Effects of methylprednisolone on intracellular bacterial growth

Clinical studies have shown positive associations among sustained and inten se inflammatory responses and the incidence of bacterial infections. Patien ts presenting with acute respiratory distress syndrome (ARDS) and high leve ls of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-a lpha), interleukin 10 (IL-1 beta), and IL-6, have increased risk for develo ping nosocomial infections attributable to organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter spp., compared to those patients with lower levels. Our previous in vitro studies have demonstrated that these bacterial strains exhibit enhanced growth extracellularly when supplemented with high concentrations of pure recombinant TNF-alpha, IL-10, or IL-6. In addition, we have shown that the intracellular milieu of phago cytic cells that are exposed to supraoptimal concentrations of TNF-alpha, I L-1 beta, and IL-6 or lipopolysaccharide (LPS) favors survival and replicat ion of ingested bacteria. Therefore, we hypothesized that under conditions of intense inflammation the host's micromilieu favors bacterial infections by exposing phagocytic cells to protracted high levels of inflammatory cyto kines. Our clinical studies have shown that methylprednisolone is capable o f reducing the levels of TNF-alpha, IL-1 beta, and IL-6 in ARDS patients. H ence, we designed a series of in vitro experiments to test whether human mo nocytic cells (U937 cells) that are activated with high concentrations of L PS, which upregulate the release of proinflammatory cytokines from these ph agocytic cells, would effectively kill or restrict bacterial survival and r eplication after exposure to methylprednisolone. Fresh isolates of S. aureu s, P. aeruginosa, and Acinetobacter were used in our studies. Our results i ndicate that, compared with the control, stimulation of U937 cells with 100 -ng/ml, 1.0-mug/ml, 5.0-mug/ml, or 10.0-mug/ml concentrations of LPS enhanc ed the intracellular survival and replication of all three species of bacte ria significantly (for all, P = 0.0001). Stimulation with less than or equa l to 10.0 ng of LPS generally resulted in efficient killing of the ingested bacteria. Interestingly, when exposed to graded concentrations of methylpr ednisolone, U937 cells that had been stimulated with 10.0 mug of LPS were a ble to suppress bacterial replication efficiently in a concentration-depend ent manner. Significant reduction in numbers of CFU was observed at greater than or equal to 150 mug of methylprednisolone per ml (P values were 0.032 , 0.008, and 0.009 for S. aureus, A aeruginosa, and Acinetobacter, respecti vely). We have also shown that steady-state mRNA levels of TNF-alpha, IL-10 , and IL-6 in LPS-activated cells were reduced by treatment of such cells w ith methylprednisolone, in a concentration-dependent manner. The effective dose of methylprednisolone was 175 mg, a value that appeared to be independ ent of priming level of LPS and type of mRNA. We therefore postulate that a U-shaped relationship exists between the level of expression of TNF-alpha, IL-1 beta, and IL-6 within the phagocytic cells and their abilities to sup press active survival and replication of phagocytized bacteria.