Biomedical subjects

C Dahlgren

Publications and source records attributed to C Dahlgren.

At least 19 recordsLinked to original sources

PubMed2003

Histamine inhibits neutrophil NADPH oxidase activity triggered by the lipoxin A4 receptor-specific peptide agonist Trp-Lys-Tyr-Met-Val-Met.

The vasoactive amine histamine is found at high concentrations in the immune and inflammatory tissues. Earlier studies have revealed that histamine regulates the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase-dependent formation of oxygen radicals by phagocytic cells. However, the effects of histamine on intracellular signal transduction mechanisms of relevance to oxidase regulation remain controversial. For this study, we investigated the effects of histamine on NADPH oxidase activity in human neutrophil granulocytes triggered by a lipoxin A4 receptor agonist [the hexapeptide Trp-Lys-Tyr-Met-Val-Met (WKYMVM), a formyl peptide receptor (FPR) agonist (the chemotactic tripeptide formylmethionyl-leucyl-phenylalanine (fMLF)) and an activator of protein kinase C (phorbol myristate acetate (PMA)]. We report that histamine, acting via H2-type histamine receptors (H2R), suppresses NADPH oxidase-dependent formation of oxygen radicals induced by WKYMVM and fMLF but not that induced by PMA. Peptide-induced mobilization of granule-localized complement receptor 3 (CR3) was unaffected by histamine suggesting that the inhibition specifically affected NADPH oxidase activation. Our data suggest that histamine downregulates FPRL1- and FPR-induced NADPH oxidase activity upstream of protein kinase C (PKC) and downstream of the separation of the peptide-induced signal into granule secretion and oxidase activation.

Chemotactic Factors↗

PubMed2002

Difference in neutrophil cytokine production induced by pathogenic and non-pathogenic mycobacteria.

This study shows differences between Mycobacterium tuberculosis and Mycobacterium avium (opportunistic) and Mycobacterium smegmatis (non-pathogenic), with respect to their abilities to induce cytokine/chemokine release from human neutrophils. Neutrophils incubated with live cells of M. tuberculosis, M. avium, or M. smegmatis produced and released TNF-alpha, IL-6, and IL-8. No or very small amounts of these cytokines/chemokines were found in resting neutrophils, suggesting that they were newly synthesised. The levels of TNF-alpha, IL-6, and IL-8 produced/released from neutrophils incubated with M. tuberculosis were markedly lower than those of the opportunistic or non-pathogenic bacterial species. The production of TNF-alpha reached a maximum level at a time (4 h) when the production of IL-8 had only just started, and this was true for all three mycobacteria tested. However, the time course for IL-6 production differed between the species, reaching a peak value after 8 h for M. tuberculosis not seen with the other bacteria. It is likely that the relatively high levels of cytokines induced by opportunistic/non-pathogenic mycobacteria are of importance for the induction of an innate immune response through which these organisms are eliminated, while the low levels of cytokines released by neutrophils interfering with M. tuberculosis might help the bacteria to persist.

Cells, Cultured↗

PubMed2002

Phagocyte activation by Trp-Lys-Tyr-Met-Val-Met, acting through FPRL1/LXA4R, is not affected by lipoxin A4.

Lipoxin A4 (LXA4) has been shown to bind to the leucocyte formyl peptide receptor (FPR) homologue, FPRL1, without triggering the biological activities induced by other FPRL1 agonists. We investigated the direct effect of LXA4 as well as the effect on agonist-induced biological responses using transfected HL-60 cells expressing FPR, FPRL1 or FPRL2. LXA4 neither induced an intracellular rise in calcium in these transfectants nor affected the response induced by the peptide Trp-Lys-Tyr-Met-Val-Met (WKYMVM), an agonist that activates cells through FPRL1 and -2. Both agonists induced Erk-2 activation; however, the eicosanoid-induced activity was independent of FPRL1 and FPRL2. Moreover, LXA4 was unable to trigger neutrophil upregulation of complement receptor 3 and respiratory burst, and it had no effect on the responses induced by triggering with WKYMVM. We conclude that LXA4 is unable to affect the WKYMVM-induced signalling through FPRL1 and suggest that it acts through a receptor different from FPRL1.

Calcium Signaling↗

PubMed2001

The synthetic peptide Trp-Lys-Tyr-Met-Val-Met-NH2 specifically activates neutrophils through FPRL1/lipoxin A4 receptors and is an agonist for the orphan monocyte-expressed chemoattractant receptor FPRL2.

Neutrophils express the G protein-coupled N-formyl peptide receptor (FPR) and its homologue FPRL1, whereas monocytes express FPR, FPRL1, and FPRL2, an orphan receptor sharing 83% amino acid identity with FPRL1. FPRL1 is a promiscuous receptor activated by serum amyloid A and by different synthetic peptides, including the hexapeptide Trp-Lys-Tyr-Met-Val-d-Met-NH(2) (WKYMVm). By measuring calcium flux in HL-60 cells transfected with FPR, FPRL1, or FPRL2, we show that WKYMVm activated all three receptors, whereas the l-conformer WKYMVM activated exclusively FPRL1 and FPRL2. The functionality of FPRL2 was further assessed by the ability of HL-60-FPRL2 cells to migrate toward nanomolar concentrations of hexapeptides. The half-maximal effective concentrations of WKYMVM for calcium mobilization in HL-60-FPRL1 and HL-60-FPRL2 cells were 2 and 80 nm, respectively. Those of WKYMVm were 75 pm and 3 nm. The tritiated peptide WK[3,5-(3)H(2)]YMVM bound to FPRL1 (K(D) approximately 160 nm), but not to FPR. The two conformers similarly inhibited binding of (125)I-labeled WKYMVm to FPRL2-expressing cells (IC(50) approximately 2.5-3 micrometer). Metabolic labeling with orthophosphoric acid revealed that FPRL1 was differentially phosphorylated upon addition of the l- or d-conformer, indicating that it induced different conformational changes. In contrast to FPRL1, FPRL2 was already phosphorylated in the absence of agonist and not evenly distributed in the plasma membrane of unstimulated cells. However, both receptors were internalized upon addition of either of the two conformers. Taken together, the results indicate that neutrophils are activated by WKYMVM through FPRL1 and that FPRL2 is a chemotactic receptor transducing signals in myeloid cells.

Animals↗

PubMed2001

Quantification of annexin I in subcellular fractions of human neutrophils reveals an exclusive cytosolic localisation.

Annexin I is an abundant cytosolic protein in human neutrophils. Besides its intracellular location, annexin I is found as an extracellular protein and the pathway for secretion has been of interest since the protein lacks a signal sequence for secretion. It was recently shown that annexin I is stored in the secretory gelatinase granules of human neutrophils, suggesting that the protein might be released through a granule mobilisation and fusion process resembling classical secretion. In this study we have determined the intracellular localisation of annexin I in human neutrophils using subcellular fractionation, protein separation by SDS-PAGE and immunoblotting, and show that virtually all annexin I is localised in the cell cytosol.

Annexin A1↗

PubMed2001

Priming of human neutrophils by mycobacterial lipoarabinomannans: role of granule mobilisation.

Lipoarabinomannans (LAMs) from mycobacteria were investigated concerning their effect on human neutrophils. Two types of LAM, the mannose-capped ManLAM from the virulent Mycobacterium tuberculosis H37Rv and the mannose-lacking AraLAM from a rapidly growing mycobacterial strain were used. Neither AraLAM nor ManLAM induced any significant direct activation of the NADPH-oxidase. Both LAMs, however, primed the neutrophils so that subsequent stimulation with the peptide chemoattractants fMet-Leu-Phe (fMLF), Trp-Lys-Tyr-Met-Val-DMet (WKYMVm) and the mammalian lactose-binding lectin galectin-3 resulted in a markedly enhanced oxidative response. The LAM-induced priming was accompanied by an increased exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the neutrophil surface, suggesting that the enhanced oxidative response could be due to upregulation of receptors on the cell surface as a result of granule mobilisation. Since LAM-primed neutrophils released 65% of the cell content of gelatinase but showed no increased release of vitamin B(12)-binding protein, mobilisation of the gelatinase granules rather than the specific granules is concluded to be responsible for the priming effects. This is in agreement with the subcellular localisation of receptors for fMLF, WKYMVm, as well as galectin-3, which are stored in the secretory vesicles and gelatinase granules. The priming effect appeared very similar to that of Escherichia coli lipopolysaccharide, and since no differences in activity could be detected between AraLAM and ManLAM, we hypothesize that the lipid anchor of the LAM is responsible for the priming effects.

Cytoplasmic Granules↗

PubMed2001

An intact cytoskeleton is required for prolonged respiratory burst activity during neutrophil phagocytosis.

The temporal relationship between phagocytosis and respiratory burst activity was investigated. Neutrophil uptake of yeast particles was synchronized and the kinetics of the oxidative burst was determined using an isoluminol/luminol amplified chemiluminescence system. The reactive oxygen species were mainly generated intracellularly (defined as the activity that remained in an luminol-enhanced system in the presence of superoxide dismutase and catalase). Following phagocytosis, the intracellular response rapidly reached a level close to the maximum and the activity was almost constant for the first 10 to 15 min. The response then slowly declined. The presence of cytochalasin B, an inhibitor of actin polymerization, greatly reduced the respiratory burst activity, and this was true also when the inhibitor was added after completion of uptake of yeast particles. Our results thus show that there is a continuous production of oxygen metabolites long after phagocytosis is completed. There is also a requirement for an intact cytoskeleton for prolonged superoxide production inside the phagosome.

Adult↗

PubMed2001

Different glycosphingolipid composition in human neutrophil subcellular compartments.

The binding of a number of carbohydrate-recognizing ligands to glycosphingolipids and polyglycosylceramides of human neutrophil subcellular fractions (plasma membranes/secretory vesicles of resting and ionomycin-stimulated cells, specific and azurophil granules) was examined using the chromatogram binding assay. Several organelle-related differences in glycosphingolipid content were observed. The most prominent difference was a decreased content of the GM3 ganglioside in plasma membranes of activated neutrophils. Gangliosides recognized by anti-VIM-2 antibodies were detected mainly in the acid fractions of azurophil and specific granules. Slow-migrating gangliosides and polyglycosylceramides with Helicobacter pylori-binding activity were found in all acid fractions. A non-acid triglycosylceramide, recognized by Gal(alpha)4Gal-binding Escherichia coli, was detected in the plasma membrane/secretory vesicles but not in the azurophil and specific granules. Although no defined roles of glycosphingolipids have yet been conclusively established with respect to neutrophil function, the fact that many of the identified glycosphingolipids are stored in granules, is in agreement with their role as receptor structures that are exposed on the neutrophil cell surface upon fusion of granules with the plasma membrane. Accordingly, we show that neutrophil granules store specific carbohydrate epitopes that are upregulated to the plasma membrane upon cell activation.

Antibodies, Monoclonal↗

PubMed2001

Proinflammatory activity of a cecropin-like antibacterial peptide from Helicobacter pylori.

Helicobacter pylori, the bacterial pathogen associated with gastritis and peptic ulcers, is highly successful in establishing infection in the human gastric mucosa, a process typically associated with massive infiltration of inflammatory cells. Colonization of the mucosa is suggested to be facilitated by H. pylori-produced cecropin-like peptides with antibacterial properties, giving the microbe a competitive advantage over other bacteria. We show that a cecropin-like antibacterial peptide from H. pylori, Hp(2-20), not only has a potent bactericidal effect but also induces proinflammatory activities in human neutrophils, e.g., upregulation of integrins (Mac-1), induction of chemotaxis, and activation of the oxygen radical producing NADPH-oxidase. Furthermore, we show that these effects are mediated through binding of Hp(2-20) to the promiscuous, G-protein-linked lipoxin A(4) receptor-formyl peptide-like receptor 1.

Anti-Bacterial Agents↗

PubMed2001

Lipopolysaccharide-induced gelatinase granule mobilization primes neutrophils for activation by galectin-3 and formylmethionyl-Leu-Phe.

We have earlier shown that galectin-3, a lactose-binding mammalian lectin that is secreted from activated macrophages, basophils, and mast cells, induces activation of the NADPH oxidase in exudated but not in peripheral blood neutrophils (A. Karlsson, P. Follin, H. Leffler, and C. Dahlgren, Blood 91:3430-3438, 1998). The alteration in responsiveness occurring during extravasation correlated with mobilization of the gelatinase and/or specific granules to the cell surface, indicating a role for mobilizable galectin-3 receptors. In this study we have investigated galectin-3-induced NADPH oxidase activation, measured as superoxide production, in lipopolysaccharide (LPS)-primed neutrophils. Upon galectin-3 challenge, the LPS-primed cells produced superoxide, both extracellularly and intracellularly. A primed extracellular response to formylmethionyl-Leu-Phe (fMLF) was also achieved. The exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the cell surface was markedly increased after LPS treatment, indicating that granule fusion with the plasma membrane had occurred. Further assessment of specific markers for neutrophil granules showed that the LPS treatment had mobilized the gelatinase granules but only a minor fraction of the specific granules. We thus suggest that the mechanism behind LPS priming lies at the level of granule (receptor) mobilization for galectin-3 as well as for fMLF.

Antigens, CD↗

PubMed2001

A proinflammatory peptide from Helicobacter pylori activates monocytes to induce lymphocyte dysfunction and apoptosis.

Infection with Helicobacter pylori causes chronic gastritis, which is characterized by a dense mucosal infiltration by inflammatory cells such as monocytes/macrophages. H. pylori-induced inflammation is a risk factor for the development of gastric adenocarcinoma, but the mechanisms involved in H. pylori-associated carcinogenesis are poorly understood. A cecropin-like H. pylori peptide, Hp(2-20), was found to be a monocyte chemoattractant and activated the monocyte NADPH-oxidase to produce oxygen radicals. The receptors mediating monocyte activation were identified as FPRL1 and the monocyte-specific orphan receptor FPRL2. Hp(2-20)-activated monocytes inhibited lymphocytes with antitumor properties, such as CD56+ natural killer (NK) cells and CD3epsilon+ T cells. The changes observed in NK cells and T cells--a reduced antitumor cytotoxicity, downregulation of CD3zeta expression, and apoptosis--were mediated by Hp(2-20)-induced oxygen radicals. Histamine, a gastric mucosal constituent, rescued NK cells and T cells from inhibition and apoptosis by suppressing Hp(2-20)-induced oxygen radical formation. We conclude that H. pylori expression of this monocyte-activating peptide contributes to its ability to attract and activate monocytes and reduces the function and viability of antineoplastic lymphocytes. These novel mechanisms may be subject to local, histaminergic regulation in the gastric mucosa.

Adenocarcinoma↗

PubMed2001

Neutrophil secretory vesicles are the intracellular reservoir for GPI-80, a protein with adhesion-regulating potential.

The subcellular localization of GPI-80, a novel, adhesion-regulating protein, was investigated in human neutrophils. Surface expression of GPI-80 was determined by FACS analysis as well as by the ability for phospholipase C to cleave the protein from the cell surface. Increasing amounts of GPI-80 were exposed on the cell surface after weak stimulation with the chemoattractant fMLF, suggesting that the protein can be translocated to the plasma membrane from intracellular stores. By subcellular fractionation of the neutrophils, GPI-80 was defined as a component of a light membrane fraction, containing secretory vesicles and plasma membranes, and it was absent from the neutrophil granule fractions. Separation of the plasma membranes from the secretory vesicles by flotation gradient fractionation confirmed that the GPI-80 was localized in the mobilizable secretory vesicles by approximately 50%, and the rest was plasma membrane-bound. Thus, we identify secretory vesicles as the reservoir of GPI-80 from which it may translocate to the plasma membrane after weak stimulation of the cells.

Amidohydrolases↗

PubMed2001

Serotonin protects NK cells against oxidatively induced functional inhibition and apoptosis.

High concentrations of the neurotransmitter serotonin can be found in inflamed and ischemic peripheral tissues, but the role of serotonin in immunoregulation is largely unknown. Here we report that serotonin protected human natural-killer (NK) cells from oxidatively induced inhibition inflicted by autologous monocytes in vitro. Serotonin protected NK cells from monocyte-mediated apoptosis and suppression of cytotoxicity and maintained the activation of NK cells induced by interleukin-2 despite the presence of inhibitory monocytes. A detailed analysis of these protective effects revealed that serotonin scavenged reactive oxygen species (ROS) derived from the H(2)O(2)-myeloperoxidase (-MPO) system. Serotonin shared this scavenger activity with its precursor, 5-hydroxytryptophan (5-HTP); however, serotonin was >10-fold more potent than 5-HTP in protecting NK cells against functional inhibition and apoptosis. We propose that serotonin, by scavenging peroxidase-derived ROS, may serve to protect NK cells from oxidative damage at inflammatory sites.

5-Hydroxytryptophan↗

PubMed2000

Endogenous cleavage of annexin I generates a truncated protein with a reduced calcium requirement for binding to neutrophil secretory vesicles and plasma membrane.

We have earlier shown that an N-terminal truncated annexin I molecule, annexin I(des1-8), is generated in human neutrophils through cleavage by a membrane localized metalloprotease. The truncated protein showed differences in membrane binding among the neutrophil granule populations as compared to full-length annexin I. In this study, we investigated the cleavage capabilities of isolated neutrophil secretory vesicles and plasma membrane, and the binding of full-length annexin I and annexin I(des1-8) to these membrane fractions. Translocations were performed in vitro to secretory vesicles and plasma membrane, respectively, at different Ca(2+) concentrations. We show that the annexin I-cleaving membrane localized metalloprotease is present both in the secretory vesicles and the plasma membrane. The N-terminal truncation of annexin I gives rise to a molecule with a decreased Ca(2+) requirement for binding, both to secretory vesicles and plasma membrane. There was, thus, no difference in binding of either full-length annexin I or annexin I(des1-8) to the secretory vesicles as compared to the plasma membrane.

Annexin A1↗

PubMed2000

Natural killer cell dysfunction and apoptosis induced by chronic myelogenous leukemia cells: role of reactive oxygen species and regulation by histamine.

Natural killer (NK) cells are deficient in patients with chronic myelogenous leukemia (CML), but the mechanisms responsible for the dysfunction are not completely understood. This study reports that CML cells effectively inhibit the baseline and interleukin-2 (IL-2)-induced NK cell cytotoxicity against a CML cell-derived line (K562). A sizable fraction of NK cells subsequently acquired features characteristic of programmed cell death/apoptosis. The CML cell-mediated inhibition of NK cells required triggering of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated formation of reactive oxygen species (ROS) and was prevented by catalase, a scavenger of ROS, and by histamine, acting via H(2)-receptor-mediated inhibition of ROS production in CML cells. In contrast, nonmalignant neutrophilic granulocytes inhibited NK cells via ROS production without the requirement of exogenous NADPH oxidase-triggering stimuli. We propose that paracrine production of ROS may contribute to the dysfunction of NK cells in CML and that histamine may serve as an autocrine inhibitor of ROS formation in leukemic granulocytes. (Blood. 2000;96:1961-1968)

Apoptosis↗

PubMed2000

The synthetic chemoattractant Trp-Lys-Tyr-Met-Val-DMet activates neutrophils preferentially through the lipoxin A(4) receptor.

A D-methionine-containing peptide, Trp-Lys-Tyr-Met-Val-D-Met-NH(2) (WKYMVm), featuring a unique receptor specificity was investigated with respect to its ability to activate neutrophil effector functions. The peptide was found to be more potent than the N-formylated peptide N-formyl-Met-Leu-Phe (fMLF) at inducing neutrophil chemotaxis, mobilization of neutrophil complement receptor 3 (CR3), and activation of the neutrophil NADPH-oxidase. The fact that binding of fML[(3)H]F was inhibited by both fMLF and WKYMVm suggests that N-formyl peptide receptor (FPR) is shared by these peptides. However, the neutrophil response induced by the WKYMVm peptide was insensitive to the fMLF antagonists, cyclosporin H, and Boc-FLFLF that specifically block the function of the FPR. These results suggest that even though WKYMVm may bind FPR the cells are activated preferentially through a receptor distinct from the FPR. Using transfected HL-60 cells expressing either the FPR or its neutrophil homologue FPRL1, also referred to as LXA(4)R because it has been shown to bind lipoxin A(4), we show that WKYMVm is about 300-fold more active at mobilizing intracellular calcium through FPRL1 than through FPR. The WKYMVm activates FPRL1-expressing cells in a cyclosporin H-independent manner with an EC(50 )of around 75 pmol/L, whereas it activates FPR-expressing cells with an EC(50 )of around 25 nmol/L. The observation that exudated cells are primed in their response to WKYMVm suggests that FPRL1/LXA(4)R like FPR is stored in mobilizable organelles. (Blood. 2000;95:1810-1818)

Calcium↗

PubMed2000

Alleviating oxidative stress in cancer immunotherapy: a role for histamine?

Interleukin-2 is a remarkable activator of lymphocytes with anti-neoplastic properties such as T-cells or natural killer cells, but tumor regression only rarely occurs in interleukin-2-treated cancer patients. In this review, we focus on interactions between monocytes/macrophages and T-cells/natural killer-cells, and in particular the role of such interactions for the outcome of cancer immunotherapy with interleukin-2. We propose that interleukin-2 therapy should be supplemented with compounds that alleviate toxicity inflicted by monocyte/macrophage-derived reactive oxygen metabolites within and around tumors. The hypothesis is founded on data demonstrating that (i) functions of intratumoral lymphocytes in many human malignant tumors are inhibited by reactive oxygen metabolites, generated by neighboring monocytes/macrophages, (ii) interleukin-2 only weakly activates T-cells or natural killer cells in an environment of oxidative stress, and (iii) inhibitors of the formation of reactive oxygen metabolites or scavengers of reactive oxygen metabolites synergize with interleukin-2 to activate these lymphocyte subsets. We also review the preclinical background to the use of histamine dihydrochloride, an inhibitor of reactive oxygen metabolite formation in monocytes/macrophages, as a supplement to cancer immunotherapy with interleukin-2.

Cell Communication↗

PubMed2000

Dynamic ca(2+)changes in neutrophil phagosomes A source for intracellular ca(2+)during phagolysosome formation?

An increase in cytosolic Ca(2+)concentration periphagosomally is critical for phagolysosomal formation and neutrophil elimination of microbes. The Ca(2+)increase could be achieved through release of Ca(2+)from mobilized intracellular stores. Alternatively, Ca(2+)that passively enter the phagosome during phagocytosis could be provided by the phagosome. Intraphagosomal Ca(2+)changes in single human neutrophils was measured during phagocytosis of serum opsonized Fura-2-conjugated zymosan particles, using a digital image processing system for microspectrofluorometry. A decrease in phagosomal Ca(2+)down to nanomolar concentrations was seen within minutes following phagosomal closure. Blockage of plasma membrane Ca(2+)channels by econazole abolished this decrease. The fluorescence properties of Fura-2 zymosan were retained after phagocytosis and stable to pH changes, reactive oxygen species, and proteolytic enzymes. We suggest that Ca(2+)ions present in the phagosome enter the cell cytosol through Ca(2+)channels in the phagosomal membrane, achieving a localized Ca(2+)rise that is important for phagosome processing.

Calcium↗