Macrophages, reactive nitrogen species, and lung injury
ABSTRACT Evidence has accumulated over the past several years demonstrating that lung injury following inhalation of irritants like ozone is due, not only to direct effects of the chemical, but also indirectly to the actions of inflammatory mediators released by infiltrating macrophages. Among the mediators involved in the cytotoxic process, reactive nitrogen species (RNS) are of particular interest because of their well-documented cytotoxic potential. Findings that macrophage suppression blocks RNS production and ozone-induced toxicity provide strong support for a role of these cells and inflammatory mediators in lung injury. Recent investigations have focused on understanding pathways by which macrophages become activated to release RNS. One protein that has attracted considerable attention is caveolin-1, a membrane scaffolding molecule that functions to negatively regulate cell signaling. The fact that expression of caveolin-1 is down-regulated in macrophages after ozone inhalation suggests a mechanism controlling the release of cytotoxic mediators by these inflammatory cells.
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ABSTRACT: Receptors for advanced glycation end-products (RAGE) are multiligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages, and expression increases following exposure to cigarette smoke extract (CSE). The present study sought to characterize the proinflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE knockout (KO) mice compared with controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and analyzed. CSE significantly increased RAGE expression by wild-type AMs. Employing ELISAs, wild-type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates proinflammatory signaling. Conversely, RAGE KO AMs had less Ras activation compared with wild-type AMs after exposure to CSE. In RAGE KO AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed that CSE-induced inflammation may occur in part via RAGE signaling. Lastly, quantitative RT-PCR revealed that the expression of proinflammatory cytokines including TNF-α and IL-1β were detectably decreased in RAGE KO AMs exposed to CSE compared with CSE-exposed wild-type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.AJP Lung Cellular and Molecular Physiology 04/2012; 302(11):L1192-9. DOI:10.1152/ajplung.00099.2012 · 4.04 Impact Factor
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ABSTRACT: T-2 toxin is known to be one of the most toxic trichothecene mycotoxins. Exposure to T-2 toxin induces many hematologic and immunotoxic disorders and is involved in immuno-modulation of the innate immune response. The objective of this work was to evaluate the effects of T-2 toxin on the activation of macrophages by different agonists of Toll-like receptors (TLR) using an in vitro model of primary porcine alveolar macrophages (PAM). Cytotoxic effects of T-2 toxin on PAM were first evaluated. An IC50 of 19.47 ± 0.9753 nM was determined for the cytotoxicity of T-2 toxin. A working concentration of 3 nM of T-2 toxin was chosen to test the effect of T-2 toxin on TLR activation; this dose was not cytotoxic and did not induce apoptosis as demonstrated by Annexin/PI staining. A pre-exposure of macrophages to 3 nM of T-2 toxin decreased the production of inflammatory mediators (IL-1 beta, TNF-alpha, nitric oxide) in response to LPS and FSL1, TLR4 and TLR2/6 agonists respectively. The decrease of the pro-inflammatory response is associated with a decrease of TLR mRNA expression. By contrast, the activation of TLR7 by ssRNA was not modulated by T-2 toxin pre-treatment. In conclusion, our results suggest that ingestion of low concentrations of T-2 toxin affects the TLR activation by decreasing pattern recognition of pathogens and thus interferes with initiation of inflammatory immune response against bacteria and viruses. Consequently, mycotoxins could increase the susceptibility of humans and animals to infectious diseases.Veterinary Research 04/2012; 43(1):35. DOI:10.1186/1297-9716-43-35 · 3.38 Impact Factor
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ABSTRACT: Abstract Aims: Prostaglandin endoperoxide H(2) synthase (PGHS) is a well-known target for peroxynitrite-mediated nitration. In several experimental macrophage models, however, the relatively late onset of nitration failed to coincide with the early peak of endogenous peroxynitrite formation. In the present work, we aimed to identify an alternative, peroxynitrite-independent mechanism, responsible for the observed nitration and inactivation of PGHS-2 in an inflammatory cell model. Results: In primary rat alveolar macrophages stimulated with lipopolysaccharide (LPS), PGHS-2 activity was suppressed after 12 h, although the prostaglandin endoperoxide H(2) synthase (PGHS-2) protein was still present. This coincided with a nitration of the enzyme. Coincubation with a nitric oxide synthase-2 (NOS-2) inhibitor preserved PGHS-2 nitration and at the same time restored thromboxane A(2) (TxA(2)) synthesis in the cells. Formation of reactive oxygen species (ROS) was maximal at 4 h and then returned to baseline levels. Nitrite (NO(2)(-)) production occurred later than ROS generation. This rendered generation of peroxynitrite and the nitration of PGHS-2 unlikely. We found that the nitrating agent was formed from NO(2)(-), independent from superoxide ((•)O(2)(-)). Purified PGHS-2 treated with NO(2)(-) was selectively nitrated on the active site Tyr(371), as identified by mass spectrometry (MS). Exposure to peroxynitrite resulted in the nitration not only of Tyr(371), but also of other tyrosines (Tyr). Innovation and Conclusion: The data presented here point to an autocatalytic nitration of PGHS-2 by NO(2)(-), catalyzed by the enzyme's endogenous peroxidase activity and indicate a potential involvement of this mechanism in the termination of prostanoid formation under inflammatory conditions. Antioxid. Redox Signal. 17, 1393-1406.Antioxidants & Redox Signaling 05/2012; 17(10):1393-406. DOI:10.1089/ars.2011.4485 · 7.67 Impact Factor