Control PMNs Research Articles

Metabolic Abnormalities in G6PC3 Deficient Human Neutrophils Result in Severe Functional Defects

IntroductionSevere congenital neutropenia, type 4 results from a mutation in the G6PC3 gene. This gene encodes the enzyme glucose-6-phosphatase (G6Pase) which is ubiquitously expressed in endoplasmic reticulum and is responsible for the final step of gluconeogenesis and glycogenolysis. G6pc3-/- mouse models have demonstrated decreased neutrophil (PMN) function including impaired chemotaxis and superoxide anion production and increased apoptosis. Metabolically, g6pc3-/-mice have impaired intracellular glucose transport and reduced levels of ATP and lactic acid. We present the most comprehensive functional and metabolic analysis to date on a patient with a novel compound heterozygous mutation in the G6PC3 gene, c.325G>A and c.758G>A, validating mouse model findings in a human.MethodsPMNs were isolated from heparinized whole blood by dextran sedimentation and hypotonic lysis of red cells. Chemotactic assays were performed under agarose techniques with zymosan activated serum (ZAS), fMLF, and buffer control. Expression of CD18 and F-actin assembly were analyzed after incubation with buffer, PMA, and fMLF using standard flow cytometric techniques. Bactericidal activity was measured using variable ratios of S Aureus:PMNs in the presence of 10% normal human serum. Superoxide anion production was measured with SOD-inhibitable chemiluminescence with Diogenes reagent. Chemiluminescence was employed to measure caspase expression. Metabolomics and tracing experiments were performed incubating cells with U-13C-glucose prior to UHPLC/MS analysis.ResultsCell motility was significantly impaired in human G6Pase deficient (patient) PMNs. Non-directed migration was reduced by over 50% compared to controls. Directed migration of patient PMNs was only 35% and 23% of control when stimulated with ZAS and 0.1 µM fMLF respectively. Surface expression of CD18 in patient cells did not increase in response to either stimulus but more than doubled in normal PMNs. F-actin was present at significantly higher levels in unstimulated patient PMNs and did not increase in the presence of 200 ng/mL PMA or 1 µM fMLF.Bactericidal activity was normal at a bacteria:PMN ratio of 1:1 but abnormal at ratios > 5:1 with over a 20% increase in viable bacteria at 120 minutes for patient PMNs. Superoxide anion production was markedly impaired with over 70% reduction compared to control when stimulated with either 200 ng/mL PMA or 1 µM fMLF. Caspase expression was not significantly different between patient and control PMNs, but the patient was on filgrastim which has antiapoptotic effects.Tracing experiments with 13C6-glucose showed decreased flux through glycolysis (significant reductions in labeled isotopologues for G6P and lactate) and marked inhibition of the NADPH-generating pentose phosphate pathway (PPP) in patient PMNs stimulated with 1µM fMLF. Glutaminolysis appears to be increased with increased glutamine consumption in patient PMNs, decreased glucogenic amino acids, and increased TCA cycle intermediates.DiscussionMetabolic reprogramming is a recently appreciated hallmark of immune cell activation. Decreases in G6P affect multiple metabolic pathways including glycolysis, PPP and TCA cycle. Deficiency of NADPH-dependent superoxide anion production likely results from impairment in the NADPH-generating PPP. Interestingly the bactericidal defect is not as severe as would be predicted given the degree of impairment in superoxide anion production. The most striking functional defect is with PMN motility. Defects in the glycolytic/PPP likely contribute to this phenotype. ATP-dependent actin assembly is critical for cell migration. Our data demonstrate that in human patient PMNs, nearly all actin is in the filamentous form and does not increase further in response to chemoattractants. This could result in impaired hydrolysis of the F-actin filament and reassembly at the leading edge. We also see reduction in surface expression of CD11b/CD18 which is vital for PMN adhesion and migration.In conclusion, metabolic defects resulting from G6PC3 deficiency contribute to global PMN dysfunction and absolute neutropenia. Patients may be at increased risk for infections in spite of correction of neutropenia with filgrastim. These novel findings in human patient cells elucidate further the pathogenesis of this disease which has largely been dependent on data from mouse models. DisclosuresNo relevant conflicts of interest to declare.

Deficient Neutrophil Extracellular Trap Formation in Patients Undergoing Bone Marrow Transplantation.

Overwhelming infection causes significant morbidity and mortality among patients treated with bone marrow transplantation (BMT) for primary immune deficiencies, syndromes of bone marrow failure, or cancer. The polymorphonuclear leukocyte (PMN; neutrophil) is the first responder to microbial invasion and acts within the innate immune system to contain and clear infections. PMNs contain, and possibly clear, infections in part by forming neutrophil extracellular traps (NETs). NETs are extensive lattices of extracellular DNA and decondensed chromatin decorated with antimicrobial proteins and degradative enzymes, such as histones, myeloperoxidase, and neutrophil elastase. They trap and contain microbes, including bacteria and fungi, and may directly affect extracellular microbial killing. Whether or not deficient NET formation contributes to the increased risk for overwhelming infection in patients undergoing BMT remains incompletely characterized, especially in the pediatric population. We examined NET formation in vitro in PMNs isolated from 24 patients who had undergone BMT for 13 different clinical indications. For these 24 study participants, the median age was 7 years. For 6 of the 24 patients, we examined NET formation by PMNs isolated from serial, peripheral blood samples drawn at three different clinical time points: pre-BMT, pre-engraftment, and post-engraftment. We found decreased NET formation by PMNs isolated from patients prior to BMT and during the pre-engraftment and post-engraftment phases, with decreased NET formation compared with healthy control PMNs detected even out to 199 days after their BMT. This decrease in NET formation after BMT did not result from neutrophil developmental immaturity as we demonstrated that >80% of the PMNs tested using flow cytometry expressed both CD10 and CD16 as markers of terminal differentiation along the neutrophilic lineage. These pilot study results mandate further exploration regarding the mechanisms or factors regulating NET formation by PMNs in patients at risk for overwhelming infection following BMT.

Nitric Oxide Synthase - Nitric Oxide Involvement in the Human Neutrophil Free Radical Generation: Role of iNOS and Rac2 Interaction

AbstractAbstract 1036 Aims:Previous reports from this lab demonstrated importance of nitric oxide (NO) in augmenting neutrophils (PMNs) free radical generation. To explore further, present study investigated interaction of nitric oxide synthase (NOS) with Rac2 in resting and PMA activated human PMNs, and also nitration and nitrosylation of BSA following phagocytosis of coated beads. Results:Rac2 and iNOS interaction was evident in the cytosolic fraction of resting cells, which was augmented significantly in the membrane fraction of PMA treated PMNs. Nitration/nitrosylation of BSA, implicated functional importance of iNOS, and interaction of NO with NADPH-oxidase (NOX) and myeloperoxidase (MPO) derived species, as it was reduced significantly by the inhibitors of NOX (VAS), MPO (ABAH) and iNOS (1400W). Moreover, studies to assess the molecular characteristics and sub-cellular distribution of iNOS using standard methodologies, demonstrated iNOS transcript (RT-PCR, Real time PCR) and protein (immunoprecipitation and WB) in PMNs. iNOS protein was distributed in the granules, mitochondria, cytosol, nucleus and plasma membrane (immuno-electron microscopy and confocal microscopy) of human PMNs.Molecular characteristics, sub-cellular distribution of iNOS and its interaction with Rac2 are herein shown for the first time in human neutrophils. This study also elucidates the functional importance of iNOS-Rac2 interaction and highlighted the role of iNOS in nitration and nitrosylation of BSA following phagocytosis. Conclusions:Altogether these results suggest the functional importance of iNOS-Rac2 interaction in the nitration and S-nitrosylation of phagocytosed pathogens. [Display omitted] Association of iNOS with Rac2 and its migration to the membrane in activated PMNs (A) Total cell lysate of human PMNs (1 × 108 cells) were immunoprecipitated (IP) with anti-Rac2 antibody and blotted with anti-iNOS (Lane 1) or anti-nNOS antibody (Lane 4). Lanes 2 and 5 – IP with rabbit IgG (negative control for anti-Rac2). iNOS recombinant protein (Lane 3) and rat brain homogenate protein (Lane 6) were used as positive controls for iNOS (130 kDa) and nNOS (160 kDa) respectively. Lanes 7 and 8 were immunoprecipitated (IP) with anti-Rac2 antibody and blotted with anti-Rac2. Inducible NOS and Rac2 interaction in the cytosol and membrane fractions of resting, PMA andNSC23766with PMA stimulated cells. Both cytosol and membrane fractions were immunoprecipitated (IP) with anti-Rac2 antibody and blotted with anti-Rac2 (B) and anti-iNOS (C). The data represent 5 independent experiments, and are presented as Mean ± SEM (For Rac2, *** p<0.001 for control cytosol vs membrane fraction in control PMNs, * p<0.01 for cytosol vs membrane fraction in PMA treated PMNs or cytosol vs membrane fraction in NSC23766 with PMA treated PMNs, @p<0.01 for PMA vs control in cytosolic fraction and @@p<0.01 for PMA vs control in membrane fraction or NSC23766 PMA vs control in membrane fraction). (For iNOS, *** p<0.01 for cytosol vs membrane fraction in control PMNs or cytosol vs membrane fraction in NSC23766 with PMA treated PMNs, * p<0.01 for cytosol vs membrane fraction in PMA treated PMNs @p<0.05 for PMA cytosolic vs control cytosolic fraction or in PMA membrane vs control membrane fraction). Disclosures:No relevant conflicts of interest to declare.

Clarithromycin Accumulation by Phagocytes and Its Effect on Killing of Aggregatibacter actinomycetemcomitans

Clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonuclear leukocytes (PMNs) and less mature myeloid cells possess a similar transporter for clarithromycin. It is feasible that clarithromycin accumulation inside PMNs could enhance their ability to kill Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans). To test the first hypothesis, purified PMNs and cultured HL-60 cells were incubated with [(3)H]-clarithromycin. Clarithromycin transport was assayed by measuring changes in cell-associated radioactivity over time. The second hypothesis was examined with PMNs loaded by incubation with clarithromycin (5 μg/ml). Opsonized bacteria were incubated at 37°C with control and clarithromycin-loaded PMNs. Mature human PMNs, HL-60 cells differentiated into granulocytes, and undifferentiated HL-60 cells all took up clarithromycin in a saturable manner. The kinetics of uptake by all yielded linear Lineweaver-Burk plots. HL-60 granulocytes transported clarithromycin with a K(m) of ≈250 μg/ml and a V(max) of 473 ng/min/10(6) cells, which were not significantly different from the values obtained with PMNs. At steady state, clarithromycin levels inside HL-60 granulocytes and PMNs were 28- to 71-fold higher than extracellular levels. Clarithromycin-loaded PMNs killed significantly more A. actinomycetemcomitans and achieved shorter half-times for killing than control PMNs when assayed at a bacteria-to-PMN ratio of 100:1 (P <0.04). At a ratio of 30:1, these differences were not consistently significant. PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could help PMNs cope with an overwhelming infection by A. actinomycetemcomitans.

Neopterin May Enhance TNF-a-mediated Mononuclear Cell Death in the Cerebrospinal Fluid of Patients with Bacterial Meningitis

Abstract It has been reported that levels of neopterin are elevated in the cerebrospinal fluid (CSF) of patients with bacterial meningitis, and that neopterin enhances cell death induced by H2O2 in mouse monocytes and macrophages. In the present study, we examined the relationship between the disappearance of inflammatory cells in the CSF of patients with bacterial meningitis and the effects of neopterin on cell death. We hypothesized that the rapid cell death of mononuclear leukocytes (MNs) and survival of polymorphonuclear leukocytes (PMNs) brought about by high levels of tumor necrosis factor-a TNF-a may be related to the pathophysiology of PMN predominance in the CSF of patients with bacterial meningitis. Peripheral blood leukocytes separated into two fractions (MNs and PMNs) were suspended in CSF samples from patients with bacterial meningitis, and the cell death rate was determined with MTT assay. The higher the CSF TNF-a level, the higher the MN mortality rate tended to be. In contrast, PMNs were able to survive high levels of TNF-a in the CSF. For control purposes, MNs and PMNs were also suspended in various conditioned media. The same association between TNF-a levels and MN mortality was observed, as was the survival of PMNs. The addition of neopterin enhanced the cytocidal effect of TNF. The present study suggests that neopterin is related to PMN predominance in the CSF of patients with bacterial meningitis via the effect of TNF-a on cell death.

Evidence for the involvement of mTOR inhibition and basal autophagy in familial Mediterranean fever phenotype

Familial Mediterranean fever (FMF) inflammatory attacks are often triggered by metabolic or physical stress. mTOR signaling and autophagy modulate cellular responses to metabolic danger signals. In this study, we investigated the implication of mTOR inhibition and autophagy in FMF pathophysiology. mTOR inhibition induced MEFV gene expression in polymorphonuclear cells (PMNs) from healthy individuals, whereas it had no effect on PMNs from attack-free FMF patients. A significant reduction in pyrin levels in PMNs from FMF patients after mTOR inhibition was also observed. Pyrin levels in control PMNs remained unaffected. Moreover, the basal autophagic status in PMNs from FMF patients was reduced, as indicated by the lower LC3B-II/I ratio and ATG mRNA expression levels. However, mTOR inhibition had similar effects on the induction of autophagy in the two groups. The differential pyrin expression after metabolic stress induction and the impaired basal autophagy suggest a potential role in the triggering of FMF attacks.

Polymorphonuclear Cell Priming Associated with NF-kB Activation in Patients with Severe Injury Is Partially Dependent on Macrophage Migration Inhibitory Factor

Severe trauma may induce alternations of cytokine response and polymorphonuclear cell (PMN) activity in patients. This study investigated the correlation of plasma migration inhibitory factor (MIF) level and PMN activation after severe injury, and their relationship with clinical outcomes. A prospective observational study was performed at the emergency department and intensive care unit of a university hospital. Thirty-two severe blunt trauma patients (Injury Severity Score greater than 16) with systemic inflammatory response syndrome (SIRS) were enrolled. Age- and gender-matched healthy persons were the controls. Patient blood samples were obtained within 24 hours of and at 72 hours after injury. PMNs were isolated and measured for NF-kBp65 translocation and respiratory burst. Plasma MIF, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-8, and IL-10 concentrations were measured. Control PMNs were incubated with patient plasma preincubated with anti-MIF antibody or anti-IL-6 antibody; cytokine blockade effects were evaluated. Twelve patients developed organ failure. Compared with patients without organ failure, patients with organ failure had lower blood pressure and a higher base deficit on admission, higher NF-kBp65 translocation and respiratory burst of PMNs, and higher plasma MIF (968 ± 246 pg/mL vs 564 ± 299 pg/mL) and IL-6 (202 ± 91 pg/mL vs 119 ± 84 pg/mL) levels within 24 hours after injury. Plasma MIF had significant positive correlation with NF-kB translocation of PMNs within 24 hours of incurring trauma (R = 0.668). The presence of anti-MIF antibody in patients' plasma obtained within 24 hours, but not at 72 hours, after injury could significantly partially block the NF-kBp65 translocation and respiratory activity of PMNs in the controls. An early increase of plasma MIF associates with NF-kB translocation and respiratory burst in PMNs of severe trauma patients and correlates with higher morbidity. MIF is one of the important factors responsible for early PMN activation and may provide a target of immunomodulation after injury.

Abstract 1346: Neutrophils facilitate metastasis by increasing cancer cell recruitment to liver sinusoids

Abstract Background: Mounting evidence suggests that acute inflammation can facilitate the implantation and growth of circulating tumor cells. Previously, we have shown that bacterial lipopolysaccharide (LPS)-induced acute systemic inflammation promotes cancer cell recruitment to liver sinusoids in a neutrophil (PMN) dependent manner. This present study focuses on the various levels at which PMNs and their products can affect metastasis. Methods: In vitro adhesion assays were conducted to test for interactions between Lewis lung carcinoma H-59 cells, isolated mouse PMNs and human umbilical vein endothelial cells (HUVECs). In vivo real-time, single-cell H-59 adhesion to liver sinusoids was measured by hepatic intravital microscopy (IVM). C57BL/6 mice received intra-arterial injections of GFP-tagged H-59 cells. PMN depletion was achieved by IV injection of RB6-8C5 mAb. Some mice received LPS IV 4 hours prior to IVM. Some PMN-depleted mice received isolated PMNs from littermates intra-arterially 10 minutes prior to cancer cell inoculation. The effect of PMNs on gross metastasis formation was assessed after intra-splenic injection of H-59 cells in control and PMN-depleted mice. Results are presented as mean +/- SEM and MWU-test determined significance (* = p&amp;lt;0.05). Results: LPS-activated PMNs adhered 2.5 fold more to a monolayer of H-59 cells than control PMNs (12.3 +/− 1.2 vs 4.9 +/− 3.1 cells/field)*. H-59 adhesion to a HUVEC monolayer was increased when HUVECs were pre-incubated with PMNs versus control RPMI (40.5 +/− 2.6 vs 27.8 cells/field)*. Similarly, HUVECs pre-incubated with LPS-activated PMN-condition media resulted in 70% increased H-59/HUVEC adhesion compared to control RPMI*. Co-incubation of H-59 with control and LPS-treated PMNs prior to co-culture with HUVECs resulted in a 2.8 and 3.5 fold increase in H-59/HUVEC adhesion. These in vitro results correlated with in vivo findings from intravital microscopy and intra-splenic injection. PMN depletion resulted in a significant decrease in H-59 adhesion in both control and LPS-inflamed mice. Pre-infusion of LPS-treated PMNs in a PMN-depleted mouse prior to H-59 infusion caused a 42% increase in H-59 adhesion to liver sinusoids as compared to PBS pre-infusion (16.5 +/− 0.5 vs 11.7 +/− 0.5)*. Finally, PMN depletion prior to intra-splenic injection of H-59 cells decreased gross surface metastases at 2 weeks by 88% from a mean of 48 +/− 12.2 to 6 +/− 2.6*. Conclusions: PMNs may facilitate liver metastasis by direct interactions with cancer cells as well as remotely via PMN derived factors. LPS-activated PMNs were sufficient to promote early cancer cell recruitment to liver sinusoids. PMNs are key participants in the development of gross metastasis. Together, these results suggest that PMNs are important contributors to the implantation and growth of circulating tumor cells in the liver and constitute a possible target for anti-metastatic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1346.

The effects of HSP72 expression on adhesion receptor activation and phagolysosome function in human PMNs (134.93)

Abstract Heat shock protein 72 (HSP72) expression is a hallmark of functionally suppressed neutrophils from pediatric burn patients. CD11b and CD15 are critical receptors that mediate neutrophil (PMN) adhesion. In this study, we examined the effects of HSP72 on these receptors as well as phagolysosome function. Expression of CD11b, CD15 and pHrodo were measured by flow cytometry. HSP72 expression in PMNs, induced by 60-90 min incubation at 42C, suppressed the upregulation of high affinity CD11b conformation induced by fMLP (~90% reduction vs controls). Additionally, CD15 expression is reduced in HSP72 positive PMNs. GM-CSF treatment (5-25 ng/ml) appears to have a modest ability to preserve PMN function in stressed PMNs. HSP72 positive PMNs also have a reduced capacity phagocytose. Opsonized pHrodo labelled Staphylococcus aureus were incubated with HSP72 positive or negative PMNs. Control samples had significantly more pHrodo positive bacteria (~2-fold increase, controls vs stressed PMNs, P&amp;lt;0.01) than the thermally stressed cells. However, the HSP72 positive PMNs appeared to acidify phagolysosomes to the same extent as control PMNs. Taken together, these data suggest that HSP72 expression reduces adhesion as well as the extent of phagocytosis. These deficits may contribute to the immune suppression found in thermally injured patients. Supported by Shriners of North America.

ClC-3 and IClswell are Required for Normal Neutrophil Chemotaxis and Shape Change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).

The involvement of secondary signaling molecules in cytochrome P-450 1A1-mediated inducible nitric oxide synthase expression in benzo( a)pyrene-treated rat polymorphonuclear leukocytes

Benzo( a)pyrene induces cytochrome P-450 1A1 (CYP1A1) expression in rat polymorphonuclear leukocytes (PMNs) that upregulates expression of inducible nitric oxide synthase (iNOS). In the present study, the involvement of secondary signaling molecules in CYP1A1-mediated augmentation of iNOS expression in benzo( a)pyrene-treated rat PMNs was investigated. PMNs were isolated from the peripheral blood of controls and benzo( a)pyrene-treated rats. The expression and/or activity of CYP1A1, iNOS, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and intracellular calcium ([Ca 2+]i) concentrations were measured in control and benzo( a)pyrene-treated rat PMNs with and without alpha-naphthoflavone, aminoguanidine, genistein, pyrrolidine dithiocarbamate (PDTC), felodipine, or SB202190 pre-treatment. A significant elevation in CYP1A1 and [Ca 2+]i was observed in benzo( a)pyrene-treated rat PMNs, which was significantly restored by alpha-naphthoflavone or genistein. Neither PDTC, SB202190, nor aminoguanidine altered the benzo( a)pyrene-mediated increase in [Ca 2+]i. Although felodipine reduced the benzo( a)pyrene-mediated increase in [Ca 2+]i, no significant change was observed in CYP1A1 expression and activity. Benzo( a)pyrene-augmented iNOS expression and activity in PMNs were significantly reverse by felodipine, genistein, or PDTC. Benzo( a)pyrene also induced TNF-α and IL-1β production in PMNs, which was significantly reversed by genistein. The results demonstrated the involvement of [Ca 2+]i, tyrosine kinase, inflammatory cytokines, and NF-κB in CYP1A1-mediated iNOS expression in benzo( a)pyrene-treated rat PMNs.

Anion Channels, Including ClC-3, Are Required for Normal Neutrophil Oxidative Function, Phagocytosis, and Transendothelial Migration

NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.

Complement activation: the missing link between ADAMTS-13 deficiency and microvascular thrombosis of thrombotic microangiopathies

Endothelial injury is the central factor in the events leading to thrombotic microangiopathy (TMA); however, the mechanisms involved are not fully understood. Here we investigate the role of neutrophils (PMNs) and of complement activation in inducing microvascular damage and loss of thromboresistance in TMA associated with ADAMTS-13 deficiency. PMNs isolated during the acute phase of the disease released excessive amounts of reactive-oxygen species (ROS), N-derived oxidants and proteinases and induced damage and thromboresistance loss in human microvascular endothelial cell line (HMEC-1) ex vivo. Endothelial cytotoxicity and thromboresistance loss was also induced by TMA serum. Complement-derived products were responsible for the above effects: in fact, TMA serum caused C3 and Membrane Attack Complex (MAC) deposition on HMEC-1 and its cytotoxic effect was abolished by complement inhibition. TMA serum caused surface expression of P-selectin on HMEC-1 which may promote PMN adhesion and resulted in increased PMN cytotoxicity, indicating that complement may have a role in PMN activation. In addition, TMA serum stimulated control PMNs to release ROS and proteinases, and to cause endothelial cell cytotoxicity. All of the above effects were abrogated by complement inactivation. These data document for the first time that complement-initiated PMN activation and endothelial injury may have a crucial role in microvascular thrombosis of TMA associated with ADAMTS-13 deficiency.

The chemotaxis defect of Shwachman-Diamond Syndrome leukocytes.

Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive, multisystem disorder presenting in childhood with intermittent neutropenia and pancreatic insufficiency. It is characterized by recurrent infections independent of neutropenia, suggesting a functional neutrophil defect. While mutations at a single gene locus (SBDS) appear to be responsible for SDS in a majority of patients, the function of that gene and a specific defect in SDS neutrophil behavior have not been elucidated. Therefore, employing 2D and 3D computer-assisted motion analysis systems, we have analyzed the basic motile behavior and chemotactic responsiveness of individual polymorphonuclear leukocytes (PMNs) of 14 clinically diagnosed SDS patients. It is demonstrated that the basic motile behavior of SDS PMNs is normal in the absence of chemoattractant, that SDS PMNs respond normally to increasing and decreasing temporal gradients of the chemoattractant fMLP, and that SDS PMNs exhibit a normal chemokinetic response to a spatial gradient of fMLP. fMLP receptors were also distributed uniformly through the plasma membrane of SDS PMNs as in control PMNs. SDS PMNs, however, were incapable of orienting in and chemotaxing up a spatial gradient of fMLP. This unique defect in orientation was manifested by the PMNs of every SDS patient tested. The PMNs of an SDS patient who had received an allogenic hematopoietic stem cell transplant, as well as PMNs from a cystic fibrosis patient, oriented normally. These results suggest that the defect in SDS PMNs is in a specific pathway emanating from the fMLP receptor that is involved exclusively in regulating orientation in response to a spatial gradient of fMLP. This pathway must function in parallel with additional pathways, intact in SDS patients, that emanate from the fMLP receptor and regulate responses to temporal rather than spatial changes in receptor occupancy.

Increased nitric oxide production by neutrophils from patients with chronic granulomatous disease on trimethoprim–sulfamethoxazole

Chronic granulomatous disease (CGD) is an inherited disease characterized by severe and recurrent bacterial and fungal infections. Phagocytic cells of CGD patients are unable to produce superoxide anion, and their efficiency in bacterial killing is significantly impaired. In these patients, the prophylactic and therapeutic validity of a long-term use of trimethoprim–sulfamethoxazole (TMP–SMX) has been well established. However a role of nitric oxide (NO) produced by phagocytic cells from CGD patients is unknown, and the mechanism of TMP–SMX in CGD is unclear. We have directly measured NO production in whole human blood by using 4,5-diaminofluorescein as a novel fluorescent indicator for intracellular NO. Intracellular NO production of gated neutrophils increased time dependently when stimulated by lipopolysaccharide (LPS) and calcium ionophore. Although all polymorphonuclear leukocyte (PMN) specimens from patients with CGD failed to generate hydrogen peroxide, NO production by CGD PMNs was significantly increased compared with that of control PMNs ( p<0.05). TMP–SMX with LPS significantly increased compared with LPS-stimulated samples at clinical ( n=5, p<0.05) and 10-fold clinical concentrations ( n=5, p<0.01). TMP–SMX with LPS in CGD PMNs significantly increased the production of NO in comparison with the LPS stimulation at 10-fold clinical concentrations ( n=5, p<0.05). In conclusion, our data indicate the possibility that NO production by neutrophils from patients with CGD treated with TMP–SMX has a role of bactericidal activity instead of O 2 − in host defense mechanism.

Effect of ciprofloxacin on killing of Actinobacillus actinomycetemcomitans by polymorphonuclear leukocytes.

Actinobacillus actinomycetemcomitans, a pathogen associated with aggressive periodontitis, resists phagocytic killing by polymorphonuclear leukocytes (PMNs). It is susceptible to ciprofloxacin, which PMNs actively accumulate. This study tested the hypothesis that ciprofloxacin-loaded PMNs are more effective at killing A. actinomycetemcomitans than control PMNs. Isolated human PMNs were loaded by brief incubation with 0.5 microg of ciprofloxacin/ml. Opsonized bacteria (ATCC 43718) were incubated at 37 degrees C with control and ciprofloxacin-loaded PMNs and in the presence and absence of 0.5 microg of ciprofloxacin/ml. When assayed at bacteria-to-PMN ratios of 30:1 and 90:1, ciprofloxacin-loaded PMNs killed significantly more bacteria and achieved significantly shorter half times for killing than control PMNs (P < 0.05; Tukey's test). At ratios of 3:1 and 10:1, these differences were not significant.

Nitric oxide mediated modulation of free radical generation response in the rat polymorphonuclear leukocytes: a flowcytometric study.

Nitric oxide (NO) synthesis and free radical generation from polymorphonuclear leukocytes (PMNs) play an important role in several pathological conditions. It is therefore important to understand the regulatory mechanisms of free radical generation from PMNs. Flowcytometry can be used to assess generation of reactive oxygen and nitrogen species from PMNs by using fluorescent probes. In the present study regulation of NO synthesis in the control and lipopolysaccharide (LPS) treated rat PMNs has been investigated. Free radical generation was assessed by flow cytometry using a dye, 2'7'-dichlorodihydrofluorescein diacetate (DCFDA), dihydrorhodamine-123 (DHR) and 4,5-diaminofluorescein diacetate (DAF). Superoxide dismutase (SOD), and catalase significantly attenuated the arachidonic acid (AA, 1 x 10(-6) M) induced free radical generation, while 4-aminobenzoicacid hydrazide (ABH), myeloperoxidase (MPO) inhibitor had no significant effect. Intracellular and extracellular calcium levels also modulated FR generation. AA induced free radical generation from PMNs was also enhanced significantly after LPS treatment. NO synthase (NOS) inhibitors, aminoguanidine (AG) and 7-nitroindazole (NI) inhibited arachidonic acid induced free radical generation from LPS treated PMNs, while in control PMNs NOS inhibition had no effect. Augmentation of free radical generation from rat PMNs following LPS treatment seems to be regulated by NO.

In vitro Effect of Amikacin, Imipenem, Cefodizime, IFNα-2a Alone and Combinations of Antibiotics with IFNα-2a on Polymorphonuclear Leukocyte Function in Chronic Hepatitis Patients

The in vitro effect of amikacin (8 µg/ml), imipenem (30 µg/ml), cefodizime (10 µg/ml), interferon α-2a (IFNα-2a) (10 IU/ml) and antibiotic combinations with IFNα-2a on polymorphonuclear leukocyte (PMN) functions (phagocytosis and intracellular killing of Candida albicans blastospores) was investigated in chronic hepatitis B patients. Phagocytosis and candidacidal activity was not affected after pretreatment of PMNs with amikacin and imipenem (p > 0.05). Phagocytic activity was enhanced after pretreatment of PMNs with cefodizime and IFNα-2a compared with that of control PMNs (p < 0.05), but candidacidal activity was not affected by the same drugs (p > 0.05). Phagocytic and intracellular activity of PMNs were not affected by combinations of IFNα-2a and antibiotics (p > 0.05).

The immunomodulatory effects of damage control abdominal packing on local and systemic neutrophil activity.

Damage control laparotomy (DCL) with abdominal packing has become commonplace after major trauma, but the immune consequences of DCL are unknown. We collected 37 fluid samples from laparotomy pads (LPF) removed from 28 patients 1 hour to 7 days after DCL. Samples from eight patients who underwent serial packing were assayed for their mediator content and effects on neutrophil (PMN) function. Respiratory burst (RB) to N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate (PMA), as well as PMN calcium ([Ca2+]i) mobilization by GRO-alpha and platelet-activating factor were studied using dihydrorhodamine and fura-2-acetoxymethyl ester fluorescence. Brief exposure to 20% LPF (LPF20) modeled LPF acting on peritoneal PMNs and 2% LPF (LPF2) modeled the systemic effects on PMNs. Endotoxin (ETX), GRO-alpha, and leukotriene B4 were assayed by enzyme-linked immunosorbent assay. Data analysis was by analysis of variance with Dunn's comparisons or the Mann-Whitney test when indicated. LPF increased N-formyl- methionyl-leucyl-phenylalanine-induced RB from 0.4 +/- 0.1 x 103 counts per second (control) to 0.7 +/- 0.1 (LPF2) to 1.3 +/- 0.3 (LPF20) (p < 0.05), with LPF2 increasingly active at later times after injury. PMA-elicited RB was primed only by LPF2 from < 24 hours. Both LPF2 and LPF20 markedly suppressed GRO-alpha [Ca2+]i flux. Suppression by LPF2 was maximal at < 24 hours, abating after 48 hours. Suppression of GRO-alpha response was dose dependent: 150 +/- 8 nmol/L in control PMNs, 97 +/- 19 after LPF2, and 59 +/- 4 after LPF20 (all p < 0.05). [Ca2+]i flux after 1 nmol/L platelet-activating factor was only suppressed (from 181 +/- 14 nmol/L to 149 +/- 15 nmol/L, p < 0.05) by LPF20. LPF contained ETX, GRO-alpha, and leukotriene B4 at 10- to 20-fold plasma concentration in trauma patients. DCL results in peritoneal ETX and mediator accumulation even when cultures are sterile. LPF exposure primes PMN RB elicited by nonreceptor- (PMA) or receptor-coupled agonists that resist receptor desensitization. Conversely, LPF suppresses PMN responses to agonists that undergo receptor desensitization at high mediator concentrations. PMN dysfunction in such circumstances probably reflects a concomitant priming of some cell functions (e.g., RB) and desensitization of other (receptor-dependent) functions after an exposure to concentrated mediators. Peritoneal mediator production after DCL may be ETX driven, and may contribute to systemic inflammatory response syndrome. DCL trades early hemostasis for later inflammation. This should be considered in planning management strategies.

Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients.

To determine the extent to which peripheral polymorphonuclear leukocytes (PMNs) contributed to oxidative stress (OS) and inflammation in type 2 diabetic patients. PMNs and plasma were separated from blood withdrawn from 18 type 2 diabetic patients and 16 age- and sex-matched normal control subjects. The rate of superoxide release from phorbol 12-myristate 13-acetate (PMA)-stimulated PMNs and the plasma glutathione (GSH) levels served as measures of OS. Inflammation was assessed by PMN recruitment, expressed by peripheral blood PMN counts, and the in vitro survival of PMNs, which reflects cell necrosis. PMA-stimulated PMNs from diabetes released superoxide significantly faster, and plasma-reduced GSH was lower in diabetic patients than in normal control subjects. The rate of superoxide release from diabetic PMNs showed no correlation with the plasma glucose concentrations, whereas a positive linear correlation with HbA1c was found. The in vitro survival of diabetic PMNs was lower than normal control PMNs when each was incubated in its own serum. The in vitro survival of normal control PMNs was reduced when incubated with diabetic serum, whereas normal control sera promoted the survival of diabetic PMNs. Peripheral PMN counts were higher in diabetic patients than in normal control patients. Type 2 diabetes is accompanied by a priming of PMNs, resulting in OS and increased self-necrosis. Necrosis starts a chain of inflammatory reactions that result in cell recruitment and in the long run, with OS, may result in endothelial dysfunction. Understanding the contribution of PMNs to OS and inflammation in diabetes may illuminate new mechanisms through which endothelial dysfunction evolves and causes angiopathy and atherosclerosis.