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.

In severe trauma, sepsis or during surgery, bacterial lipopolysaccharide (LPS) frequently enters the circulation. Persons with high levels of high-density lipoprotein (HDL) have previously demonstrated higher monocyte procoagulant activity (PCA) when whole blood is challenged with LPS. The aim of the study was to investigate the distribution of radiolabelled LPS (125I-LPS) in plasma from six persons with high (2.14-2.82 mmol l-1) and six persons with low (0.54-1.04 nmol l-1) HDL, subjecting plasma to fast protein liquid chromatography (FPLC), or agarose electrophoresis followed by quantitative autoradiography. In heparin plasma 125I-LPS was located mainly in parts of plasma containing low-density lipoprotein (LDL) or very low-density lipoprotein (VLDL) and the immunoglobulins, and located to a lesser extent in HDL. However, persons with high HDL showed significantly higher binding of 125I-LPS to HDL and the immunoglobulins, probably to IgG, and significantly lower binding to LDL/VLDL. In calcium-depleted plasma (EDTA) 125I-LPS demonstrated a sharp increase in the binding to HDL, combined with a persistently high binding to LDL/VLDL and binding to the immunoglobulins was almost eliminated in all subjects investigated. Likewise, the binding of 125I-LPS to HDL in EDTA plasma was also significantly higher and to LDL/VLDL significantly lower in persons with high HDL. This study demonstrates that the distribution of 125I-LPS in heparin and EDTA plasma from persons with high or low HDL is different, which is presumed to be of importance concerning the various bioactivities of LPS.

Endotoxin interacts with several plasma protein systems and blood cells, causing release of a multitude of endogenous mediators that contribute to the pathophysiological process of sepsis. Binding of 125I-labelled lipopolysaccharide, LPS, to human blood in vitro showed that the major part of the 125I-LPS was recovered in plasma, whereas only small amounts were retained in washed suspensions of granulocytes, erythrocytes, monocytes and lymphocytes, respectively. Whole leukocyte preparations or isolated subpopulations incubated with 125I-LPS or fluorescein-conjugated LPS followed by autoradiography, flow cytometry or immunofluorescence microscopy showed unequivocally that monocytes bound much more LPS than did granulocytes and lymphocytes. Lipoprotein electrophoresis followed by autoradiography showed that 125I-LPS bound to all the purified lipoprotein fractions, which was also confirmed by gel filtration chromatography. These findings demonstrate that monocytes represent the most important blood cell for LPS binding and that radiolabelled LPS is able to bind to lipoproteins as well as to other serum constituents.

Lung alveolar macrophages (LAM), obtained by bronchoalveolar lavage of healthy donors, were separated into four subfractions on discontinuous gradients of Percoll and subjected to light microscopic, transmission (TEM) and scanning electron microscopic (SEM) studies. Alveolar macrophage morphometric analysis was performed on cytocentrifuged preparations. TEM of subpopulations revealed considerable morphologic heterogeneity. By SEM, cells of the most dense (D) subfraction were small, round, and, typically, the surface was highly ruffled with small membrane pseudopods. Cells of the least dense subfraction (A) showed a low degree of membrane folding or filopodia and were often totally disorganized. In smokers, macrophages of fraction A had a greater area and perimeter compared with non-smokers, whereas the inverse relationship was observed for C and D cells. Also, the number of electron-dense inclusions and the level of acid phosphatase were higher in smokers than in non-smokers. Coupled with functional heterogeneity the morphologic differences described in this paper suggest that density-separated subpopulations of LAM may represent different stages of differentiation or maturation.