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Compared with the Toll-like receptor 4 (TLR4) ligand LPS restricted to Gram-negative bacteria, few studies have addressed induction of lung inflammation and concomitant leukocyte recruitment in response to TLR2 ligands. This study is the first report showing that selective TLR2 stimulation by its ligand Pam3-Cys-Ser-Lys-Lys-Lys-Lys-OH (Pam3CSK4) within the alveolar compartment promoted lung inflammation in mice and induced the migration of circulatory immune cells including mononuclear phagocytes into the inflamed alveolar space. By using the transgenic CX3CR1+/GFP mouse strain for high-purity sorting of circulating and alveolar recruited mononuclear phagocytes together with SMART preamplification and whole genome oligonucleotide microarray techniques, we found that alveolar trafficking of mononuclear phagocytes was associated with profound changes of their gene expression profiles (∼900 differentially regulated genes postrecruitment). In particular, alveolar recruited mononuclear phagocytes showed upregulated transcripts of genes encoding cytokines/chemokines and pattern recognition receptor (PRR)-associated molecules. Notably, we observed a dynamic change of the genetic program of recruited mononuclear phagocytes obtained from bronchoalveolar lavage fluid at different time points (24 vs. 48 h) post-Pam3CSK4 challenge. In early alveolar recruited mononuclear phagocytes, mRNA levels of both proinflammatory (e.g., TNF-α, CCL2, and IL-6) and central anti-inflammatory/ proresolution [e.g., IL-1-receptor antagonist (IL-1RN), CD200 receptor (CD200R), IL-1 receptor-associated kinase (IRAK-M), IL-10, and Bcl-2-associated X protein (Bax)] mediators were found to be highly upregulated simultaneously. In corresponding cells recruited until later time points, transcript levels of anti-inflammatory/proresolution molecules persisted at the same level, whereas mRNA levels of proinflammatory mediators were found to decline. Collectively, our in vivo study identifies genetic programs by which alveolar recruited mononuclear phagocytes may contribute to the development and termination of pneumonia caused by Gram-positive bacteria.

The double-stranded RNA (dsRNA)-activated serine/threonine kinase R (PKR) is well characterized as an essential component of the innate antiviral response. Recently, PKR has been implicated in Toll-like receptor (TLR) signal transduction in response to bacterial cell wall components. Its contribution to pulmonary immunity, however, has not yet been elucidated. In this report we investigated whether PKR is involved in TLR2/TLR4-mediated immune responses of primary alveolar macrophages (AM). We found that both TLR2 (Pam3CSK4) and TLR4 (LPS) ligands induced rapid phosphorylation of PKR. Moreover, this activation was strictly dependent on the functionality of the respective TLR. Pharmacologic inhibition of PKR activity using 2-aminopurine (2-AP) and PKR gene deletion was found to reduce the TLR2/TLR4-induced activation of the JNK signaling pathway (MKK4/JNK/c-Jun), but did not affect p38 and extracellular signal-regulated kinase 1/2 activation. Moreover, inhibition of PKR phosphorylation severely impaired TNF-alpha and IL-6 production by AM in response to LPS and Pam3CSK4. In addition, we found that PKR phosphorylation plays a major role in LPS- but not Pam3CSK4-induced activation of the p65 subunit of NF-kappaB. Collectively, these results indicate that functional PKR is critically involved in inflammatory responses of primary AM to gram-positive as well as gram-negative bacterial cell wall components.

Background Acute peritonitis developing in response to gram-negative bacterial infection is known to act as a trigger for the development of acute lung injury which is often complicated by the development of nosocomial pneumonia. We hypothesized that endotoxin-induced peritonitis provokes recruitment of monocytes into the lungs, which amplifies lung inflammatory responses to a second hit intra-alveolar challenge with endotoxin. Methods Serum and lavage cytokines as well as bronchoalveolar lavage fluid cells were analyzed at different time points after intraperitoneal or intratracheal application of LPS. Results We observed that mice challenged with intraperitoneal endotoxin developed rapidly increasing serum and bronchoalveolar lavage fluid (BALF) cytokine and chemokine levels (TNFα, MIP-2, CCL2) and a nearly two-fold expansion of the alveolar macrophage population by 96 h, but this was not associated with the development of neutrophilic alveolitis. In contrast, expansion of the alveolar macrophage pool was not observed in CCR2-deficient mice and in wild-type mice systemically pretreated with the anti-CD18 antibody GAME-46. An intentional two-fold expansion of alveolar macrophage numbers by intratracheal CCL2 following intraperitoneal endotoxin did not exacerbate the development of acute lung inflammation in response to intratracheal endotoxin compared to mice challenged only with intratracheal endotoxin. Conclusion These data, taken together, show that intraperitoneal endotoxin triggers a CCR2-dependent de novo recruitment of monocytes into the lungs of mice but this does not result in an accentuation of neutrophilic lung inflammation. This finding represents a previously unrecognized novel inflammatory component of lung inflammation that results from endotoxin-induced peritonitis.