Morphine-induced changes in the activity of proopiomelanocortin and prodynorphin systems in zymosan-induced peritonitis in mice.

We have shown that supplementation of proinflammatory agent with a high dose of morphine not only abolishes inflammation-related pain symptoms but also inhibits influx of leukocytes to the inflamed peritoneal cavity. Present investigations focused on effects of morphine on proopiomelanocortin and prodynorphin systems during zymosan-induced peritonitis. Males of SWISS mice were ip injected with zymosan (Z, 40 mg/kg) or zymosan with morphine (ZM, 20 mg/kg). At time 0 (controls) and 4 and 24h after stimulation, peritoneal leukocytes (PTLs) were counted, PTL levels of opioid peptides (beta-endorphin and dynorphin) measured by radioimmunoassays, while mRNAs coding their respective precursors (POMC and PDYN) and receptors (MOR and KOR) determined by QRT-PCR. Influx of inflammatory PTLs, mainly PMNs, was significantly delayed by morphine co-injection. Total levels of beta-endorphin and dynorphin corresponded with PTL numbers, while levels per cell were similar in all groups except of beta-endorphin, decreased in ZM at 4h. Levels of both peptides in peritoneal fluid were increased in Z and ZM groups at 4h, while at 24h only in case of beta-endorphin in Z group. POMC was increased only in ZM group at 4h of peritonitis, while PDYN in both Z and ZM groups at the same time. MOR mRNA was increased 24h after injection in Z and ZM groups, while KOR mRNA was similar in all groups except of decrease in Z at 24h. In conclusion, endogenous opioids and their receptors are involved in zymosan-induced peritonitis and affected in various ways by morphine co-injection.

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.

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.

Inhibitory effects of morphine on some inflammation-related parameters in the goldfish Carassius auratus L.

Acute peritonitis induced in the goldfish by intraperitoneal injection of a sterile Thioglycollate solution shows a typical pattern with intraperitoneal exudation of serum proteins followed by influx of leucocytes (mainly heterophils/macrophages) correlated with elevated levels of chemotactic factors in peritoneal fluid and blood plasma. Supplementation of Thioglycollate with morphine (20 mg kg(-1) b.w.) does not affect the leakage of serum proteins into peritoneum. In contrast, it reduces the number of exudate peritoneal leucocytes (among them heterophils/macrophages) to the control level and decreases the level of peritoneal fluid/plasma chemoattractants, both effects being reversed by naltrexone pretreatment. Morphine itself acts as a chemokinetic factor for fish leucocytes as it increases their random movements. Therefore inhibitory effects of morphine on accumulation of exudate cells might be explained by inhibition of the production/release of chemotactic factors and/or reduced sensitivity of leucocytes to chemotactic signals. The effects of morphine on the goldfish peritonitis are in concordance with those described recently in Atlantic salmon and CB6 mice.

Expression of proenkephalin (PENK) mRNA in inflammatory leukocytes during experimental peritonitis in Swiss mice.

Zymosan- or thioglycollate-induced experimental peritoneal inflammation in mice may serve as a convenient model for investigations of involvement of opioid peptides derived from exudatory leukocytes in the inflammatory processes. During peritonitis, the influx of neutrophils and monocytes/macrophages correlated with a sequential appearance of proinflammatory cytokines (IL-1beta and TNFalpha). After both kinds of stimulation, the expression of PENK mRNA was much higher in exudatory peritoneal leukocytes than its basal level in steady state.