Being the most aggressive human skin cancers, melanoma has always occurred with a poor prognosis. It is responsible for 80% of skin cancer. Treatments for melanoma include surgical removal, and radio- and chemotherapy, which are not effective toward the advanced stages of the disease. Only three chemotherapy drugs, hydroxyurea, dacarbazine and interleukin-2, are currently approved by the Food and Drug Administration for metastatic melanoma, and the therapeutic response rate is only 5%-20%. Thus, there is a need for novel therapies that can target tumours, especially when the tumour cells become refractory to chemotherapy. ELR–CXC chemokines with a Glutamine – leucine – arginine (ELR) motif (for example, interleukin-8/CXCL8) are able to chemoattract neutrophils during inflammation responses via their receptors, CXCR1 and CXCR2, which can be expressed by human malignant tumour cells, keratinocytes, endothelial cells, and fibroblasts. CXCR1 and CXCR2 play very important roles in melanoma by promoting tumour cell proliferation, angiogenesis, and metastasis. They are also involved in the tumour’s becoming refractory to chemotherapy. An ELR–CXC chemokine antagonist developed by our lab, CXCL8(3-72)K11R/G31P (G31P), effectively blocks CXCR1- and CXCR2- induced inflammatory responses, and further antagonizes the functions of heterologous G protein–coupled receptor’s (GPCR). The tumour–associated GPCRs, along with ELR–CXC chemokines and their receptors, have been shown to simultaneously increase in several tumour models, including melanoma. Thus, given the knowledge regarding the importance of the ELR-CXC chemokines and heterologous GPCRs’ in melanoma and G31P’s ability to block ELR-CXC chemokines and at least some heterologous GPCRs, we hypothesize that G31P is a viable therapeutic option for melanoma cancers by virtue of its success in blocking tumour progression in mouse models. Our data indicated that ELR-CXC chemokine antagonism with G31P had no significant impact on tumour growth or tumour-induced angiogenesis, which suggested that blockade of CXCR1 and CXCR2 alone was insufficient to block tumour development in this melanoma mouse model. Evaluation of other tumour-related parameters (e.g., angiogenic patterns and stress protein level) are recommended as a means of determining what parameters beyond CXCR1 and CXCR2 signaling are important in tumour progression in our matrigel model.

Thesis (Ph. D.)--University of Oklahoma. / Vita. Bibliography: leaves 141-165.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 98-130) Also available in print.

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on Mar. 2, 2009). Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Endothelial cells form the inner lining of blood and lymphatic vessels. In mice only tumours of the blood vessel endothelium (haemangiomas) have been thus far reported. In the first part of this thesis is described a highly reproducible method for the induction of benign tumours of the lymphatic endothelial cells (lymphangioma) in mice, by intraperitoneal injection of incomplete Freund's adjuvant. Different criteria have been used in order to establish the nature of the induced lesion. Morphological and histophatological studies of the tumour developed in the peritoneal cavity revealed the presence of cells at various levels of vascular development. Expression of the endothelial markers PECAM/CD31, ICAM-l/CD54, ICAM 2/CDI02 as well as the vascular endothelial growth factor (VEGF) receptor Flk-I, the endothelial cell specific receptors Tie-I, Tie-2, and the lymphatic endothelial specific Flt-4 was identified. When the lesion was induced in ~- galactosidase knock-in Flt-4 +/- mice, the tumour endothelia could be stained blue in a number of tumour cells. Tumour-derived cells were propagated in vitro where they spontaneously differentiated, forming vessel-like structures. This evidence leads to the conclusion that this is the first experimental protocol for the induction of a lymphatic endothelium hyperplasia in mice peritoneum. The second part of this thesis describes the use of this model system to investigate the profile of chemokine expression in murine lymphangiomas and in lymphangioma-derived lymphatic endothelial primary cultures. Chemokines are a superfamily of small, secreted chemoattractant molecules that plays a key role in the immune cell trafficking. Although production of chemokines by vascular endothelial cells has been extensively documented, there is much less information regarding the lymphatic endothelium. The reported results are the first detailed analysis of chemokine production by lymphatic endothelial cells. Chemokines belonging to all three subfamilies (CXC, CC and C), were found to be expressed in lymphangioma. Among these molecules is remarkable the identification of CIO, a molecule previously identified only in the bone marrow. The molecular as well as functional assays performed provide an indication of the signals that mediate the recruitment of leukocytes into lymphatic vessels.

The atypical chemokine receptor-2 (ACKR2) is a chemokine decoy receptor that recognises pro-inflammatory CC chemokines. Many studies showed up-regulated inflammation and delayed resolution of inflammatory responses in ACKR2-/- mice. Furthermore, in the absence of ACKR2, lymphatic endothelial cells (LEC) fail to regulate the expression of pro-inflammatory CC chemokines leading to the excessive peri-lymphatic accumulation of leukocytes. As a result, the migration of antigen presenting cells (APC) through lymphatic vessels may be impaired due to lymphatic congestion. In addition, ACKR2 was shown to regulate lymphatic vessel density in the embryonic skin by regulating the proximity of pro-lymphangiogenic macrophages to LEC. Therefore, to address the role of ACKR2 and its significance in 1) APC migration and 2) inflammation-associated lymphangiogenesis, three models of ocular inflammation were used in this work, experimental autoimmune uveoretinitis (EAU), corneal graft rejection and herpes simplex keratitis (HSK). With regard to APC migration, in both EAU and HSK models, this process was fine-tuned to the level of disease in that migration was significantly compromised in ACKR2-/- mice during severe inflammation, but not under mild inflammatory conditions. Furthermore, while the severity of EAU was associated with the migration of APC, this was not so in HSK. In order to study lymphangiogenesis, the transparent avascular cornea provides a good substrate and corneal lymphangiogenesis was studied using both corneal graft model and HSK model. I found that lymphatic vessel density was increased in ACKR2-/- mice compared to wild type mice in corneal graft induced lymphangiogenesis (macrophage mediated), but not altered during early stages of HSK associated lymphangiogenesis (non-macrophage mediated). These findings confirmed that ACKR2 indirectly regulates the process of lymphangiogenesis in a macrophage dependent manner. Although the severity of HSK correlated with the level of lymphangiogenesis, this does not seem to correlate with viral load but rather associated with inflammatory infiltrations in the cornea.

published_or_final_version / Pharmacology / Doctoral / Doctor of Philosophy

No description available.

We used Experimental Autoimmune Uveoretinitis (EAU) as a model system to investigate the involvement of CCR2 and CX₃CR1 in regulating the trafficking and function of monocytes and microglia in an autoimmune context. METHODS: W.T. or CX₃CR1^GFP/GFPmonocytes were adoptively transferred into mice with EAU.  At 48 hours post transfer their phenotype was examined by flow cytometry and monocytes trafficking to the retina was imaged using Scanning Laser Ophthalmoscopy. An anti-CCR2 antibody (MC21) or antagonist (JE(9-76)) was used to examine the effect of CCR2 blockade on W.T. monocytes trafficking.  Infiltration of monocytes into the inflamed retina and activation of retinal microglia were examined by confocal microscopy on retinal flatmounts from W.T. and CX₃CR1^GFP/GFP mice and immunohistochemistry on cryosections from eyes. RESULTS: CCR2 increased on W.T. monocytes at 48 hours post transfer and at 24 hours on CX₃CR1^GFP/GFP monocytes.  However, blocking CCR2 by either method did not reduce the number of W.T. monocytes rolling along retinal vessels and infiltrating the retina.  Lack of CX₃CR1 did not alter microglial activation but infiltrating monocytes lacking the receptor could not migrate through the retina and clustered around vessels. CONCLSIONS: CCR2 may  not always be needed for recruitment into an inflammatory site.  CX₃CR1 has a role in neuroprotection in the retina by enhancing the migratory ability and distribution of infiltrating monocytes within inflamed tissue.  This work stresses the importance for careful dissection of the chemokine receptors’ mechanism of action before therapeutic possibilities are explored.