Inflammatory response: participation of inflammatory mediator in pain, leukocytes activation/migration and septicaemia

Abstract

The common objective of our project is to evaluate the contribution of inflammatory mediators for the induction and control of inflammatory pain (nociceptor hipersensitization), migration/activation of leukocytes and septicaemia. Our experimental approach involves a) the temporal evaluation of the mediators released by several types of inflammatory stimuli; b) the definition of the net mediators released from resident or migrating cells or from plasma, stimulated by different injuriating stimuli; c) the characterisation of cytoplasmic second messager pathways responsible for an specific inflammatory event; d) elucidation of the site of action of anti-inflammatory agents and the development of drugs based in new mechanism of action. It is becoming clear that the relative contribution of inflammatory mediators may vary with the type of injuriating stimuli (chemical or immunological) duration of the process (acute and delayed response) and tissue or migrating cells involved. In the present project we shall further investigate the cell origin and contribution of nociceptor hipersensitizing agonists (Ieukotrienes, endothelin) and cytokines (NGF, IFN-y, IL-15, IL-18) and tachykinin (CGRP, Substance P, neurokin A) in models of inflammatory pain induced by various chemical or pathological stimuli (carrageenin, streptozotocin induced diabetic neuropathy , ovalbumin-induced immunological hypersensitivity). We are particularly interested to understand the role neutrophils for the development of nociceptor hypersensitivity, since this phenomenon does not occur in their absence. Our laboratory greatly contributed to initiate and strengthen the concept that the previous release of a cascade of cytokines is responsible for the liberation of the final nociceptor hyperalgesic agonist mediators directly responsible for nociceptor activation. At present, the two more studied pathways are: TNF alpha »IL6»IL- 1? » eicosanoides and TNF alpha »IL-8/CINC» sympathomimetic amines). Parallel to the proposed study with new cytokines, we shall also evaluate their importance in the promotion of persistent nociceptor hypersensitisation by their repeated daily injections (see below). Stimulation of receptor agonists of primary sensory neurones triggers an intracellular secondary messenger pathway, which may command either hypersensitising or antinociceptive second messager pathways. Nociceptor hipersensitizing agonists interacts with receptors associated with G-proteins which triggers cAMP> PKA or PLC>PKC pathways which via the modulation of ion channels involves ali the primary sensory neuron. Increase in Ca++ cytosolic concentration via intracellular Ca++ mobilisation, opening Ca++ and NMDA channels and the closure of KATP channels as well as the mobilisation of Na+ channels. Our laboratory has been involved in the development of this concept with the demonstration of the importance of the glutamate retrograde sensitisation of the primary neurone and the blockade of spinal PGE2 induced nociceptive hypersensitivity by peripheral nociceptor morphine administration. Mobilisation of Na+ channels may result of its phosphorilation or neosynthesis of Na+ channels. This latter event may explain the previous described persistent nociceptor sensitisation, phenomenon that may participate of chronic pain. In the present project we shall investigate in more detail the molecular basis of nociceptor hypersensitization by using new available inhibitors of those pathways. We have previously investigated the mode of action o peripheral and anti-inflammatory agents that acts either preventing the various steps of the generation/release/action of cytokines (glucocorticoids, talidomide, IL-1ra), or action of receptor agonists (antagonists of SHT, endothelin or leukotrienes). We proposed earlier a new class of peripheral acting analgesics which acts via stimulation of the arginine/ NO/ cGMP/ KATP channels. (NO donors, peripheral opiates, dipyrone). Kappa opiates which does not the cross the blood brain barrier are now in clinical tests and we intend to investigate if these agents have the same mode of action of ? opiates and the molecular base of the closure and opening K+ATP. Parallel to these studies we have also investigated specific central neuronal antinociceptive pathways, the anterior pretectal nucleus. In continuation we are proposing to study their various connections in the CNS, as well as, the involvement of opiate, serotoninergic and colinergic action in the mechanism of antinociception in the pos-incisional nociceptive test in rats. Based in our animal observations our associated clinical pain group of HC-FMRP, investigated with success the usefulness NO donors for analgesia in cancer and surgical pain in men. In addition to those studies was shown that colinergic agonists and Ca++ channels inhibitors potentiated opiate pos-surgical analgesia. In continuation we shall investigate the effectiveness of combination of agents administered in the epidural space (opiate/neostigmine, clonidine/ketamine) or in chronic pain the combined transdermic administration of ketamine, clonidine and phentanil. The advantage of these protocols is the possibility of a better efficacy and diminution of side effects. Nowadays epiduroscopy has facilitated the correct diagnosis of the cause of chronic lumbar pain and due to direct visualisation facilitates the epidural administration o analgesics and anti-inflammatory drugs. We intend to introduce this technique in our studies. We are also concerned with the development of nociceptive techniques which new end points or instruments allow either simple and objective measurements or the use of new experimental animals (transgenic and/or knockout mice for example). In this line we have standardised an eletronic tensiomer test for mice paw withdraw (modification of the Von Frey hair test). The end point of our modification of the Randall-Selitto rat paw mechanical test is now being adapted for measurement using computational image techniques. The success of this technique will constitute a major advancement for studies of inflammatory nociception, since the end point is independent of the experimenter, thus bypassing the learning difficulties of our technique variation. Our group contributed to conceive the concept that the resident cells are important source of chemotactic factor involved in the neutrophil migration. Now, to continue these studies, we are proposing to identify the chemotactic mediators (cytokines, chemokines, L TB4 and other) and the mechanism by which they mediate the neutrophil and eosinophil migration in non-immune (LPS, carragenin) and in immune inflammation (ovalbumin in sensitised animals). In the immune inflammation, the determination of resident cells type that release the chemotactic mediators and the mechanism involved in their activation will also be addressed. It will be also evaluated in our model of allergic immune inflammation the mechanism involved in the eosinopoiesis at level of bane marrow. Our laboratory also contributed to built the concept that NO is the key mediator for the microbicidal activity of macrophages and neutrophil against the majority of the micro-organisms. Now, we are investigating the possible participation of NO on microbicidal activity of eosinophils in parasite disease and on citotoxicity effect of this cell to epithelial cells in allergic diseases. Despite of the fact that NO produced in the infection focus is important for the microbicidal activity of the neutrophils, the production of this mediator in the circulation mediates the impairment of the neutrophil migration to the infection focus, phenomenon observed in lethal septicaemia. In order to understand the mechanism by which NO inhibits the neutrophil migration in sepsis, we pretend to investigate the cellular source, and the NOS isoform that produces the NO involved in the failure of neutrophil migration in lethal septicaemia, induced by Gram Negative and Positive bacteria, and in CLP-(caecum ligation and puncture) model. In the CLP septicaemia it will also evaluated the mechanism by which NO mediates the impairment of neutrophil migration. Specifically, we will analysis if NO is inhibiting the neutrophil/endothelium adhesion mechanism and/or the neutrophil locomotion mechanism. In those experiments the involvement of NO will be evaluated by use of specific inhibitors of NOS isoforms and also NOS inducible knockout animals. In addition, the failure of neutrophil migration will be evaluated in sepsis induced by pneumonia, since lung has a peculiarity concerning lhe neutrophil migration. The rolling step is unnecessary to the neutrophil adhesion and migration. The relevance of neutrophil migration impairment for the lethal of septic patients will be also analysed, correlating the in vitro chemotaxis activity of the neutrophil obtained from patients stimulated by various chemotactic mediators ( leukotrienes, FMLP, IL-8). In the eventual occurrence of neutrophil impairment, inhibitors of NO synthesis will be used in an attempt to restore its activity. (AU)