Expression of lactoferrin on human granulocytes: analysis with polyclonal and monoclonal antibodies

Abstract

SUMMARYLactoferrin (LF), an iron-binding protein present in specific granules of neutrophils, is expressed on membrane after granulocyte activation. It may represent a target for anti-neutrophil cytoplasmic antibodies (ANCA) in patients affected by some immunomediated diseases. We recently produced two MoAbs, AGM 2.29 and AGM 10.14, that recognize two spatially distant epitopes of human LF. In this study we perform a cytometric analysis in order to evaluate the expression of LF on the surface of granulocytes obtained from freshly drawn blood or after purification, in both the presence and absence of stimuli. Our results demonstrate that LF is not constitutively expressed on membrane of circulating neutrophils. After priming with phorbol myristate acetate (PMA) or tumour necrosis factor-alpha (TNF-a), an increased mean fluorescence intensity (MFI) was obtained on neutrophils stained with polyclonal anti-LF antibodies and with AGM 2.29. The kinetics of LF expression during activation demonstrated a progressive increase in MFI within 45 min. No increase in MFI was documented when primed granulocytes were stained with MoAb AGM 10.14, thus indicating that the epitope recognized by AGM 10.14 is not exposed at the cell surface. Following membrane permeabilization, performed in order to analyse the binding of anti-LF MoAbs to cytoplasmic LF, a marked increase in MFI was obtained by staining granulocytes with both anti-LF MoAbs. Indirect immunofluorescence (IIF) analysis confirmed that AGM 2.29 and AGM 10.14 reacted with human granulocytes, showing a cytoplasmic pattern on formalin-acetone-fixed neutrophils and a perinuclear one on ethanol-fixed cells.

Cervicovaginal Levels of Lactoferrin, Secretory Leukocyte Protease Inhibitor, and RANTES and the Effects of Coexisting Vaginoses in Human Immunodeficiency Virus (HIV)-Seronegative Women with a High Risk of Heterosexual Acquisition of HIV Infection

“…All of the samples used in this study were screened for Chlamydia infection and gonorrhea as determined by GenProbe and BDProbeTec tests (Gen-Probe Inc., San Diego, CA; Becton Dickinson, Sparks, MD). Samples were screened for BV by use of the criteria of Amsel et al (3), which require the presence of three of the following four factors: (i) a vaginal pH above 4.5; (ii) a thin, homogeneous vaginal discharge; (iii) a positive potassium hydroxide odor; and (iv) clue cells on a saline wet mount (1).…”

Characterization of the psycho-neuro-immunological state in patients with coronary artery disease

“…Activation state of neutrophils can be assessed by detecting cell surface lactoferrin, L-selectin and/or CD15 parameters, as their increased appearance on the cell surface can be considered as a sign of cell activation. From these, only lactoferrin can be considered as a specific neutrophil activation marker, its appearance on neutrophil cell surface and in blood plasma reflects degranulation that leads to its release from secondary granules to the circulation following activation [46][47][48].…”

“…Lactoferrin, a specific activation marker of the neutrophil cell surface, can gain access to the blood and can be regarded as a soluble inflammatory marker as well; its appearance in plasma reflects neutrophil activation/degranulation, which results in the release of proinflammatory cytokines in addition to oxygen radicals [46].…”

“…LL-37 concentration was measured by an ELISA kit (HK321, Hycult Biotech Inc., Uden, the Netherlands; intra-and inter-assay CVs: 10%; reference range: 25-250 ng/ml). were identified as activated neutrophils, because lactoferrin is specific for neutrophils and can get the cell surface only after degranulation followed by cell activation [46][47][48]. 10 000 events/tube were recorded in granulocyte populations.…”

“…Lactoferrin is a specific activation marker of neutrophils: following cell activation and release form secondary granules, it can bound to cell surface and gain access to the circulation [46][47][48]. Similarly to the changes in the ratio of CD15-bearing cells, the proportion of lactoferrin-carrying neutrophils increased mildly directly after PCI.…”