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Seven successive 20 ml aliquots of pre-warmed 0. This procedure was repeated in the LPS- challenged subsegment of the contralateral lung. Of note, our laboratory previously reported that bronchial LPS instillation does not influence CD71 expression by alveolar macrophages Total and viable cell counts were determined before and after the isolation procedure using a Burker-Turk haemocytometer and trypan blue Emergo, Landsmeer, the Netherlands.

Hence, in each subject alveolar macrophages were harvested from a lung segment instilled with LPS further indicated by LPS-AM and from a contralateral lung segment instilled with sterile saline further indicated by saline-AM. All samples were tested with the same batch of reagents.

Data are presented as the ratio of phosphoprotein to total cellular protein. Primers used are indicated in Table 4. For quantification, standard curves were constructed by PCR on serial dilutions of a concentrated cDNA and data were analyzed using the LightCycler software as described by the manufacturer. Data were checked for normal distribution and equal variances using the residuals. Statistical comparisons were made by paired t-test or Wilcoxon signed rank test to establish significance between separate datasets.

These analyses were performed using SPSS version A P-value of less than 0. Table 4. Ex vivo stimulation with 0. IL and ILp70 levels were below detection in all samples data not shown.

Ex vivo stimulation of alveolar macrophages with 0. Open dots indicate in vivo saline challenged alveolar macrophages, filled dots indicate in vivo LPS challenged alveolar macrophages. Data are presented as the ratio -2 phosphoproteins to total protein levels x To address this possibility we analyzed the surface expression of TLR4 and TLR2, together with the ligand binding part of the LPS receptor complex CD14, on alveolar macrophages harvested from lung segments instilled with saline or LPS in vivo by flow cytometry.

Differences between LPS and saline challenged alveolar macrophages were not significant. Therefore, we were interested in the potential impact of bronchial LPS instillation on the expression of these surface receptors on alveolar macrophages.

Expression patterns of intracellular regulators of TLR-cascade in alveolar macrophages Several intracellular regulators have been identified that serve to inhibit TLR signaling. Discussion The airways are continuously exposed to respiratory pathogens, which may cause bacterial pneumonia when pathogens pass innate defenses in the upper airways and enter the alveolar space.

Our results demonstrate that LPS instillation into a pulmonary segment did not render alveolar macrophages less responsive to either LPS or LTA, but rather primed these cells, as manifested by enhanced cytokine production.

The results of the current study should be considered in the context of previously reported investigations. Endotoxin tolerance has been well-described in various animal and human models and in a number of cell types, including monocytes and macrophages7. The vast majority of in vivo studies reported on tolerance of circulating immune cells upon induction of systemic inflammation, Evidence indicates that alveolar macrophages may respond differently to repeated exposure to bacterial antigens when compared with macrophages from other body sites.

Indeed, whereas intravenous administration of LPS to humans results in a diminished capacity of circulating leukocytes to produce proinflammatory cytokines upon restimulation with LPS ex vivo, it has been found to lead to priming of alveolar macrophages, as reflected by increased ex vivo LPS-induced IL-1 release Similarly, Fitting et al.

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