Patients with cystic fibrosis (CF) develop chronic bacterial infections in the respiratory tract, reflecting dysfunctional innate immunity. Neutrophil gelatinase–associated lipocalin (NGAL) has 2 functions: one as an antibacterial host defense protein and the other as a physiological iron carrier. Neutrophil gelatinase–associated lipocalin (NGAL) has been validated as a biomarker for early kidney disease. The aim of this study was to determine whether NGAL could serve as a marker of acute pulmonary exacerbations in CF by measuring NGAL levels in serum samples from subjects with CF during stable clinic visits and during hospitalization for pulmonary exacerbations and comparing these levels to healthy controls and to patients without CF with significant infection. In addition, we aimed to determine if innate immunity cells other than neutrophils act as a source of NGAL.
Methods
Circulating NGAL levels were measured by enzyme-linked immunosorbent assay in serum samples from 30 subjects with CF when hospitalized for an acute pulmonary exacerbation, 33 subjects with CF during stable clinic visits, 33 patients with sepsis, and 21 healthy controls. Secreted NGAL from CF and healthy control peripheral monocytes infected with Pseudomonas aeruginosa was also measured by enzyme-linked immunosorbent assay.
Results
Serum NGAL levels were elevated in the patients with CF compared to the healthy controls (P < 0.001). However, no difference in serum NGAL levels was found between subjects with CF with stable disease compared to subjects with CF undergoing pulmonary exacerbation. Furthermore, peripheral monocytes from CF and subjects without CF secreted NGAL upon infection with Pseudomonas aeruginosa and thereby may be contributing to the circulating NGAL levels observed.
Conclusions
Our data show that peripheral monocytes secrete NGAL, and serum NGAL levels are widely distributed among subjects with CF, being elevated both when clinically stable and during a pulmonary exacerbation and thus NGAL is not a sensitive biomarker for pulmonary exacerbations.
RiordanJR, RommensJM, KeremB, . Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989; 245: 1066–1073.
2.
McCartyNA.Permeation through the CFTR chloride channel. J Exp Biol. 2000; 203: 1947–1962.
3.
JordanIK, KotaKC, CuiG, . Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters. Proc Natl Acad Sci U S A. 2008; 105: 18865–18870.
4.
SandersDB, BittnerRC, RosenfeldM, . Pulmonary exacerbations are associated with subsequent FEV1 decline in both adults and children with cystic fibrosis. Pediatr Pulmonol. 2011; 46: 393–400.
5.
de BoerK, VandemheenKL, TullisE, . Exacerbation frequency and clinical outcomes in adult patients with cystic fibrosis. Thorax. 2011; 66: 680–685.
6.
Schmidt-OttKM, MoriK, LiJY, . Dual action of neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol. 2007; 18: 407–413.
7.
KjeldsenL, JohnsenAH, SengelovH, . Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem. 1993; 268: 10425–10432.
8.
HolmesMA, PaulseneW, JideX, . Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration. Structure. 2005; 13: 29–41.
9.
AbergelRJ, CliftonMC, PizarroJC, . The siderocalin/enterobactin interaction: a link between mammalian immunity and bacterial iron transport. J Am Chem Soc. 2008; 130: 11524–11534.
10.
Moreau-MarquisS, O'TooleGA, StantonBA.Tobramycin and FDA-approved iron chelators eliminate Pseudomonas aeruginosa biofilms on cystic fibrosis cells. Am J Respir Cell Mol Biol. 2009; 41: 305–313.
11.
LamontIL, KoningsAF, ReidDW.Iron acquisition by Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis. Biometals. 2009; 22: 53–60.
12.
FloTH, SmithKD, SatoS, . Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature. 2004; 432: 917–921.
13.
ChanYR, LiuJS, PociaskDA, . Lipocalin 2 is required for pulmonary host defense against Klebsiella infection. J Immunol. 2009; 182: 4947–4956.
14.
BergerT, TogawaA, DuncanGS, . Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury. Proc Natl Acad Sci U S A. 2006; 103: 1834–1839.
15.
DraperDW, BetheaHN, HeYW.Toll-like receptor 2-dependent and -independent activation of macrophages by group B streptococci. Immunol Lett. 2006; 102: 202–214.
16.
SaigaH, NishimuraJ, KuwataH, . Lipocalin 2-dependent inhibition of mycobacterial growth in alveolar epithelium. J Immunol. 2008; 181: 8521–8527.
17.
AbergelRJ, MooreEG, StrongRK, . Microbial evasion of the immune system: structural modifications of enterobactin impair siderocalin recognition. J Am Chem Soc. 2006; 128: 10998–10999.
18.
PeekME, BhatnagarA, McCartyNA, . Pyoverdine, the major siderophore in Pseudomonas aeruginosa, evades NGAL recognition. Interdiscip Perspect Infect Dis. 2012; 2012: 843509.
19.
TirouvanziamR, GernezY, ConradCK, . Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways. Proc Natl Acad Sci U S A. 2008; 105: 4335–4339.
20.
YanL, BorregaardN, KjeldsenL, . The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase–associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL. J Biol Chem. 2001; 276: 37258–37265.
21.
RatjenF, HartogCM, PaulK, . Matrix metalloproteases in BAL fluid of patients with cystic fibrosis and their modulation by treatment with dornase alpha. Thorax. 2002; 57: 930–934.
22.
ProvatopoulouX, GounarisA, KalogeraE, . Circulating levels of matrix metalloproteinase-9 (MMP-9), neutrophil gelatinase–associated lipocalin (NGAL) and their complex MMP-9/NGAL in breast cancer disease. BMC Cancer. 2009; 9: 390.
23.
EaganTM, DamasJK, UelandT, . Neutrophil gelatinase–associated lipocalin: a biomarker in COPD. Chest. 2010; 138: 888–895.
24.
HaaseM, Haase-FielitzA, BellomoR, . Neutrophil gelatinase-associated lipocalin as a marker of acute renal disease. Curr Opin Hematol. 2010; 6: 526–532.
25.
SmertkaM, ChudekJ.Using NGAL as an early diagnostic test of acute kidney injury. Ren Fail. 2012; 34: 130–133.
26.
NymoSH, UelandT, AskevoldET, . The association between neutrophil gelatinase–associated lipocalin and clinical outcome in chronic heart failure: results from CORONA*. J Intern Med. 2012; 271: 436–443.
27.
WheelerDS, DevarajanP, MaQ, . Serum neutrophil gelatinase–associated lipocalin (NGAL) as a marker of acute kidney injury in critically ill children with septic shock. Crit Care Med. 2008; 36: 1297–1303.
28.
EichlerI, NilssonM, RathR, . Human neutrophil lipocalin, a highly specific marker for acute exacerbation in cystic fibrosis. Eur Respir J. 1999; 14: 1145–1149.
29.
ZughaierSM, ZimmerSM, DattaA, . Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins. Infect Immun. 2005; 73: 2940–2950.
BulittaJB, YangJC, YohonnL, . Attenuation of colistin bactericidal activity by high inoculum of Pseudomonas aeruginosa characterized by a new mechanism-based population pharmacodynamic model. Antimicrob Agents Chemother. 2010; 54: 2051–2062.
32.
SoniSS, CruzD, BobekI, . NGAL: a biomarker of acute kidney injury and other systemic conditions. Int Urol Nephrol. 2009; 42: 141–150.
33.
Bachorzewska-GajewskaH, MalyszkoJ, SitniewskaE, . Could neutrophil-gelatinase-associated lipocalin and cystatin C predict the development of contrast-induced nephropathy after percutaneous coronary interventions in patients with stable angina and normal serum creatinine values?Kidney Blood Press Res. 2007; 30: 408–415.
34.
YangJ, MoriK, LiJY, . Iron, lipocalin, and kidney epithelia. Am J Physiol Renal Physiol. 2003; 285: F9–F18.
35.
DiA, BrownME, DeriyLV, . CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol. 2006; 8: 933–944.
36.
Del PortoP, CifaniN, GuarnieriS, . Dysfunctional CFTR alters the bactericidal activity of human macrophages against Pseudomonas aeruginosa. PLoS One. 2011; 6: e19970.
37.
DonnellyLE, BarnesPJ.Defective phagocytosis in airways disease. Chest. 2012; 141: 1055–1062.
38.
ZughaierSM, RyleyHC, JacksonSK.Lipopolysaccharide (LPS) from Burkholderia cepacia is more active than LPS from Pseudomonas aeruginosa and Stenotrophomonas maltophilia in stimulating tumor necrosis factor alpha from human monocytes. Infect Immun. 1999; 67: 1505–1507.
39.
TanBK, AdyaR, ShanX, . Ex vivo and in vivo regulation of lipocalin-2, a novel adipokine, by insulin. Diabetes Care. 2009; 32: 129–131.
40.
WurfelMM, ParkWY, RadellaF, . Identification of high and low responders to lipopolysaccharide in normal subjects: an unbiased approach to identify modulators of innate immunity. J Immunol. 2005; 175: 2570–2578.
41.
SvegerT, OhlssonK, MorseH, . Plasma neutrophil lipocalin, elastase-alpha1-antitrypsin complex and neutrophil protease 4 in preterm infants with respiratory distress syndrome. Scand J Clin Lab Invest. 2003; 63: 89–92.
42.
Schmidt-OttKM, MoriK, KalandadzeA, . Neutrophil gelatinase-associated lipocalin-mediated iron traffic in kidney epithelia. Curr Opin Nephrol Hypertens. 2006; 15: 442–449.
43.
MoretonRE, KennedyCR.C-reactive protein concentrations in cystic fibrosis. Arch Dis Child. 1988; 63: 958–960.
44.
GlassS, HaywardC, GovanJR.Serum C-reactive protein in assessment of pulmonary exacerbations and antimicrobial therapy in cystic fibrosis. J Pediatr. 1988; 113: 76–79.
45.
FriesenCA, WiensLA, BurryVF, . C-reactive protein in acute pulmonary exacerbations of patients with cystic fibrosis. Pediatr Pulmonol. 1995; 20: 215–219.
46.
ChakrabortyS, KaurS, GuhaS, . The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer. Biochim Biophys Acta. 2012; 1826: 129–169.
47.
MoriK, LeeHT, RapoportD, . Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest. 2005; 115: 610–621.
48.
CaiL, RubinJ, HanW, . The origin of multiple molecular forms in urine of HNL/NGAL. Clin J Am Soc Nephrol. 2010; 5: 2229–2235.
49.
HuL, HittelmanW, LuT, . NGAL decreases E-cadherin–mediated cell-cell adhesion and increases cell motility and invasion through Rac1 in colon carcinoma cells. Lab Invest. 2009; 89: 531–548.
50.
MakrisK, RizosD, KafkasN, . Neurophil gelatinase–associated lipocalin as a new biomarker in laboratory medicine. Clin Chem Lab Med. 2012; 50: 1519–1532.
51.
ViscaP, ImperiF, LamontIL.Pyoverdine siderophores: from biogenesis to biosignificance. Trends Microbiol. 2007; 15: 22–30.
52.
RoganMP, TaggartCC, GreeneCM, . Loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fibrosis. J Infect Dis. 2004; 190: 1245–1253.
53.
BritiganBE, HayekMB, DoebbelingBN, . Transferrin and lactoferrin undergo proteolytic cleavage in the Pseudomonas aeruginosa–infected lungs of patients with cystic fibrosis. Infect Immun. 1993; 61: 5049–5055.
54.
HvidbergV, JacobsenC, StrongRK, . The endocytic receptor megalin binds the iron transporting neutrophil gelatinase–associated lipocalin with high affinity and mediates its cellular uptake. FEBS Lett. 2005; 579: 773–777.
55.
BolignanoD, DonatoV, LacquanitiA, . Neutrophil gelatinase–associated lipocalin (NGAL) in human neoplasias: a new protein enters the scene. Cancer Lett. 2009; 288: 10–16.
56.
ZografosT, HaliassosA, KorovesisS, . Association of neutrophil gelatinase–associated lipocalin with the severity of coronary artery disease. Am J Cardiol. 2009; 104: 917–920.
YangYH, HeXJ, ChenSR, . Changes of serum and urine neutrophil gelatinase–associated lipocalin in type-2 diabetic patients with nephropathy: one year observational follow-up study. Endocrine. 2009; 36: 45–51.
59.
WooHJ, ParkJC, BaeCH, . Up-regulation of neutrophil gelatinase–associated lipocalin in cholesteatoma. Acta Otolaryngol. 2009; 129: 624–629.
60.
BaoG, CliftonM, HoetteTM, . Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex. Nat Chem Biol. 2010; 6: 602–609.
61.
DevireddyLR, HartDO, GoetzDH, . A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell. 2010; 141: 1006–1017.
62.
RoudkenarMH, HalabianR, BahmaniP, . Neutrophil gelatinase–associated lipocalin: a new antioxidant that exerts its cytoprotective effect independent on heme oxygenase-1. Free Radic Res. 2011; 45: 810–819.
63.
BahmaniP, HalabianR, RouhbakhshM, . Neutrophil gelatinase–associated lipocalin induces the expression of heme oxygenase-1 and superoxide dismutase 1, 2. Cell Stress Chaperones. 2010; 15: 395–403.
