The role of cybr during leukocyte recruitment and angiogenesis in vivo

Abstract

Trafficking, adhesion and trans-migration are important properties of leukocytes which allow them to perform their immune function. However, the molecular mechanism that allows the extracellular signal to be transduced intracellularly to control leukocyte behaviour or coordinate their intercellular response is not well understood. Cytohesin binder and regulator (Cybr) whose physiological function remains unknown is a scaffold protein containing various protein-protein interaction domains, and is exclusively expressed in hematopoietic cells. Studies in vitro have shown that Cybr can regulate LFA-1 which is a key molecule in leukocyte adhesion and trans-migration. Cybr can also bind cytohesin-1 which affects cell spreading, vesicular transport and actin cytoskeleton remodelling during leukocyte migration. In order to shed light on the immunoregulatory role of Cybr in vivo, we investigated the effect of Cybr deletion on leukocyte trafficking, adhesion, transmigration and angiogenesis in the normal vasculature, in addition to during an aseptic inflammatory response, wound healing and tumour progression.

In the present study, Cybr-/- mice have normal viability, fertility and immunocompetence. Apart from reduction of cell numbers in lymph nodes and Peyer‟s patches and reduction of intravascular leukocyte velocity, Cybr-/- mice have histologically normal development of immune organs/tissues. In an aseptic peritonitis model, Cybr-/- have fewer peritoneal exudate cells than Cybr+/+ littermates at 6 hours post-thioglycolate i.p. injection, as well as fewer circulating leukocytes leaving the bloodstream at 3 days post-thioglycolate i.p. injection. During local inflammatory response to LPS (through a micro-instillation in the panniculus carnosus muscle in the DSC), the numbers of trans-migrated leukocytes are significantly fewer in Cybr-/- compared to Cybr+/+.

Coupling intravital microscopy with an unbiased stereological analysis, the spatio-temporal distribution of morphological parameters (such as capillary branch points, density, length, diameter and anisotropy index) have been quantitatively assessed in both pre-existing and newly formed capillaries within the heat-induced injured panniculus carnosus muscle of C57Bl/6J mice, in addition to leukocyte trafficking, adhesion and trans-migration. Cybr-/- does not increase the non-perfused area post wounding. Capillary parameters within the regenerating vascular plexus are not significantly different to Cybr+/+ littermates, with the exception of a decrease in capillary diameters in Cybr-/- mice. The time-course of wound healing in Cybr-/- mice did not change in comparison to Cybr+/+. However, the numbers of adherent (1 day post injury) and trans-migrated (1−3 day post injury) leukocytes are significantly lower in Cybr-/- mice.

The area of tumour growth and various blood vessel parameters are not significantly different between Cybr+/+ and Cybr-/- littermates during tumour progression. However, Cybr-/- mice have fewer circulating leukocytes exiting the blood stream at 22 days post-MCA38 cell inoculation. Moreover, in the model of implanted tumour fragment in the DSC, the leukocyte velocity in the tumour bloodstream is significantly lower in Cybr-/- mice than in Cybr+/+ littermates. In addition, the numbers of adherent and trans-migrated leukocytes in the periphery of tumours were much lower in Cybr -/- mice than in Cybr+/+ mice over the time period of observation.

Taken together, Cybr is an importment intracellular factor combination with Cytohesin-1 to control leukocyte motility through regulating actin cytoskeletal rearrangement and dynamic pseudopod protrusion by PI3K-Cytohesin-1/Cybr-Rac signalling transduction pathway. Cybr deletion not only impedes lymphocytes access into peripheral lymphatic system but also affects leukocyte recruitment in pathological situation, particularly in acute inflammation, in addition to wound healing and tumour progression.

Thesis is embargoed until 31st October 2013

Date of Award	Dec 2011
Original language	English
Sponsors	Vice Chancellor's Research Scholarship (VCRS)
Supervisor	Christopher Mitchell (Supervisor)