Annexin 1 localisation in tissue eosinophils as detected by electron microscopy

Background: Human and rodent leukocytes express high levels of the glucocorticoid-inducible protein annexin 1 (ANXA1) (previously referred to as lipocortin 1). Neutrophils and monocytes have abundant ANXA1 levels.Aim: We have investigated, for the first time, ANXA1 ultrastructural expression in rat eosinophils and compared it with that of extravasated neutrophils. The effect of inflammation (carrageenin peritonitis) was also monitored.Methods: Electron microscopy was used to define the sub-cellular localisation of ANXA1 in rat eosinophils and neutrophils extravasated in the mesenteric tissue. A pair of antibodies raised against the ANXA1 N-terminus (i.e. able to recognise intact ANXA1, termed LCPS1) or the whole protein (termed LCS3) was used to perform the ultrastructural analysis.Results: The majority of ANXA1 was localised in the eosinophil cytosol (~60%) and nucleus (30-40%), whereas a small percentage was found on the plasma membrane (< 10%). Within the cytosol, the protein was equally distributed in the matrix and in the granules, including those containing the typical crystalloid. The two anti-ANXA1 antibodies gave similar results, with the exception that LCPS1 gave a lower degree of immunoreactivity in the plasma membrane. Inflammation (i.e. carrageenin injection) produced a modest increase in eosinophil-associated ANXA1 reactivity (significant only in the cytoplasm compartment). Extravasated neutrophils, used for comparative purposes, displayed a much higher degree of immunoreactivity for the protein.Conclusion: We describe for the first time ANXA1 distribution in rat eosinophil by ultrastructural analysis, and report a different protein mobilisation from extravasated neutrophils, at least in this acute model of peritonitis.

Influence of Timing of Administration on the Analgesic Effect of Bupivacaine Infiltration in Carrageenin-injected Rats

Background Recent evidence has suggested that the timing of administration of analgesic drugs could influence their efficacy by reducing the sensitization of the nervous system induced by the nociceptive inputs, but this concept of preemptive analgesia is still debated in both clinical and basic research. Methods The model of acute inflammatory pain induced by carrageenin was used to study the influence of timing of administration of bupivacaine (0.2 ml of a 0.5% solution with 0.005 mg/ml epinephrine) on the development of hyperalgesia, edema, and increase in temperature. The animals received bupivacaine 5 min before (BUPI PRE group, n = 20) or 60 min after (BUPI POST group, n = 20) carrageenin (1 ml/kg of 1% solution) was injected into the left hind paw. Two control groups (n = 15 in each) received saline 5 min before or 60 min after administration of carrageenin. Hyperalgesia of the injected paw was evaluated by the vocalization threshold to paw pressure, edema by measuring paw circumference with a thread, and plantar temperature with a thermocouple thermometer. All measurements were done before carrageenin injection then every 30 min thereafter for 240 min. Another series (n = 24), with the same four groups was also evaluated at 24 h. Results Local injection of bupivacaine 60 min after carrageenin partially reduced the edema and hyperalgesia. The injection of bupivacaine 5 min before carrageenin was more efficient than the delayed injection and reduced hyperalgesia, edema and the increase in temperature temporarily, but did not totally prevent their development. All groups were similar at 240 min and 24 h. Conclusions These results show that a slight advantage of infiltration with bupivacaine before injury exists in this carrageenin model of acute inflammatory pain. However, this benefit is limited in time and bupivacaine did not have any preemptive analgesic effect.

An increase in nitric oxide produced by rat peritoneal neutrophils is not involved in cell apoptosis

Polymorphonuclear neutrophils (PMN) obtained from carrageenin-stimulated peritoneal cavities of rats, but not blood PMN, spontaneously produced nitric oxide (NO) when incubatedin vitro. Incubation of the cells with the NO synthase inhibitors, L-imino-ethyl-L-ornithine (L-NIO) or NG-monomethyl-L-arginine (L-NMMA), inhibited NO production. This inhibition could be reversed by L-arginine. Incubation of PMN with lipopolysaccharide (LPS) failed to enhance NO production. Pretreatment of the rats with dexamethasone (DEXA) prior to carrageenin injection or incubation of PMN with the glucocorticoidin vitropartially inhibited the spontaneous release of NO. On the other hand, when PMN obtained from DEXA pretreated rats were incubatedin vitrowith DEXA, NO synthase activity and hence NO generation were almost abolished. A similar inhibition was also observed following the addition of L-NIO or cycloheximide to cultures of carrageenin-elicited PMN. The NO production by PMN did not appear to be related to cell viability or apoptosis. Indeed, neither the blockade of NO generation by L-NIO nor the incubation of the neutrophils with a NO donor, S-nitroso-acetylpenicillamine (SNAP) modified the pattern of LDH release or DNA fragmentation. In summary, it appears that PMN migration triggers a continuous NO synthesis, and that NO produced by these cells is not related to their apoptosis.

Participation of the adrenal gland in the anti-inflammatory effect of polyunsaturated diets

The effect of an n-3 (fish) and n-6 (soybean) fatty acid-rich diet on carrageenin paw oedema in rats, and the participation of adrenal gland, corticosterone and α2-macroglobulin (α2-M) in this process were studied. A significant inhibition of carrageenin oedema was observed not only in rats fed a diet rich in fish oil but also in the soybean group. α2-M was not detectable before carrageenin injection, suggesting that this putative antiinflammatory factor does not participate in the observed anti-inflammatory effect. Corticosterone levels were higher in fat-fed than in control rats, before carrageenin stimulus and adrenalectomy abolished the anti-inflammatory response in fat-fed animals, showing the important role of the adrenocortical hormones in this process.