Using a specific enzyme-linked immunosorbent assay, plasminogen activator inhibitor-2 (PAI-2) was quantitated in cultures of human monocytes. Lipopolysaccharide (LPS) increased both extracellular and cell-associated PAI-2 levels, as well as PAI-2 mRNA measured by Northern analysis. Both the lymphokine, interleukin-4 (IL-4) (greater than or equal to 10 pmol/L), and the glucocorticoid, dexamethasone (100 nmol/L), inhibited PAI-2 formation and PAI-2 mRNA induction. Another lymphokine, interferon-gamma (IFN-gamma) (100 U/mL), as for IL-4 alone, did not stimulate PAI-2 formation; however, in contrast to IL-4, IFN- gamma did not reverse the LPS effect but could potentiate it. The suppression of PAI-2 formation by IL-4 and glucocorticoid in stimulated human monocytes extends the list of monocyte products whose synthesis can be downregulated in these cells by the two agents. The findings could have relevance to the control by monocytes/macrophages of connective tissue resorption, including that of fibrin, at sites of inflammation.
Intraabdominal abscesses were induced in mice by intraperitoneal inoculation of Bacteroides fragilis and Escherichia coli plus bran as the abscess-potentiating agent. Sixor seven-day-old abscesses were mechanically disaggregated in buffer, and the cells obtained were fractionated on discontinuous Percoll density gradients. Neutrophil populations of different density, each approximately 90% pure, were isolated. When the abscess-derived neutrophils were subsequently incubated with normal serum in vitro under aerobic conditions, the viability of the gram-negative bacteria that had been phagocytosed within the abscess did not change significantly. This anergy to intracellular bacteria (on subsequent incubation in vitro under optimal conditions for phagocytic killing) was also found for neutrophils that had been obtained from abscesses induced by a mixture that included Proteus mirabilis plus B. fragilis and from those induced by E. coli plus P. mirabilis. While unable to significantly kill intracellular organisms that had been phagocytosed in vivo, the abscessderived neutrophils could engulf and kill organisms to which they were exposed in vitro. Neutrophils from abscesses induced by P. mirabilis only plus bran killed that organism introduced in vitro significantly more effectively than the organisms that had been engulfed in vivo. In contrast, neutrophils from abscesses induced by the gram-positive organism Staphylococcus aureus plus bran were able to kill their intracellular organisms on subsequent incubation in vitro as effectively as they could kill added S. aureus. Neutrophils isolated from the peripheral blood and from induced peritoneal exudates of abscess-bearing mice were able to phagocytose and kill organisms in vitro with greater efficiency than abscess-derived neutrophils. The mechanism whereby neutrophils from abscesses induced by the gram-positive organism S. aureus can kill the organisms phagocytosed in vivo on subsequent in vitro incubation, in contrast to the relative anergy to their intracellular organisms displayed by neutrophils derived from abscesses induced by combinations of gram-negative bacteria, is not known.
Summary. Elicited, mouse peritoneal exudate cells were fractionated by centrifugation on discontinuous Percoll density gradients. TVo subpopulations of neutrophils, each of greater than 90% purity, were isolated at discontinuous density gradient interfaces different from the region of mononuclear ceU enrichment (i.e., 1-0694-1 0871 and 1-0872-1-1002 g/ml for neutrophils and less than 1-0694 g/ml for mononuclear cells). Peritoneal exudate cells were mixed with Proteus mirabitis in the presence of 1% normal mouse serum for 30 min. The mixtures were fractionated on gradients of Percoll diluted with a clacium-free medium. Populations of cells banding at densities greater than 1 -0693 g/ml were washed free of gradient material, and neutrophil suspensions containing intracellular bacteria and which were relatively free of extracellular bacteria were isolated. Less than 7% of the total bacteria present was extracellular. The continuing extracellular presence of a heat-labile component of normal mouse serum was essential for maximal intracellular kill of P. mirabitis by mouse peritoneal neutrophils.
Summary. In the absence of antimicrobial therapy, bacteria such as Bacteroides fragilis, Escherichia coli and Proteus mirabilis may persist within an intra-abdominal abscess in the presence of large numbers of neutrophils which, under optimal conditions in vitro, can readily phagocytose and kill the same bacterial strains. Neutrophils taken from abscesses induced by gram-negative bacteria such as those above contain viable organisms. On incubation in vitro in the presence of serum, these neutrophils kill the bacteria phagocytosed in the abscess poorly, if at all, yet can readily kill organisms added in vitro. To determine possible mechanisms that might explain this, we examined the bactericidal activity in vitro of neutrophils from a range of abscesses induced by one or two species of bacteria plus an abscess-potentiating agent, bran. The organisms studied were B. fragilis, E. coli, P . mirabilis and Staphylococcus aureus. The killing in vitro of E. coli and P . mirabilis, engulfed within an abscess, was significantly less than that of the same organisms when they were added to the in-vitro assay. In contrast, the killing of S. aureus was similar, whether engulfed in vivo or in vitro. However, S. aureus was less susceptible to phagocytosis and killing in vitro than P . mirabilis or E. coli, and the killing of S. aureus during in-vitro incubation of neutrophils that had engulfed the organism within the abscess was similar to that of the gram-negative bacteria engulfed within the abscess. We examined whether organisms phagocytosed in vivo were within neutrophils which had diminished or no expression of cell surface Fc and C3 receptors, and might, therefore, be unresponsive to extracellular serum factors such as complement which stimulate intracellular killing. No population of neutrophils lacking CR3 complement receptors was detected. Furthermore, the expression of these receptors was enhanced on abscess neutrophils compared with peripheral blood neutrophils. FcyRII receptor expression was equivalent in the two populations. Extracellular serum was necessary for any abscess neutrophil bactericidal activity. To determine whether the bacteria within abscess neutrophils were a subpopulation selected for resistance to intracellular killing, we lysed the neutrophils and added the released bacteria to bactericidal assays with peritoneal exudate neutrophils. The bacteria were killed readily, but not as efficiently as log-phase organisms grown in vitro. As these studies have shown that neutrophils from abscesses are functional, with enhanced CR3 expression, and that bacteria persisting within abscesses are not resistant to intracellular killing, it is possible that the poor killing in vitro of bacteria engulfed in uivo by abscess neutrophils reflects depleted intracellular killing mechanisms in those particular neutrophils, or results from the phagocytosis of organisms under conditions prevailing within abscesses, or is due to as yet undefined bacterial virulence factors.
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