During infection or certain metabolic disorders, neutrophils can escape from blood vessels, invade and attach to other tissues. The invasion and adhesion of neutrophils is accompanied and maintained by their own secretion. We have previously found that adhesion of neutrophils to fibronectin dramatically and selectively stimulates the release of the free amino acid hydroxylysine. The role of hydroxylysine and lysyl hydroxylase in neutrophil adhesion has not been studied, nor have the processes that control them. Using amino acid analysis, mass spectrometry and electron microscopy, we found that the lysyl hydroxylase inhibitor minoxidil, the matrix metalloproteinase inhibitor doxycycline, the PI3K/Akt pathway inhibitors wortmannin and the Akt1/2 inhibitor and drugs that affect the actin cytoskeleton significantly and selectively block the release of hydroxylysine and partially or completely suppress spreading of neutrophils. The actin cytoskeleton effectors and the Akt 1/2 inhibitor also increase the phenylalanine release. We hypothesize that hydroxylysine release upon adhesion is the result of the activation of lysyl hydroxylase in interaction with matrix metalloproteinase, the PI3K/Akt pathway and intact actin cytoskeleton, which play important roles in the recruitment of neutrophils into tissue through extracellular matrix remodeling.

Neutrophils can phagocytose microorganisms and destroy them intracellularly using special bactericides located in intracellular granules. Recent evidence suggests that neutrophils can catch and kill pathogens extracellularly using the same bactericidal agents. For this, live neutrophils create a cytoneme network, and dead neutrophils provide chromatin and proteins to form neutrophil extracellular traps (NETs). Cytonemes are filamentous tubulovesicular secretory protrusions of living neutrophils with intact nuclei. Granular bactericides are localized in membrane vesicles and tubules of which cytonemes are composed. NETs are strands of decondensed DNA associated with histones released by died neutrophils. In NETs, bactericidal neutrophilic agents are adsorbed onto DNA strands and are not covered with a membrane. Cytonemes and NETs occupy different places in protecting the body against infections. Cytonemes can develop within a few minutes at the site of infection through the action of nitric oxide or actin-depolymerizing alkaloids of invading microbes. The formation of NET in vitro occurs due to chromatin decondensation resulting from prolonged activation of neutrophils with PMA (phorbol 12-myristate 13-acetate) or other stimuli, or in vivo due to citrullination of histones with peptidylarginine deiminase 4. In addition to antibacterial activity, cytonemes are involved in cell adhesion and communications. NETs play a role in autoimmunity and thrombosis.

Leukotriene synthesis in neutrophils is critical for host survival during infection. In particular, leukotriene B4 (LTB4) is a powerful neutrophil chemoattractant that plays a crucial role in neutrophil swarming. In this work, we demonstrated that preincubation of human neutrophils with Salmonella typhimurium strongly stimulated LTB4 production induced by the bacterial chemoattractant, peptide N-formyl-L-methionyl-L-leucyl-l-phenylalanine (fMLP), while the reverse sequence of additions was ineffective. Preincubation with bacterial lipopolysaccharide or yeast polysaccharide zymosan particles gives weaker effect on fMLP-induced LTB4 production. Activation of 5-lipoxygenase (5-LOX), a key enzyme in leukotrienes biosynthesis, depends on rise of cytosolic concentration of Ca2+ and on translocation of the enzyme to the nuclear membrane. Both processes were stimulated by S. typhimurium. With an increase in the bacteria:neutrophil ratio, the transformation of LTB4 to ω-OH-LTB4 was suppressed, which further supported increased concentration of LTB4. These data indicate that in neutrophils gathered around bacterial clusters, LTB4 production is stimulated and at the same time its transformation is suppressed, which promotes neutrophil swarming and elimination of pathogens simultaneously.

Timely neutrophil apoptosis is an essential part of the resolution phase of acute inflammation. Ceruloplasmin, an acute-phase protein, which is the predominant copper-carrying protein in the blood, has been suggested to have a marked effect on neutrophil life span. The present work is a comparative study on the effects of intact holo-ceruloplasmin, its copper-free (apo-) and partially proteolyzed forms, and synthetic free peptides RPYLKVFNPR (883–892) and RRPYLKVFNPRR (882–893) on polymorphonuclear leukocyte (PMNL, neutrophil) oxidant status and apoptosis. The most pronounced effect on both investigated parameters was found with copper-containing samples, namely, intact and proteolyzed proteins. Both effectively reduced spontaneous and tumor necrosis factor-α (TNF-α)-induced extracellular and intracellular accumulation of superoxide radicals, but induced a sharp increase in the oxidation of intracellular 2′,7′-dichlorofluorescein upon short exposure. Therefore, intact and proteolyzed ceruloplasmin have both anti- and pro-oxidant effects on PMNLs wherein the latter effect is diminished by TNF-α and lactoferrin. Additionally, all compounds investigated were determined to be inhibitors of delayed spontaneous apoptosis. Intact enzyme retained its pro-survival activity, whereas proteolytic degradation converts ceruloplasmin from a mild inhibitor to a potent activator of TNF-α-induced neutrophil apoptosis.

The interaction between ceruloplasmin (CP), the multicopper oxidase of human plasma, and 5-lipoxygenase (5-LO), the key enzyme of leukotriene synthesis, is shown for the first time. By Western-blotting and mass spectrometry of tryptic fragments, it is shown that 5-LO from protein extract of human leukocytes binds with immobilized CP. Dose-dependent influence of intact CP on leukotrienes synthesis is found: CP reduced leukotrienes synthesis in leukocytes in a dose above 50 µg/ml (normal CP concentration in plasma is about 300-400 µg/ml). Proteolyzed CP and apo-form of CP is unable to inhibit activity of 5-LO. CP increased activity of 5-LO at low doses (5-10 µg/ml). On the whole, the influence of CP on phagocytosis index of leukocytes coordinates with influence on activity of 5-LO: the index increased in the range of 2-10 µg/ml CP and decreased at doses of CP above 40 µg/ml. The dual role of CP in regulation of cellular response of leukocytes is discussed.