Effect of prostate cancer cell line supernatant on functional polarization in macrophages.

OBJECTIVES: We aimed on effect of supernatant derived from prostate cancer cell line PC-3 on M1/M2 functional polarization in macrophages. BACKGROUND: Cytokines play an important role in carcinogenesis. Most of them are produced by macrophages. Macrophages are divided into groups M1 or M2. Classical phenotype macrophages M1 support pro-inflammatory effects and produce pro-inflammatory cytokines, such as interleukin 6 (IL-6), interleukin 12 (IL-12), tumor necrosis factor α (TNF-α). Macrophages exhibiting a phenotype M2 secrete anti-inflammatory cytokines, e. g. interleukin 10 (IL-10), transforming growth factor ß (TGF-ß). METHODS: Peripheral blood monocytes were cultivated for 7 days and during this time went through a differentiation into macrophages. Macrophages were stimulated for 24 hours by lipopolysaccharide (LPS) as a positive control and cultivated with supernatant for another 24 hours. RESULTS: Macrophages cultivated without LPS and without supernatant were used as negative control. Relative expression of IL-6, IL-10, IL-12 and TNF-α was measured by Quantitative real-time PCR. Expression of pro-inflammatory cytokines was lower in macrophages with supernatant compared to positive control. CONCLUSION: Expression of pro-inflammatory cytokines was lower in macrophages with supernatant (MΦ+sup) compared to positive control (MΦ+LPS). Effect of the supernatant on expression of IL-6, IL-10, IL-12 and TNF-α was not confirmed (Tab. 1, Fig. 5, Ref. 15).

Effect of dietary Schizochytrium microalga oil on selected markers of low-grade inflammation in rats.

The objective of the present study was to evaluate a potential of Schizochytrium microalga oil to alleviate possible negative effects of high-fat-high-energy diets. Forty adult male rats (Wistar Albino) were fed 7 weeks the diet containing beef tallow + evaporated sweetened milk (diet T) intended to cause mild obesity and low-grade systemic inflammation. Consequently, the animals were divided into four groups by 10 animals each and fed either the T-diet (control) or the diet containing 6% of safflower oil (S), 6% of fish oil (F) and 6% of Schizochytrium microalga oil (A), respectively, for another 7 weeks. The A-diet decreased (p < 0.05) live weight to 86% and glycaemia to 85% of control, respectively; an effect of the S- and F-diet on these markers was insignificant (p > 0.05). In comparison with control, higher (p < 0.05) deposition of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in the epididymal adipose tissue (EAT) of the A-rats correlated with increased (p < 0.05) plasma adiponectin concentration, but it was without the effect (p > 0.05) on cellular adiponectin content in the EAT. Higher (p < 0.05) EPA+DHA deposition in the liver of the A-rats correlated with higher expression (149% of control; p < 0.05) of the gene coding for peroxisome proliferator-activated receptor gamma, and with lower expression (82% and 66%; p < 0.05) of the genes coding for adiponectin receptors AdipoR1 and AdipoR2; no relationship to the expression of receptor GPR120 was found. The A-diet did not affect amount of the nuclear fraction of the nuclear factor kappa B in the liver, but increased plasma level of anti-inflammatory cytokine TGF-ß1 (p < 0.05). The presented data agree with results of other in vivo rodent and human studies, but not with literature data regarding in vitro experiments: it can be concluded that the effects of dietary oils on inflammatory markers need further investigation.

The thermal treatment effects on bovine blood neutrophil granulocytes apoptosis and necrosis in vitro.

The aim of the study was to evaluate the effect of selected temperatures on viability (apoptosis and necrosis) of bovine blood neutrophil granulocytes (neutrophils) in vitro. The following temperatures were tested: -80, -20, 4, 23, 37 degrees C. Heparinised bovine blood was incubated for 1, 4 and 24 h under respective temperature. Apoptosis and necrosis of neutrophils were detected by light microscopy, transmission electron microscopy (TEM) and flow cytometry (FCM). From selected temperatures, 4 degrees C impaired the neutrophil viability least. The proportion of apoptotic and necrotic neutrophils amounted to (mean +/- SD) 5.25 +/- 3.53% and 0.83 +/- 0.38%; 7.09 +/- 2.07% and 1.64 +/- 0.50%; 35.39 +/- 12.53% and 5.46 +/- 1.46%; after 1, 4 and 24 h incubation, respectively. The temperature (4 degrees C) is the best alternative for short-term storage.

The role of CD14 during resolution of experimentally induced Staphylococcus aureus and Streptococcus uberis mastitis.

This study was undertaken to investigate the time course of surface expression of CD14 on neutrophils and macrophages and to determine their association with resolution of inflammatory responses during Staphylococcus aureus and Streptococcus uberis experimental mastitis. Infections of the mammary gland induce a local immune response characterized by an increase in the total counts of CD14+ neutrophils and CD14+ macrophages particularly. On the other hand, resolution is accompanied by an increase in relative counts of CD14+ neutrophils, CD14+ vacuolized macrophages and apoptotic neutrophils. Following the immune reaction of mammary gland against Gram-negative/positive bacteria is very similar. Between the apoptotic and CD14+ neutrophils a high correlation was measured during the whole experimental period (S. aureus: r=0.64; S. uberis: r=0.61; P<0.05). Using anti-CD14 monoclonal antibodies in vitro suggested the involving of the CD14 surface receptor in recognition of apoptotic neutrophils by macrophages.

The image of exocytosis during neutrophils and macrophages phagocytic activities in inflammation of mammary gland triggered by experimental Staphylococcus aureus infection.

The experiments were carried out in five clinically normal virgin heifers. Before the experimental infection and at 24, 48, 72 and 168 h after the infection, respective mammary glands were rinsed with phosphate buffered saline. Neutrophils as well as macrophages underwent a classic exocytosis accompanied by translocation of lysosomal granules. The granules filled the protuberances of the plasmalemma and after the protuberances separated from the cell, they entered the extracellular space in the shape of round bodies of different sizes. After exocytosis, neutrophils displayed a smaller nucleus/cytoplasm ratio, a greater chromatin density of the nucleus, and an overall smaller size. Macrophages phagocytosed bacteria and/or neutrophils with and without signs of apoptosis (early and late apoptotic respectively) and neutrophils after exocytosis. Macrophages underwent cytolysis that was accompanied by extrusion of granules, phagosomes and phagolysosomes containing phagocytosed bacteria or neutrophils. Confluences were formed in which the process of digestion continued. Apoptosis of neutrophils gradually appeared and intensified in resolution of inflammation. The macrophages contributed to the inactivation of bacterial noxa as well as of histotoxic contents of neutrophils. Nevertheless, macrophages often underwent cytolysis at the site of inflammation.

Macrophages of the bovine heifer mammary gland: morphological features during initiation and resolution of the inflammatory response.

This work characterizes macrophage morphological features during initiation and resolution of an inflammatory response by the bovine mammary gland. The study has been carried out in 20 mammary glands of five virgin heifers by using light microscopy of natural and stained cells and by using transmission electron microscopy (TEM). The inflammatory reaction was induced by an intramammary administration of phosphate-buffered saline (PBS). It has been found that both the initial as well as the resolution phases of the inflammatory reaction are characteristic of the presence of various morphologically different macrophage forms. During the initial phase of the inflammatory response, the major proportion of the macrophage population consisted of monocyte-like macrophages, which represented newly migrated cells. These macrophages were 12-15 mum in size, with spherical or ovoidal shapes, and contained homogenous, fine-granular cytoplasm rich in Golgi complexes, numerous mitochondria, and no lysosomes. The nuclei of the macrophages were kidney-shaped, and surrounded by dark chromatin along the peripheries. Macrophages with phagocytosed apoptotic neutrophils in the cytoplasm were detected already during the initial phase. These macrophages reached the highest proportion 48-72 h after the influx induction and participated in the resolution of the inflammatory reaction. Other cells, also detected during the resolution of the inflammatory reaction, were vacuolized macrophages that formed the largest cells in the lavages of the mammary glands and that were structurally characteristic for the presence of vacuoles in the cytoplasm. In TEM the macrophage vacuoles formed both phagolysosomes with residues of pre-digested material of phagocytosed apoptotic neutrophils and vacuoles that were less electon-dense. Morphologically different forms of macrophages reflected their real-time functions in the inflammation process.

Tissue pool neutrophils of the bovine mammary gland: ultrastructural features during in vitro senescence.

The aim of the study was to verify whether the in vitro senescence process of tissue pool neutrophils of the bovine mammary gland is accompanied by similar changes of ultrastructure as typically occurs in in vivo conditions. The experiments were carried out in four clinically healthy, Holstein x Bohemian Red-Pied crossbred heifers aged 14-16 months. With the aid of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and flow cytometry (FCM), neutrophil apoptosis in vivo was detected and during senescence it was monitored in vitro. The neutrophil apoptosis comprised three ultrastructurally different stages: (1) karyopyknosis, (2) zeiosis, and (3) apoptotic bodies. These stages were obvious in the apoptotic neutrophils both in vivo and in vitro. In addition to the common morphological signs, however, ultrastructural differences were also detected in apoptotic neutrophils in vitro. These in vitro ultrastructural differences mostly comprised hyper-vacuolation of the cytoplasm with mega-vacuoles and secondary necrosis of apoptotic neutrophils. Morphological features of apoptosis during in vitro senescence of tissue pool neutrophils of the bovine mammary gland were shown to be in close accordance with these in vivo signs.

Effects of certain inducers of leukocytes migration into the bovine mammary gland on neutrophil apoptosis manifestation in a subsequent in vitro cultivation.

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.

Variations among unbred heifers in the activities of polymorphonuclear leucocytes from the mammary gland and blood.

The phenotypic characteristics are described for the activity of polymorphonuclear leucocytes NMN) obtained by either lavage of the cavity system of juvenile mammary glands stimulated with a synthetic muramyl dipeptide analogue or isolation from the peripheral blood. Attention was paid to the variability of characteristics and its sources, and to correlations among them. The following characteristics were investigated in 27 clinically healthy, unbred Bohemian Red Pied x Holstein heifers: migration activity in situ, number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and unstimulated and zymosan-stimulated luminol-dependent chemiluminescence. Considerable individual variation was found in the characteristics. Significant differences between blood PMN and PMN from lavages after influx induction were found for bactericidal activity (P < 0.05) and chemiluminescence (P < 0.01). A significant correlation between blood PMN and mammary gland PMN was found only for the number of phagocytosing cells (r = 0.329; P < 0.01). Highly significant positive correlations (P < 0.01) were demonstrated between the number of phagocytosing PMN [a], phagocytotic index [b], and bactericidal activity [c] in both blood PMN (r(ab) = 0.602; r(ac) = 0.565; r(bc) = 0.529) and mammary gland PMN (r(ab) = 0.730, r(ac) = 0.618, r(bc) = 0.589). No significant correlation was demonstrated for non-stimulated (NS), zymosan-stimulated (ZS), or opsonized zymosan-stimulated (OZS) chemiluminescence with any of the other characteristics of phagocytotic activity, in either blood PMN or mammary gland PMN (P > 0.05). The animal was a highly significant source of variability for all the phagocytotic activity characteristics (P < 0.01). Udder quarter was a non-significant source of variability for all the characteristics of phagocytotic activity except for NS chemiluminescence (P < 0.05) and ZS or OZS chemiluminescence (P < 0.01). However, udder quarter was a non-significant source of variability of chemiluminescence indices ZS/NS and OZS/NS (P > 0.05). It has been demonstrated that in situ migration activity, the number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and chemiluminescence indices of PMN collected from juvenile mammary glands of unbred heifers after influx induction can be regarded as candidate early markers of resistance to mammary infections.

Neutrophil apoptosis during the resolution of bovine mammary gland injury.

The role of neutrophil apoptosis in the resolution of bovine mammary gland injury induced by intramammary administration of physiological buffered saline (PBS) or lipopolysaccharide (LPS) was investigated. Twenty mammary glands of five non-pregnant heifers were used in the two studies and each animal received both stimuli. Samples of cell populations were collected by mammary gland lavages before and 24, 48, 72 and 96 hours after treatment and examined by light microscopy and staining for myeloperoxidase (MPO). A marked influx of neutrophils into the mammary gland was observed 24 hours after stimulation. At the same time, apoptotic neutrophils and MPO-positive macrophages (MAC) were identified in the samples. The numbers increased to reach maximum values at 48 hours after stimulation with PBS and at 72 hours after stimulation with LPS. The observed differences in the length of the resolution period indicate that neutrophil viability can be modulated by delaying the apoptotic process. Apoptosis of neutrophils and their subsequent phagocytosis by MAC can be regarded as a significant mechanism in the removal of neutrophils from the acutely injured mammary glands and, hence, in the resolution of bovine mastitis.

Apoptosis of polymorphonuclear leukocytes of the juvenile bovine mammary gland during induced influx.

The dynamics of apoptosis of polymorphonuclear leukocytes (PMN) during induced influx of PMN into the cavity system of the juvenile bovine mammary gland in order to investigate the role of apoptosis of PMN in the resolution of mastitis was studied. The instillation of a synthetic analogue of muramyl dipeptide into teat sinus of the sixteen mammary glands was followed by a massive influx of PMN culminating after 24 h and resolving after 96 h. Every 24 h following the influx, apoptotic PMN were microscopically detected, based on morphological characteristics. Twenty four hours after the stimulation, apoptotic PMN were already observed, and peak counts of apoptotic PMN were reached 48 h after the stimulation. The lowest differential count of apoptotic PMN, corresponding to the pre-stimulation value, was found 96 h after the stimulation. The presence of macrophages (MAC) containing phagocytized apoptotic PMN was observed by histochemical staining for myeloperoxidase (MPO) and electron microscopy. The percentage of MPO-positive macrophages increased during the resolution phase to reach peak values 48 h after the stimulation. Apoptosis of PMN and phagocytosis by macrophages may represent a removal mechanism that is important in the resolution of the induced influx of PMN in the cavity system of juvenile bovine mammary gland.

Ultrastructure of phagocytes from mammary glands of non-pregnant heifers.

Results of ultramicroscopic investigations of phagocytes isolated from non-secreting and aberrantly secreting juvenile mammary glands of non-pregnant heifers are presented. The two types of phagocytes observed in cell suspensions obtained by lavage of mammary gland cavities were polymorphonuclear leukocytes and macrophages. Polymorphonuclear leukocytes were spherical or irregular in shape and contained segmented nuclei. Azurophilic and specific electron-dense granules, mitochondria, glycogen particles, phagosomes and phagolysosomes in cytoplasma and characteristic pseudopodia on the cell surface were observed. In addition to these normal polymorphonuclear leukocytes, degenerating cells, characterized by spherical nuclei, total absence of pseudopodia, merged nuclear segments and altered granules, other cellular organelles and plasmalemma were present. Two types of macrophages, i.e. vacuolized and non-vacuolized, could be distinguished. Typical of the non-vacuolized type was a kidney-shaped nucleus, a rich Golgi complex and a large amount of lysosomes in the cytoplasm. The vacuolized macrophages contained a large amount of electron-dense vacuoles in the cytoplasm. Unlike non-secreting glands, the cell suspensions collected from aberrantly secreting juvenile mammary glands contained only vacuolized macrophages. The vacuolization results from phagocytosis of corpuscular particles of aberrant milk plasma.