Stimulus-Dependent Impairment of the Neutrophil Oxidative Burst Response in Lactoferrin-Deficient Mice
2008; Elsevier BV; Volume: 172; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2008.061145
ISSN1525-2191
AutoresPauline P. Ward, Marisela Mendoza-Meneses, Pyong Woo Park, Orla M. Conneely,
Tópico(s)Neonatal Respiratory Health Research
ResumoLactoferrin (LF) is an iron-binding protein found in milk, mucosal secretions, and the secondary granules of neutrophils in which it is considered to be an important factor in the innate immune response against microbial infections. Moreover, LF deficiency in the secondary granules of neutrophils has long been speculated to contribute directly to the hypersusceptibility of specific granule deficiency (SGD) patients to severe, life-threatening bacterial infections. However, the exact physiological significance of LF in neutrophil-mediated host defense mechanisms remains controversial and has not yet been clearly established in vivo using relevant animal models. In this study, we used lactoferrin knockout (LFKO) mice to directly address the selective role of LF in the host defense response of neutrophils and to determine its contribution, if any, to the phenotype of SGD. Neutrophil maturation, migration, phagocytosis, granule release, and antimicrobial response to bacterial challenge were unaffected in LFKO mice. Interestingly, a stimulus-dependent defect in the oxidative burst response of LFKO neutrophils was observed in that normal activation was seen in response to opsonized bacteria whereas an impaired response was evident after phorbol myristate-13-acetate stimulation. Taken together, these results indicate that although LF deficiency alone is not a primary cause of the defects associated with SGD, this protein does play an immunomodulatory role in the oxidative burst response of neutrophils. Lactoferrin (LF) is an iron-binding protein found in milk, mucosal secretions, and the secondary granules of neutrophils in which it is considered to be an important factor in the innate immune response against microbial infections. Moreover, LF deficiency in the secondary granules of neutrophils has long been speculated to contribute directly to the hypersusceptibility of specific granule deficiency (SGD) patients to severe, life-threatening bacterial infections. However, the exact physiological significance of LF in neutrophil-mediated host defense mechanisms remains controversial and has not yet been clearly established in vivo using relevant animal models. In this study, we used lactoferrin knockout (LFKO) mice to directly address the selective role of LF in the host defense response of neutrophils and to determine its contribution, if any, to the phenotype of SGD. Neutrophil maturation, migration, phagocytosis, granule release, and antimicrobial response to bacterial challenge were unaffected in LFKO mice. Interestingly, a stimulus-dependent defect in the oxidative burst response of LFKO neutrophils was observed in that normal activation was seen in response to opsonized bacteria whereas an impaired response was evident after phorbol myristate-13-acetate stimulation. Taken together, these results indicate that although LF deficiency alone is not a primary cause of the defects associated with SGD, this protein does play an immunomodulatory role in the oxidative burst response of neutrophils. The initial level of surveillance and host defense against microbial challenge is provided by the innate immune system. 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Various reports indicate that LF may influence neutrophil recruitment to sites of infection by altering aggregation, adhesion, attachment, and/or motility of this cell type.35Oseas R Yang HH Baehner RL Boxer LA Lactoferrin: a promoter of polymorphonuclear leukocyte adhesiveness.Blood. 1981; 57: 939-945Crossref PubMed Google Scholar, 36Boxer LA Bjorksten B Bjork J Yang HH Allen JM Baehner RL Neutropenia induced by systemic infusion of lactoferrin.J Lab Clin Med. 1982; 99: 866-872PubMed Google Scholar, 37Gahr M Speer CP Damerau B Sawatzki G Influence of lactoferrin on the function of human polymorphonuclear leukocytes and monocytes.J Leukoc Biol. 1991; 49: 427-433PubMed Google Scholar, 38Boxer LA Haak RA Yang HH Wolach JB Whitcomb JA Butterick CJ Baehner RL Membrane-bound lactoferrin alters the surface properties of polymorphonuclear leukocytes.J Clin Invest. 1982; 70: 1049-1057Crossref PubMed Scopus (66) Google Scholar In addition, it has been shown that LF stimulates phagocytosis.39Kai K Komine K Komine Y Kuroishi T Kozutsumi T Kobayashi J Ohta M Kitamura H Kumagai K Lactoferrin stimulates A Staphylococcus aureus killing activity of bovine phagocytes in the mammary gland.Microbiol Immunol. 2002; 46: 187-194Crossref PubMed Scopus (48) Google Scholar, 40Miyauchi H Hashimoto S Nakajima M Shinoda I Fukuwatari Y Hayasawa H Bovine lactoferrin stimulates the phagocytic activity of human neutrophils: identification of its active domain.Cell Immunol. 1998; 187: 34-37Crossref PubMed Scopus (111) Google Scholar Finally, an enhancement of superoxide production by neutrophils was demonstrated in the presence of LF37Gahr M Speer CP Damerau B Sawatzki G Influence of lactoferrin on the function of human polymorphonuclear leukocytes and monocytes.J Leukoc Biol. 1991; 49: 427-433PubMed Google Scholar whereas contradictory reports in neutrophils and cell-free systems support either an enhancement41Vercellotti GM van Asbeck BS Jacob HS Oxygen radical-induced erythrocyte hemolysis by neutrophils. Critical role of iron and lactoferrin.J Clin Invest. 1985; 76: 956-962Crossref PubMed Scopus (63) Google Scholar, 42Bannister JV Bannister WH Hill HA Thornalley PJ Enhanced production of hydroxyl radicals by the xanthine-xanthine oxidase reaction in the presence of lactoferrin.Biochim Biophys Acta. 1982; 715: 116-120Crossref PubMed Scopus (74) Google Scholar, 43Ambruso DR Johnston JR, RB Lactoferrin enhances hydroxyl radical production by human neutrophils, neutrophil particulate fractions, and an enzymatic generating system.J Clin Invest. 1981; 67: 352-360Crossref PubMed Scopus (226) Google Scholar or no effect44Sibille JC Doi K Aisen P Hydroxyl radical formation and iron-binding proteins. 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In the present study, we have used LF knockout (LFKO) mice46Ward PP Mendoza-Meneses M Cunningham GA Conneely OM Iron status in mice carrying a targeted disruption of lactoferrin.Mol Cell Biol. 2003; 23: 178-185Crossref PubMed Scopus (92) Google Scholar to analyze the specific consequences of LF ablation on the host defense response of the neutrophil. We show that although LF is dispensable for many neutrophil functions, an impairment of phorbol myristate-13-acetate (PMA)-stimulated neutrophil oxidative burst is clearly associated with LF deficiency. The generation of LFKO mice by targeted disruption of the LF gene in embryonic stem cells has been reported.46Ward PP Mendoza-Meneses M Cunningham GA Conneely OM Iron status in mice carrying a targeted disruption of lactoferrin.Mol Cell Biol. 2003; 23: 178-185Crossref PubMed Scopus (92) Google Scholar LFKO mice used for these studies were backcrossed at least 10 generations onto the C57BL/6 genetic background. Wild-type (WT) C57BL/6 mice were obtained from Harlan-Sprague Dawley (Indianapolis, IN) and bred in the same facility as the LFKO mice. Mice were maintained in microisolator cages under specific pathogen-free conditions in a 12-hour light/dark cycle and were fed a basal rodent chow ad libitum (LabDiet; PMI, Richmond, IN). Age- and sex-matched adult mice were used for experiments and all animal research complied with National Institutes of Health and Baylor College of Medicine guidelines for research with experimental animals. Blood was obtained by cardiac puncture and blood smears were stained with Wright-Giemsa to examine neutrophil morphology. Images were acquired using bright-field microscopy (Zeiss Axioscope; Carl Zeiss, Thornwood, NY). Peripheral blood cells were collected from mice by cardiac puncture and were analyzed using a Bayer Advia 120 hematology analyzer (Bayer Diagnostics, Dallas, TX). Mature bone marrow neutrophils were isolated and purified from mice essentially as described previously with minor modifications.47Lowell CA Fumagalli L Berton G Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions.J Cell Biol. 1996; 133: 895-910Crossref PubMed Scopus (327) Google Scholar, 48Hart PH Spencer LK Nulsen MF McDonald PJ Finlay-Jones JJ Neutrophil activity in abscess-bearing mice: comparative studies with neutrophils isolated from peripheral blood, elicited peritoneal exudates, and abscesses.Infect Immun. 1986; 51: 936-941PubMed Google Scholar Briefly, bone marrow cells were isolated from femurs and tibias and mature neutrophils were purified using a discontinuous Percoll gradient (81%, 62%, 55%, 50%, 45%). Cells at the interphase of the 81% and 62% layer were isolated and washed in NM buffer (Hanks' buffered saline solution minus calcium and magnesium (HBSS−/−)/0.1% bovine serum albumin). Contaminating red blood cells were lysed in ACK buffer (BioWhittaker, Walkersville, MD) and the resulting cell population was washed twice in NM buffer. Viable cells were counted with a hemocytometer using trypan blue exclusion and cells were resuspended in HBSS−/−/5 mmol/L glucose. Purified bone marrow neutrophils (5 × 105) were incubated with anti Gr-1 (1 μg/ml; clone RB6-8C5; eBioscience, Inc., San Diego, CA) for 30 minutes on ice. Cells were washed and resuspended in 1% paraformaldehyde for flow cytometric analysis using a Coulter EPICS XL-MCL flow cytometer and System 2 version 3.0 software (Beckman Coulter, Hialeah, FL). Cells were gated on the neutrophil population based on characteristic forward and side light scatter. Quantitative determination of the oxidative burst of purified bone marrow neutrophils was determined using a DHR123/R123 assay essentially as described.49Smith JA Weidemann MJ Further characterization of the neutrophil oxidative burst by flow cytometry.J Immunol Methods. 1993; 162: 261-268Crossref PubMed Scopus (161) Google Scholar DHR123 is the nonfluorescent substrate used in the assay and R123 is the fluorescent oxidation reporter product of this substrate formed during the oxidative burst response of the neutrophil. Briefly, 5 × 105 cells in HBSS−/−/5 mmol/L glucose were incubated with DHR123 (1 μmol/L; Molecular Probes, Eugene, OR) for 5 minutes at 37°C with gentle horizontal agitation. Cells were incubated in PMA (0 to 600 ng/ml; Sigma Chemical Co., St. Louis, MO) at 37°C to stimulate the oxidative burst or incubated with buffer vehicle alone (nonstimulated cells). After 30 minutes, the reactions were stopped by placing the tubes on ice and neutrophil cells were analyzed for conversion of DHR123 to the fluorescent R123 by flow cytometry. S. aureus Wood 46 was opsonized with 50% serum (obtained from WT C57BL6) for 15 minutes at room temperature as described.50Ellson CD Davidson K Ferguson GJ O'Connor R Stephens LR Hawkins PT Neutrophils from p40phox−/− mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing.J Exp Med. 2006; 203: 1927-1937Crossref PubMed Scopus (150) Google Scholar Neutrophils (5 × 105) were incubated with DHR123 (1 μmol/L) for 5 minutes at 37°C followed by an incubation with a 100-fold excess of opsonized bacteria for 1 hour at 37°C. The reaction was stopped by placing tubes on ice and neutrophils were analyzed for conversion of DHR123 to the fluorescent R123 as described above. Purified bone marrow neutrophils (2 to 4 × 106 cells/ml) were sonicated in 1× sodium dodecyl sulfate loading buffer and proteins corresponding to 1 to 2 × 105 cells were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5 to 15%). Immunoblots were probed with antibodies directed against p22phox (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p40phox, p47phox, p67phox (Millipore, Billerica, MA), GP91phox (a generous gift from Al Jesaitis, Montana State University, Billings, MT), and GAPDH (loading control; Chemicon International, Temecula, CA) following the manufacturer's recommendations. Signal was detected by ECL Plus detection reagents (GE Health Care, Piscataway, NJ) and relative levels of proteins were determined by using densitometric scanning and ImageQuant software (version 5.2; Amersham Biosciences, Piscataway, NJ). Analysis of neutrophil migration to the peritoneal cavity after thioglycollate administration was performed as described by Lu and colleagues51Lu H Smith CW Perrard J Bullard D Tang L Shappell SB Entman ML Beaudet AL Ballantyne CM LFA-1 is sufficient in mediating neutrophil emigration in Mac-1-deficient mice.J Clin Invest. 1997; 99: 1340-1350Crossref PubMed Scopus (263) Google Scholar with minor modifications. Briefly, WT and LFKO mice were injected intraperitoneally with 3% thioglycollate media (1 ml) and were euthanized at 0 or 4 hours after injection. Peritoneal exudates were harvested by lavage using 4 ml of cold EB buffer [1× phosphate-buffered saline (PBS) minus calcium and magnesium/40 U/ml heparin] and total cell count was determined using a hemocytometer. The peritoneal extract was washed twice in WB buffer (1× PBS, pH 7.2, minus calcium and magnesium/0.1% bovine serum albumin) and differential cell counts were obtained using CamcoQuik-stained (Fisher Scientific, Pittsburgh, PA) cytospin samples. At least 500 cells were counted per slide under oil immersion microscopy (×100). Phagocytic analysis of blood neutrophils was quantitatively analyzed using a commercially available kit (Phagotest; Orpegen, Heidelberg, Germany). Heparinized whole blood (100 μl) obtained from WT and LFKO mice was cooled on ice and mixed with ice-cold opsonized fluorescein isothiocyanate-labeled bacteria (20 μl). Samples were incubated on ice (control samples) or 37°C (test samples) for 60 minutes. After bacterial incubation, samples were placed on ice and neutralization solution was added to quench the fluorescence of surface-bound bacteria while leaving internalized bacteria unaltered. Erythrocytes were lysed and the remaining cells were resuspended in DNA staining solution for flow cytometric analysis. Neutrophils were distinguished using forward and light scatter patterns and the percentage of cells undergoing phagocytosis and the number of bacteria ingested per cell was determined. Purified mature bone marrow neutrophils (5 × 105 cells) were suspended in phenol red-free RPMI 1640 media and stimulated with 5 μg/ml of cytochalsin B (Sigma) and 5 μmol/L fMLP (Sigma) to induce primary granule exocytosis and myeloperoxidase (MPO) secretion.52Abdel-Latif D Steward M Macdonald DL Francis GA Dinauer MC Lacy P Rac2 is critical for neutrophil primary granule exocytosis.Blood. 2004; 104: 832-839Crossref PubMed Scopus (132) Google Scholar MPO in the supernatants was detected using a tetramethylbenzidine assay as described.52Abdel-Latif D Steward M Macdonald DL Francis GA Dinauer MC Lacy P Rac2 is critical for neutrophil primary granule exocytosis.Blood. 2004; 104: 832-839Crossref PubMed Scopus (132) Google Scholar, 53Lacy P Mahmudi-Azer S Bablitz B Hagen SC Velazquez JR Man SF Moqbel R Rapid mobilization of intracellularly stored RANTES in response to interferon-gamma in human eosinophils.Blood. 1999; 94: 23-32PubMed Google Scholar The activation status and secondary/secretory granule release of WT and LFKO neutrophils were assessed by measuring the cell surface expression of CD11b in resting and stimulated neutrophils. Purified bone marrow neutrophils (5 × 105 cells in HBSS−/−/5 mmol/L glucose) were incubated at 37°C with 100 ng/ml of PMA (stimulated cells) or buffer vehicle alone (nonstimulated cells). After 10 minutes, the reactions were stopped by placing the tubes on ice. Cells were analyzed by flow cytometry for cell surface expression of CD11b using phycoerythrin-conjugated anti-CD11b (BD PharMingen, San Diego, CA). Cells were gated on the neutrophil population based on characteristic forward and side light scatter and dead cells were excluded using propidium iodide staining. Isotype-specific antibodies were used to assess nonspecific staining. Swabs of bacterial abscesses were obtained and submitted to Antech Diagnostics (Irvine, CA) or Radil (University of Missouri, Columbia, MO) for bacterial species identification. The bactericidal response of WT and LFKO neutrophils was assessed using a bactericidal assay with S. aureus (Wood strain) as described by Clemens and colleague54Clemens RA Newbrough SA Chung EY Gheith S Singer AL Koretzky GA Peterson EJ PRAM-1 is required for optimal integrin-dependent neutrophil function.Mol Cell Biol. 2004; 24: 10923-10932Crossref PubMed Scopus (27) Google Scholar with minor modifications. Purified bone marrow neutrophils (2 × 106) were incubated with 2 × 105 log phase opsonized bacteria in HBSS/10 mmol/L HEPES/10 mmol/L dextrose/1 mmol/L Ca/3. mmol/L Mg, pH 7.44 containing 10% mouse serum. At 0-, 15-, 30-, and 60-minute time points, 20-μl aliquots were removed and combined
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