De Filippo, Katia;
(2009)
The role of Macrophages and Mast cells in Neutrophil recruitment.
Doctoral thesis (Ph.D), University of London.
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Abstract
Neutrophils are the first immune cells to migrate into infected tissue sites. Therefore, an important step in the initiation of an immune response is the synthesis of neutrophil-recruiting chemokines. Using an LPS-induced peritonitis model of inflammation, we studied the biosynthetic process of the major neutrophil chemoattractants, KC and MIP-2 and found that resident tissue macrophages are a major source of these mediators. Macrophages rapidly regulate the synthesis of KC and MIP-2 at the transcriptional level by signalling through Toll-like receptor (TLR) 4. The TLR4 signalling pathway is characterized by the two adaptor proteins MyD88 and TRIF, with specific mediators produced via the two pathways. We found that KC and MIP-2 are both produced by signalling through MyD88. However, only MIP-2 is also synthesized through the TRIF adaptor protein, identifying it as a new product of this alternative TLR pathway. Use of both pathways by TLR4 ensures maximal levels of KC and MIP-2 that leads to robust neutrophil recruitment. However, MIP-2 generated exclusively by the TRIF pathway is still sufficient to cause a limited but significant influx of neutrophils. Furthermore, analysis of peritoneal wall tissue reveals that another cell type can also contribute to the total level of KC and MIP-2 production. These cells, preferentially located next to the blood vessels and positively stained for toluidine blue, were revealed to be mast cells. However, unlike macrophages, mast cells contain pre-formed KC and MIP-2 proteins, which they release when stimulated with LPS. W/WV mice, which lack mast cell population, showed a severe defect in recruiting neutrophils. However, the lack of neutrophils recruitment is not due to a problem in KC and MIP-2 produced in the peritoneum because it is comparable with WT mice. Furthermore, the number of circulating neutrophils is comparable with the WT. All these results together point to an essential function for mast cells in the process of neutrophil recruitment. There is evidence in the literature suggesting a cooperative role between adhesion through β2 integrins and TLR4 via the MyD88 adaptor. Because we found a different requirement for MyD88 and TRIF in the synthesis of KC and MIP-2, we investigated the role of adhesion in this process. We found that KC, but not MIP-2, requires the adhesion step for its synthesis upon LPS stimulation. Furthermore, the lack of all of the four β2 integrin members, showed a reduced ability in the production of KC, whereas MIP-2 was not affected. Moreover to identify the member of the β2 integrin family involved in this adhesion step, we made use of the single knock-out mice, called LFA-1-/- , Mac-1-/- , CD11c-/-, and CD11d-/-, we showed that the absence of CD11a, did not cause any effect on the synthesis of either KC or MIP-2, whereas the lack of CD11b showed a reduced ability to produce KC but not problem for the production of MIP-2. The other two members CD11c and CD11d are under investigation. These results suggest different regulatory processes for KC and MIP-2 synthesis, not only from the point of view of the TLR- adaptor molecules involved, but also from the requirement for adhesion.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | The role of Macrophages and Mast cells in Neutrophil recruitment |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10144048 |
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