Background and objectives: Several molecular forms of human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), a novel biomarker for acute kidney injury (AKI), have been found in urine. The origin of these different forms and the effect of antibody configuration on assay performances were investigated in this report. Design, setting, participants, & measurements: The molecular forms of HNL/NGAL from human neutrophils and present in urine obtained from cardiac surgery patients and patients with urinary tract infection (UTI), as well as secreted from HK-2 cells, were studied by Western blotting. The levels of HNL/NGAL in urine were measured by ELISAs. Kidney injury was simulated by incubation of HK-2 cells under stressful conditions. Results: The major molecular form of HNL/NGAL secreted by neutrophils is dimeric, whereas the major form secreted by HK-2 cells is monomeric. This was reflected by a predominance of the monomeric form in urine from patients with AKI and the dimeric form in patients with UTIs. The epitope specificities of the antibody used in the ELISAs had a profound effect on assay performance and paralleled differences of the antibodies to identify the different forms of urine HNL/NGAL. Conclusions: The monomeric form is the predominant form secreted by tubular epithelial cells, and the dimeric form is the predominant form secreted by neutrophils. The development of molecular form-specific assays for HNL/NGAL may be a means to identify the origin of HNL/NGAL in urine and construct more specific tools for the diagnosis of AKI.

A phospholipase B (PLB) precursor was purified from normal human granulocytes using Sephadex G‐75, Mono‐S cation‐exchange and hydroxyapatite columns. The molecular mass of the protein was estimated to be ∼ 130 kDa by gel filtration and 22 and 42 kDa by SDS/PAGE. Tryptic peptide and sequence analyses by MALDI‐TOF and tandem mass spectrometry (MS/MS) identified the protein as a FLJ22662 (Homo sapiens) gene product, a homologue of the amoeba Dictyostelium discoideum PLB. The native protein needed modifications to acquire deacylation activity against phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and lysophospholipids. Enzyme activity was associated with fragments derived from the 42 kDa fragment. The enzyme revealed a PLB nature by removing fatty acids from both the sn‐1 and sn‐2 positions of phospholipids. The enzyme is active at a broad pH range with an optimum of 7.4. Immunoblotting of neutrophil postnuclear supernatant using antibodies against the 42 kDa fragment detected a band at a molecular mass of 42 kDa, indicating a neutrophil origin of the novel PLB precursor. The existence of the PLB precursor in neutrophils and its enzymatic activity against phospholipids suggest a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation.

Granulocyte colony‐stimulating factor (G‐CSF) is a haematopoietic growth factor required for the proliferation and differentiation of haematopoietic precursors of neutrophil granulocytes and is now used to overcome congenital and acquired neutropenia. In addition to increasing the numbers of neutrophils in vivo and modulating neutrophil functions, G‐CSF may induce the production of cytokines such as tumour necrosis factor α (TNF‐α). In the present study, the plasma levels of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) in six healthy volunteers given G‐CSF at 10 μg/kg once daily for 6 d were measured and found to be elevated. The elevated levels (P < 0·05) were detected on day 2, peaked on days 6–7 and returned to baseline on day 12. In vitro, G‐CSF did not enhance the secretion of TNF‐α and GM‐CSF from mononuclear cells, whole blood or endothelial cells. However, in the co‐presence of whole blood and endothelial cells, the secretion of TNF‐α was significantly enhanced by G‐CSF at low concentrations. The GM‐CSF secretion, however, was unaltered. G‐CSF pretreatment of whole blood suppressed lipopolysaccharide (LPS)‐induced secretion of TNF‐α and GM‐CSF in a dose‐dependent manner. These results together with our previous findings suggest that G‐CSF induces the production of TNF‐α and GM‐CSF in vivo, and that this production may be due to the co‐effects of endothelial cells and whole blood under the influence of G‐CSF through an as yet unknown network of cells and cytokines. Treatment of whole blood with G‐CSF suppresses LPS‐induced secretion of TNF‐α and GM‐CSF.

A 75 kDa protein was purified to homogeneity from granule extracts of normal human granulocytes using Sephadex G-75 chromatography, Mono-S cation exchange chromatography and chromatofocusing. The protein consisted of one chain with a molecular mass of 75 kDa, as determined by SDS/PAGE. Tryptic peptide analysis by MALDI-TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS and sequence analysis by MS/MS identified the protein to be N-acetylglucosamine-6-sulphatase (EC 3.1.6.14). The identity of the protein was confirmed by demostrating enzymatic activity towards the substrate N-acetylglucosamine 6-sulphate. The enzyme was active over a broad pH range with an optimum of pH 7.0, and showed a K_m value of 13.0 mM and a V_max value of ∼1.8 μM/min per mg. The enzyme also showed O-desulphation activity towards heparan sulphate-derived saccharides. Subcellular fractionation of neutrophil organelles showed the presence of enzymatic activity mainly in the same fractions as primary granules. Furthermore, PMA treatment of the neutrophils induced release of the enzyme, indicating its matrix protein nature. The presence of N-acetylglucosamine-6-sulphatase in human neutrophils implies that neutrophils may play a role in the modulation of cell surface molecules and extracellular matrix by O-desulphation.