Microvesicles released from pneumolysin-stimulated lung epithelial cells carry mitochondrial cargo and suppress neutrophil oxidative burst.

Microvesicles (MVs) are cell-derived extracellular vesicles that have emerged as markers and mediators of acute lung injury (ALI). One of the most common pathogens in pneumonia-induced ALI is Streptococcus pneumoniae (Spn), but the role of MVs during Spn lung infection is largely unknown. In the first line of defense against Spn and its major virulence factor, pneumolysin (PLY), are the alveolar epithelial cells (AEC). In this study, we aim to characterize MVs shed from PLY-stimulated AEC and explore their contribution in mediating crosstalk with neutrophils. Using in vitro cell and ex vivo (human lung tissue) models, we demonstrated that Spn in a PLY-dependent manner stimulates AEC to release increased numbers of MVs. Spn infected mice also had higher levels of epithelial-derived MVs in their alveolar compartment compared to control. Furthermore, MVs released from PLY-stimulated AEC contain mitochondrial content and can be taken up by neutrophils. These MVs then suppress the ability of neutrophils to produce reactive oxygen species, a critical host-defense mechanism. Taken together, our results demonstrate that AEC in response to pneumococcal PLY release MVs that carry mitochondrial cargo and suggest that these MVs regulate innate immune responses during lung injury.

Spleen tyrosine kinase inhibition blocks airway constriction and protects from Th2-induced airway inflammation and remodeling.

BACKGROUND: Spleen tyrosine kinase (Syk) is an intracellular nonreceptor tyrosine kinase, which has been implicated as central immune modulator promoting allergic airway inflammation. Syk inhibition has been proposed as a new therapeutic approach in asthma. However, the direct effects of Syk inhibition on airway constriction independent of allergen sensitization remain elusive. METHODS: Spectral confocal microscopy of human and murine lung tissue was performed to localize Syk expression. The effects of prophylactic or therapeutic Syk inhibition on allergic airway inflammation, hyperresponsiveness, and airway remodeling were analyzed in allergen-sensitized and airway-challenged mice. The effects of Syk inhibitors BAY 61-3606 or BI 1002494 on airway function were investigated in isolated lungs of wild-type, PKCα-deficient, mast cell-deficient, or eNOS-deficient mice. RESULTS: Spleen tyrosine kinase expression was found in human and murine airway smooth muscle cells. Syk inhibition reduced allergic airway inflammation, airway hyperresponsiveness, and pulmonary collagen deposition. In naïve mice, Syk inhibition diminished airway responsiveness independently of mast cells, or PKCα or eNOS expression and rapidly reversed established bronchoconstriction independently of NO. Simultaneous inhibition of Syk and PKC revealed additive dilatory effects, whereas combined inhibition of Syk and rho kinase or Syk and p38 MAPK did not cause additive bronchodilation. CONCLUSIONS: Spleen tyrosine kinase inhibition directly attenuates airway smooth muscle cell contraction independent of its protective immunomodulatory effects on allergic airway inflammation, hyperresponsiveness, and airway remodeling. Syk mediates bronchoconstriction in a NO-independent manner, presumably via rho kinase and p38 MAPK, and Syk inhibition might present a promising therapeutic approach in chronic asthma as well as acute asthma attacks.

Moxifloxacin is not anti-inflammatory in experimental pneumococcal pneumonia.

OBJECTIVES: Anti-inflammatory functions of antibiotics may counteract deleterious hyperinflammation in pneumonia. Moxifloxacin reportedly exhibits immunomodulatory properties, but experimental evidence in pneumonia is lacking. Therefore, we investigated moxifloxacin in comparison with ampicillin regarding pneumonia-associated pulmonary and systemic inflammation and lung injury. METHODS: Ex vivo infected human lung tissue and mice with pneumococcal pneumonia were examined regarding local inflammatory response and bacterial growth. In vivo, clinical course of the disease, leucocyte dynamics, pulmonary vascular permeability, lung pathology and systemic inflammation were investigated. In addition, transcellular electrical resistance of thrombin-stimulated endothelial cell monolayers was quantified. RESULTS: Moxifloxacin reduced cytokine production in TNF-α-stimulated, but not in pneumococci-infected, human lung tissue. In vivo, moxifloxacin treatment resulted in reduced bacterial load as compared with ampicillin, whereas inflammatory parameters and lung pathology were not different. Moxifloxacin-treated mice developed less pulmonary vascular permeability during pneumonia, but neither combination therapy with moxifloxacin and ampicillin in vivo nor examination of endothelial monolayer integrity in vitro supported direct barrier-stabilizing effects of moxifloxacin. CONCLUSIONS: The current experimental data do not support the hypothesis that moxifloxacin exhibits potent anti-inflammatory properties in pneumococcal pneumonia.

PKC(alpha) and PKC(epsilon) differentially regulate Legionella pneumophila-induced GM-CSF.

Legionella pneumophila is an important causative agent of severe pneumonia in humans. The human alveolar epithelium is an effective barrier for inhaled microorganisms and actively participates in the initiation of innate host defense. Although secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) is essential for the elimination of invading Legionella spp., mechanisms of Legionella pneumophila-induced release of this cytokine are widely unknown. In this study, we have demonstrated a toll-like receptor (TLR)2- and TLR5-dependent release of GM-CSF in L. pneumophila-infected human alveolar epithelial cells. GM-CSF secretion was not dependent on the bacteria type II or type IV secretion system. Furthermore, an increase in protein kinase C (PKC) activity, particularly PKC(alpha) and PKC(epsilon), was noted. Blocking of PKC(alpha) and PKC(epsilon) activity or expression, but not of PKC(beta), PKC(delta), PKC(eta), PKC(theta), and PKC(zeta), significantly reduced the synthesis of GM-CSF in infected cells. While PKC(alpha) was critical for the initiation of a nuclear factor-kappaB-mediated GM-CSF expression, PKC(epsilon) regulated GM-CSF production via activator protein 1. Thus, differential regulation of GM-CSF, production by PKC isoforms, contributes to the host response in Legionnaires' disease.

Differential regulation of Moraxella catarrhalis-induced interleukin-8 response by protein kinase C isoforms.

Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease. In pulmonary epithelial cells, M. catarrhalis induces release of the pro-inflammatory cytokine interleukin (IL)-8, which plays a pivotal role in orchestrating airway inflammation. The present study demonstrated that protein kinase (PK)C was activated by Moraxella infection and positively regulated M. catarrhalis-triggered nuclear factor (NF)-kappaB activation and subsequent IL-8 release. Activation of the PKC/NF-kappaB signalling pathway was found to be dependent on expression of the Moraxella-specific ubiquitous surface protein A2. In addition, it was shown that specific isoforms of PKC play differential roles in the fine-tuning of the M. catarrhalis-induced NF-kappaB-dependent gene expression through controlling il8 promoter activity. Inhibition of PKCalpha and epsilon with chemical inhibitors or using short interfering RNA-mediated gene silencing significantly suppressed, whereas inhibition of PKCtheta increased, the M. catarrhalis-induced IL-8 transcription and cytokine release. In conclusion, it was shown that Moraxella catarrhalis infection activates protein kinase C and its isoforms alpha, epsilon and theta, which differentially regulate interleukin-8 transcription in human pulmonary epithelial cells.

Allergic lung inflammation induces pulmonary vascular hyperresponsiveness.

Pulmonary arterial vasoconstriction is an important early component of pulmonary hypertension. Inflammatory mechanisms play a prominent role in the pathogenesis of pulmonary hypertension. The present authors investigated the potential role of acute allergic lung inflammation for alterations in pulmonary haemodynamics. BALB/c mice were intraperitoneally sensitised to ovalbumin and challenged by ovalbumin inhalation. Subsequently, lungs were ventilated and perfused ex vivo, and pulmonary arterial pressure (P(pa)) was continuously monitored. Isolated perfused lungs of allergen-sensitised and -challenged mice showed five-fold enhanced P(pa) responses to serotonin, which is reported to be a significant contributor to pulmonary hypertension in humans. This increase in P(pa) was abolished by the serotonin receptor-2A antagonist ketanserin, but not the serotonin receptor-1B antagonist GR127935. Intracellular signalling to serotonin involved phosphatidylcholine-specific phospholipase C and protein kinase C, as well as Rho-kinase, as assessed by employing the specific inhibitors D609, bisindolylmaleimide and Y27632, respectively. In addition to serotonin, impressively enhanced P(pa) increases in allergic lungs were also evoked by the thromboxane receptor agonist U46619, angiotensin II and endothelin-1. In conclusion, allergic lung inflammation was accompanied by impressive pulmonary vascular hyperresponsiveness. These results suggest a possible role for allergic inflammation in the development of pulmonary arterial hypertension.

Differences in cell activation by Chlamydophila pneumoniae and Chlamydia trachomatis infection in human endothelial cells.

Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.

Comparison of different detection methods in quantitative microdensitometry.

Quantitative evaluation of immunohistochemical staining has become a focus of attention in research applications and in pathological diagnosis, such as Her-2/neu assessment in mammary carcinoma. Reproducibility of immunostaining techniques and microscopical evaluation are prerequisites for a standardized and reliable quantitation of immunostaining intensity. In the present study, different staining and microscopical techniques, including fluorescence microscopy, epipolarization microscopy of immunogold-silver, and absorbance microdensitometry were compared concerning suitability for quantitative evaluation. We describe a staining procedure using alkaline phosphatase-based immunohistochemistry with the substrate Vector Red and subsequent microdensitometry with a custom-designed absorbance filter. We have characterized linearity of the staining intensity in dependence of development time, antibody concentration, and section thickness by means of artificial standards consisting of agarose blocks into which immunogold- or alkaline phosphatase-conjugated antibodies were incorporated. Applicability of the different techniques was tested by anti-CD45 immunostaining of leukocytes within rat lung tissue detected by immunofluorescence, immunogold-silver epipolarization microscopy, as well as alkaline phosphatase-based Vector Red absorbance or fluorescence measurement. Excellent qualities of Vector Red for quantitative microdensitometric evaluation of staining intensity were particularly obvious for absorbance microscopy. Applicability in paraffin-embedded tissue as well as in cryosections, excellent segmentation, linearity over a wide range, light stability, and feasibility for permanent mounting and for long-term storage are the outstanding features of this technique for use in routine quantitative evaluation.