Date of Award
Doctor of Philosophy (PhD)
Dr. Jack Gauldie
Lipopolysaccharide (LPS), a component of the outer membrane of gramnegative bacteria, is a potent inflammatory stimulus responsible for a number of clinically critical airway conditions including gram-negative pneumonia, gram-negative septic lung injury and septic adult respiratory distress syndrome (ARDS). These conditions are all characterized by activation of alveolar monocytes/macrophages and massive infiltration of polymorphonuclear leukocytes (PMNs). While it is generally agreed that alveolar macrophages have a central role in the initiation of neutrophil accumulation and that the contents released from PMNs play a major part in causing tissue injury, the molecular mechanisms underlying these processes remain incompletely understood. Recently, some cytokines (polypeptide hormones) such as interleukin-1 (IL-1), tumor necrosis factor α (TNFα), interleukin-8 (IL-8) and interleukin-6 (IL-6), have been implicated in these processes, yet the expression of these cytokines by airway cells in response to LPS still remains to be fully elucidated. Thus, both in vitro and in vivo studies were undertaken to investigate the effect of LPS on cytokine gene expression and protein production by airway cells and tissues.
To investigate the regulation of cytokine expression in alveolar macrophages by LPS and extracellular matrix (ECM) components, LPS stimulated rat alveolar macrophages were maintained on different substrates: plastic, collagen and airways fibroblast-derived ECM (fECM). It was found that adherence to plastic induced IL-1β expression and that LPS further enhanced this expression in a time-dependent manner. In contrast, significant expression of IL-6 mRNA was observed only in LPS-stimulated alveolar macrophages. Adherence to collagen or fECM evoked a stronger IL-1β mRNA expression as compared to adherence to plastic. Only cells cultured on fEeM, however, retained maximal responsiveness to LPS stimulation over a period of five days in culture.
To determine whether LPS can directly act on airway-derived fibroblasts, nasal, bronchial and lung fibroblasts were exposed to LPS and expression of several cytokines including granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-8 and IL-6 was assessed. While fibroblasts of all three anatomic sites produced significantly increased amounts of cytokines in response to IL-1, only upper airway-derived nasal fibroblasts could synthesize significant quantities of cytokines upon LPS stimulation.
Furthermore, the interaction of airways structural epithelial cells and fibroblasts with monocytes/macrophages was examined. Human peripheral blood monocytes were cultured with either nasal epithelial cell- or fibroblast-derived conditioned medium, and survival, proliferation and differentiation of monocytes then analyzed. Both macrophage colony-stimulating factor (M-CSF) and particularly, GM-CSF released by airway structural cells were found to dramatically promote survival and differentiation, but not proliferation, of monocytes.
To investigate the temporal sequence of cytokine induction upon LPS exposure in vivo, a rat model of acute lung inflammation was established. Kinetics and cellular origin of cytokines were examined. LPS markedly evoked an early expression of TNFα and macrophage inflammatory protein-2 (MIP-2; the rodent functional equivalent of human IL-8), followed by IL-1β and IL-6 but not RANTES (a T cell/monocyte chemotactic cytokine) or transforming growth factor β₁ (TGFB₁) expression. Alveolar macrophages represented the most significant source of cytokines shortly after LPS challenge. At later times, the infiltrating PMNs were the most significant source of cytokines in the lung.
In conclusion, these data suggest that (a) the alveolar macrophage can respond directly to LPS stimulation by elaborating and releasing cytokines; (b) this cytokine response can be modulated by the extracellular environment in which alveolar macrophages reside; (c) only the fibroblast population in the upper airway, in close proximity to the external environment, is capable of responding to LPS exposure by cytokine release. This particular fibroblast population may be thus directly involved in the initiation of acute airways inflammation; (d) monocytes/macrophages and airway structural cells communicate with each other. This interaction is accomplished in part through cytokines released from the structural cells; and (e) LPS evokes a sequential and specific cytokine response in the airways in vivo. TNFa and MIP-2 appear to play a major early role in eliciting the neutrophilic response. Consistent with in vitro findings, alveolar macrophages serve as a significant source of cytokines in vivo prior to recruitment of PMNs. Thereafter, PMNs serve as the other significant source of cytokines in vivo, suggesting that these cells likely contribute to the amplification of the response to LPS in an autocrine and paracrine fashion.
Xing, Zhou, "Investigation of Endotoxin-induced Cytokine Expression in the Airways in Vitro and in Vivo" (1994). Open Access Dissertations and Theses. Paper 1732.