Workshop 1: Phagocyte biology: from gene to function - 3



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Ligation of leukotriene B4 receptor BLT1 in human endothelial cells generates a signal via the MAP kinase pathway leading to gradually increases of adhesive events and of release of MCP-1, IL-8 and nitric oxide

^ A-S Workshop 1: Phagocyte biology: from gene to function - 3. Johansson1, J.Z. Haeggström2 & J. Palmblad1

1Center for Inflammation and Hematology Research, 3Dept. of Medicine, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden; 2Dept. of Medical Biochemistry and Biophysics, Division Workshop 1: Phagocyte biology: from gene to function - 3 of Chemistry II, Karolinska Institutet, Stockholm, Sweden

Leukotriene B4 (LTB4), a powerful chemotactic and immune modulating lipid, signals via distinct G-protein-coupled surface receptors, denoted BLT. Recently, we reported that BLT Workshop 1: Phagocyte biology: from gene to function - 31 is the predominating BLT expressed on human umbilical vein endothelial cells (HUVEC). Here, we found that LTB4 stimulation of HUVEC causes adhesion of neutrophils, up-regulation of E-selectin, ICAM-1 and Workshop 1: Phagocyte biology: from gene to function - 3 VCAM-1, release of the granule-stored proteins MCP-1 and IL-8, and of nitrite. Since L-NAME inhibited the nitrite release, this is suggested to reflect NO production. Adhesion of neutrophils and release Workshop 1: Phagocyte biology: from gene to function - 3 of MCP-1, IL-8 and NO required only 15 min to be expressed, but robust increases, similar in magnitude to what lipopolysaccharide conferred, were observed also after 3-7 h, whereas up-regulation and the adhesion Workshop 1: Phagocyte biology: from gene to function - 3 molecules peaked at 4-6 h. Using BLT1 and -2 specific blockers we found that these responses were mediated by BLT1. Moreover, they were mediated by the MAP kinase/Erk pathway, whereas no activation of Workshop 1: Phagocyte biology: from gene to function - 3 NKκB p65, c-jun or Elk signaling was observed. Since IL-8 and MCP-1 plays a role in neutrophil and monocytes recruitment our findings may have functional consequences in the early vascular responses Workshop 1: Phagocyte biology: from gene to function - 3 to inflammation. Moreover, the results point to BLT receptors as potential targets for pharmacological intervention in vasculitides of various causes.


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The acute phase reactant serum amyloid A delays neutrophil apoptosis independently Workshop 1: Phagocyte biology: from gene to function - 3 of formyl peptide receptor like 1

K. Christenson, L. Björkman, C. Tängemo & J. Bylund

Department of Rheumatology and Inflammation Research, Göteborg, Sweden

Background: Neutrophil apoptosis is important for termination of inflammatory reactions in that Workshop 1: Phagocyte biology: from gene to function - 3 it ensures placid clearance of potently cytotoxic inflammatory cells. Delay in neutrophil apoptosis can result in cell accumulation, sometimes accompanied by tissue destruction, potentially leading to inflammatory disease states, e.g., rheumatoid arthritis Workshop 1: Phagocyte biology: from gene to function - 3 (RA). RA is frequently characterized by elevated levels of the acute phase reactant serum amyloid A (SAA), both in circulation and in inflamed joints. The purpose of this study was to Workshop 1: Phagocyte biology: from gene to function - 3 investigate how SAA affected neutrophil apoptosis.

^ Materials and methods: Human neutrophils were cultured with or without stimuli and apoptosis assessed on the basis of Annexin-V binding and mitochondrial membrane potential using FACS Workshop 1: Phagocyte biology: from gene to function - 3. Intracellular calcium transients were monitored with the fluorescent calcium probe Fura-2.

Results: Recombinant SAA potently suppressed apoptosis in human neutrophils at concentrations well below those found in RA circulation. SAA Workshop 1: Phagocyte biology: from gene to function - 3 is emerging as a cytokine-like molecule with ability to activate various proinflammatory processes, many of which involve signaling via the chemotactic receptor formyl peptide receptor like 1 (FPRL1). Specific blocking of Workshop 1: Phagocyte biology: from gene to function - 3 FPRL1 or FPRL1-mediated signaling did not diminish SAA´s antiapoptotic effects, indicating that FPRL1 was not involved in this process. Instead, oxidized ATP, an antagonist of the nucleotide receptor P2X7, completely abrogated Workshop 1: Phagocyte biology: from gene to function - 3 the effect of SAA, implying that this receptor was involved in SAA signaling.

Conclusions: SAA is a potent inhibitor of neutrophil apoptosis employing a signaling pathway independent of FPRL1. The extended neutrophil survival Workshop 1: Phagocyte biology: from gene to function - 3 mediated by SAA could be of importance in understanding the pathology of RA.


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Piroxicam, a non-steroidal anti-inflammatory drug, antagonizes binding to the formyl peptide receptor

A. Stenfeldt, J Workshop 1: Phagocyte biology: from gene to function - 3. Karlsson, J. Bylund, H. Fu & C. Dahlgren

Department of Rheumatology and Inflammation Research, Göteborg University, Göteborg, Sweden

Background: Piroxicam, classified as a cyclo-oxygenase inhibitor, is an anti-inflammatory drug Workshop 1: Phagocyte biology: from gene to function - 3 that has been reported to affect the production of reactive oxygen species in phagocytes. In this study we compare the effect of piroxicam on the neutrophil activity triggered by two different G-protein coupled Workshop 1: Phagocyte biology: from gene to function - 3 receptors. Agonists that trigger the formyl peptide receptor (FPR; fMLF), the formyl peptide receptor like-1 (FPRL1; WKYMVM) or both these receptors (WKYMVm) were used to activate the cells.

Materials and methods: Human neutrophils Workshop 1: Phagocyte biology: from gene to function - 3 were treated with piroxicam and then stimulated with the FPR/FPRL1 agonists. The transient rise in intracellular calcium was measured by flow cytometry and the release of superoxide was determined Workshop 1: Phagocyte biology: from gene to function - 3 with a chemiluminescence technique. A competitive receptor-binding assay was preformed by flow cytometry using the fluorescent labeled FPR agonist fNLPNTL.

Results: Piroxicam reduced the release of superoxide anion from neutrophils stimulated with the Workshop 1: Phagocyte biology: from gene to function - 3 FPR agonist fMLF. Piroxicam had no or little effect on the activity induced by the FPRL1 agonist (WKYMWM). The neutrophil response induced by WKYMVm (an agonist that binds both FPR and Workshop 1: Phagocyte biology: from gene to function - 3 FPRL1) was inhibited by piroxicam only when signaling through FPRL1 was blocked by a receptor specific antagonist (WRWWWW). Binding to the neutrophil cell surface of the FPR specific agonist fNLPNTL was competed Workshop 1: Phagocyte biology: from gene to function - 3 for by both piroxicam and fMLF.

Conclusions: Our data show that piroxicam inhibits the neutrophil responses triggered through FPR but not through the closely related FPRL1, and this inhibition is due Workshop 1: Phagocyte biology: from gene to function - 3 to a reduced binding of the activating ligand to its cell surface receptor.


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The ligand binding and signalling domains of the formyl peptide receptor like-2, share properties with another member, formyl peptide receptor Workshop 1: Phagocyte biology: from gene to function - 3 like-1, of the same receptor family

^ J. Karlsson1, H. Fu1, J. Bylund1, M.J. Rabiet2, F. Boulay2 & C. Dahlgren1

1Dept. of Rheumatology and Inflammation Research, Göteborg, Sweden; 2CEA-Grenoble, France

Background: The peptides WRWWWW Workshop 1: Phagocyte biology: from gene to function - 3 and PBP10 have been shown to function as receptor antagonist and inhibitor of signalling, respectively, for formyl peptide receptor like-1(FPRL1). Since there is a large homology between FPRL1 and formyl Workshop 1: Phagocyte biology: from gene to function - 3 peptide receptor like-2(FPRL2), we set out to determine if these peptides affects also the FPRL2 triggered response.

^ Materials and methods: The members of the formyl peptide receptor(FPR)-family were stably expressed Workshop 1: Phagocyte biology: from gene to function - 3 in undifferentiated HL-60 cells. The cells were activated by WKYMVm and the rise in intracellular Ca2+ was monitored as a change in Fura-2 fluorescence.

Results: The previously described differences between FPR and Workshop 1: Phagocyte biology: from gene to function - 3 FPRL1 signalling in sensitivity to PBP10 and to defined receptor antagonists were confirmed for NADPH-oxidase activity, intracellular Ca2+ and actin polymerization in neutrophils.

The response induced by WKYMVm in FPRL Workshop 1: Phagocyte biology: from gene to function - 31 expressing cells was inhibited by PBP10, as was the response induced in cells expressing FPRL2. The earlier described FPRL1 specific antagonist WRWWWW inhibited also the response induced in FPRL2 expressing Workshop 1: Phagocyte biology: from gene to function - 3 cells. As revealed from binding data using an FPRL1 specific antibody no spontaneous expression of FPRL1 was observed in FPRL2 expressing cells.

Conclusions: We show that WRWWWW antagonizes binding of an activating peptide not only Workshop 1: Phagocyte biology: from gene to function - 3 to FPRL1 but also to FPRL2. In addition, we show that PBP10 that has no effect on FPR signalling fully inhibits signalling through FPRL1 and FPRL2. The mechanism by which it Workshop 1: Phagocyte biology: from gene to function - 3 occurs remains to be investigated. The ligand binding and signalling domains of FPRL1 and FPRL2 are thus very similar.


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Role of different Fc receptors in neutrophil responses induced by immune complexes

T. Németh Workshop 1: Phagocyte biology: from gene to function - 3, Z. Jakus & A. Mócsai

Department of Physiology, Semmelweis University School of Medicine, Budapest, Hungary

Background: During autoimmune diseases (e. g. rheumatoid arthritis), antibodies against host antigens are generated, leading to immune complex deposition Workshop 1: Phagocyte biology: from gene to function - 3 in the host tissues. These immune complexes trigger activation of neutrophil effector functions that will be targeted against the host tissues. We wanted to identify which Fc receptors (FcR Workshop 1: Phagocyte biology: from gene to function - 3) are crucial for inducing neutrophil responses stimulated by plate bound immune complexes.

Methods: Human neutrophils or murine FcR γ-chain–/–, FcγRIII–/–, FcγRI–/–, FcγRI–/–/ FcγRIII–/– neutrophils were used. Neutrophils were preincubated Workshop 1: Phagocyte biology: from gene to function - 3 with or without FcR blocking antibodies, and the cells were activated by plate bound immune complexes. Effector responses (superoxide production, degranulation and spreading) of neutrophils were detected.

Results: In human neutrophils Workshop 1: Phagocyte biology: from gene to function - 3 blocking antibodies against FcγRIIA (which has its own ITAM and does not associate with the FcR γ-chain) completely inhibited the effector responses induced by immune complexes. In murine neutrophils the three activating Workshop 1: Phagocyte biology: from gene to function - 3 Fc receptors (FcγRI, FcγRIII and FcγRIV) are associated with Fc receptor γ-chain. FcR γ-chain–/– cells failed to produce superoxide on immune complex surface. Surprisingly, effector responses were not defective in Fc Workshop 1: Phagocyte biology: from gene to function - 3γRIII–/–, FcγRI–/– and FcγRI–/–/ FcγRIII–/– double knockout neutrophils.

Conclusions: We found that plate bound immune complex induced effector responses are mediated through FcγRIIA in human Workshop 1: Phagocyte biology: from gene to function - 3 neutrophils. Our results indicate that in murine neutrophils these responses are initiated by a FcR γ-chain associated receptor, but not through FcγRIII and FcγRI. These findings raise the possible role of the Fc Workshop 1: Phagocyte biology: from gene to function - 3γRIV in immune complex activated murine neutrophil responses.


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Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization

R. Blomgran & O. Stendahl

Division of Medical Microbiology, Link Workshop 1: Phagocyte biology: from gene to function - 3öping University, Sweden

Background: Lysosomal membrane permeabilization (LMP) is emerging as an important regulator of cell apoptosis. We previously observed that intracellular, non-phagosomal generation of reactive oxygen species (ROS) triggered by adherent and fimbriated Workshop 1: Phagocyte biology: from gene to function - 3 bacteria induced neutrophil apoptosis, whereas intraphagosomal production of ROS during phagocytosis had no such effect (IAI, Aug. 2004 p 4570-4578). The present study dissects the effect of phagosomal versus non-phagosomal-derived Workshop 1: Phagocyte biology: from gene to function - 3 ROS on LMP.

^ Material and methods: The lysosomotropic fluorophore acridine orange, and the mitochondrial transmembrane potential marker TMRE was used to analyze organelle stability. Detection of expression, activation, or interaction of Bcl-2 proteins was Workshop 1: Phagocyte biology: from gene to function - 3 performed by immunoprecipitation and immunoblotting. Apoptosis was evaluated by measuring phophatidyl serine exposure and internucleosomal DNA fragmentation.

Results: Adherent, non-ingested type 1 fimbriated ^ Escherichia coli rapidly decreased lysosomal membrane Workshop 1: Phagocyte biology: from gene to function - 3 stability and induced leakage of cathepsins in a NADPH oxidase-dependent manner. Induction of lysosomal destabilization triggered cathepsin-dependent cleavage of the pro-apoptotic Bcl-2 protein Bid, and a decrease in the anti Workshop 1: Phagocyte biology: from gene to function - 3-apoptotic protein Mcl-1. Also, Bax dissociated from its heterodimerization to Mcl-1 before a decrease could be seen in Mcl-1. Involvement of LMP in the initiation of apoptosis was supported by the observation Workshop 1: Phagocyte biology: from gene to function - 3 that both Bid-cleavage and the concomitant drop in mitochondrial membrane potential required activation of cysteine cathepsins but not caspases. In contrast, intraphagosomal production of ROS during immune receptor -mediated phagocytosis did Workshop 1: Phagocyte biology: from gene to function - 3 not cause LMP, mitochondrial damage or apoptosis.

Conclusions: We conclude that in microbe-induced apoptosis in neutrophils, ROS-dependent LMP represents an early event that is followed by Bid cleavage, mitochondrial Workshop 1: Phagocyte biology: from gene to function - 3 damage and caspase activation.


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The Fc receptor γ-chain ITAM is required for antibody immunotherapy of melanoma

S. de Haij1, J.H.M. Jansen1, J.E. Bakema1, B. Gilissen1, J.S. Verbeek2, J.G.J Workshop 1: Phagocyte biology: from gene to function - 3. van de Winkel1,3 & J.H.W. Leusen1

^ 1Dept. Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; 2Dept. of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands; 3Genmab, Utrecht, The Netherlands

Background Workshop 1: Phagocyte biology: from gene to function - 3: In vitro studies have indicated antibody-dependent cellular cytotoxicity (ADCC) as the primary mechanism for tumor-kill by therapeutic antibodies. In vivo studies with FcR γ-chain deficient mice have demonstrated the importance of FcR Workshop 1: Phagocyte biology: from gene to function - 3 for antibody therapy. However, as these mice lack expression of activating FcR, it is not possible to discriminate between ADCC and apoptosis induced by FcR cross-linking. Therefore, the mechanism Workshop 1: Phagocyte biology: from gene to function - 3 of FcR-mediated tumor kill in vivo is still unknown and aim of the present study.

^ Materials and methods: We have generated a novel transgenic mouse expressing a mutated human FcR γ-chain in Workshop 1: Phagocyte biology: from gene to function - 3 which the ITAM-associated tyrosines at positions 65 and 76 have been replaced by phenylalanines. These so-called NOTAM transgenic mice were crossed with FcR γ-chain deficient mice. Expression of the transgene was determined Workshop 1: Phagocyte biology: from gene to function - 3 by western blot and FcR expression by FACS analysis. The ADCC capacity of isolated neutrophils was determined by chromium release assays. Tumor therapy in vivo was determined in the protective B16F Workshop 1: Phagocyte biology: from gene to function - 310 melanoma model in mice as described (Van Spriel, Blood 2003).

Results: Strong expression of the NOTAM γ-chain was observed in isolated neutrophils, macrophages and dendritic cells. Furthermore, NOTAM transgenic mice showed reconstitution Workshop 1: Phagocyte biology: from gene to function - 3 of FcγRI and III to wild-type levels. Neutrophils from NOTAM mice were unable to kill different tumor cell-lines ex vivo. In vivo, antibody treatment resulted in protection of wild-type Workshop 1: Phagocyte biology: from gene to function - 3 mice but not of NOTAM transgenic mice.

Conclusions: We have developed a novel mouse model expressing a signalling defective FcR γ-chain with intact FcR expression levels. With this transgenic mouse, we Workshop 1: Phagocyte biology: from gene to function - 3 have established for the first time that FcR γ-chain signalling via the ITAM-associated tyrosines is required for tumor therapy of mouse melanoma in vivo.


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Toll-like receptor-induced gene expression patterns Workshop 1: Phagocyte biology: from gene to function - 3 may not accurately reflect human immunity

K.L. Brown1, R. Falsafi1, J. Bylund2, K.L. MacDonald2, S. Turvey2, D.P. Speert2 & R.E.W. Hancock1

^ 1Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver Workshop 1: Phagocyte biology: from gene to function - 3, BC, Canada; 2 The Child & Family Research Institute, Vancouver, BC, Canada

Background: The current study aimed to identify defective gene responses that are potentially responsible for susceptibility to infection by particular Workshop 1: Phagocyte biology: from gene to function - 3 bacteria and to identify gene responses that are consistent with relatively normal immunity.

^ Materials and methods: Toll-like receptor (TLR)-induced cellular responses were evaluated by microarray analysis, quantitative polymerase chain reaction Workshop 1: Phagocyte biology: from gene to function - 3 (qPCR) and enzyme-linked immunosorbent assay (ELISA) on peripheral blood monocytes from patients with a clinically described predisposition to particular bacterial infections and inflammatory disorders.

Results: The expression of TLR-induced genes and Workshop 1: Phagocyte biology: from gene to function - 3 associated biological processes were severely distorted in the patients’ monocytes. However this global perturbation in the classic cellular response to TLR ligands did not correlate with the patients’ susceptibility to infection by Workshop 1: Phagocyte biology: from gene to function - 3 particular microorganisms.

Conclusions: These results caution against extrapolation of in vitro TLR-responsiveness to etiology of infection and suggest that particular genes, rather than global patterns, may be better determinants of Workshop 1: Phagocyte biology: from gene to function - 3 host defense.


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Targeting of soluble tumor necrosis factor receptor to secretory lysosomes in murine hematopoietic cells in vivo

M. Hansson, K.S. Nandakumar, A.M. Persson, R. Holmdahl & I. Olsson

Departments of Hematology and Medical Inflammation Workshop 1: Phagocyte biology: from gene to function - 3 research, Lund University, Lund, Sweden

Background: Our research regarding sorting mechanisms of granule proteins have led to a new therapeutic idea – targeted therapy with hematopoietic cells as carriers. Exogenous proteins are expressed Workshop 1: Phagocyte biology: from gene to function - 3 and the gene product is directed for storage into secretory lysosomes. These organelles are unique storage compartments in neutrophils for biological active proteins, designated for inflammatory foci. We have earlier shown Workshop 1: Phagocyte biology: from gene to function - 3 that granule-sorting of therapeutic protein is possible in cell-lines and in primary cells in vitro.

^ Materials and methods: Bone marrow cells are transduced retrovirally with sTNFR1-tm-Y (a fusion Workshop 1: Phagocyte biology: from gene to function - 3 gene with soluble TNF-receptor, allowing granule sorting and storage). The transduced cells are transplanted to irradiated mice and the hematopoesis are allowed to reconstitute. Peripheral blood cells and plasma were recovered Workshop 1: Phagocyte biology: from gene to function - 3 weekly from transplanted mice.

Results: The transduction efficiency for bone marrow cells was 80-90% before transplantation. After two weeks, transduced cells were found in peripheral blood. Furthermore, the sTNFR1 was detected in several populations Workshop 1: Phagocyte biology: from gene to function - 3 of cells in peripheral blood.

Conclusions: We have shown that a fusion protein with sTNFR1 for granule storage could be retrovirally transferred into bone marrow cells and transplanted into mice. The expression Workshop 1: Phagocyte biology: from gene to function - 3 of was stable over months in transplanted mice and the sTNFR1 protein was found in peripheral blood cells. This indicates that granule storage of therapeutic proteins may be used as a targeted therapy.
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