Susanne Rolle

Integrins and Cytokines Activate Nuclear Transcription Factor-κB in Human Neutrophils

Neutrophil adhesion to extracellular matrix is necessary for an effective inflammatory response. Adhesion may accelerate neutrophil activation by affecting intracellular signaling pathways. The nuclear transcription factor κB (NF-κB) controls several cellular functions, including inflammation, proliferation, and cell survival. We explored the role of adhesion in NF-κB activation in human neutrophils. Cells were stimulated with tumor necrosis factor-α (TNF-α), granulocyte macrophage-colony-stimulating factor (GM-CSF), interleukin-8 (IL-8), and formyl-methionyl-leucyl-phenylalanine (fMLP). All four initiated neutrophil adherence to and spreading on fibronectin. GM-CSF and IL-8 did not activate NF-κB in suspended neutrophils but rapidly activated NF-κB under adherent conditions on matrix, as shown by IκB kinase activity assay, IκBα degradation, electromobility shift assay, and quantitative reverse transcriptase-PCR. In contrast, TNF–α activated NF-κB both in suspended cells and adherent cells. fMLP did not activate NF-κB in either suspended or adherent cells. Specific β2 integrin blockade prevented NF-κB activation by GM-CSF and IL-8 on fibronectin. Co-stimulating CD18 and CD11b with activating antibodies resulted in NF-κB activation by GM-CSF and IL-8 in suspended cells. We inhibited actin polymerization with cytochalasin and blocked the non-receptor kinase Syk with piceatannol. Both maneuvers prevented the co-stimulatory NF-κB-activating signal by β2 integrins. Thus, in addition to β2 integrin ligand binding, NF-κB activation depended on the formation of the receptor-associated intracellular focal adhesion complex. We conclude that β2 integrins may provide co-stimulatory signals allowing some soluble mediators to activate the NF-κB pathway even when they are not capable of doing so in suspension. This effect may become important when human neutrophils leave the circulating blood and migrate through extracellular matrix during inflammation.

Phosphatidylinositol 3-Kinase Controls Antineutrophil Cytoplasmic Antibodies—Induced Respiratory Burst in Human Neutrophils

Antineutrophil cytoplasmic antibodies (ANCA) activate human polymorphonuclear neutrophils (PMN) primed with tumor necrosis factor alpha (TNF-alpha) in vitro. Phosphatidylinositol 3-kinase (PI3-K) and the protein-serine/threonine kinase Akt have been implicated in the control of the phagocyte respiratory burst. The hypothesis that PI3-K controls the ANCA-induced respiratory burst was tested. TNF-alpha-primed PMN were stimulated with a monoclonal antibody to myeloperoxidase (MPO) and with PR3- and MPO-ANCA, respectively. Akt activation was assessed with phospho-specific antibodies. Superoxide release was measured with ferricytochrome. ANCA antigen translocation was assessed by fluorescence-activated cell sorter. The effect of TNF-alpha and MPO-ANCA on Akt signaling was studied with immunoprecipitation and glutathione S-transferase pull-down assays. Western blotting revealed rapid transient Akt phosphorylation during TNF-alpha priming and a second phosphorylation after ANCA. PI3-K inhibition by LY294002 blocked both Akt phosphorylation and superoxide generation. A total of 20 +/- 3 nmol O(2)(-)/0.75 x 10(6) PMN/45 min was released after stimulation with PR3-ANCA. LY294002 (5 microM) decreased this amount to 0.3 +/- 2.6 nmol (n = 10, P < 0.05); the MPO-ANCA values were 23 +/- 3 versus 1.6 +/- 3.6 (n = 10, P < 0.05). p38 MAPK inhibition with 10 microM SB202190 that also decreased ANCA-induced superoxide generation prevented S473 phosphorylation of Akt in response to TNF-alpha and to ANCA. However, SB202190 but not LY294002 abrogated TNF-alpha-mediated ANCA antigen surface translocation, demonstrating that superoxide generation and ANCA antigen translocation proceed by separate mechanisms. Akt, PAK1, and Rac1 existed as cytosolic complex in resting PMN. TNF-alpha stimulation increased association of PAK1 with Akt. An MPO monoclonal antibody did not alter the Akt signaling complex further. The data demonstrate the importance of PI3-K for the ANCA-induced PMN oxidant production.

Complement Receptor Mac-1 Is an Adaptor for NB1 (CD177)-mediated PR3-ANCA Neutrophil Activation

The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1pos/PR3high neutrophils, but not in the mNB1neg/PR3low subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1pos/mPR3high neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when β2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.

Phosphoinositol 3-kinase-γ mediates antineutrophil cytoplasmic autoantibody-induced glomerulonephritis

Antineutrophil cytoplasmic autoantibodies (ANCA) are associated with necrotizing crescentic glomerulonephritis (NCGN) and systemic vasculitis. We examined the role of phosphoinositol 3 kinase-gamma isoform (PI3Kgamma) in ANCA-activated neutrophil functions. Further, we tested whether its inhibition protects a mouse model of ANCA NCGN from developing NCGN. We transplanted bone marrow from wild-type mice or PI3Kgamma-deficient mice into myeloperoxidase-deficient mice immunized with myeloperoxidase. Bone marrow from PI3Kgamma(-/-) mice protected against development of the disease. Similarly, bone marrow transplanted from wild-type mice followed by treatment with the specific PI3Kgamma inhibitor AS605240 also protected these mice against NCGN in this model. AS605240 significantly abrogated myeloperoxidase- or proteinase 3-ANCA-stimulated superoxide production in vitro. Furthermore, ANCA-induced degranulation and GM-CSF-stimulated migration in a transwell assay of isolated human neutrophils were also abrogated by the drug. We found that PI3Kgamma plays a pivotal role in ANCA-induced NCGN and suggest that its specific inhibition may provide a novel treatment target.

β2-Integrins and Acquired Glycoprotein IIb/IIIa (GPIIb/IIIa) Receptors Cooperate in NF-κB Activation of Human Neutrophils

Microparticles from various cells are generated during inflammation. Platelet-derived microparticles (PMPs) harbor receptors that are not genuinely expressed by neutrophils. We tested whether or not functional glycoprotein IIb/IIIa (GPIIb/IIIa) receptors can be acquired by neutrophils via PMPs and whether these receptors participate in pro-inflammatory signaling. Surface expression was analyzed by flow cytometry and confocal microscopy. NF-κB activation was analyzed by Western blot experiments, electrophoretic mobility shift assays, and reverse transcription-PCR. Cell adhesion and spreading were estimated by myeloperoxidase assay and light microscopy. We found that PMPs transfer GPIIb/IIIa receptors to isolated and whole blood neutrophils via PMPs. We used specific antibodies in granulocyte macrophage colony-stimulating factor-treated neutrophils and observed that acquired GPIIb/IIIa receptors co-localized with β2-integrins and cooperated in NF-κB activation. We show that Src and Syk non-receptor tyrosine kinases, as well as the actin cytoskeleton, control NF-κB activation. In contrast to NF-κB, acquisition of GPIIb/IIIa receptors was not necessary to induce adhesion to fibronectin or phosphatidylinositol 3-kinase/Akt signaling. When granulocyte macrophage colony-stimulating factor-stimulated neutrophils were incubated on fibronectin, strong NF-κB activation was observed, but only after loading with PMPs. Blocking either β2-integrins or GPIIb/IIIa receptors abrogated this effect. Therapeutic GPIIb/IIIa inhibitors were similarly effective. The compounds also inhibited NF-κB-dependent tumor necrosis factor-α mRNA up-regulation. The data implicate GPIIb/IIIa receptors as new therapeutic targets in neutrophil-induced inflammation.

BK channels in innate immune functions of neutrophils and macrophages

Oxygen-dependent antimicrobial activity of human polymorphonuclear leukocytes (PMNs) relies on the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate oxidants. As the oxidase transfers electrons from NADPH the membrane will depolarize and concomitantly terminate oxidase activity, unless there is charge translocation to compensate. Most experimental data implicate proton channels as the effectors of this charge compensation, although large-conductance Ca2+-activated K+ (BK) channels have been suggested to be essential for normal PMN antimicrobial activity. To test this latter notion, we directly assessed the role of BK channels in phagocyte function, including the NADPH oxidase. PMNs genetically lacking BK channels (BK(-/-)) had normal intracellular and extracellular NADPH oxidase activity in response to both receptor-independent and phagocytic challenges. Furthermore, NADPH oxidase activity of human PMNs and macrophages was normal after treatment with BK channel inhibitors. Although BK channel inhibitors suppressed endotoxin-mediated tumor necrosis factor-alpha secretion by bone marrow-derived macrophages (BMDMs), BMDMs of BK(-/-) and wild-type mice responded identically and exhibited the same ERK, PI3K/Akt, and nuclear factor-kappaB activation. Based on these data, we conclude that the BK channel is not required for NADPH oxidase activity in PMNs or macrophages or for endotoxin-triggered tumor necrosis factor-alpha release and signal transduction BMDMs.

β2 Integrin-mediated Cell-Cell Contact Transfers Active Myeloperoxidase from Neutrophils to Endothelial Cells

Background: How endothelial cells (ECs) could acquire exogenous neutrophil myeloperoxidase (MPO) is unknown. Results: ECs acquired enzymatically active MPO directly from neutrophils, via β2 integrin-mediated cell-cell contact independent of extracellular MPO release. Conclusion: Neutrophils directly transfer MPO to ECs by cell-cell contact. Significance: Direct delivery of MPO to ECs may contribute to the vascular pathology and dysfunction seen in atherosclerosis and vasculitides. Atherosclerosis and vasculitis both feature inflammation mediated by neutrophil-endothelial cell (EC) contact. Neutrophil myeloperoxidase (MPO) can disrupt normal EC function, although the mechanism(s) by which MPO is transferred to ECs are unknown. We tested the hypothesis that close, β2 integrin-dependent neutrophil-EC contact mediates MPO transfer from neutrophils to ECs. We used sensitive MPO assays and flow cytometry to detect MPO in ECs and demonstrate that ECs acquired MPO when contacted by neutrophils directly but not when ECs and neutrophils were separated in Transwells. The transfer was dependent on neutrophil number, exposure time, and incubation temperature. Transfer occurred in several EC types, increased with endotoxin, was not accompanied by MPO release into the medium, and was not abrogated by inhibiting degranulation to secretagogues. Confocal microscopy showed MPO internalization by ECs with cytoplasmic and nuclear staining. Neutrophils and ECs formed intimate contact sites demonstrated by electron microscopy. Blocking CD11b or CD18 β2 integrin chains, or using neutrophils from CD11b gene-deleted mice, reduced MPO transfer. EC-acquired MPO was enzymatically active, as demonstrated by its ability to oxidize the fluorescent probe aminophenyl fluorescein in the presence of a hydrogen peroxide source. The data suggest an alternative to EC uptake of soluble MPO, namely the cell contact-dependent, β2 integrin-mediated transfer from neutrophils. The findings could be of therapeutic relevance in atherosclerosis and vasculitis.

Aldosterone Abrogates Nuclear Factor κB–Mediated Tumor Necrosis Factor α Production in Human Neutrophils via the Mineralocorticoid Receptor

Mineralocorticoid receptor (MR) activation by aldosterone controls salt homeostasis and inflammation in several tissues and cell types. Whether or not a functional MR exists in polymorphonuclear neutrophils is unknown. We investigated the hypothesis that aldosterone modulates inflammatory neutrophil responses via the MR. By flow cytometry, Western blot analysis, and microscopy, we found that neutrophils possess MR. Preincubation with aldosterone (10−11 to 10−6 M) dose-dependently inhibited nuclear factor &kgr;B activation in interleukin (IL)-8– and granulocyte/macrophage colony-stimulating factor–treated neutrophils on fibronectin by I&kgr;B&agr; Western blotting, electrophoretic mobility shift assay, and RT-PCR for I&kgr;B&agr; mRNA. Aldosterone had no effect on tumor necrosis factor &agr;– and lipopolysaccharide-mediated nuclear factor &kgr;B activation or on IL-8– and granulocyte/macrophage colony-stimulating factor–induced extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, or phosphatidylinositol 3-kinase/Akt activation. Spironolactone prevented nuclear factor &kgr;B inhibition, indicating an MR-specific aldosterone effect. By RT-PCR, we found that neutrophils have 11&bgr;-hydroxysteroid dehydrogenase. Tumor necrosis factor &agr;, which is controlled by nuclear factor &kgr;B, increased in the cell supernatant with IL-8 treatment. Aldosterone completely prevented this effect. RT-PCR showed a strong tumor necrosis factor &agr; mRNA increase with IL-8 that was blocked by aldosterone, excluding the possibility that the tumor necrosis factor &agr; increase was merely a consequence of secretion. Finally, conditioned medium from IL-8–treated neutrophils increased intercellular adhesion molecule–1 expression on endothelial cells and subsequently the adhesion of IL-8–treated neutrophils to endothelial cells. These effects were reduced when conditioned medium from aldosterone-pretreated neutrophils was used, and spironolactone blocked the aldosterone effect. Our data indicate that a functional MR exists in neutrophils mediating antiinflammatory effects that are at work when neutrophils interact with endothelial cells. These data could be relevant to MR-blockade treatment protocols.

The effect of fever-like temperatures on neutrophil signaling

The effect of fever on neutrophils has not been explored. We tested the hypothesis that fever‐like temperature spikes affect neutrophil signaling and function. Prior 60 min, 42°C heat exposure inhibited p38 MAPK, ERK, PI3‐Kinase/Akt, and NF‐κB activation in TNF‐α‐challenged suspended neutrophils. Using pharmacological inhibitors and an inhibitory peptide transduced into neutrophils by a HIV‐TAT sequence, we found that p38 MAPK and NF‐κB mediate TNF‐α‐mediated delayed apoptosis in suspended neutrophils. Heat exposure (39–42°C) did not affect constitutive apoptosis but abrogated TNF‐α‐delayed apoptosis in these suspended cells. In contrast, adhesion‐dependent functions were not inhibited. Furthermore, we found that heat exposure neither blocked p38 MAPK, ERK, and NF‐κB activation in neutrophils on fibronectin nor prevented delayed apoptosis by TNF‐α when cells interacted with fibronectin. Above and beyond apoptosis, TNF‐α initiated NF‐κB‐dependent gene transcription. Heat exposure blocked this effect in suspended neutrophils but not in neutrophils on fibronectin. Finally, we show that β2‐integrins, which are not necessary for TNF‐α‐induced NF‐κB activation at 37°C, transduce costimulatory signals allowing NF‐κB activation after heat exposure. The effect could protect circulating neutrophils from TNF‐α activation, while not interfering with activation of adherent neutrophils. Fever could make neutrophils more parsimonious.

Fever-Like Temperatures Affect Neutrophil NF-κB Signaling, Apoptosis, and ANCA-Antigen Expression

The neutrophil is pivotal to ANCA vasculitis pathogenesis. Fever frequently complicates ANCA diseases. This study investigated the effects of short-term heat exposure on apoptosis in neutrophils that were treated with LPS, GM-CSF, IL-8, and dexamethasone. All compounds delayed apoptosis. Heat abrogated the apoptosis-delaying effect of LPS without affecting constitutive apoptosis or delayed apoptosis by GM-CSF, IL-8, or dexamethasone. The heat effect was dose dependent over the 39 to 42 degrees C range. NF-kappaB but not extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or phosphatidylinositol 3-kinase/Akt controlled LPS-delayed apoptosis. Furthermore, LPS-induced IkappaBalpha degradation, DNA binding, and NF-kappaB-dependent gene transcription activation were abrogated by short-term heat. When core temperatures were raised to 40.5 degrees C for 30 min in mice, LPS-induced neutrophil NF-kappaB activation also was prevented. Short-term heat removed heat-shock protein 90 from the IkappaB kinase complex, resulting in failure of LPS-induced IkappaB kinase activation. Despite delayed apoptosis, ANCA antigen expression was increased in LPS-treated neutrophils. ANCA antigen increase was prevented by p38 MAPK inhibition and by heat exposure. Heat exposure did not inhibit LPS-induced p38 MAPK phosphorylation. Instead, apoptosis-mediated p38 MAPK degradation was accelerated, thereby decreasing the p38 MAPK that was available for LPS-mediated ANCA antigen upregulation. These data suggest that fever-like temperatures modulate neutrophil behavior in this disease.