Effective activation of antioxidant system by immune-relevant factors reversely correlates with apoptosis of Eisenia andrei coelomocytes.

Oxidative stress is harmful to the microbes but also to the host, and may result in bystander damage or death. Because of this, respiratory burst triggered in phagocytes by pathogens is counteracted by production of antioxidative factors. The aim of this work was to examine effectiveness of the latter system in earthworms Eisenia andrei by induction of reactive oxygen species, lipofuscin and phenoloxidase by natural (LPS, zymosan, Micrococus luteus) and synthetic (phorbol ester, PMA) stimulants. The compounds impaired numbers, viability (increased apoptosis) and composition of coelomocytes, and triggered the antioxidant activity of catalase and selenium-dependent glutathione peroxidase. The natural pathogenic compounds, unlike PMA, strongly activated antioxidative responses that diminished cell apoptosis. Moreover, repeated exposure to the same or different pathogenic compounds did not induce respiratory burst exhausted phenotype showing that coelomocytes are constantly at bay to withstand numerous infections. The current study reveals importance and efficiency of the oxidative-antioxidative systems in annelids but also confirms its evolutionary conservatism and complexity even in lower taxa of the animal kingdom.

Carp neutrophilic granulocytes form extracellular traps via ROS-dependent and independent pathways.

Neutrophil extracellular traps (NETs) have recently been described as an important innate defense mechanism that leads to immobilization and killing of invading pathogens. NETs have been identified in several species, but the mechanisms involved in NET formation and their role in infection have not been well determined yet. Here we show that upon in vitro stimulation with different immunostimulants of bacterial, fungal or viral origin, carp neutrophilic granulocytes rapidly release NET structures. We analyzed the composition of these structures and the kinetics of their formation by confocal microscopy, by quantifying the levels of extracellular DNA and the release of enzymes originating from neutrophilic granules: myeloperoxidase, neutrophil elastase and matrix metalloproteinase 9 (MMP-9). Profiles of NET release by carp neutrophils as well as their enzyme composition are stimulus- and time-dependent. This study moreover provides evidence for a stimulus-dependent selective requirement of reactive oxygen species in the process of NET formation. Collectively the results support an evolutionary conserved and strictly regulated mechanism of NET formation in teleost fish.

Ceramic modifications of porous titanium: effects on macrophage activation.

Porous titanium is one of the most widely used implant materials because of its mechanical properties, however, it is also characterised by low bioactivity. To improve the above parameter we prepared three modifications of the porous (30 wt%) titanium (Ti) surface by covering it with bioactive hydroxyapatite (HA), bioglass (BG) and calcium silicate (CS). Subsequently we tested the impact of the modifications on macrophages directing the inflammatory response that might compromise the implant bioactivity. In the study we investigated the in vitro effects of the materials on murine cell line RAW 264.7 macrophage adherence, morphology and activation (production/release of metalloproteinase MMP-9 and pro- and anti-inflammatory cytokines). CS Ti decreased the macrophage adherence and up-regulated the release of several pro-inflammatory mediators, including TNF-α, IL-6, IL-12. Also HA Ti reduced the cell adherence but other parameters were generally not increased, except of TNF-α. In contrast, BG Ti improved macrophage adherence and either decreased production of multiple mediators (MMP-9, TNF-α, IFN-γ, MCP-1) or did not change it in comparison to the porous titanium. We can conclude that analyzing the effects on the inflammatory response initiated by macrophages in vitro, calcium silicate did not improve the biological properties of the porous titanium. The improved bioactivity of titanium was, however, achieved by the application of the hydroxyapatite and bioglass layers. The present in vitro results suggest that these materials, HA Ti and especially BG Ti, may be suitable for in vivo application and thus justify their further investigation.

Effects of aliphatic polyesters on activation of the immune system: studies on macrophages.

There is a constant search for biodegradable polymers with biocompatible characteristics. However, the reported materials are rarely tested for their immunostimulatory properties, which is an important issue as immune cells activated by the polymers might cause their rejection and lead to further injury to the host tissues. Therefore, the aim of the present study was to determine if biodegradable polymers are able to activate RAW 264.7 macrophages. Aliphatic polyesters, poly(L-lactide) (PLLA), poly(L-lactide-co-trimethylene carbonate) (PLTMC), poly(glycolide-co-L-lactide) (PGLA), poly(glycolide-co-L-lactide-co-ε-caprolactone) (PGLCap) and poly(glycolide-co-ε-caprolactone) (PGCap), processed into foils by slip-casting, were characterized in terms of their structure ((1)H-NMR, GPC, DSC) and surface properties (chemical composition, water contact angle, surface free energy, topography and roughness). RAW 264.7 cells were cultured on the materials for 3 or 5 days and their adherence, numbers of apoptotic/necrotic cells, as well as production of several cytokines/chemokines and other inflammation-related molecules (matrix metalloproteinases, nitric oxide) was evaluated. The study demonstrated that PLLA and PGLA did not influence macrophage activation and survival. In contrast, PLTMC, PGLCap and PGCap significantly decreased macrophage adherence, increased ratio of apoptosis and up-regulated synthesis/release of numerous inflammatory mediators. Thus, the latter materials might initiate an undesired inflammatory reaction. The above effects of the polymers were attributed to their high hydrophobicity and low polarity due to the presence of ε-caproyl blocks (PGLCap and PGCap), and/or high flexibility and susceptibility to mechanical deformation due to low glasstransition temperature (PLTMC, PGLCap and PGCap). In conclusion, while PLLA and PGLA do not affect macrophage functioning, the other materials (PLTMC, PGLCap, PGCap) up-regulate macrophage activity.

Role of lymphocytes in the course of murine zymosan-induced peritonitis.

OBJECTIVE AND DESIGN: To investigate a putative role of lymphocytes in a murine model of zymosan peritonitis. MATERIAL OR SUBJECTS: Rag-deficient mice (KO) and their counterparts (WT) (13 animals in each group). TREATMENT: Mice were injected i. p. with zymosan (2 mg/ml, 0.5 ml/mouse) and sacrificed either 30 min or 6 h post-treatment. METHODS: At 30 min of inflammation vascular permeability was assessed by peritoneal leakage of i. v. injected Evans blue. At 6 h of peritonitis leukocyte numbers were estimated (Turk's staining), and MMP-2 and -9 presence (zymography). Levels of inflammatory mediators were evaluated by either ELISA (PGE(2), KC) or Cytometric Bead Array (IL-6, IL-10, MCP-1, IFN-gamma, TNF-alpha, and IL-12p70). The Amount of nitric oxide (NO) was measured by the Greiss reaction. Differences between WT and KO mice were analyzed by Student's t-test (p

Shedding light on vascular permeability during peritonitis: role of mast cell histamine versus macrophage cysteinyl leukotrienes.

The inflammatory response consists of sequential steps that are essentially the same whatever the cause and wherever the site. The main purpose of inflammation is to bring fluid, proteins, and cells from the blood into the damaged tissues. Therefore there are mechanisms that allow cells and proteins to gain access to extravascular sites, where and when they are needed if damage and infection has occurred. A critical process for formation of inflammatory exudate is an increase in permeability of local blood vessels. Vasopermeability changes can be usually attributed to mast cells and their mediators but recent studies reveal that also macrophages can be involved in this process. This short commentary discusses new data on cellular origin of major vasoactive mediators, and their receptors during peritoneal inflammation in mice.

Critical role of mast cells in morphine-mediated impairment of zymosan-induced peritonitis in mice.

OBJECTIVE AND DESIGN: Zymosan-induced peritoneal inflammation is significantly inhibited in mice injected with an irritant supplemented with morphine. The aim of the present study was to examine the putative mast cell involvement in this inhibition. SUBJECTS: Peritonitis was induced in WBB6FI mice (genetically mast cell-deficient W/Wv and their control littermaters +/+) and in Balb/c mice, with normal mast cells (MC) and mast cell-depleted (MCx) by pretreatment with compound 48/80. Bone marrow leukocytes from intact Balb/c mice were tested for their sensitivity to chemoattractants after in vitro incubation with morphine (10(-6) M), with or without preincubation with naltrexone (10(-8) M). Control cells were incubated in medium only. TREATMENT: Peritonitis was induced by i.p. injection of either zymosan only (Z, 2 mg/ml) or zymosan supplemented with morphine (ZM, M: 20 mg/kg), without or with pretreatment with naltrexone (NZM, N: 5 mg/kg). METHODS: Thirty minutes after induction of peritonitis, the histamine levels (ELISA) and vascular permeability (Evans blue leakage) were measured. At 6 h, the number of exudatory leukocytes (haemocytometer) and chemotaxis/chemoattractant level (48-well chemotactic chamber) were estimated. RESULTS: (1) At 6 h of peritonitis, the number of exudatory leukocytes and levels of plasma chemoattractants were significantly lower in animals injected with zymosan supplemented with morphine (ZM) than in Z and NZM groups of WBB6F1 and Balb/c mice, but only in those with normal mast cells, and not in their mast cell-deficient/depleted counterparts. (2) In contrast, at 30 minutes, vascular permeability and histamine levels were higher in ZM than in Z group of mice with normal mast cells (MC), but not in those depleted of mast cells (MCx). (3) In vitro preincubation of leukocytes with morphine inhibited their migratory activity only towards peritoneal fluid from zymosan-treated MC mice but not from their MCx counterparts. CONCLUSIONS: Mast cell-derived factors are involved in morphine-mediated impairment of zymosan-induced peritonitis in mice.

Role of mast cells in zymosan-induced peritoneal inflammation in Balb/c and mast cell-deficient WBB6F1 mice.

Zymosan-induced peritonitis was investigated in mast cell-deficient WBB6F1 mice and in Balb/c mice pretreated with mast cell stabilizer (cromolyn) or antagonists of histamine receptors (mepyramine, triprolidine, cimetidine, or ranitidine). The inherited mast cell deficiency in W/Wv knockouts of WBB6F1 mice impaired significantly the level of histamine and plasma exudation (measured 30 min after stimulation) as well as the influx of exudatory leukocytes, accumulation of plasma and exudate chemoattractants, and the release of proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-6) measured at 6 h of inflammation. All of those factors were fully restored after selective intraperitoneal reconstitution of W/Wv mice with bone marrow-derived mast cells from their control +/+ counterparts. Cromolyn pretreatment of Balb/c mice reduced exclusively the early plasma exudation and histamine influx. Blocking of histamine receptors inhibited not only the early plasma exudation but also temporarily diminished primary leukocyte influx and levels of MCP-1 and IL-1beta. In conclusion, mast cells play an important role in the initiation of zymosan-induced peritonitis and modulate its further course.

Strain differences in some immune parameters can be obscured by circadian variations and laboratory routines: studies of male C57BL/6J, Balb/c and CB6 F1 mice.

This is a report on the potential influence of circadian changes and laboratory routines on some immune parameters (thymic and splenic weights, the numbers of bone marrow, peripheral blood, and peritoneal leukocytes) in: (1) males of C57BL/6J, Balb/c, and CB6 F1 mice kept under identical laboratory conditions; (2) males of CB6 mice kept under the same laboratory conditions, except for opposite light/dark regimes, either light/dark (LD) or dark/light (DL). All the animals were purchased from the same supplier and adapted for 4-5 weeks to strictly controlled housing conditions. Some parameters were similar at certain time points but statistically significantly different at others due to strain-specific daily variations. In order to make the interstrain comparisons more reliable, the data collected around the day/night cycle were pooled for calculations of mean values. Several immune parameters of CB6 mice kept under DL conditions were significantly different than those in mice under the conventional LD conditions. In conclusion, the extrapolation of results (especially in the field of neuroimmunology) to other strains (or species) should be done with great caution; and all interstrain (interspecific) comparisons, especially those from various laboratories, should always be related to specific time points and laboratory conditions.

Experimental peritonitis in anuran amphibians is not suppressed by morphine treatment.

Morphine-induced inhibition of peritoneal inflammation, consistently recorded in several murine strains and in fish (salmon and goldfish), was not observed in the investigated species of anuran amphibians (Rana temporaria, Rana esculenta and Bombina bombina).

Morphine modulation of peritoneal inflammation in Atlantic salmon and CB6 mice.

Peritoneal inflammation is a convenient model for comparisons of modulatory effects of morphine in phylogenetically distant vertebrates. Both in salmon and mice morphine injected intraperitoneally together with an irritant (thioglycollate) significantly inhibits inflammation as estimated by the number of peritoneal leukocytes. The low number of exudate cells in morphine-treated animals seems to be compensated by their high activity, as evidenced by the enhanced phorbol myristate acetate-induced respiratory burst. The morphine-inhibited influx of leukocytes into the irritated peritoneal cavity correlates with the morphine-lowered level of plasma chemotactic factors both in fish and mice. It implies that morphine impairs the level of plasma chemotactic factor either directly (affecting their release from the resident peritoneal cells) or indirectly (decreasing the number of inflammatory leukocytes by inhibition of their migration from hemopoietic sites). The inhibitory effects of morphine on both the cell number and chemoattractant level are completely reversed by the naltrexone pretreatment, which implicates the involvement of opioid receptors.

Modification of adenylate cyclase by photoaffinity analogs of forskolin.

Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking [125I]PF-M (see Figure 1 for structure) to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by [125I]PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that [a] no other AC-regulatory proteins are known to be of this size, [b] the catalytic unit of bovine brain enzyme is in the same range and [c] this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.