Mechanisms and immune consequences of Neutrophil trafficking via lymphatics

Following injury and infection, the first line of defence is provided by white blood cells termed neutrophils that respond to the presence of invading microbes by emigrating in large numbers from the blood circulation to the tissues. Here, these short-lived professional killers recognize general features shared by all pathogens and kill them by releasing an arsenal of toxic products. The next line of defence is provided by lymphocytes, which respond to the invading microbes by multiplying in the lymph nodes and generating protective antibodies and pathogen specific T cells. Normally this adaptive response relies on the transport of pathogens to the nodes via lymphatic vessels by means of professional sentinels termed dendritic cells which present the pathogen in the appropriate way for lymphocyte activation. Until recently it was thought that the neutrophil and lymphocyte arms of the response (termed innate and adaptive immunity) were essentially independent. However it is now becoming clear that they are not; neutrophils not only act as sentinels carrying pathogens to the lymph nodes but can also communicate with dendritic cells to manipulate antibody and T cell responses. We will investigate how neutrophils enter the lymphatic system and migrate to the lymph nodes, which cells they interact with when they get there and to what extent they can alter conventional immune responses generated by dendritic cells.

Emerging evidence indicates that neutrophils - the key phagocytes of the innate immune system can also modify and even initiate adaptive immune responses either by cross-talk with antigen presenting cells or by directly functioning as antigen presenting cells themselves. These properties are critical in generating protective immune responses to important intracellular pathogens such as mycobacteria and Toxoplasma and protozoal parasites such as Leishmania where neutrophils induce Th1 responses that promote effective killing by macrophages. The capacity of neutrophils to modify such responses requires their exit from peripheral tissues and trafficking to the lymph nodes via afferent lymphatics. Just recently such trafficking has been observed in vivo using fluorescence microscopic imaging. However, the molecular mechanisms orchestrating the entry of neutrophils to lymphatics including the adhesion receptors chemokines and proteinases involved have yet to be elucidated. Moreover, the interactions of neutrophils with resident lymph node populations, their influence on ongoing immune responses and their fate within the lymph nodes are also obscure. This grant proposal seeks to investigate each of these areas using a combination of in vitro transmigration assays with primary lymphatic endothelial cells and function blocking antibodies together with in vivo assays measuring lymph node trafficking of fluorescent BCG treated neutrophils in appropriate transgenic and gene knockout animals by flow cytometry and confocal microscopy. In addition we will assess the cytokine profile of neutrophils having trafficked to the lymph nodes and investigate whether the presence of such neutrophils can influence or override the polarity of neighbouring responses in the same location. This is a straightforward proposal designed to understand the basic biology of a potential rate-limiting step connecting innate and adaptive immunity. The results of this research could well be exploited for the optimization of neutrophil-based vaccination strategies or for reshaping protective immune responses.