| Abstract |
Corticotropin- releasing factor (CRF) is the principal regulator of the stress response. Hypothalamic CRF acts indirectly as an anti- inflammatory agent through the production of glucocorticoids. CRF as well as the structurally related peptides Urocortin 1 and Urocortin 2 (UCN1 and UCN2) are also secreted locally at the inflammatory sites from peripheral nerves or inflammatory cells themselves directly affecting the immune system. In vivo and in vitro studies describe peripheral CRF as a pro- inflammatory factor while anti- inflammatory effects of CRF and UCN have also been observed. Among inflammatory cells, macrophages are important players in innate immunity because of their non selective response to almost all infectious microorganisms. Activated macrophages produce cytokines such as TNF-α, IL-6 and NO, which are crucial mediators of the inflammatory response.
We have previously shown that CRF augments LPS- induced pro-inflammatory cytokine secretion from Raw264.7 macrophages. In this thesis we demonstrate that CRF, as well as its related peptides UCN1 and UCN2, can induce the transcription and surface expression of TLR4 in Raw264.7 and murine primary peritoneal macrophages through the activation of PU.1 and AP-1 transcription factors. The up-regulation of TLR4 by the peptides provides a mechanism for the increased sensitivity to LPS observed in macrophages treated with CRF.
Using antagonists of CRF1 and CRF2 receptors we found that CRF2 mediates the effect of CRF peptides on TLR4. Thus, the CRF2 ligands UCN1 and UCN2 also augmented LPS- induced pro- inflammatory cytokine secretion in primary macrophages. Moreover, using primary macrophages from wild type or CRF2 -/- mice, we demonstrated that CRF2 is involved in the induction of pro-inflammatory cytokine in macrophages following LPS activation. In the absence of CRF2 macrophages produced significantly reduced levels of IL-6 and TNF-α. On the contrary, CRF1-/- macrophages produced higher levels of IL-6 and TNF-α in response to LPS, possibly due to glucocorticoid insufficiency of CRF1-/- mice. Interestingly, both CRF1-/- and CRF2-/- macrophages expressed significantly higher levels of Cox-1 than wild type.
CRF peptides induced Cox-1 and Cox-2 expression and PGE2 production from macrophages, transiently inhibiting TNF-α production. PI3K mediated the inhibitory effect of CRF peptides on TNF-α. These data suggest an additional mechanism for the pro-inflammatory effect of CRF peptides, as well as a mechanism of a transient anti-inflammatory effect on macrophages.
Along with the effect of CRF peptides on pro-inflammatory cytokine secretion we demonstrated that CRF peptides affect apoptosis of LPS- activated macrophages in a time dependent manner. More specifically, prolonged exposure to CRF augments apoptosis of activated macrophages, while shortly after stimulation the LPS-induced apoptosis is transiently inhibited by CRF. CRF augments NO production from activated macrophages simultaneous with the induction of apoptosis. The early inhibitory effect of CRF on apoptosis on the other hand is consistent with the transient inhibitory effect of CRF on TNF-α.
UCN1 and UCN2, via CRF2, induced apoptosis in naïve, non- activated macrophages, suggesting another anti- inflammatory effect of CRF peptides. Moreover, macrophages expressed high levels of UCN1 which was reduced upon LPS simulation. In contrast to the mechanism of LPS- induced apoptosis in macrophages, we found that UCNs induce apoptosis via Bax and Bad up-regulation.
In conclusion, these data provide supporting evidence on the peripheral role of CRF peptides as modulators of the immune response acting directly on macrophages, the primary and principal coordinators of the inflammatory cascade.
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