Competitively disrupting the neutrophil-specific receptor-autoantigen CD177:proteinase 3 membrane complex reduces anti-PR3 antibody-induced neutrophil activation.

CD177 is a neutrophil-specific receptor presenting the proteinase 3 (PR3) autoantigen on the neutrophil surface. CD177 expression is restricted to a neutrophil subset, resulting in CD177pos/mPR3high and CD177neg/mPR3low populations. The CD177pos/mPR3high subset has implications for antineutrophil cytoplasmic autoantibody (ANCA)-associated autoimmune vasculitis, wherein patients harbor PR3-specific ANCAs that activate neutrophils for degranulation. Here, we generated high-affinity anti-CD177 monoclonal antibodies, some of which interfered with PR3 binding to CD177 (PR3 "blockers") as determined by surface plasmon resonance spectroscopy and used them to test the effect of competing PR3 from the surface of CD177pos neutrophils. Because intact anti-CD177 antibodies also caused neutrophil activation, we prepared nonactivating Fab fragments of a PR3 blocker and nonblocker that bound specifically to CD177pos neutrophils. We observed that Fab blocker clone 40, but not nonblocker clone 80, dose-dependently reduced anti-PR3 antibody binding to CD177pos neutrophils. Importantly, preincubation with clone 40 significantly reduced respiratory burst in primed neutrophils challenged with either monoclonal antibodies to PR3 or PR3-ANCA immunoglobulin G from ANCA-associated autoimmune vasculitis patients. After separating the two CD177/mPR3 neutrophil subsets from individual donors by magnetic sorting, we found that PR3-ANCAs provoked significantly more superoxide production in CD177pos/mPR3high than in CD177neg/mPR3low neutrophils, and that anti-CD177 Fab clone 40 reduced the superoxide production of CD177pos cells to the level of the CD177neg cells. Our data demonstrate the importance of the CD177:PR3 membrane complex in maintaining a high ANCA epitope density and thereby underscore the contribution of CD177 to the severity of PR3-ANCA diseases.

ß2 integrin-mediated cell-cell contact transfers active myeloperoxidase from neutrophils to endothelial cells.

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, ß2 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 ß2 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, ß2 integrin-mediated transfer from neutrophils. The findings could be of therapeutic relevance in atherosclerosis and vasculitis.

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 mNB1(pos)/PR3(high) neutrophils, but not in the mNB1(neg)/PR3(low) 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 mNB1(pos)/mPR3(high) neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when ß2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.

Aldosterone abrogates nuclear factor kappaB-mediated tumor necrosis factor alpha 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 kappaB activation in interleukin (IL)-8- and granulocyte/macrophage colony-stimulating factor-treated neutrophils on fibronectin by IkappaBalpha Western blotting, electrophoretic mobility shift assay, and RT-PCR for IkappaBalpha mRNA. Aldosterone had no effect on tumor necrosis factor alpha- and lipopolysaccharide-mediated nuclear factor kappaB 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 kappaB inhibition, indicating an MR-specific aldosterone effect. By RT-PCR, we found that neutrophils have 11beta-hydroxysteroid dehydrogenase. Tumor necrosis factor alpha, which is controlled by nuclear factor kappaB, increased in the cell supernatant with IL-8 treatment. Aldosterone completely prevented this effect. RT-PCR showed a strong tumor necrosis factor alpha mRNA increase with IL-8 that was blocked by aldosterone, excluding the possibility that the tumor necrosis factor alpha 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.

Phosphoinositol 3-kinase-gamma 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.

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.

Short-term heat exposure inhibits inflammation by abrogating recruitment of and nuclear factor-{kappa}B activation in neutrophils exposed to chemotactic cytokines.

Cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-8 attract neutrophils into inflammatory sites. During emigration from the blood neutrophils interact with extracellular matrix proteins such as fibronectin. Fibronectin provides beta2-integrin co-stimulation, allowing GM-CSF and IL-8 to activate nuclear factor (NF)-kappaB, an effect that does not occur in suspension. We tested the hypothesis that exposure of mice to fever-like temperatures abrogates neutrophil recruitment and NF-kappaB activation in a mouse model of skin inflammation. Mice that were exposed to 40 degrees C for 1 hour showed strongly reduced GM-CSF- and IL-8-induced neutrophilic skin inflammation. In vitro heat exposure did not interfere with neutrophil adhesion or spreading on fibronectin but strongly inhibited migration toward both cytokines. Using specific inhibitors, we found that PI3-K/Akt was pivotal for neutrophil migration and that heat down-regulated this pathway. Furthermore, neutrophils on fibronectin showed abrogated NF-kappaB activation in response to GM-CSF and IL-8 after heat. In vivo heat exposure of mice followed by ex vivo stimulation of isolated bone marrow neutrophils confirmed these results. Finally, less NF-kappaB activation was seen in the inflammatory lesions of mice exposed to fever-like temperatures as demonstrated by in situ hybridization for IkappaBalpha mRNA. These new findings suggest that heat may have anti-inflammatory effects in neutrophil-dependent inflammation.

Febrile temperatures control antineutrophil cytoplasmic autoantibody-induced neutrophil activation via inhibition of phosphatidylinositol 3-kinase/Akt.

OBJECTIVE: Neutrophil activation by antineutrophil cytoplasmic autoantibodies (ANCAs) is central to the pathogenesis of the ANCA-associated vasculitides. Febrile infections occur frequently during these diseases, often in the context of immunosuppressive treatment. Heat exposure may affect the underlying pathophysiologic processes of the vasculitis. In this study we tested the hypothesis that short-term exposure to heat inhibits ANCA-induced neutrophil activation. METHODS: After exposure to temperatures from 37 degrees C to 42 degrees C, human neutrophils were primed with either tumor necrosis factor alpha (TNFalpha) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and stimulated with monoclonal antibodies to myeloperoxidase or to proteinase 3. Respiratory burst activity was assayed using rhodamine and a nitroblue tetrazolium reduction assay. Specific inhibition experiments against p38 MAPK, ERK, and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt, and Western blotting with phospho-specific antibodies were used to identify key components in the antibody-induced respiratory burst. RESULTS: A temperature-dependent reduction in ANCA-induced respiratory burst was observed over a range of heat exposures from 37 degrees C to 42 degrees C. Inhibition of human ANCA-induced neutrophil stimulation was significant at 40 degrees C (after priming with 2 ng/ml TNFalpha, mean [+/- SEM] fluorescence intensity [MFI] 114 +/- 12 at 37 degrees C versus 53 +/- 6 at 40 degrees C; after priming with 20 ng/ml GM-CSF, MFI 92 +/- 16 at 37 degrees C versus 35 +/- 6 at 40 degrees C; both P < 0.01). In the priming phase, the transient activation of the p38 MAPK, ERK, and PI 3-kinase/Akt pathways by TNFalpha was blocked by prior exposure of the neutrophils to heat, but GM-CSF-induced activation was unaltered by heat. However, in the second, antibody-induced wave of kinase activation, exposure to heat inhibited only the PI 3-kinase/Akt pathway, and these effects were independent of the priming agent used. CONCLUSION: Short-term spikes of modest heat abrogate ANCA-induced activation of neutrophils via inhibition of PI 3-kinase/Akt signaling. Febrile responses in ANCA-mediated diseases may therefore have a physiologic purpose.

Beta2-integrins and acquired glycoprotein IIb/IIIa (GPIIb/IIIa) receptors cooperate in NF-kappaB 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-kappaB 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 beta2-integrins and cooperated in NF-kappaB activation. We show that Src and Syk non-receptor tyrosine kinases, as well as the actin cytoskeleton, control NF-kappaB activation. In contrast to NF-kappaB, 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-kappaB activation was observed, but only after loading with PMPs. Blocking either beta2-integrins or GPIIb/IIIa receptors abrogated this effect. Therapeutic GPIIb/IIIa inhibitors were similarly effective. The compounds also inhibited NF-kappaB-dependent tumor necrosis factor-alpha mRNA up-regulation. The data implicate GPIIb/IIIa receptors as new therapeutic targets in neutrophil-induced inflammation.

Fever-like temperatures affect neutrophil NF-kappaB 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.

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 degrees C heat exposure inhibited p38 MAPK, ERK, PI3-Kinase/Akt, and NF-kappaB activation in TNF-alpha-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-kappaB mediate TNF-alpha-mediated delayed apoptosis in suspended neutrophils. Heat exposure (39-42 degrees C) did not affect constitutive apoptosis but abrogated TNF-alpha-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-kappaB activation in neutrophils on fibronectin nor prevented delayed apoptosis by TNF-alpha when cells interacted with fibronectin. Above and beyond apoptosis, TNF-alpha initiated NF-kappaB-dependent gene transcription. Heat exposure blocked this effect in suspended neutrophils but not in neutrophils on fibronectin. Finally, we show that beta2-integrins, which are not necessary for TNF-alpha-induced NF-kappaB activation at 37 degrees C, transduce costimulatory signals allowing NF-kappaB activation after heat exposure. The effect could protect circulating neutrophils from TNF-alpha activation, while not interfering with activation of adherent neutrophils. Fever could make neutrophils more parsimonious.

Integrins and cytokines activate nuclear transcription factor-kappaB 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 kappaB (NF-kappaB) controls several cellular functions, including inflammation, proliferation, and cell survival. We explored the role of adhesion in NF-kappaB activation in human neutrophils. Cells were stimulated with tumor necrosis factor-alpha (TNF-alpha), 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-kappaB in suspended neutrophils but rapidly activated NF-kappaB under adherent conditions on matrix, as shown by IkappaB kinase activity assay, IkappaBalpha degradation, electromobility shift assay, and quantitative reverse transcriptase-PCR. In contrast, TNF-alpha activated NF-kappaB both in suspended cells and adherent cells. fMLP did not activate NF-kappaB in either suspended or adherent cells. Specific beta(2) integrin blockade prevented NF-kappaB activation by GM-CSF and IL-8 on fibronectin. Co-stimulating CD18 and CD11b with activating antibodies resulted in NF-kappaB 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-kappaB-activating signal by beta(2) integrins. Thus, in addition to beta(2) integrin ligand binding, NF-kappaB activation depended on the formation of the receptor-associated intracellular focal adhesion complex. We conclude that beta(2) integrins may provide co-stimulatory signals allowing some soluble mediators to activate the NF-kappaB 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.

Inhibition of NF-kappaB by a TAT-NEMO-binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis.

Delivery of biologically active peptides into human polymorphonuclear neutrophils (PMNs) has implications for studying cellular functions and may be therapeutically relevant. The transcription factor nuclear factor-kappaB (NF-kappaB) regulates the expression of multiple genes controlling inflammation, proliferation, and cell survival. PMNs play a crucial role in first-line defense. Targeting NF-kappaB in these cells may promote apoptosis and therefore facilitate resolution of inflammation. We used an 11-amino acid sequence NEMO-binding domain (NBD) that selectively inhibits the IKKgamma (NEMO)/IKKbeta interaction, preventing NF-kappaB activation. An HIV-TAT sequence served as a highly effective transducing shuttle. We show that lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), and dexamethasone (DEX) significantly reduced apoptosis after 20 hours. LPS, but not GM-CSF or DEX, activated NF-kappaB as shown by IkappaBalpha degradation, NF-kappaB DNA binding, and transcriptional activity. The TAT-NBD blocked LPS-induced NF-kappaB activation and NF-kappaB-dependent gene expression. TAT-NBD accelerated constitutive PMN apoptosis dose dependently and abrogated LPS-delayed apoptosis. These results provide a proof of principle for peptide delivery by TAT-derived protein transduction domains to specifically inhibit NF-kappaB activity in PMNs. This strategy may help in controlling various cellular functions even in short-lived, transfection-resistant primary human cells.

Extracellular signal-regulated kinase inhibition by statins inhibits neutrophil activation by ANCA.

BACKGROUND: 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) may modulate cellular inflammatory functions independent of serum cholesterol. We tested the hypothesis that statins decrease respiratory burst activity of human polymorphonuclear neutrophils (PMN) in response to anti-neutrophil cytoplasmic antibodies (ANCA). METHODS: Neutrophils were isolated from healthy human volunteers, human immunoglobulins were isolated from patients with proteinase-3 (PR3)- and myeloperoxidase (MPO)-ANCA. Superoxide generation was measured by the ferricytochrome C assay and the nitro blue tetrazolium (NBT) test. ANCA antigen expression was measured by flow cytometry and phosphorylation of mitogen-activated protein kinase (MAPK) was assessed by Western blotting. RESULTS: Cerivastatin and simvastatin inhibited respiratory burst activity to ANCA dose-dependently (1 to 25 micromol/L). Tumor necrosis factor-alpha (TNF-alpha)-primed neutrophils released 26.7 +/- 2.8 nmol O2-/0.75 x 106 PMN/45 min and 10 micromol/L simvastatin reduced this amount to 18.0 +/- 2.1 nmol. The inhibitory effect was confirmed by the NBT test. The respiratory burst decrease could not be reversed by 500 micromol/L mevalonic acid (MVA). In this assay, both statins also inhibited the response to human ANCA. PR3-ANCA resulted in 19.4 +/- 2.0 nmol O2- nmol. This amount was decreased to 6.0 +/- 1.2 nmol by preincubation with 10 micromol/L simvastatin (P < 0.01). For MPO-ANCA, the values were 22.6 +/- 2.8 nmol for controls versus 16.7 +/- 3.1 nmol with statin (P < 0.01). By FACS, simvastatin decreased TNF-alpha-mediated ANCA antigen translocation (from 219 +/- 33 to 180 +/- 35 MFI for PR3 and 24.0 +/- 2.4 to 18.3 +/- 1.1 for MPO). Finally, since p38 MAPK and ERK control TNF-alpha priming, we studied the effects of both statins on MAPK. Western blotting showed that statins inhibited TNF-alpha-induced ERK phosphorylation in a dose dependent fashion, but had no effect on p38. CONCLUSION: These findings demonstrate that HMG-CoA reductase inhibitors decrease respiratory burst activity of human PMN in response to ANCA. This effect was independent of mevalonate, but involved inhibition of ERK activation during TNF-alpha priming. Our data suggest that HMG-CoA reductase inhibitors may help limit inflammatory responses.

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.