Endotoxin Priming of Neutrophils Requires NADPH Oxidase-generated Oxidants and Is Regulated by the Anion Transporter ClC-3

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 is a described priming agent. We that NADPH oxidants as during endotoxin-mediated priming and that ClC-3 as a of NADPH oxidase The of the primed phenotype in response to endotoxin, including enhancement of the respiratory burst to fMLF, of of and phosphorylation of p38 MAPK, required NADPH oxidase activation and ClC-3 also found that of NADPH ROS were required for the of the that ClC-3 is involved in in the PMN, including endotoxin priming and the of cellular quiescence. was from was from to ClC-3 was from MAPK and and p38 MAPK were from was from and was from the the of and were from was from and was from to the component of the flavocytochrome was by Nauseef Lipooligosaccharide (LOS) from Neisseria meningitidis and lipopolysaccharide-binding protein were from was from in a of and used a of were from Clcn3–/– and Leukocyte of the Clcn3–/– with a has been previously described B. Barna T.J. Bailey M.C. R.A. Lamb F.S. Res. 2002; PubMed Scopus Google Scholar). to and were and by the Care and the of of PMNs from were and the and were and of and of were a was the and was used to were for in of was in a by of and on of of were on of were for and PMNs were and and to PMN PMNs were to from from in with a by the for the of PMNs were and by of as previously described A. J. Clin. Scholar). of NADPH of NADPH oxidase activity were in a the of a PMN in with and was to with a of were by the of LOS in the of fMLF as was as a with for of generation was as the of in a the PMN were and to the as described was to the in the In was a of The of generation and the was as the of with for after of the stimulus as of on ClC-3 of ClC-3 was and the ClC-3 the and enhanced green fluorescent protein the were and by the of cells were from the and in with were with in for to the was to for were with an by were and and were was and were to from to in were and The with and was to by were by for cell with was and was were as cells were a of and p38 by a of were a surface of flavocytochrome and PMNs were in or in the of in the or of or as PMNs were and in with and normal for on were after or concentrations of and for on were and in and for on were in of PMN p38 were on and Clcn3–/– PMNs that were to as described and in for and on for by and with on for were for of p38 and the of p38 MAPK were with for the PMNs were with the with cells were and in PMN for with were for and to and were to for by p38 MAPK, were and with a phosphorylation MAPK and were in an by and to were for or p38 MAPK and by enhanced were the and were for the LOS Priming in Clcn3–/– explore the contribution of ClC-3 to LOS priming, the respiratory burst in and Clcn3–/– PMNs by fMLF fMLF is a of NADPH oxidase activity in otherwise unstimulated to a priming in a of subsequent ROS We used a highly LOS from N. meningitidis as the priming T. A. L. A. M.A. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were ROS in response to fMLF fMLF elicited similar of NADPH oxidase activity in both and Clcn3–/– PMNs In LOS elicited NADPH oxidase Clcn3–/– PMNs of the NADPH oxidase activity by We a but in ROS in response to fMLF in PMNs and primed burst was in Clcn3–/– Priming of the in PMNs after of the and PMNs W.M. J. Clin. PubMed Google J. J. Immunol. 2004; PubMed Scopus Google Scholar), the anion to of NADPH oxidase activity in Clcn3–/– PMNs to In response to fMLF PMN a burst of NADPH oxidase activity in both and PMNs In response to the priming stimulus ROS were but in a and with production by the PMNs in to PMNs fMLF after priming elicited a enhanced respiratory burst in PMN, the PMNs ROS after fMLF the by the priming stimulus is for superoxide and be used the effect of on of NADPH oxidase activity the of superoxide generation in response to LOS was PMNs primed for with LOS and with fMLF in with previous of NADPH oxidase activation in 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 a for anion and ClC-3 during LOS priming. The ClC-3 previously that ClC-3 (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), that that the ClC-3 was a ClC-3 the of on ClC-3 in The of the by ClC-3 were similar to previously for and T. T. T.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and with ClC-3 as a S. T.J. PubMed Scopus Google A. M. PubMed Scopus Google Scholar). ClC-3 were from the to the and J. M. S. A. M. M. J. G. and F. S. for inhibited with a inhibition of a of inhibited ClC-3 and be used to the contribution of ClC-3 to PMN in to LOS Priming and to of PMN NADPH oxidase the oxidase the plasma in response to soluble and ROS but the to ROS after a particulate stimulus. studies that the oxidase on or granular and ROS an T. J.M. H. J. Sci. 1998; PubMed Google H. Full Text Full Text PDF PubMed Scopus Google N. G. 2004; PubMed Scopus Google Scholar). with that ROS were during the priming response to LOS, but superoxide was by the ROS during LOS priming, investigated the effect of on the of ROS in response to inhibited NADPH oxidase activity by LOS as by LUC-CL, and the ROS LOS priming in the of or cells were and to and with The primed fMLF burst in PMN that were in the of was to PMN but was inhibited in cells primed in the of with and normal to PMA, that was of the that ROS during priming are required for the of the primed We that priming ROS, cells primed in an anaerobic in the respiratory primed cells with LOS in an anaerobic and the cells to stimulation with PMN primed in the anaerobic inhibition of the burst in to PMN primed in NADPH oxidase activity was to cells in PMN from the anaerobic after suggesting that the the of the NADPH oxidase to in the was to be on superoxide generation as by the of as with previous studies under anaerobic B.M. Blood. PubMed Google Scholar). these the that priming of the respiratory burst is an process and generation of Impaired Mobilization of the and in and Clcn3–/– the of ClC-3 on LOS priming, of cell surface of a of the flavocytochrome as a of priming F. J. S. M. M. J. Nauseef J. Clin. 1998; 101: PubMed Scopus Google Scholar) and of PMNs with LOS for surface of cyt by PMNs after LOS was of PMNs in the of other stimulation elicited a basal cyt and a in and the of the of LOS to of cyt and in cells was to the that were by the it that the effect of LOS and are of p38 MAPK in response to LOS in PMNs as by of with LOS PMNs of cells by for p38 from PMNs enhancement in p38 phosphorylation after in LOS for from Clcn3–/– PMNs also a of cells with enhanced but was in to the the in intensity for was in PMNs as with from the of cells with of was also in PMNs as with Clcn3–/– from the of on from inhibition of in and Clcn3–/– In LOS priming a in surface and Clcn3–/– PMNs also demonstrated an in after it was from that seen in the PMNs and In addition, similar to basal surface of were in Clcn3–/– PMNs as with in the and a or for the anion transporter ClC-3 during LOS priming. NADPH ROS for Mobilization of cyt and in to previous have described low oxidant generation in response to priming stimuli P. H. R.A. J. Leukocyte Biol. 1990; 97-104Crossref PubMed Scopus Google Scholar), it is these oxidants are to of the primed the anaerobic that of priming required an the of ROS in the of the primed the for NADPH ROS in of of cyt and an of was used to the NADPH of was after studies demonstrated inhibition of superoxide generation in response to PMA as by of inhibited of with previously J. 1986; PubMed Scopus Google Scholar). inhibited the LOS-mediated enhancement of surface cyt was also investigated in PMNs from patients with CGD, PMN lack of the have NADPH oxidase The to of after of with LOS, and the by LOS that NADPH ROS are required for after priming with In addition, similar to and Clcn3–/– elicited of both surface cyt and surface and suggesting that in the of basal ROS, cells of the primed The also basal of on the cell surface as with p38 MAPK in to the of NADPH oxidants in signaling relevant to LOS priming, p38 MAPK, a required signaling in endotoxin-mediated priming D. R. 2002; PubMed Scopus Google Scholar) and a that both and J. J. E. B.M. 1996; PubMed Scopus Google P.M. F. Gougerot-Pocidalo M.A. J. PubMed Scopus Google Scholar), necessary for and activation of the NADPH oxidase PMNs in low of p38 phosphorylation with in phosphorylation after In response to LOS, was phosphorylation of p38 in PMNs with NFA, or enhanced of p38 in the of other and p38 phosphorylation in response to LOS PMNs a of phosphorylation that was to that seen in the of or N-Ac, with basal of p38 MAPK phosphorylation and in response to LOS The effect on basal or LOS phosphorylation of p38 MAPK in response to LOS in PMNs enhanced phosphorylation of p38 MAPK in response to LOS the course as In PMNs with or basal of phosphorylation of p38 MAPK were and LOS elicited enhancement for in is CGD, the involvement of ClC-3 in p38 MAPK p38 in and Clcn3–/– PMNs In unstimulated cells was of cells by for p38 The intensity of was in unstimulated cells and Clcn3–/– LOS priming, of fluorescent PMNs were with cells and a with and In PMNs was enhancement in the after LOS for with the of PMNs of and In PMNs was enhanced phosphorylation in of cells and but phosphorylation was as with PMNs p38 were in the cell these a for both ClC-3 and NADPH oxidase ROS in the activation of p38 MAPK in response to LOS priming Furthermore, these that basal ROS contribute to the of the or PMN by the phosphorylation of p38 and PMNs for an of as an of ClC-3 or to NADPH oxidase elicited a phenotype similar to that of PMNs with to enhanced cell surface and phosphorylation of p38 these NADPH oxidase activity was primed. the cells have NADPH oxidase the PMN oxidants in response to certain 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), the of the of NFA, in PMNs that were and after the priming to PMNs were and to for of on NADPH oxidase PMNs marked enhancement of the respiratory burst in response to subsequent stimulation with fMLF, with seen in primed PMNs PMNs with for by an enhancement in the respiratory burst in response to fMLF the surface of cyt and of the PMN that was with the primed the NADPH oxidase activity in the PMNs was after LOS priming in to PMN with suggesting that the LOS priming effect is to the priming effect of the of to priming of the respiratory burst is by inhibition of basal oxidant PMNs with for by and to NFA, primed PMN for an enhanced respiratory burst in response to fMLF as by PMNs that were ROS, that was an in inhibition of oxidant production by or in a PMN phenotype that the primed these that basal oxidants are required to maintain the cell in a and that ClC-3 is involved in the regulation of priming by a of soluble mediators, including endotoxin, occurs both in and in vivo and PMNs to a of enhanced to to subsequent stimuli release of of in response to the priming stimulation. underlying PMN priming by endotoxin of of the flavocytochrome to the plasma and enhanced phosphorylation of of the of the F. J. S. M. M. J. Nauseef J. Clin. 1998; 101: PubMed Scopus Google Scholar), of other receptors to the cell surface (13Blouin E. Halbwachs-Mecarelli L. Rieu P. Eur. J. Immunol. 1999; 29: 3419-3431Crossref PubMed Scopus (57) Google Scholar), and involvement of MAPK signaling D. R. 2002; PubMed Scopus Google Scholar). The on a for the NADPH oxidase in priming and for that NADPH ROS in signaling that are required during endotoxin-mediated priming but as of a of signaling that the primed of the that the anion transporter ClC-3 is required for normal NADPH oxidase both during LOS priming and in the of the the of priming that is and activation of the oxidase by the priming and other P. H. R.A. J. Leukocyte Biol. 1990; 97-104Crossref PubMed Scopus Google Scholar) have demonstrated that ROS are during priming. that these oxidants were required for the primed the primed PMN phenotype in response to LOS in cells lacking NADPH oxidase oxidants or ROS have been demonstrated to in in a of cell J. 2000; Scholar), the and of oxidant species the identity of ROS that as and the protein elicited by the oxidants under In both integrin activation (13Blouin E. Halbwachs-Mecarelli L. Rieu P. Eur. J. Immunol. 1999; 29: 3419-3431Crossref PubMed Scopus (57) Google Scholar) and apoptosis (14Watson R.W. Antioxid. Redox Signal. 2002; 4: 97-104Crossref PubMed Scopus (37) Google Scholar) are by oxidant with involvement of NADPH ROS during apoptosis A. S. S. M. Y. Clin. Exp. Immunol. 2002; PubMed Scopus Google Scholar). Redox regulation of p38 MAPK is during PMN priming by (15Bouaouina M. Blouin E. Halbwachs-Mecarelli L. Lesavre P. Rieu P. J. Immunol. 2004; 173: 1313-1320Crossref PubMed Scopus (42) Google Scholar) and during priming D. R. 2002; PubMed Scopus Google Scholar). p38 and and in J. J. E. B.M. 1996; PubMed Scopus Google P.M. F. Gougerot-Pocidalo M.A. J. PubMed Scopus Google Scholar), and the on that is by these was recently P.M. A. T. H. C. Y. G. Gougerot-Pocidalo M.A. J. J. Clin. 2006; PubMed Scopus Google Scholar). of phosphorylation inhibited priming of PMNs by and In the study NADPH phosphorylation of p38 MAPK in response to LOS priming. In the setting of or absent ROS, basal phosphorylation of p38 MAPK was of basal of p38 MAPK phosphorylation in response to inhibition of NADPH oxidase has previously been in cells R. A. J. PubMed Scopus Google Scholar). that basal of NADPH oxidase ROS a in p38 MAPK in the might stimulation of a or inhibition of an and is an of in oxidant signaling has been described in and the that is NADPH oxidase in The includes a to of or with by R.A. Nauseef W.M. J. Clin. 1990; PubMed Scopus Google M. J.M. B.M. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). basal oxidant signaling is described in a of other cell Jr., F.J. Am. J. Circ. PubMed Scopus Google J.M. A.M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google J.M. S. A.M. 2004; PubMed Scopus Google Scholar), and the phagocyte NADPH oxidase is the of ROS in of these The that a of the phagocyte NADPH oxidase is and low oxidants under basal to a signaling has been is that the flavocytochrome might low of involved in of these for The also a for the anion transporter ClC-3 in PMN priming by ClC-3 protein is in PMNs and is required for normal PMN NADPH oxidase activity (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). The of ClC-3 has been a of with studies suggesting that it was a cell surface by D. C. S. J.R. B. PubMed Scopus Google Scholar), protein P. J. A. M.A. J. Biol. Chem. Scholar), or the T. T. T. Y. H. N. J. 2002; 16: PubMed Scopus Google Scholar). ClC-3 has been to be a an on the of ClC-3 to and as that a and are S. T.J. PubMed Scopus Google A. M. PubMed Scopus Google Scholar). of ClC-3 in cells that ClC-3 are similar to of the other The on a for ClC-3 during priming from in Clcn3–/– vascular smooth muscle cells signaling in response to and (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 M. Huss G. Barna T. F. Lamb F. J. 2006; Scholar). We found that the LOS priming of NADPH oxidase activity was inhibited in PMNs and inhibited in Clcn3–/– The of Clcn3–/– PMNs to inhibition of the primed fMLF burst changes in the In addition, the and for priming are as previous have that cells have for priming J. R.A. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). PMNs and PMNs to that the anion transporter was involved in regulation of NADPH oxidase during basal oxidant signaling and during LOS priming. We that ClC-3 in during ROS generation a ClC-3 were as a in an the by the respiratory burst with and the The NADPH to a and is required to M. A. L. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google A. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). be that a of ClC-3 in an that ClC-3 has a similar the plasma previous that and PMNs have normal ROS production in response to PMA (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) that an a In addition, of ClC-3 in PMNs or cell is required to that in cells are to the of ClC-3 in to be that ClC-3 has a in of the NADPH oxidase on of these are In summary, the that NADPH ROS are involved in signaling both during endotoxin priming of neutrophils and in the of the or We that patients with have impaired priming to endotoxin, a that contribute to the or of sepsis in these In addition, the anion transporter ClC-3 also to contribute to LOS-mediated priming and cellular quiescence. We that ClC-3 in for the NADPH oxidase under certain oxidants are an is under as as studies to the of oxidant generation during priming by