The National Nosocomial Infection Surveillance indicates that P. aeruginosa is the second most common cause of nosocomial pneumonia after Staphylococcus aureus. Ventilator-associated pneumonia (VAP) caused by P. aeruginosa is a severe complication of intensive care, with mortality rates of 34 to 48% [28–30]. Therefore, it is critical to study the pathogenesis of P. aeruginosa. In recent years, with the development of technologies such as the gene chip and the protein chip, and the clarification of the genome sequence of the P. aeruginosa strain, it has been found that many elements such as pro-inflammatory cytokines, antimicrobial peptides, complements and epithelial cell receptors and their signal transduction systems (TLR2, 4, 5, CFTR, GM1, and its downstream NF-κB) participate in host defense and immune response induced by P. aeruginosa. It has also been found that P. aeruginosa components (flagella and pili) and virulence factors (such as the density-sensing system, type secretion system, toxins, alginate and cell toxin) play important roles in the pathogenesis [2, 16]. Among them, most P. aeruginosa strains secrete PCN (N-methyl-1-hydroxyphenazine), the pigment that gives blue-green color to the bacterial colonies . High concentrations of PCN are detected in pulmonary secretions of patients with cystic fibrosis, where it triggers inflammation, disrupts the bronchial epithelium and impairs ciliary function. PCN also interferes with the antioxidant defenses in the lung and facilitates oxidative damage to the lung epithelium [31–35]. PCN has been detected at concentrations as high as 100 μM in pulmonary secretions from patients with P. aeruginosa-associated airway disease , and its production is increased when the organism is in the biofilm form [4, 37]. Therefore, PCN plays an important role in acute and chronic invasive infections.
Pseudomonas infections are characterized by a marked influx of polymorphonuclear cells (PMNs) (neutrophils) . Activated PMNs release a variety of oxidants and proteases that may contribute to the tissue injury that is observed in Pseudomonas-infected airways [12, 38]. Little is known about the stimuli that are responsible for the influx and activation of PMNs into the presence of this bacterium. IL-8 is the major PMN chemoattractant responsible for PMN influx and activation in a variety of disease states and thus likely plays an important role in P. aeruginosa infections as well. It has been found that culture supernatants and various purified secretion factors of P. aeruginosa such as pili protein, flagellin, self-sensing materials, elastase, PCN and nitrite reductase [4, 13, 36, 39, 40] increase IL-8 secretion in airway epithelial cells, primary bronchial gland epithelial cells both in vivo and in vitro. It was found that with NF-κB activation, rapid and sustained IL-8 mRNA expression was induced .
Recent studies have also further confirmed that in a variety of respiratory cell lines and primary cultures of cells, PCN stimulation can cause the release of IL-8, accompanied by increased IL-8 mRNA expression. PCN also acts in synergy with IL-1α, IL-1β and TNF-α to induce IL-8 expression [5, 6, 8]. After PCN was injected into animals and the respiratory tracts, bronchial lavage fluid and neutrophil (PMN) levels were increased significantly . However, there are few reports on PCN effect on macrophages.
Our experimental results show that PCN induced expression of IL-8 in PMA-differentiated U937 cells, as well as IL-8 protein secretion and mRNA expression in a concentration- and time- dependent manner. It is also found that PCN synergizes with TNF-α to induce the expression of IL-8 in PMA-differentiated U937 cells. So far, most studies only observe the pro-inflammatory effects of the P. aeruginosa bacterial products on epithelial cells and macrophages, and their effects on U937 cells are less than well defined. The present study extends these findings by demonstrating that MAPKs and NF-κB signalings lie behind PCN-induced IL-8 production in differentiated U937 cells.
The MAPK family has an important role in signal transduction, and the pathway is activated by a variety of stimuli such as growth factors and cellular stresses [42, 43]. Activated MAPKs can regulate the expression of inflammatory cytokines. In mammalian cells, it has been found that there are at least three major MAP kinase (MAPK) pathways including the extracellular signal-regulated kinase pathway (ERK), c-Jun N-terminal kinase/stress-activated protein kinase pathway (JNK), and the P38 MAPK pathway. A unique feature of the MAPKs is that they become activated after phosphorylation of both their tyrosine and threonine amino acids . They are different activated extracellular signals that produce different biological effects. It has been found that MAPKs can modulate the expression of IL-8 in human peripheral blood mononuclear cells, granulocytes, mast cells, intestinal epithelial cells, and pulmonary vascular endothelial cells and that the use of P38 inhibitors can reduce the IL-8 mRNA and protein expression [19, 23, 41, 45].
We used PCN to stimulate PMA-differentiated U937 cells and found that PCN could induce ERK and P38 MAPK protein phosphorylation, thus indicating the possible participation of ERK and p38 MAPK pathways in the regulation of IL-8. Our further investigation using MAPK pathway inhibitors PD98059 and SB203580 demonstrated that they may partially inhibit the phosphorylation and reduce IL-8 synthesis induced by PCN in a concentration-dependent manner, indicating that PCN may stimulate PMA-differentiated U937 cells to express cytokine IL-8 by MAPK signaling pathways.
NF-κB is a ubiquitous pleiotropic transcription factor, and studies have shown that NF-κΒ activation is critically involved in a variety of lung diseases and lung inflammation [19–21]. NF-κB activation can regulate a series of lung gene expression related to inflammatory and immune responses: pro-inflammatory cytokines such as TNF-α, IL-1β, chemokines MCP-1, IL-8, and many other molecules. Therefore, its activity is closely related with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) . In most cell types, NF-kB is retained usually in the cytoplasm of the unstimulated cells by I-kBα family proteins. Upon stimulation, the I-kBα kinase complex is activated, resulting in the phosphorylation of I-kBs [47, 48] The phosphorylated IkBs are ubiquitinated and subsequently degraded, which will release the transcription factor NF-kB [36, 37]. In this study, we also found that PCN stimulation was associated with a significant increase in the level of phosphorylated I-kBα in total cell lysates. We further demonstrated that I-kBα decrease was accompanied by increased nuclear localization of p65 protein. These results suggest that PCN induces degradation of I-κBα and the subsequent translocation of NF-κB to the nucleus. The results also showed that different blockers (SB203580,PD98059 and PDTC) can reduce the expression of NF-κB p65 expression in cytosol and IL-8 expression, indicating that PCN may stimulate PMA-differentiated U937 cells to express cytokines IL-8 by MAPK and NF-κB signaling pathways.
Acute and chronic pulmonary infection with P. aeruginosa is associated with an intense neutrophil inflammatory response that contributes to lung injury . A previous study has shown that PCN enhances airway epithelial cell release of IL-8 , a neutrophil chemokine whose production is regulated by oxidant-sensitive transcription factors [50, 51]. Our data indicated that PCN could induce oxidative damage in U937 cells and antioxidant NAC inhibited PCN-induced IL-8 protein expression. In most cases, PCN’s cytotoxicity has been strongly linked to its potential effects on redox cycle. When entering into cells, PCN oxidizes intracellular pools of NADPH, NADH and GSH directly by accepting electrons, and it passes these electrons to oxygen leading to sustained generation of ROS (O2
_ and H2O2) under aerobic condition . Oxidative damage results in unbalance between the oxidant and antioxidant processes. Antioxidant defense system (enzymatic scavengers SOD, CAT and so on and some smal1 molecule antioxidants including NAC, GSH, vitamin C and vitamin E) plays an important role in the elimination of oxygen radical . Cellular GSH levels have been reported to influence the activity of a number of transcription factors, including NF-κB, AP-1, and HIF-1α [53, 54]. NAC is a thiol compound that has direct antioxidant properties and also is converted to GSH by cells and thereby limits oxidant-mediated cell injury. By demonstrating the inhibitory effect of NAC on PCN-induced IL-8 production, we indicate that NAC can act as a protective factor that mitigates PCN pro-inflammatory effect on differentiated U937 cells.
In short, in this study, we found that PCN could induce PMA-differentiated U937 cells to produce IL-8 by activating MAPKs and NF-κB signaling pathways. Our further studies will focus on understanding the interaction between p38 MAPK, ERK and other cytokine regulators. Knowledge of the mechanisms by which PCN induces PMA-differentiated U937 cells to produce cytokines may provide better understanding and rational approaches for the control of PCN-induced inflammatory processes.