Stimulation Of Swine Alveolar Macrophages With Lipopolysaccharide Alters The Balance Between Protein Kinase C And Protein Tyrosine Phosphatase: Modulation By Aspirin

Acute lung injury (ALI) is a pulmonary manifestation of uncontrolled systemic inflammatory response syndrome (SIRS), in which activation of numerous inflammatory effector cells such as polymorphonuclear leukocytes and macrophages triggers excessive release of inflammatory mediators and cytokines. Mortality is still high in the patients with severe ALI ,especially in association with multiple organ dysfunction (MOD) or multiple organ failure (MOF). Unfortunately, the mechanisms of the pathologic processes remain unclear. It has been found, however, that nuclear factor-kappa B (NF-kappa B) might play a central role in the cellular signaling of pathological processes including ALI in which the activation of NF-kappa B could lead to the synthesis and secretion of numerous pre-inflammatory cytokines. The phosphorylation and degradation of inhibitory kappa B alpha is the most important step in the activation of NF-kappa B. Resolution of an excessive inflammatory response, therefore, should be achieved by restoring the dynamic balance of phosphorylation and dephosphorylation in inflammatory cells such as macrophages. The balance of phosphorylation and dephosphorylation in inflammatory cells would prevent sustained activation of NF-kappa B and excessive release of inflammatory mediators involved in the pathological process of ALI. In the present study, the methods as fluorescence assay, Western blot assay, electrophoretic mobility shift assay (EMSA), reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) were used to determine the dynamic changes of the activity of protein kinase C (PKC) and protein tyrosine phosphatase (PTP), the levels of inhibitory kappa B alpha and cyclooxygenase-2 (COX-2) mRNA expression in swine alveolar macrophages (s-AM) stimulated with lipopolysaccharide (LPS). The concentrations of prostaglandin E2 in the s-AM culture supernatant were measured. After pretreatment with aspirin, peroxovanadium and calphostin C alone or in different combinations for 30 minutes, all the above mentioned parameters were also measured. A summary of the main results are as follows: 1) After stimulation of the alveolarmacrophages with LPS, the activity of PKC, the activated NF-kappa B in the nuclear extract, the level of COX-2 mRNA expression and the concentration of prostglandin E2 reached peak levels, while the activity of PTP and the inhibitory kappa B alpha level in the cytoplasm decreased to their basal levels. The changes in PKC and PTP activity appeared much earlier than the other changes. 2) The non-steroidal anti-inflammatory drug aspirin can inhibited the increase in PKC activity, the activation level of NF-kappa B, the expression level of COX-2 mRNA and the concentration of prostaglandin E2 in the cytoplasm or culture supernatant of alveolar macrophages stimulated with LPS. Conversely, it has a negative effect on the degradation of inhibitory kappa B alpha and the down-regulation of PTP induced by the stimulation with lipopolysaccharde in alveolar macrophages. 3) The non-specific inhibitor of PKC, calphostin C, enhanced the inhibitory effects of aspirin on inflammatory responses induced by lipopolysaccharide in alveolar macrophages, while the PTP inhibitor, peroxovanadium, exerted the oppsite effects. Our results suggest that there is a disturbance of the PKC and PTP system in swine alveolar macrophages stimulated with lipopolysaccharide, and aspirin is effective in restoring the balance of the system, which might be useful as a reference in clinical therapy of ALI.