Calprotectin (MRP8/14, S100A8/S100A9, 27E10 antigen) is a heterodimer of two calcium-binding proteins present in the cytoplasm of neutrophils and expressed on the membrane of monocytes. Upon neutrophil activation or endothelial adhesion of monocytes, calprotectin is released and may be detected in serum or body fluids as potentially useful clinical inflammatory marker. The soluble form of calprotectin provides both bacteriostatic and cytokine-like effects in the local environment. When calprotectin metabolism is affected on a systemic level, the zinc-binding properties of protein may induce severe dysregulation of zinc homeostasis with severe clinical symptoms. The distribution of membrane form of calprotectin is restricted to monocytes and immature macrophages and the presence of calprotectin-positive infiltrating cells reflects the influx of mononuclear phagocytes to the site of inflammation. Calprotectin expression and release seems to be of particular importance in immune and immunopathological reactions.
Polymorphonuclear neutrophils (PMN) are thought to play a role in reperfusion injury and ischemia. These effects are partly mediated by toxic oxygen species (superoxide anion, hydrogen peroxide and hydroxyl radical) acting at the level of the endothelium. It was demonstrated recently that the superoxide anion reacts with nitric oxide (NO) and that interaction leads to the generation of highly toxic peroxynitrite. Several drugs were tested so far in order to affect PMN function. It was demonstrated that dipyridamole (2,6-bis-diethanolamino-4,8-dipiperidinopyrimido-(5,4-d)-pyrimidine) can influence neutrophil function by inhibiting adenosine uptake. However, this action can not fully explain all of the observed effects of dipyridamole action on PMN metabolism. The aim of our study was to evaluate the influence of dipyridamole on nitric oxide production by activated polymorphonuclear neutrophils. Incubation of PMNs with hydroxylamine (HA) and phorbol myristate acetate (PMA) generated nitrite (36.4±4.2 nmol/h 2x106 PMN), dipyridamole at 100 μmol/l, 50 μmol/l and 10 μmol/l caused a considerable drop in nitrite production (11.8±1.8, 19.7±2.7 and 27.4±3.2 nmol/h, respectively). Neither adenosine nor the adenosine analogue could mimic the dipyridamole effect. Moreover theophylline, an adenosine inhibitor could not reverse the dipirydamole action on PMN metabolism. We also found that dipyridamole inhibited hydrogen peroxide release from neutrophils. Catalase that scavenges hydrogen peroxide also largely abolished nitric oxide release from PMN. It is evident that dipyridamole inhibits hydroxylamine-augmented nitric oxide production by activated polymorphonuclear neutrophils through an adenosine-independent mechanism.
The aim of the study was to elucidate the effects of induced leukocyte migration into the bovine mammary gland on the manifestations of early and late apoptotic features of neutrophils cultivated in vitro. The Latin square design was used in two experiments, each involving four experimental repetitions in 4 clinically healthy virgin heifers. The neutrophil early apoptotic features were detected by flow cytometric detection (FCM) of phosphatidyl-serine translocation. Late neutrophil apoptotic features were detected by ELISA quantitation of histone-complexed DNA fragments. Leukocyte influx induction was accomplished by using four inducers:
i) sterile buffered saline solution (PBS); ii) 5 % glucose solution (GLU); iii) synthetic muramyl dipeptide analogue (MDP); and iv) lipopolysaccharide (LPS), administered into the mammary gland lumen. Leukocytes from mammary glands were obtained by mammary gland lumen lavages after
influx induction. The total cell counts in lavages increased after treatment by all inducers in comparison to the counts before influx induction (P<0.001). Cell counts were higher and differed significantly by MDP and LPS (P<0.01) in contrast to PBS. The highest proportion of neutrophils was induced by LPS (P<0.01). After three-hour incubation, light
microscopy examination revealed the highest manifestation of neutrophil apoptosis after induction by GLU (P<0.05). The lowest apoptosis manifestation, though statistically non-significant, was detected after induction by MDP and LPS. Determination of early manifestation of neutrophil apoptosis revealed the lowest manifestation of neutrophil apoptosis
after induction by LPS (P<0.01). The results of late manifestation of neutrophil apoptosis revealed the highest proportion of apoptotic neutrophils after induction by GLU (P<0.05). The manifestation of secondary necrosis of apoptotic neutrophils or neutrophil lysis after 3 h of incubation was low and not significant. In conclusion, certain
inducers of neutrophil migration into the lumen of bovine mammary glands (GLU and LPS in the present experiments) significantly influence the manifestation of neutrophil apoptosis during their subsequent in vitro incubation.