Edinburgh Napier University

  This thesis investigates the effects of various inflammatory stimuli, including cytokines and air pollution particles, on the expression and secretion of various proinflammatory proteins in the lung epithelial cell line A549. The pro-inflammatory proteins investigated were C-reactive protein (CRP), fibrinogen, and heat shock protein 70 (Hsp70), all of which are known to be produced during inflammation and
are also known risk factors for cardiovascular disease. These proteins were investigated because inhalation of particulate air pollution has been associated with increased cardiovascular disease and mortality. The production of these proteins in the lung may be involved in a systemic inflammatory response which increases the plasma levels of these proteins and other cardiovascular risk factors therefore
increasing the susceptibility for cardiovascular events. Alternatively, localised CRP functions as an opsonin, while fibrinogen and its degradation products are involved in the recruitment of neutrophils and leukocytes to the lung.

Both CRP and fibrinogen are known to be produced in hepatocytes in response to cytokines. Chapter 3 investigates the effect of cytokine treatment of the A549 cells
which shows that CRP and fibrinogen can be induced in this cell line by various cytokines. CRP was found to be induced by the cytokines ILI b, IL6, IL8, TNF(a and IFN7 with increased effects shown by simultaneous treatment with two cytokines. Fibrinogen was found to be induced mainly by IL6, but fibrinogen levels were also
slightly increased by ILlb; simultaneous treatments of IL6 with either ILlb or TNF(a reduced the effect of IL6 treatment. Treatment of A549 cells with IL6 and IL8 simultaneously induced a synergistic effect.

After establishing that cytokines induce the expression of CRP in A549 cells, the effects of particulate air pollution on pro-inflammatory protein expression were then investigated (chapter 4). The cells were treated with carbon black (CB), ultrafine CB (ufCB), and iron chloride (FeC13) to find out what effect these air pollution components and PM10 would have on the expression of CRP, fibrinogen, Hsp70 and the transcription factor NFKB and its inhibitor IKB. NFKB is known be activated by PM10 and is involved in the signalling of pro-inflammatory responses. All the particulate treatments induced a pro-inflammatory response with expression and secretion of CRP, fibrinogen, and Hsp70, whereas the soluble metal treatment had little effect. The metal salt FeC13w as used to treat the cells since it has been suggested that PM10 may mediate its effect through the presence of transition metals which are implicated in oxidant generation. The particulate exposures were also associated with the activation and nuclear translocation of NFKB indicating the involvement of NFKB and IxB in the induction of the pro-inflammatory response.

Investigations into the mechanisms by which the particles induced the proinflammatory response are discussed in chapter 5. Firstly, investigations into the effect of particles on the secretion of the pro-inflammatory proteins were carried out; these indicated that particles were capable of inducing the secretion of CRP, flbrinogen and Hsp70. Investigations into the transcriptional mechanisms of proinflammatory protein expression were carried out using specific inhibitors. CRP and fibrinogen were induced in an NFKB dependent manner, while Hsp70 was produced as a result of activation of the JAK/STAT pathway. The effect of oxidative stress being induced as a result of particle exposure was investigated using antioxidants. This showed a reduction in the amount of intracellular and secreted CRP, fibrinogen and Hsp70 in the presence of antioxidants, indicating that oxidative stress was involved. This correlated with a reduction in the levels of intracellular ATP.

Finally in chapter 6 the effects of proteins secreted from A549 cells on the monocytelike cell line MM6 were examined. This was carried out as a model of the effects of secreted pro-inflammatory proteins from the lung epithelium on other cells. It was found that CRP and fibrinogen were able to induce the activation of NFkB in MM6 cells, indicating that secretion of these proteins during lung inflammation could have an effect on other resident lung cells. Conditioned medium from A549 cells was also found to induce the expression of pro-inflammatory proteins including CRP, fibrinogen, Hsp70, IL6, IL8 and TNFcc. The A549 conditioned medium also appeared to induce a stress response and cellular damage in MM6 cells, thereby potentially exacerbating the inflammatory response.

These results indicate that pro-inflammatory proteins can be produced in lung epithelial cells in response to particle exposure as a result of oxidative stress. They may be involved in the progression of localised lung inflammation and in the systemic response to particulate air pollution.