexed with neutrophil gelatinase. However, both NGAL and gelatinase exist mainly in forms not associated with each other. An explanation for this phenomenon might be that the unassociated proteins reside in different subcellular compartments. The aim of the present study was to determine the subcellular localization of NGAL. An enzyme-linked immunosorbent assay (ELISA) for NGAL was developed using specific anti-NGAL antibodies. The assay was applied on subcellular fractions of neutrophils obtained after centrifugation of a postnuclear supernatant on a two-layer Percoll gradient. The distribution profile of NGAL was found to colocalize strictly with the distribution profile of lactoferrin. This was confirmed by immunogold double-labeling of frozen thin sections of neutrophils that showed a high degree of colocalization of NGAL and lactoferrin, and by exocytosis experiments, which showed lactoferrin, vitamin B12-binding protein, and NGAL to be similarly released upon stimulation. Therefore, NGAL is a novel matrix protein of specific granules and thus partly segregated from gelatinase, the major part of which is located in a separate compartment, the gelatinase granules. An ELISA specific for the NGAL/gelatinase complex was developed and the subcellular distribution and release of this complex was determined. The distribution and mobilization of the complex allowed us to confirm the existence of differentially mobilized granule subpopulations among peroxidase negative granules.

Acute-Phase Proteins↗