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• W1968492024 abstract "Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes. Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes. Polymorphonuclear leukocyte (PMN) 2The abbreviations used are: PMN, polymorphonuclear leukocytes; ROS, reactive oxygen species; fMLF, formyl-Met-Leu-Phe; CGD, chronic granulomatous disease; LUC-CL, lucigenin-enhanced chemiluminescence; LOS, lipooligosaccharide; NFA, niflumic acid; SOD, superoxide dismutase; cyt b558, flavocytochrome b558; DPI, diphenyleneiodonium; N-Ac, N-acetylcysteine; LBP, lipopolysaccharide-binding protein; PMA, phorbol 12-myristate 13-acetate; CGD, chronic granulomatous disease; MAPK, mitogen-activated protein kinase; eGFP, enhanced green fluorescent protein; PEG, polyethylene glycol. 2The abbreviations used are: PMN, polymorphonuclear leukocytes; ROS, reactive oxygen species; fMLF, formyl-Met-Leu-Phe; CGD, chronic granulomatous disease; LUC-CL, lucigenin-enhanced chemiluminescence; LOS, lipooligosaccharide; NFA, niflumic acid; SOD, superoxide dismutase; cyt b558, flavocytochrome b558; DPI, diphenyleneiodonium; N-Ac, N-acetylcysteine; LBP, lipopolysaccharide-binding protein; PMA, phorbol 12-myristate 13-acetate; CGD, chronic granulomatous disease; MAPK, mitogen-activated protein kinase; eGFP, enhanced green fluorescent protein; PEG, polyethylene glycol. activation occurs during ingestion of microorganisms and includes the release of preformed granular proteins, including proteolytic enzymes, and de novo generation of reactive oxygen species (ROS) by the NADPH oxidase (1Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar). There are multiple regulatory mechanisms that modulate PMN activation so that the amplitude of the cellular response matches the intensity of the stimulus. One such mechanism is priming, whereby the functional response of PMN to an activating stimulus is amplified by previous interaction with a priming agent. Diverse agents prime PMN, including microbial components and soluble host proteins, and both the underlying mechanisms of priming and the phenotype of the primed PMN are dependent on the identity of the stimulus (for review, see Ref. 2Condliffe A.M. Kitchen E. Chilvers E.R. Clin. Sci. (Lond.). 1998; 94: 461-471Crossref PubMed Scopus (326) Google Scholar). Endotoxin or lipopolysaccharide, a component of the cell wall of Gram-negative bacteria and a potent inflammatory mediator, primes PMN for marked enhancement of the respiratory burst in response to formyl-Met-Leu-Phe (fMLF) (3Aida Y. Pabst M.J. J. Immunol. 1990; 145: 3017-3025PubMed Google Scholar), opsonized zymosan (4Follin P. Dahlgren C. Inflammation. 1992; 16: 83-91Crossref PubMed Scopus (20) Google Scholar), and PMA (5Forehand J.R. Bomalski J.S. Johnston Jr., R.B. Adv. Exp. Med. Biol. 1991; 297: 65-73Crossref PubMed Google Scholar). Lipopolysaccharide stimulation also primes PMN for degranulation and elastase release in response to fMLF (6Fittschen C. Sandhaus R.A. Worthen G.S. Henson P.M. J. Leukocyte Biol. 1988; 43: 547-556Crossref PubMed Scopus (49) Google Scholar) and for changes in adhesion (7Condliffe A.M. Chilvers E.R. Haslett C. Dransfield I. Immunology. 1996; 89: 105-111Crossref PubMed Scopus (127) Google Scholar). Priming occurs in vivo and primed PMNs have been demonstrated in the circulation of patients after traumatic injury (8Ogura H. Tanaka H. Koh T. Hashiguchi N. Kuwagata Y. Hosotsubo H. Shimazu T. Sugimoto H. J. Trauma. 1999; 46: 774-783Crossref PubMed Scopus (111) Google Scholar), during the course of the acute respiratory distress syndrome (9Chollet-Martin S. Montravers P. Gibert C. Elbim C. Desmonts J.M. Fagon J.Y. Gougerot-Pocidalo M.A. Am. Rev. Respir. Dis. 1992; 146: 990-996Crossref PubMed Scopus (102) Google Scholar), and in the setting of sepsis (10Bass D.A. Olbrantz P. Szejda P. Seeds M.C. McCall C.E. J. Immunol. 1986; 136: 860-866PubMed Google Scholar). In addition, low concentrations of endotoxin are present in the plasma of patients with Gram-negative bacterial infections (11Venet C. Zeni F. Viallon A. Ross A. Pain P. Gery P. Page D. Vermesch R. Bertrand M. Rancon F. Bertrand J.C. Intensive Care Med. 2000; 26: 538-544Crossref PubMed Scopus (44) Google Scholar). Therefore, the study of endotoxin-mediated priming is highly relevant to disease pathogenesis. The phagocyte NADPH oxidase is a multicomponent enzyme complex that produces substantial quantities of ROS necessary for optimal microbicidal activity against certain pathogens. Patients with chronic granulomatous disease (CGD) lack a functional NADPH oxidase and are prone to frequent and serious infections (12Segal B.H. Leto T.L. Gallin J.I. Malech H.L. Holland S.M. Medicine (Baltimore). 2000; 79: 170-200Crossref PubMed Scopus (718) Google Scholar). Oxidant signaling in PMN has also been demonstrated to be involved in the regulation of integrin activation (13Blouin E. Halbwachs-Mecarelli L. Rieu P. Eur. J. Immunol. 1999; 29: 3419-3431Crossref PubMed Scopus (57) Google Scholar), apoptosis (14Watson R.W. Antioxid. Redox Signal. 2002; 4: 97-104Crossref PubMed Scopus (37) Google Scholar), and priming by select agents (15Bouaouina M. Blouin E. Halbwachs-Mecarelli L. Lesavre P. Rieu P. J. Immunol. 2004; 173: 1313-1320Crossref PubMed Scopus (42) Google Scholar). Impaired oxidant signaling in PMN might contribute to the complex phenotype of CGD. We recently described the involvement of the anion transporter ClC-3 in NADPH oxidase activation of PMNs in response to both particulate and soluble stimuli (16Moreland J.G. Davis A.P. Bailey G. Nauseef W.M. Lamb F.S. J. Biol. Chem. 2006; 281: 12277-12288Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Concurrently, studies conducted in vascular smooth muscle cells have demonstrated impaired cytokine-initiated redox signaling in Clcn3–/– cells (17Miller Jr., F.J. Filali M. Huss G.J. Stanic B. Chamseddine A. Barna T.J. Lamb F.S. Circ. Res. 2007; 101: 663-671Crossref PubMed Scopus (170) Google Scholar). We investigated priming in PMNs as a process similar to cytokine-induced signaling in other cell types, as tumor necrosis factor-α is a well described priming agent. We hypothesized that NADPH oxidase-derived oxidants serve as signals during endotoxin-mediated priming and that ClC-3 participates as a regulator of NADPH oxidase function. The development of the primed phenotype in response to endotoxin, including enhancement of the respiratory burst to fMLF, up-regulation of intracellular stores of receptors, and phosphorylation of p38 MAPK, required NADPH oxidase activation and ClC-3 function. Unexpectedly, we also found that low, tonic levels of NADPH oxidase-derived ROS were required for the maintenance of the non-primed quiescent state. Our data suggest that ClC-3 is proximally involved in oxidant-dependent events in the PMN, including endotoxin priming and the maintenance of cellular quiescence. Materials—Hanks' balanced salt solution was obtained from BioWhittaker (Walkersville, MD). Fetal bovine serum was obtained from HyClone (Logan, UT). Polyclonal antibody to ClC-3 was from Sigma. Rabbit anti-p38 MAPK and anti-phosphorylated threonine and tyrosine p38 MAPK were from Cell Signaling (Beverly, MA). Murine anti-human CD11b was purchased from Pharmingen, and rat-anti-mouse CD11b was purchased from the Developmental Hybridoma bank at the University of Iowa. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG+IgM and FITC-conjugated donkey anti-rabbit F(ab′)2 were from Jackson ImmunoResearch Laboratories (West Grove, PA). Mouse IgG1 was purchased from Sigma, and rat IgG2 was purchased from BIOSOURCE. Antibody to the gp91phox component of the flavocytochrome b558 was generated by Dr. Nauseef (clone 7D5). Lipooligosaccharide (LOS) purified from Neisseria meningitidis and lipopolysaccharide-binding protein (LBP) were generous gifts from Dr. Jerrold Weiss. Niflumic acid (NFA) was purchased from Sigma, dissolved in Me2SO at a concentration of 10–1 m, and used at a final concentration of 10–3 m. Additional reagents were all obtained from Sigma. Clcn3–/– Mice and Murine Leukocyte Isolation—Generation of the Clcn3–/– mice with a C57Bl/6J-SV129 background has been previously described (18Dickerson L.W. Bonthius D.J. Schutte B.C. Yang B. Barna T.J. Bailey M.C. Nehrke K. Williamson R.A. Lamb F.S. Brain Res. 2002; 958: 227-250Crossref PubMed Scopus (80) Google Scholar). All animals had free access to food and water. Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Iowa. For isolation of murine PMNs from bone marrow, mice were euthanized, and then the femur, tibia, and iliac bones were removed and stripped of all tissue. Distal and proximal ends of each bone were transected, a 25-gauge needle was inserted into the shaft, and cold buffered Hanks' balanced salt solution was used to flush out bone marrow cells. Cells were pelleted at 600 × g for 10 min at 4 °C then suspended in 3 ml of 45% Percoll. Percoll density gradient was made in a 15-ml tube by layering successively 2 ml each of 62, 55, and 50% Percoll on top of 3 ml of 81% Percoll. Leukocytes were then gently loaded on top of gradient. Cells were centrifuged at 1600 × g for 30 min at 10 °C, and PMNs were harvested between 81 and 62% layers and washed to remove Percoll. Human PMN Purification—Human PMNs were isolated according to standard techniques from heparin anti-coagulated venous blood from healthy consenting adults in accordance with a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa. PMNs were isolated using dextran sedimentation and Hypaque-Ficoll density-gradient separation followed by hypotonic lysis of erythrocytes as previously described (19Boyum A. Scand. J. Clin. Lab. Investig. 1968; 97: 77-89Google Scholar). Measurement of NADPH Oxidase Activity Chemiluminescence—Lucigenin-enhanced chemiluminescence (LUC-CL) assays of NADPH oxidase activity were performed in a 96-well plate using the Wallac Victor3 luminometer (PerkinElmer Life Sciences). 200 μl of a PMN suspension containing 2.5 × 106 PMNs/ml in Hanks' balanced salt solution with 1% human serum albumin and 0.1% dextrose was added to each well with a final concentration of lucigenin 100 μm. Cells were stimulated by the addition of LOS (10 ng/ml) in the presence of LBP (100 ng/ml) ± fMLF as specified. Chemiluminescence was quantitated as relative luminescence units using a kinetic assay with readings every minute for 30–90 min. Reduction of Ferricytochrome c—Extracellular O2 generation was measured as the superoxide-dismutase (SOD)-inhibitable reduction of ferricytochrome c in a 96-well microplate using the SPECTRmax plus (Molecular Devices). PMN suspensions were diluted and added to the microplate as described above. Cytochrome c (100 μm) was added to the suspension just before loading in the microplate. In duplicate wells, SOD was added at a final concentration of 50 μg/ml. The maximum rate (Vmax) of O2 generation and the total nmol O2 was calculated as the SOD-inhibitable reduction of ferricytochrome c, with readings at absorbance 550 nm every 15 s for 30 min after injection of the stimulus as specified. Effect of Anion Channel Inhibition on ClC-3 Current—The short (see GenBank™ X78520) N-terminal isoform of human ClC-3 was PCR-amplified and cloned into the adenovirus shuttle plasmid pacAd5 CMV. Bicistronic adenoviruses co-expressing ClC-3 (Ad-ClC-3) behind the cytomegalovirus promoter and enhanced green fluorescent protein (eGFP) behind the Rous sarcoma virus promoter were prepared and titrated by the University of Iowa Vector core. Control adenovirus expressed only eGFP (Ad-eGFP). HEK293 cells (HEK293T, adenoviral propagation-resistant) were obtained from the American Tissue Culture Collection and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were infected with adenovirus in serum-free Dulbecco's modified Eagle's medium for 16 h before being returned to their standard serum concentration where the virus was allowed to express for 48 h before experimentation. Chloride ion currents were measured at room temperature (22 °C) using standard whole-cell voltage-clamp techniques with an Axopatch 200B patch clamp amplifier driven by pClamp 9 software (Molecular Devices Corp., Sunnyvale, CA). Pipette resistances were 3–5 megaohms. Pipette and whole-cell capacitance and series resistance were compensated. Holding potential was –40 mV and 1-s test pulses were delivered every 3 s to test potentials from –100 to +100 mV in 20-mV increments. Currents were sampled at 5 kHz and filtered at 1 kHz. The standard bath solution contained 120 mm NaCl, 2.5 mm MgCl2, 2.5 mm CaCl2, 10 mm HEPES, 5.5 mm glucose, pH 7.2 with NaOH, and osmolality was titrated to 300 mosmol by osmometer (μ OSMETTE) using 1 m mannitol. Liquid junction potentials were minimized by using 3 m KCl agar bridges. Pipette solutions for standard whole cell recordings contained 120 mm CsCl, 4 mm triethylammonium chloride, 5 mm EGTA, 1.187 mm CaCl2, 2 mm MgCl2, 5 mm Na-ATP, 0.5 mm Na-GTP, 10 mm HEPES, pH 7.2 with CsOH, osmolality was 290 mosmol, and free [Ca2+] was 55 nm (calculated using WEBMAXC). Currents were expressed as current density (pA/picofarads). GFP-positive cells were identified using a fluorescence-equipped inverted microscope (Zeiss Axiovert 25). Analysis of Cell Surface Receptor Expression and Intracellular p38 Levels by Flow Cytometry—PMNs at a concentration of 1 × 106/ml were analyzed using a FACScalibur flow cytometer (BD Biosciences). For surface expression of flavocytochrome b558 and CD11b, PMNs were incubated in Hanks' balanced salt solution alone or in the presence of LOS/LBP in the presence or absence of NFA or diphenyleneiodonium (DPI) as specified. After incubation, PMNs were centrifuged and resuspended in blocking buffer containing phosphate-buffered saline with 2% nonfat dry milk and 1% normal goat serum for 20 min on ice. Primary antibodies were added after blocking; mouse IgG1 control, anti CD11b, or anti-gp91phox, all at final concentrations of 10 μg/ml and incubated for 1 h on ice. Cells were centrifuged and resuspended in fluorescein isothiocyanate-conjugated goat anti-mouse at 1:1000 dilution and incubated for 30 min on ice. Cells were resuspended in buffer containing 5 μg/ml propidium iodide before analysis. Analysis of murine PMN p38 levels were performed on Clcn3+/+ and Clcn3–/– PMNs that were exposed to LOS/LBP as described and then fixed in 2% formaldehyde for 10 min and placed on ice for 1 min followed by centrifugation and permeabilization with 90% methanol on ice for 30 min. Samples were analyzed for levels of anti-total p38 and anti-phospho-p38 using the manufacturer's recommended dilutions. Analysis of p38 MAPK Phosphorylation—PMNs (2 × 107) were treated with purified LOS/LBP for the specified time points. Some PMNs were treated with inhibitors (DPI, N-acetylcysteine (N-Ac), rotenone, NFA) at the specified concentration before incubation with LOS. After incubation, cells were centrifuged and lysed in PMN lysis buffer (100 mm Tris, 150 mm NaCl, 2 mm MgCl2, 1% Triton, 1 mm phenylmethylsulfonyl fluoride, 2% leupeptin/pepstatin A) for 45 min at 4 °C with tumbling. Lysates were centrifuged at 14,000 rpm for 7 min and removed to fresh tubes, and samples were heated to 103 °C for 3 min before analysis by SDS-PAGE. To quantify total p38 MAPK, blots were stripped and reprobed with a phosphorylation state-independent anti-p38 MAPK antibody. Protein Electrophoresis and Immunoblotting—Samples were resolved in an 11% gel by SDS-PAGE and then transferred to nitrocellulose. Immunoblots were processed using polyclonal antibody specific for phospho-p38 or total p38 MAPK and horseradish peroxidase-labeled donkey anti-rabbit followed by enhanced chemiluminescence detection (Super Signal Substrate, Pierce). Immunoblots were scanned using the Typhoon imager (Amersham Biosciences), and relative abundances were quantitated using ImageQuant software for the PhosphorImager (Sunnyvale, CA). Diminished LOS Priming in Murine Clcn3–/– PMNs—To explore the contribution of ClC-3 to LOS priming, we measured the respiratory burst in Clcn3+/+ and Clcn3–/– murine PMNs by fMLF using LUC-CL. fMLF is a relatively weak activator of NADPH oxidase activity in otherwise unstimulated PMNs; however, exposure to a priming agent resulted in a 5–10-fold augmentation of subsequent fMLF-stimulated ROS production. We used a highly purified LOS from N. meningitidis as the priming agent (20Giardina P.C. Gioannini T. Buscher B.A. Zaleski A. Zheng D.S. Stoll L. Teghanemt A. Apicella M.A. Weiss J. J. Biol. Chem. 2001; 276: 5883-5891Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Although there were minimal ROS generated in response to fMLF at 1–10 μm, 100 μm fMLF elicited similar levels of NADPH oxidase activity in both Clcn3+/+ and Clcn3–/– PMNs (Fig. 1A). In Clcn3+/+ PMNs, LOS exposure (10 ng/ml) elicited robust NADPH oxidase activity, whereas Clcn3–/– PMNs had less than 50% of the NADPH oxidase activity by comparison (Fig. 1B). We detected a transient but significant increase in ROS in response to fMLF 100 μm in LOS-primed murine PMNs (Fig. 1C and inset). This primed burst was 2-fold increased in Clcn3+/+ versus Clcn3–/– PMNs. Abnormal Priming of the Respiratory Burst in PMNs after Anion Transporter Inhibition—In view of the well defined differences between human and murine PMNs (21Nauseef W.M. J. Clin. Investig. 2001; 107: 401-403Crossref PubMed Google Scholar, 22Mestas J. Hughes C.C. J. Immunol. 2004; 172: 2731-2738Crossref PubMed Scopus (2269) Google Scholar), we utilized the anion channel inhibitor NFA to extend our findings of abnormal NADPH oxidase activity in Clcn3–/– PMNs to human PMNs. In response to fMLF (1 μm) alone, naïve PMN exhibited a small burst of NADPH oxidase activity detected in both control and NFA-treated PMNs (Fig. 2A). In response to the priming stimulus alone (LOS 10 ng/ml), ROS were generated, but in a delayed fashion and with significantly diminished production by the NFA-treated PMNs in comparison to control PMNs (Fig. 2B). fMLF (1 μm) injected after priming elicited a markedly enhanced respiratory burst in control PMN, whereas the NFA-treated PMNs did not generate ROS after fMLF above the background levels generated by the priming stimulus (Fig. 2C). Because lucigenin is not specific for superoxide detection and cannot be used quantitatively, we examined the effect of NFA treatment on LOS-priming of NADPH oxidase activity using the SOD-inhibitable reduction of ferricytochrome c. Extracellular superoxide generation in response to LOS alone was very low. Control PMNs primed for 30 min with LOS and then stimulated with fMLF generated 1.38 ± 0.109 nmol O2 PMN/10 min versus 0.15 ± 0.007 nmol O2 PMN/10 min in NFA-treated PMNs. Taken together with our previous findings of decreased NADPH oxidase activation in NFA-treated PMNs after opsonized zymosan or PMA stimulation (16Moreland J.G. Davis A.P. Bailey G. Nauseef W.M. Lamb F.S. J. Biol. Chem. 2006; 281: 12277-12288Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), these data suggested a role for anion transport and ClC-3 during LOS priming. The Anion Channel Inhibitor Niflumic Acid Inhibits ClC-3 Current—Although we previously suggested that NFA inhibits ClC-3 function (16Moreland J.G. Davis A.P. Bailey G. Nauseef W.M. Lamb F.S. J. Biol. Chem. 2006; 281: 12277-12288Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), we recognized that electrophysiological evidence that NFA blocked the ClC-3 current was required. Using a heterologous adenoviral ClC-3 expression system, we assessed the effects of NFA on ClC-3 currents in HEK293 cells. The basic biophysical properties of the currents induced by ClC-3 overexpression were very similar to those previously published for ClC-4 and ClC-5 (23Friedrich T. Breiderhoff T. Jentsch T.J. J. Biol. Chem. 1999; 274: 896-902Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) and consistent with ClC-3 functioning as a chloride proton exchanger at physiologic pH (24Scheel O. Zdebik A.A. Lourdel S. Jentsch T.J. Nature. 2005; 436: 424-427Crossref PubMed Scopus (397) Google Scholar, 25Picollo A. Pusch M. Nature. 2005; 436: 420-423Crossref PubMed Scopus (385) Google Scholar). ClC-3 currents were sharply outwardly rectifying, strongly favoring Cl– movement from the extracellular to the intracellular compartment (Fig. 3, A and B). 3J. J. Matsuda, M. S. Filali, K. A. Volk, M. M. Collins, J. G. Moreland, and F. S. Lamb, submitted for publication. NFA (1 mm) significantly inhibited this current (Fig. 3C) with a mean percent inhibition of 57 ± 4.2% (mean ± S.E., n = 7) at a test potential of +80 mV. Thus, NFA clearly inhibited ClC-3 current and can be used to assess the contribution of ClC-3 currents to PMN function. Oxidants Generated in Response to LOS Priming Are Both Extracellular and Intracellular—According to current understanding of PMN NADPH oxidase activity, the oxidase assembles at the plasma membrane in response to soluble agonists and generates extracellular ROS but assembles at the phagosomal membrane to generate intraphagosomal ROS after a particulate stimulus. However, several studies suggest that the oxidase can assemble on vesicular or granular membranes and generate ROS into an intracellular, non-phagosomal compartment (26Kobayashi T. Robinson J.M. Seguchi H. J. Cell Sci. 1998; 111: 81-91Crossref PubMed Google Scholar, 27Brown G.E. Stewart M.Q. Liu H. Ha V.L. Yaffe M.B. Mol. Cell. 2003; 11: 35-47Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 28Ambruso D.R. Cusack N. Thurman G. Mol. Genet. Metab. 2004; 81: 313-321Crossref PubMed Scopus (30) Google Scholar). Our data with LUC-CL suggested that ROS were generated during the priming response to LOS, but minimal extracellular superoxide was measured by ferricytochrome c. To locate the ROS produced during LOS priming, we investigated the effect of added extracellular SOD on the recovery of ROS produced directly in response to LOS. Extracellular SOD significantly inhibited NADPH oxidase activity stimulated by LOS as detected by LUC-CL, and the membrane-permeable PEG-SOD reduced detectable ROS levels below background (Fig. 4A). After LOS priming in the presence of either extracellular SOD or PEG-SOD, cells were centrifuged and washed to remove SOD and then stimulated with fMLF. The primed fMLF burst in PMN that were LOS-primed in the presence of extracellular SOD was identical to control PMN but was completely inhibited in cells primed in the presence of PEG-SOD (Fig. 4B). Cells treated with PEG-SOD and then washed had normal responses to PMA, indicating that there was adequate removal of the permeable dismutase (data not shown). These data suggest that intracellular ROS generated during priming are required for the development of the primed burst. We reasoned that if priming requires ROS, cells primed in an anaerobic environment would not demonstrate amplification in the fMLF-stimulated respiratory burst. To test this hypothesis, we primed cells with LOS in an anaerobic chamber and then removed the cells to room air immediately before stimulation with fMLF. PMN primed in the anaerobic chamber had near complete inhibition of the fMLF-induced burst in comparison to PMN primed in room air (Fig. 4C). PMA-stimulated NADPH oxidase activity was identical to control cells in PMN removed from the anaerobic chamber after 30 min, suggesting that the conditions had not permanently altered the ability of the NADPH oxidase to function (data not shown). Oxygen concentration in the chamber was estimated to be <1% based on PMA-stimulated superoxide generation as measured by the reduction of ferricytochrome c as compared with previous studies performed under anaerobic conditions (29Gabig T.G. Bearman S.I. Babior B.M. Blood. 1979; 53: 1133-1139Crossref PubMed Google Scholar). Taken together, these data strongly support the hypothesis that endotoxin-elicited priming of the respiratory burst is an oxygen-dependent process and requires intracellular generation of ROS. Impaired Mobilization of the Flavocytochrome b558 and CD11b in NFA-treated and Clcn3–/– PMNs—To examine the impact of ClC-3 on LOS priming, we examined up-regulation of cell surface expression of gp91phox, a subunit of the flavocytochrome b558 (cyt b558), as a manifestation of priming (30DeLeo F. Renee J. McCormick S. Nakamura M. Apicella M. Weiss J. Nauseef W. J. Clin. Investig. 1998; 101: 455-463Crossref PubMed Scopus (268) Google Scholar) and of CD11b/CD18. PMNs incubated with LOS for 30 min increased surface expression of cyt b558 by 128 ± 40.3%, whereas NFA-treated PMNs showed virtually no increase after LOS (Fig. 5A). CD11b expression was similarly affected (Fig. 5B). Interestingly, NFA treatment of PMNs in the absence of other stimulation elicited a 2.2-fold increase above basal cyt b558 levels and a 2.9-fold increase in CD11b expression (Fig. 5, C and D). These data raised the issue of whether the failure of LOS to elicit up-regulation of cyt b558 and CD11b in NFA-treated cells was secondary to the fact that intracellular stores were fully mobilized by the NFA alone. However, it appears that the “priming” effect of LOS and NFA are additive (see Fig. 9).FIGURE 9Phosphorylation of p38 MAPK in response to LOS in murine PMNs as assessed by flow cytometry. After 10 min of incubation with LOS (10 ng/ml), murine PMNs displayed two populations of cells by flow cytometry for phosphorylated p38 MAPK. A, representative histogram from Clcn3+/+ PMNs showing significant enhancement in p38 phosphorylation after incubation in LOS for 10 min. FITC, fluorescein isothiocyanate. B, representative histogram from Clcn3–/– PMNs also displayed a population of cells with enhanced phosphorylation, but this was reduced in comparison to the wild-type PMNs. C, the increase in mean fluorescence intensity (MFI) for phospho-p38 was significantly greater in Clcn3+/+ PMNs as compared with Clcn3–/–PMNs. n = 5 mice/strain. *, significantly different from wild type, p < 0.05. D, the percentage of cells with high levels of phospho-p38 was also increased in Clcn3+/+ PMNs as compared with Clcn3–/– PMNs. n = 5 mice/strain. *, significantly different from wild-type, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether the effects of NFA on receptor expression resulted from inhibition of ClC-3, we compared CD11b expression in Clcn3+/+ and Clcn3–/– murine PMNs. In Clcn3+/+ PMNs, LOS priming induced a 288% increase in surface CD11b expression (Fig. 6, A and C). Clcn3–/– PMNs also demonstrated an increase in CD11b after LOS; however, it was significantly reduced from that seen in the wild-type PMNs (Fig. 6, B and C). In addition, similar to NFA-treated human PMNs, basal surface levels of CD11b were 1.5-fold increased in Clcn3–/– PMNs as compared with Clcn3+/+ (Fig. 6D). Considered in combination, the human and murine data suggest a spec" @default.
• W1968492024 created "2016-06-24" @default.
• W1968492024 creator A5001849343 @default.
• W1968492024 creator A5019806106 @default.
• W1968492024 creator A5034849583 @default.
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• W1968492024 date "2007-11-01" @default.
• W1968492024 modified "2023-10-15" @default.
• W1968492024 title "Endotoxin Priming of Neutrophils Requires NADPH Oxidase-generated Oxidants and Is Regulated by the Anion Transporter ClC-3" @default.
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