We previously documented that strains of P. aeruginosa
can be classified as cytotoxic or noncytotoxic to the lungs (19
). The airspace instillation of a cytotoxic strain, PA103, consistently produces alveolar epithelial lung injury and bacteremia (14
). This experimental paradigm is unique in that septic shock and bacteremia are consistently produced, thereby allowing investigation of the mechanisms causing their development, as well as the relationship of these conditions to lung epithelial injury. Our previous studies documented that instillation of noncytotoxic P. aeruginosa
strains into the lungs of mice or rats did not produce lung injury or death when compared with the instillation of cytotoxic strains (10
). An isogenic mutant bacteria lacking specific cytotoxins (PA103ΔUT
) was instilled to create lung inflammation without epithelial injury or bacteremia.
These investigations showed that instillation of PA103ΔUT
led to significantly elevated levels of TNF-α in the BALF of rabbit lungs — levels 10,000-fold higher than in the plasma of the same animals. These experiments support our prior observations that lung inflammation can occur without increased epithelial permeability (27
). The difference between TNF-α levels in the airspaces and the plasma is consistent with the containment of TNF-α within the lung by an intact epithelial barrier. Further evidence for this comes from the experiments in which radiolabeled TNF-α was instilled along with PA103 or PA103ΔUT
. The efflux of labeled TNF-α was significantly increased only when the cytotoxic PA103 was instilled into the airspaces. However, plasma TNF-α levels resulting from PA103 instillation were much higher than those predicted assuming that the only source of circulating TNF-α was the lung. This suggests that leakage of proinflammatory mediators from the lung into the circulation stimulates systemic TNF-α production.
The instillation of PA103ΔUT
also led to the generation of levels of IL-8 in the airspaces that were comparable to those generated in response to instilled PA103. However, IL-8 was undetectable in the circulation of the animals instilled with PA103ΔUT
. This can be explained by the fact that little lung injury occurred, or by the fact that IL-8 coming from the lung was absorbed onto circulating receptors (29
). Plasma IL-8 levels were detectable in the animals instilled with PA103; this was consistent with the efflux of airspace IL-8 across the injured epithelium in amounts that exceeded the binding capacity of circulating receptors.
A previous investigation by Tutor et al. documented leakage of TNF-α from isolated, perfused rat lungs injured with α-naphthylthiourea (30
). Clinical investigations measuring proinflammatory mediators in patients with acute lung injury have documented high concentrations of inflammatory mediators in BALF and in pulmonary-edema fluid (31
). One investigation that compared mediator levels in a patient’s pulmonary-edema fluid to levels in the same patient’s plasma found increased concentrations of the inflammatory mediators in the lung fluids, and low concentrations of the same mediators in the plasma (33
). Plasma cytokine levels vary markedly in the course of experimental and clinical septic shock; therefore the timing of plasma sampling may explain the discrepancy between those results and our own. Our investigation documents that significant bacterial-induced alveolar epithelial injury causes a progressive increase in circulating TNF-α and IL-8 that is most likely due to leakage from the airspaces.
The systemic administration of anti–TNF-α serum or rhIL-10 blocked the increase of proinflammatory mediators in the circulation, and prevented hypotension and decreased cardiac output. Pretreatment with anti–TNF-α serum did not significantly decrease bacteremia; however, shock was prevented. The rhIL-10 pretreatment significantly decreased bacteremia without affecting the degree of alveolar epithelial injury. This suggests that the presence of rhIL-10 led to improved clearance of circulating bacteria. Thus, despite the presence of bacteria in the lung and in the circulation, and the development of lung injury, septic physiology did not develop when the systemic effects of the lung-generated inflammatory mediators had been blocked by anti–TNF-α or by rhIL-10.
Finally, intravenous infusion of PA103 did not cause septic shock. The presence of P. aeruginosa
in the circulation was not sufficient for the development of septic physiology, even though the concentration of circulating bacteria was greater than that found circulating in the blood of septic patients (34
). Notably, the circulatory levels of some of the proinflammatory mediators were different in the PA103-infused animals compared with the PA103-instilled animals. Plasma TNF-α levels in the animals infused with PA103 transiently increased early and then significantly declined later. This phenomenon is similar to the changes observed in serum TNF-α levels after LPS injections in rats (35
) and in primates (36
). Plasma IL-8 levels were significantly different between treatments; there was no detectable plasma IL-8 in the animals infused with PA103, whereas there was a large increase in plasma IL-8 in the animals instilled with PA103.
These results show the crucial importance of a tissue infection, as opposed to bacteremia, in the pathogenesis of septic shock. The results are consistent with the interpretation that mediators leaked from the lung in response to bacterial cytotoxicity and contributed to the development of septic shock. We recently demonstrated that the toxin secretion system of P.aeruginosa
could be blocked by antibody therapy (37
). Therapeutic strategies that decrease or prevent epithelial permeability in patients with pneumonia or lung injury should limit the development of septic shock.