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

AbstractWe have earlier shown that an N-terminal truncated annexin I molecule, annexin Ides1–8, 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 Ides1–8 to these membrane fractions. Translocations were performed in vitro to secretory vesicles and plasma membrane, respectively, at different Ca2+ 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 Ca2+ 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 Ides1–8 to the secretory vesicles as compared to the plasma membrane

Leukocyte oxygen radical production determines disease severity in the recurrent Guillain-Barré syndrome

<p>Abstract</p> <p>Background</p> <p>The recurrent Guillain-Barré syndrome (RGBS) is characterized by at least two GBS episodes with intervening remission. In a previous study of monophasic GBS, we reported that the magnitude of oxygen radical production ("respiratory burst") in peripheral blood leukocytes was inversely correlated to disease severity. The present study sought to establish a similar correlation in patients with RGBS.</p> <p>Methods</p> <p>Oxygen radical production in leukocytes was induced by formyl-Met-Leu-Phe (fMLF), Trp-Lys-Tyr-Met-Val-Met-NH₂(WKYMVM), or phorbol myristate acetate (PMA) and assessed by quantifying superoxide anion formed by the leukocyte NADPH oxidase.</p> <p>Results</p> <p>Disease severity, assessed using the MRC score, was negatively correlated to superoxide anion production triggered by fMLF or WKYMVM (p = 0.001 and 0.002, respectively; n = 10). Superoxide anion production also was significantly lower in RGBS patients with incomplete recovery after stimulation with fMLF (p = 0.004) or WKYMVM (p = 0.003).</p> <p>Conclusion</p> <p>We conclude that a lower respiratory burst in leukocytes is strongly associated with a severe course of RGBS.</p

Role of regulatory T cells in acute myeloid leukemia patients undergoing relapse-preventive immunotherapy

Regulatory T cells (Tregs) have been proposed to dampen functions of anti-neoplastic immune cells and thus promote cancer progression. In a phase IV trial (Re:Mission Trial, NCT01347996, http://www.clinicaltrials.gov ) 84 patients (age 18-79) with acute myeloid leukemia (AML) in first complete remission (CR) received ten consecutive 3-week cycles of immunotherapy with histamine dihydrochloride (HDC) and low-dose interleukin-2 (IL-2) to prevent relapse of leukemia in the post-consolidation phase. This study aimed at defining the features, function and dynamics of Foxp3+CD25highCD4+ Tregs during immunotherapy and to determine the potential impact of Tregs on relapse risk and survival. We observed a pronounced increase in Treg counts in peripheral blood during initial cycles of HDC/IL-2. The accumulating Tregs resembled thymic-derived natural Tregs (nTregs), showed augmented expression of CTLA-4 and suppressed the cell cycle proliferation of conventional T cells ex vivo. Relapse of AML was not prognosticated by Treg counts at onset of treatment or after the first cycle of immunotherapy. However, the magnitude of Treg induction was diminished in subsequent treatment cycles. Exploratory analyses implied that a reduced expansion of Tregs in later treatment cycles and a short Treg telomere length were significantly associated with a favorable clinical outcome. Our results suggest that immunotherapy with HDC/IL-2 in AML entails induction of immunosuppressive Tregs that may be targeted for improved anti-leukemic efficiency

The function of annexin I in human neutrophils. Studies of membrane binding

The neutrophil granulocyte plays an important role in host defense against infections induced by invading microorganisms. The defense mechanism is dependent on the migration of neutrophils from the blood stream to the infected area, where the intruder is phagocytosed and killed. Neutrophil activation is associated with a mobilization of intracellular granules and vesicles. These organelles contain both matrix proteins that upon activation are secreted to the extracellular milieu or into the phagosome, and membrane localized receptors that become exposed on the cell surface. Neutrophil secretion is highly regulated and depends on intracellular Ca2+-levels, suggesting that Ca2+-regulated proteins may be part of the regulatory network.The aim of this study was to investigate the role of Ca2+-regulated proteins in the secretion of neutrophil organelles. The study was focused on the role of the Ca2+-dependent and membrane-binding proteins annexin I and grancalcin, found in the cytosol of resting neutrophils.To study the Ca2+-regulated binding of annexin I to different neutrophil membrane compartments (i.e. azurophil granules, specific granules, gelatinase granules and secretory vesicles/plasma membrane) in vitro, these organelles were isolated by subcellular fractionation on different types of Percoll gradients. The binding properties of annexin I and grancalcin to specialized detergent insoluble membrane domains, or lipid rafts, were studied by isolating these domains from the plasma membrane/secretory vesicles by cold Triton X-100 extraction and Percoll gradient centrifugation.Annexin I has beside its cytosolic localization also been found extracellularly, secreted via an unknown pathway. By using the subcellular fractionation technique, annexin I was shown to be exclusively localized to the cytosol of human neutrophils and absent from the granules. This implies that the secretion of annexin I to the extracellular milieu is independent of degranulation.Annexin I has earlier been shown to bind to all subcellular organelles isolated from neutrophils in the presence of Ca2+. The present study shows that the binding is accompanied by the cleavage of the annexin I molecule in the N-terminal domain, generating a truncated annexin I molecule, annexin Ides1-8. The cleavage is mediated by a membrane-localized metalloprotease present in some subcellular organelles, but excluded from others. The cleavage of annexin I affects the binding of the protein to membranes by decreasing the Ca2+ requirement. This suggests that the membrane-binding properties of annexin I is regulated by the N-terminal cleavage of the protein by effecting the Ca2+ affinity.Annexin I and grancalcin both bind to detergent insoluble membrane domains within the plasma membrane in the presence of Ca2+, suggesting that lipid rafts are of regulatory importance in neutrophil secretion.In conclusion, the Ca2+-dependent binding of annexin I and grancalcin to detergent insoluble membrane domains and the functional modification achieved through endogenous cleavage of annexin I suggest an involvement of these processes during regulation of neutrophil secretion

Neutrophil NADPH-oxidase activation by an annexin AI peptide is transduced by the formyl peptide receptor (FPR), whereas an inhibitory signal is generated independently of the FPR family receptors.

International audienceTruncation of the N-terminal part of the calcium-regulated and phospholipid-binding protein annexin AI has been shown to change the functional properties of the protein and to generate immunoregulatory peptides. Proinflammatory as well as anti-inflammatory signals are triggered by these peptides, and the two formyl peptide receptor (FPR) family members expressed in neutrophils, FPR and FPR-like 1 (FPRL1), have been suggested to transduce these signals. We now report that an annexin AI peptide (Ac9-25) activates, as well as inhibits, the neutrophil release of superoxide anions. Results obtained from experiments with receptor antagonists/inhibitors, desensitized cells, and transfected cells reveal that the Ac9-25 peptide activates the neutrophil reduced nicotinamide adenine dinucleotide phosphate oxidase through FPR but not through FPRL1. The Ac9-25 peptide also inhibits the oxidase activity in neutrophils triggered, not only by the FPR-specific agonist N-formyl-Met-Leu-Phe but also by several other agonists operating through different G protein-coupled receptors. Our data show that the two signals generated by the Ac9-25 peptide are transmitted through different receptors, the inhibitory signal being transduced by a not-yet identified receptor distinct from FPR and FPRL1

Oxygen radical production in leukocytes and disease severity in multiple sclerosis.

This study investigated the relationship between the formation of NADPH oxidase-dependent oxygen radicals in peripheral blood leukocytes (\u27respiratory burst\u27) and disease severity in patients with multiple sclerosis (MS). Oxygen radical production was induced by formyl-Met-Leu-Phe (fMLF), Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) or phorbol myristate acetate (PMA) and was assessed by quantifying superoxide anion, i.e. the initial radical formed by the NADPH oxidase. Disease severity was evaluated using the Multiple Sclerosis Severity Score (MSSS). In patients with severe disease, the production of superoxide anion was significantly lower for all three inducers of radical formation (p=0.04-0.004). Our findings are supportive of a protective role of oxygen radicals in autoimmunity

The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide

Abstract Background The formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites. Results We report that a cell permeable ten amino acid peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate (PIP2) binding region of gelsolin (an uncapper of actin filaments) blocks granule mobilization as well as secretion of oxygen radicals. The inhibitory effect of PBP10 is, however, receptor specific and affects the FPRL1-, but not the FPR-, induced cellular response. The transient rise in intracellular calcium induced by the active receptors is not affected by PBP10, suggesting that the blockage occurs in a parallel, novel signaling pathway used by FPRL1 to induce oxygen radical production and secretion. Also the FPR can activate neutrophils through a PBP10-sensitive signaling pathway, but this signal is normally blocked by the cytoskeleton. Conclusions This study demonstrates that the two very closely related chemoattractant receptors, FPR and FPRL1, use distinct signaling pathways in activation of human neutrophils. The PIP2-binding peptide PBP10 selectively inhibits FPRL1-mediated superoxide production and granule mobilization. Furthermore, the activity of this novel PBP10 sensitive pathway in neutrophils is modulated by the actin cytoskeleton network.</p

The recurrent Guillain-Barre syndrome: a long-term population-based study

Objectives To describe a population-based material of patients with recurrent GuillainBarre syndrome (RGBS), examine the long time course, and search for factors predisposing to recurrence. Materials and methods We performed a follow-up study of the neurology and neurophysiology and a systematic study of the acute microbial serology of patients with RGBS. These parameters were compared with the results of a previous study of monophasic GBS. Results The patients with RGBS (n = 15) were retrieved from admissions of 229 patients with GBS during a 17-year period. They had 27 (median 3) episodes occurring at irregular intervals over decades. Of the 11 patients who accepted a follow-up examination, six were in full remission, and five had moderate sequelae. Nine had a demyelinating subtype, one had an axonal motor variant, and one patient with incomplete Miller Fisher syndrome had associated arachnoiditis. Two patients showed ultimate transition to a course similar to chronic inflammatory demyelinating polyneuropathy. Episodes were generally shorter in RGBS than in GBS, and an initial episode duratio