Effects of recombinant human lactoferrin on calcium signaling and functional responses of human neutrophils

Daria V Grigorieva,Irina V Gorudko,Ekaterina V Shamova,Maria S Terekhova,Elena V Maliushkova,Igor V Semak,Sergey N Cherenkevich,Alexey V Sokolov,Alexander V Timoshenko

doi:10.1016/j.abb.2019.108122

Abstract

Lactoferrin is a non-heme iron-binding glycoprotein with multiple health-beneficial functions including antimicrobial, antioxidant, anticarcinogenic, and immunomodulatory effects. There is emerging evidence that neutrophils may serve as targets of lactoferrin in vivo, and here we show how recombinant human lactoferrin (rhLf) can contribute to this regulation. Indeed, our results demonstrate that rhLf binds efficiently to human neutrophils and induces a variety of early cellular responses such as mobilization of intracellular Ca2+, remodeling of actin cytoskeleton, and degranulation (release of lysozyme and myeloperoxidase). In addition, rhLf facilitates lectin-induced H2O2 production and stabilization of lectin-induced cellular aggregates. The role of calcium signaling seems to be essential for rhLf-induced activation of neutrophils, as Ca2+-chelators inhibit degranulation response while lectin-induced H2O2 production correlates significantly with cytoplasmic Ca2+ elevation. Taken together, our findings justify that rhLf can activate neutrophil functions in a calcium-dependent manner and hence, can potentiate innate immune responses.

Highlights

Lactoferrin (Lf) is a cationic iron-binding glycoprotein (M ~ 78 kDa) of the transferrin family, which is found at high concentration in exocrine fluids and specific granules of neutrophils (~15 μg/106 neutrophils) [1,2]
Earlier findings demonstrated that neutrophils exposed to human Lf exhibited an increase in Abbreviations: Lf, lactoferrin; rhLf, recombinant human lactoferrin; EDTA, ethylenedinitrilotetraacetatic acid; HRP, horseradish peroxidase; fMLP, N-formyl-MetLeu-Phe; o-DA, o-dianisidine; PMA, phorbol 12-myristate 13-acetate; GlcNAc, N-acetyl-D-glucosamine; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); PNA, Arachis hypogaea agglutinin; Con A, Canavalia ensiformis agglutinin; CABA, Caragana arborescens agglutinin; SBA, Glycine hispida agglutinin; PHA-L, Phaseolus vulgaris agglutinin; SNA, Sambucus nigra agglutinin; STA, Solanum tuberosum agglutinin; VSA, Vicia sativa agglutinin; WGA, Triticum vulgaris agglutinin; PBS, phosphate-buffered saline; MPO, myeloperoxidase; [Ca2+]i, cytoplasmic Ca2+; HSR contacts, haptenic sugar resistant contacts; ROS, reactive oxygen species
As the elevation of cytoplasmic calcium can be due to Ca2+-release from the intracellular depot and/or Ca2+-entry from the extracellular medium [39,40], we suggest that binding rhLf to human neutrophils and subsequent transmembrane signaling may involve the store-operated Ca2+ entry (SOCE) pathway and Ca2+ ATP-powered P-pumps of the endoplasmic reticulum [41,42]

Summary

Introduction

Lactoferrin (Lf) is a cationic iron-binding glycoprotein (M ~ 78 kDa) of the transferrin family, which is found at high concentration in exocrine fluids (e.g. breast milk) and specific granules of neutrophils (~15 μg/106 neutrophils) [1,2]. Lf is a multifunctional protein, which is involved in regulation of iron homeostasis and has many health-beneficial functions including antimicrobial, antioxidant, anticarcinogenic, and immunomodulatory effects [1,2,6,7,8]. These functions depend on Lf binding to specific receptors on the surface of many effector cells (neutrophils, monocytes, macrophages, lymphocytes, epithelial and endothelial cells) and the subsequent transmembrane signaling [9,10]. Earlier findings demonstrated that neutrophils exposed to human Lf (from colostrums) exhibited an increase in Abbreviations: Lf, lactoferrin; rhLf, recombinant human lactoferrin; EDTA, ethylenedinitrilotetraacetatic acid; HRP, horseradish peroxidase; fMLP, N-formyl-MetLeu-Phe; o-DA, o-dianisidine; PMA, phorbol 12-myristate 13-acetate; GlcNAc, N-acetyl-D-glucosamine; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); PNA, Arachis hypogaea agglutinin; Con A, Canavalia ensiformis agglutinin; CABA, Caragana arborescens agglutinin; SBA, Glycine hispida agglutinin; PHA-L, Phaseolus vulgaris agglutinin; SNA, Sambucus nigra agglutinin; STA, Solanum tuberosum agglutinin; VSA, Vicia sativa agglutinin; WGA, Triticum vulgaris agglutinin; PBS, phosphate-buffered saline; MPO, myeloperoxidase; [Ca2+]i, cytoplasmic Ca2+; HSR contacts, haptenic sugar resistant contacts; ROS, reactive oxygen species

Methods

Results

Conclusion