Increased production of oxidants subsequent to phagocyte stimulation has been associated with tissue damage in lung inflammatory disorders. The overall oxidative burden of the lung may vary with inflammatory cell composition.Flow cytometry using three different dyes, dihydroethidium (DHE), dichlorofluorescein diacetate (DCFH-DA) and dihydrorhodamine 123 (DHR), all compounds that by interaction with oxidants are transformed to fluorescent products, was used to examine the production of intracellular oxidants in alveolar macrophages (AMs), including size-defined subpopulations, monocytes (Ms) and polymorphonuclear neutrophils (PMNs) during in vitro incubation in the presence or absence of phorbol myristate acetate (PMA).PMA stimulation led to slightly increased (two-fold) (p<0.05) DHE-induced fluorescence in AMs, whereas it was greatly increased in Ms and PMNs (13-fold and 113-fold, respectively). The levels of DCFH-DA-and DHR-induced fluorescence were significantly (p<0.05) increased (four-fold and 110-fold, respectively) by PMA stimulation of PMNs, but not of AMs and Ms. Significant differences (p<0.05) in the levels of DHE-and DCFH-DA-induced fluorescence in small and large AMs were also demonstrated.The results show that the potential to increase the generation of various oxidants upon stimulation was: PMNs> Ms>AMs, suggesting that the total oxidative burden of the lungs is dependent on the type of inflammatory cells present, as well as on their state of activation.

Endothelin‐1 (ET‐1) is a strong bronchoconstrictor which possesses pro‐inflammatory properties and is claimed to be an important mediator in bronchial asthma. The present study was undertaken to investigate whether ET‐1 synthesis, in an inflammation dominated by neutrophilic granulocytes, is as pronounced as previously demonstrated in an airway inflammation dominated by eosinophils. Moreover, the authors compared the production of ET‐1 and tumour necrosis factor (TNF)‐α in rat lungs following intratracheal instillation of either lipopolysaccharide (LPS) (neutrophilic inflammation) or Sephadex (SDX) (eosinophilic). The lung tissue ET‐1 messenger ribonucleic acid (mRNA) expression was not increased in LPS treated animals whereas a six‐fold increase was measured after 30 min in the SDX group (p<0.05). TNF‐α mRNA signals increased early following LPS instillation, peaking at 2 h, whereas elevated TNF‐α mRNA in the SDX model was observed at 24 h. The ET‐1 concentrations in bronchoalveolar lavage fluid (BALF) rose slightly, but significantly, 3 h after both LPS and SDX exposure. At 24 h no further rise in ET‐1 levels was observed in the LPS model, while a substantial increase in the ET‐1 concentration was measured in the SDX group (p<0.05). The TNF‐α concentrations in BALF rose considerably at 3 h in the LPS group, but was nearly abolished at 24 h. In SDX challenged animals however, an increase in BALF‐TNF‐α did not occur until 24 h postchallenge. In conclusion, intratracheal instillation of lipopolysaccharide, leading to a purely neutrophilic lung inflammation, does not induce synthesis of endothelin‐1. This is in contrast to observations during an eosinophilic airway inflammation, indicating a specific role of endothelin‐1 in lung inflammations dominated by eosinophils. In contrast to in vitro experiments, no evidence for induction of endothelin‐1 synthesis was observed by high levels of tumour necrosis factor‐αin vivo.

Background: Cell adhesion molecules are believed to be essential for blood cell recruitment to the lung and for the movement of alveolar macrophages (AM) within the lung. Objective: To investigate the expression pattern of L-selectin and β₂ integrins on blood leukocytes and AM, including AM of various maturity. Methods: Flow cytometry was used to study the expression of L-selectin (CD62L) and of the β₂ integrins CD11a, CD11b, and CD11c on AM (including density-defined subpopulations), monocytes (Mo), polymorphonuclear neutrophils (PMN) and lymphocytes (Ly) sampled from healthy individuals, during incubation with and without lipopolysaccharide (LPS). Results: A significantly different modulation pattern of β₂ integrins and L-selectin was demonstrated on Mo and AM, cells of the same differentiation lineage. In contrast to AM, Mo had a marked ability to respond to LPS stimulation by increased expression of CD11a, CD11b and CD11c and decreased expression of L- selectin. These molecules were expressed to a similar degree on AM, whereas the basal levels of CD11b and L-selectin were considerably higher on Mo than on AM. A significantly different expression of CD11a as well as differences in the regulation of L-selectin during incubation were also demonstrated between density-defined subpopulations of AM. CD11a could not be upregulated on PMN, otherwise the modulation patterns of CD11b, CD11c and L-selectin were similar to that on Mo. The expression of CD11a on Ly was 3- to 6-fold higher than on Mo, PMN and AM. The level of CD11b decreased significantly upon incubation (uninfluenced by LPS stimulation), and CD11c was hardly expressed on Ly. The level of L-selectin on Ly was higher than on Mo, AM and PMN and was not decreased during incubation. Conclusion: Developmental origin, degree of cell differentiation (maturity) as well as different environmental conditions all heavily influence the expression and modulation pattern of β₂ integrins and L-selectin on leukocytes and Mo-derived AM.

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We have studied the expression of the lipopolysaccharide (LPS) receptor CD14 on monocytes (Mo) and alveolar macrophages (AM), including density- and size-defined subpopulations. Bronchoalveolar lavage (BAL) was performed on eleven healthy non-smokers and blood sampled from 5 of them, and the levels of cell CD14 expression was investigated using flow cytometry. The influence of LPS stimulation on the CD14 expression of AM was studied at various intervals during prolonged incubation. Further, the relationship between CD14 expression and LPS binding to Mo and subpopulations of AM was studied by measuring fluorescein isothiocyanate (FITC)-LPS binding (flow cytometry) and binding of radioiodinated LPS (125I-LPS). The CD14 expression was 13-fold higher (P < 0.02) on Mo than on unfractionated and high density AM. The CD14 level on the latter was higher than on low density AM, and also higher (P < 0.05) on small AM compared to large (flow cytometrically defined) AM. LPS stimulation had a downregulating effect on AM CD14 level, but after several hours of continuing decreased expression, an increased (P < 0.05) CD14 expression was demonstrated, indicating de novo synthesis. The binding of LPS to subpopulations of AM and isolated Mo was not significantly different, but the binding of FITC-LPS to Mo in whole blood was higher than to AM (P < 0.02). The presented results indicate that AM of different size and maturity have different and variable (activation dependent) CD14 levels. The LPS binding capacity was, however, not proportional to the CD14 expression, indicating that LPS binding mechanisms unrelated to CD14 levels were also operable.