No description available.

No description available.

No description available.

Graduation date: 1997

Major injury leads to host immune dysregulation and increased susceptibility to infectious challenge. Toll-like receptors (TLRs) are archetypical pattern-recognition receptors that, in addition to a role in mediating innate immune responses to components of Gram-positive and Gram-negative pathogens, have been implicated in the recognition of endogenous mediators, released during host tissue injury. A murine model of thermal injury was employed to examine the impact of injury on TLR-mediated immune cell responses. Lymph node and spleen cell suspensions were prepared from wild type, TLR4-/- and IL-1RI-/- mice at 24 hours or 7 days after injury/sham injury, cultured for 48 hours with lipid A (LA), lipopolysaccharide (LPS), lipoteichoic acid (LTA) or peptidoglycan (PGN) and production of IL-1<span style='font-family:Symbol'>b, IL-6, IL-10 and TNF<span style='font-family:Symbol'>a measured by ELISA.  Cell subset localisation of cytokine production was assessed by intracellular cytokine detection and immunomagnetic bead T-cell depletion.  TLR4/MD2 cell surface expression was measured by flow cytometry and TLR gene induction by Real Time RT-PCR. Injury caused augmented wild type splenocyte production of IL-1<span style='font-family:Symbol'>b and TNF<span style='font-family: Symbol'>a at 24 hours and of IL-1<span style='font-family:Symbol'>b and TNF<span style='font-family:Symbol'>a at 7 days in response to all stimuli. Cytokine production was localised to macrophages and dendritic cells and the injury-augmented reactivity was independent of T-cells. Responses to LA, LPS and LTA required TLR4 whereas PGN responses were TLR4-independent.  TLR4 was not required for the <i>in vivo</i> establishment of injury-augmented proinflammatory responses. Injury did not substantially change TLR gene expression assessed by Real Time RT-PCR or TLR4/MD2 cell surface expression. IL-1 signalling was not essential for the injury-augmented proinflammatory response but was required for injury-augmented production of IL-6 and IL-10 and therefore may be important for the development of anti-inflammatory responses.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

The neutrophil is the first haemopoetic cell to arrive at the site of infection. In acute respiratory distress syndrome (ARDS), dense neutrophilic infiltrates are found in the lung in response to bacterial infection as well as generalised inflammatory stimuli, such as pancreatitis. At sites of infection, phagocytosis of bacteria by neutrophils enhances their subsequent apoptosis and clearance by macrophages however at inflammatory sites, the lifespan of the neutrophil is influenced by both pro- and antiapoptotic factors in the inflammatory milieu. Furthermore subsequent macrophage phagocytosis of apoptotic neutrophils induces the macrophage to switch to an antiinflammatory phenotype thereby hastening resolution of inflammation. The Fas death receptor pathway is important in T lymphocyte apoptosis but its role in neutrophil apoptosis is controversial. We have shown that neutrophils express the Fas receptor (CD95) on their surface but there is no evidence of expression of its natural ligand (FasL). An agonistic anti-Fas monoclonal antibody (CH-11) accelerated neutrophil apoptosis under certain culture conditions. Lipopolysaccharide (LPS) originating from Gram-negative bacteria is often found at sites of inflammation. We have shown that LPS attenuated CH-11 - induced neutrophil apoptosis unless the Fas/FasL death receptor pathway was activated prior to the LPS signalling pathway. This LPS-mediated attenuation did not involve the p42/44 ERK, protein kinase C or phosphatidylinositol 3-kinase signalling pathway however the p38 MAPK and NF-κB pathway appeared to be partially involved. We have shown that neutrophils express the protein cFLIPs and that CH-11 and inflammatory mediators altered its expression. Although macrophages are principally phagocytes, they are also important in determining the composition of the milieu at an inflammatory site. Macrophages have been shown to express FasL which can be shed and may contribute to the pools of sFasL found in the bronchoalveolar lavage fluid (BALF) in ARDS patients. We have shown that the conditioned supernatants from LPS-activated macrophages induced neutrophil apoptosis at early time points. The pro-apoptotic activity was mediated by TNF-α and was found in the fraction containing proteins with molecular weights greater than 50kD. Macrophage phagocytosis of apoptotic neutrophils suppressed TNF-α production by LPS-activated macrophages and this was associated with loss of the pro-apoptotic activity. In summary, our data suggest that Fas/FasL fratricide does not appear to be involved in spontaneous neutrophil apoptosis. However LPS attenuates Fas-induced apoptosis unless the Fas/FasL death receptor pathway is activated prior to LPS signalling pathways. The signalling pathways involved in this attenuation are not clear but may involve cellular FLIP. Furthermore, activated macrophages secrete inflammatory mediators and at early time points, TNF-α appears to be the most important in inducing neutrophil apoptosis.

published_or_final_version / abstract / Pharmacology / Master / Master of Philosophy

No description available.