The inflammatory response is a complex condition that can be visualised in terms of leukocyte activity at the microcirculatory level. Recent data show that leukocyte recruitment can be prevented by both the endogenous protein annexin A1 (AnxA1) and activation of its receptor FPR2/ALX (murine orthologue: Fpr2), thus aiding resolution of the inflammation. Furthermore, AnxA1 is sensitive to oestrogen (E2) and so is a candidate mediator of the sex differences seen in many inflammatory diseases. Using intravital microscopy to quantify leukocyte-endothelial cell interactions in the murine mesentery in real-time and in vivo, we aimed to establish a model of the systemic inflammatory response and determine the involvement of the AnxA1-Fpr2 system in effecting anti-inflammation. Lipopolysaccharide (LPS; 10 g/mouse I.P.) induced leukocyte rolling, adhesion and emigration, and plasma protein extravasation, observable at 2, 6 and 24 h after injection in mesenteric venules. At 2 h, leukocyte infiltration was also seen in the liver and plasma concentrations of TNF- , IL-6 and IL-10 were raised, indicating a systemic response. When given 20 min into, or at the end of, a 2 h LPS challenge, the Fpr2 ligands AnxA1Ac2-26 and LXA4 reduced LPS-induced adhesion, an effect that was blocked by both antagonists that were either pan-FPR (Boc2) or Fpr2-specific (WRW4). Our model also showed sexual dimorphisms, in that LPS-induced leukocyte-endothelial cell interactions and plasma TNF- and IL-10 concentrations were heightened in females. Ovariectomy revealed a particular role for ovarian hormones besides E2 in the manifestation of these differences, and the use of AnxA1-/- mice suggests that AnxA1 reduces the animals’ sensitivity to E2. These data suggest firstly that FPR2/ALX presents an attractive target for novel anti-inflammatory therapeutics, and secondly that ovarian function is important in the regulation of inflammation.

Mitogen Activated Protein 3 Kinase 8 (MAP3K8) is a member of the serine/threonine protein kinase family and has been demonstrated to be involved in the mitogen activated protein kinase (MAPK) pathway. The latter pathway plays an important role in many aspects of the immune mediated inflammatory response. Inhibition of MAP3K8 in primary human cell types decreases the production of TNF-α and other pro-inflammatory mediators during inflammatory events. Pharmacologic inhibition of MAP3K8 has been shown to be an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases. Inhibition of MAP3K8 may therefore potentially be a new therapeutic strategy for airway inflammation. Investigation of the role of MAP3K8 in regulation of the inflammatory response was conducted using epithelial cell lines. A MAP3K8 gene knockdown experiment using small interfering RNA (siRNA) was carried out to establish the effect of MAP3K8 on the inflammatory response following IL-1β stimulation. In addition the effect of MAP3K8 gene knockdown on the therapeutic outcome of dexamethasone (Dex) was investigated. ELISAs were used to establish whether knockdown of MAP3K8 resulted in inhibition of IL-1β-induced IL-6, IL-8 and RANTES. The effect of treatment with Dex in combination with MAP3K8 gene silencing on ERK and MEK phosphorylation was determined by Western Blotting. The impact of MAP3K8 gene silencing on global gene expression was also assessed using Affymetrix Human Gene 1.1ST arrays. Differential expression and network analyses were performed on the data generated. A significant rise in MAP3K8 gene expression was found 2 hours after IL-1β stimulation in both A549 and normal human bronchial epithelial (NHBE) cells. In addition, siRNA against MAP3K8 resulted in approximately 40% inhibition of IL-6, IL-8 and RANTES expression after IL-1β stimulation. Furthermore, MAP3K8 gene silencing enhanced by approximately 40% the decrease in IL-8 and RANTES release seen at different concentrations of Dex. The combination of MAP3K8 gene silencing and Dex also resulted in a greater inhibition of phosphorylated ERK compared to that seen with Dex alone. Data from global gene expression arrays revealed that the MAP3K8 regulated inflammatory response was predominantly involved in the ERK/MAPK and SAPK/JNK pathways but not the p38 MAPK pathway. Among the genes regulated by MAP3K8, it is interesting to note that IL-6, MMP-1, MMP-3, MMP-12 and CCL20 have previously been reported to be linked with airway diseases, such as COPD and asthma. Furthermore, MAP3K8 regulated the AP-1 transcription factor up-regulation of IL-1β-induced IL-6 and MMP-1 through the ERK1/2 and JNK signaling pathways. MAP3K8 however appears to regulate NF-kB activation through IKKα/β activation independent of the MAPK pathway regulated by itself. In conclusion, MAP3K8 plays a key role in the inflammatory cytokine response post IL-1β stimulation. MAP3K8 gene silencing by siRNA not only suppresses these key inflammatory cytokines but also enhances the therapeutic effect of steroids. MAP3K8 inhibitors might therefore provide a new therapeutic strategy for airway inflammation.

Stroke is a leading cause of death in the UK however there is only one current treatment, intravenous thrombolysis via administration of tissue plasminogen activator (tPA). The paucity of available treatments is not simply due to a lack of research as there have been many successful preclinical studies that have failed to translate to success in the clinic. The Stroke Therapy Academic Industry Roundtable (STAIR) have published several articles that outline a number of possible reasons for this lack of translation between pre-clinical and clinical research. One major reason highlighted is the failure to consider clinically relevant co-morbidities in preclinical studies. Therefore the key objectives of this thesis were to; (1) determine whether central nervous system (CNS) changes occur in both animal models and patients with risk factors for stroke, (2) determine how neuroinflammatory changes induced in response to peripheral atherosclerosis are affected by the deletion of IL-1 signalling (3) establish whether a peripheral infection in atherosclerotic mice induces any cerebral ischaemic events and to determine the inflammatory response in both the periphery and the brain. Neuroinflammation was assessed in patients at risk of stroke, the co-morbid JCR-LA rat and the atherosclerotic ApoE-/- mouse. PET imaging revealed microglial activation in the brain of JCR-LA (corpulent) rats and patients at risk of stroke. Microglial activation, vascular activation, leukocyte infiltration and focal lipid deposition were observed in the brains of atherosclerotic ApoE-/- mice. These findings show brain inflammation occurs in animals, and tentatively in humans, harbouring risk factors for stroke. Neuroinflammation was assessed in ApoE-/- mice crossed with IL-1 type 1 receptor deficient mice (ApoE-/-/IL-1R1-/-) and both neuroinflammation and systemic atherosclerosiswas assessed in ApoE-/- mice treated with an anti-IL-1β antibody. ApoE-/- mice fed Paigen or Western diet develop vascular inflammation, microglial activation and leukocyte recruitment in the brain, which are absent in ApoE-/-/IL-1R1-/-. Systemic neutralisation of IL-1β with an anti-IL-1β antibody reversed aortic plaque formation and reduced inflammatory cytokin eexpression in peripheral organs. In the brain, vascular inflammation and leukocyte infiltration into the choroid plexus were reversed by IL-1β blockade in animals fed a Paigen diet. Theseresults indicate that IL-1 is a key driver of systemically-mediated cerebrovascularinflammation and that interventions against IL-1β could be therapeutically useful in atherosclerosis, dementia or stroke. ApoE-/- and C57BLJ/6 mice infected with Streptococcus (S.) pneumoniae were assessed for spontaneous stroke events, neuroinflammation and systemic inflammatory responses to infection. Infection with S. pneumoniae in atherosclerotic mice did not induce spontaneous stroke. Raised levels of vascular activation were observed in all mice and an alteration in leukocyte accumulation in infected atherosclerotic ApoE-/- mice. T cell, B cell and granulocyte responses to both diet and infection were found to differ between ApoE-/-mice and control, C57BLJ/6, mice. Levels of the proinflammatory cytokines IL-1, IL-6 andIL-17 were also increased in response to S. pneumoniae infection in plasma, spleen and liver. These data indicate that atherosclerosis and S. pneumoniae infections not only have systemic inflammatory mechanisms but also effects that extend to the brain. Overall these findings demonstrate that risk factors for stroke cause alterations in inflammation in the brain. Therefore modelling of these risk factors is essential in future preclinical stroke research if new treatments for stroke are to be identified.

Glucocorticoids are used therapeutically for their anti-inflammatory and immunosuppressive actions. One of the ways they produce their potent effects is via regulation of the anti-inflammatory protein Annexin I (ANX-AI; 37 kDa protein). Whereas there is sufficient information on the pharmacological properties of ANX-Al and its bioactive peptides, as they have been studied in several models of experimental inflammation, much less is known about the functions of the endogenousp rotein. In this thesis I have had accesst o the recently generatedA NX-Al knockout (ANX-AI'4-) mice with the scope of elucidating the role of the endogenous mediator on the process of leukocyte (mainly neutrophil) recruitment. Two main models were used to assess leukocyte recruitment: a) a flow cytometry protocol was implemented to quantify the extent and the nature of the cellular influx into the inflamed peritoneal cavity and b) intravital microscopy of the inflamed cremaster muscle was used to identify which step of leukocyte recruitment was potentially altered. Overall, ANX-A14- mice were found to have a heightened sensitivity to inflammation. In the peritonitis model, ANX-AI4- mice displayed a higher degree of neutrophil recruitment into the peritoneal cavity, however this occurred in a stimulus specific manner. The functional role of the protein in this model was confirmed by using transgenic mice that over-express ANX-Al. These mice were shown to have the opposite response to the ANX-A14- mice, since they had a reduced influx of neutrophils into the peritoneal cavity in response to zymosan. Intravital microscopy studies i pinpointed the emigration phase of leukocyte recruitment to be significantly altered in ANX-AI-1- mice, and this defect was seen with application of different inflammatory agents. In conclusion, this thesis has demonstrated, for the first time, that there are altered leukocyte-endothelium interactions in ANX-Al-- mice, and these results indicate a pivotal role for endogenous ANX-Al in the process of neutrophil recruitment that is fundamental to the host inflammatory response.

Inflammation is a consequence of the activation of innate immunity and represents an important component of several pathological conditions, including not only the complication of infections but also sterile and autoimmune diseases. An early event in inflammation is represented by the production of proinflammatory cytokines and both their production and action have often been associated to oxidative stress. The redox status of the cell is therefore a key regulator of inflammation and glutathionylation (formation of mixed disulphides between cysteine residues of proteins and glutathione) is considered an important mechanism of this regulation. While most of the studies in the past focused on glutathionylation of intracellular proteins and transcription factors, the main goal of this project was to verify whether glutathionylated proteins are released by inflammatory cells and if these have a biological role. Using redox proteomics, we identified several proteins in the supernatants from Raw 264.7 cells (murine macrophages) stimulated with bacterial lipopolysaccharide (LPS). Among the identified proteins, we focused our attention on Peroxiredoxin 2 (Prx2), an antioxidant enzyme involved in cells protection against oxidative stress by removing H2O2. Released Prx2 was also detected in supernatant from human peripheral blood mononuclear cells (PBMC) and human macrophages. Prx2 levels were also increased in the serum of LPS-treated mice. We could confirm that Prx2 is released in the glutathionylated form. Moreover it was observed that the intracellular level of glutathione affects Prx2 release suggesting a role for glutathionylation in the mechanism of its release. The second part of the project was to verify whether released glutathionylated proteins may act as mediators of inflammation. To this purpose, the possible inflammatory role of released Prx2 was studied. The results showed that extracellular Prx2 induced an increase of TNF-α production in Raw 264.7 cells and in human macrophages. In conclusion, Prx2 is released during inflammation in a redox-dependent manner, in addition to its well-known intracellular role as enzyme, Prx2, in its released form, can also play a role in inflammatory response.

Females are relatively protected from inflammatory diseases, particularly conditions that are characterised by excessive tissue infiltration of neutrophils (PMNs), such as ischaemia/reperfusion (I/R) injury. Therefore, understanding sex-differences is very important particularly for appropriate treatment of inflammatory disorders in men and women. Unfortunately, efforts to exploit sex-differences therapeutically have been unsuccessful since the precise mechanisms that confer the protective advantage in females over males are unclear. Many fundamental aspects of the nature of sex-differences have not been investigated, particularly in the regulation of PMN mobilisation from the bone marrow during acute inflammation. The aim of this thesis is to determine the nature and mechanism of leukocyte activation and recruitment in males and females, particularly in I/R. This thesis demonstrates for the first time that regulation of PMN mobilisation during acute inflammation is distinct in females. In comparison to males, females demonstrate reduced expression of mediators that cause the release of PMNs, including GCSF, CXCR2 and CXCL5, and increased expression of CXCR4 and CXCL12, which mediate PMN retention in the bone marrow. Reduced granulopoiesis, PMN mobilisation and recruitment into tissues in response to inflammogens protect females from collateral damage incurred by PMN-derived mediators that contribute to tissue injury and loss of function. This thesis has also revealed a novel, and possibly predominant, role for the ELR+ CXC chemokine CXCL5 in mobilisation of tissue-damaging PMNs from the bone marrow, whereby CXCL5 production, PMN mobilisation and tissue infiltration was profoundly greater in males during acute inflammation. CXCL5 appears to stimulate PMN mobilisation by 1) upregulating CXCR2 v expression on bone marrow cells and simultaneously downregulating CXCR4 expression; 2) inducing its own expression; 3) stimulating GCSF expression; and 4) increasing PMN expression of β2 integrin. Thus, the thesis proposes that reduced CXCL5 expression, and increased CXCR4/CXCL12 expression, in females during acute inflammation may be a novel mechanism underlying the protection against tissue injury. The fact that these sex-differences were apparent in different species (rat, mouse), tissues (mesenteric, lung, kidney, heart), in response to different stimuli (mesenteric, renal, myocardial I/R and carrageenan-induced pleurisy) and shown both in vivo and in vitro, suggests that this is a fundamental, and more generalised, phenomenon in males and females following acute inflammation. The inherent differences between the sexes have important clinical implications in that they demonstrate the need of considering sex-differences in research. This thesis demonstrates that sex-differences must be taken into account when developing such therapies for specific inflammatory diseases such as I/R injury as there are major differences in the temporal profile of chemokines and the extent of PMN infiltration.

D6 is a novel chemokine receptor, homologous to other members of the CC-chemokine receptor family, which recognises a number of inflammatory CC-chemokines with high affinity. The aims of this thesis were to further our understanding of the biology of D6, chiefly through characterisation of immune responses in D6-deficient animals. Firstly, as described in Chapter 3, I analysed the cellular composition of lymphoid tissues of D6 KO mice. These studies revealed higher proportions of CD11c+ and F4/80+ cells in the D6 KO spleen compared with WT controls, suggesting that increased accumulation of myeloid lineage cells was occurring at this site. In Chapter 4, I examined the role of D6 in myeloid cell responses, by comparing monocyte recruitment to the inflamed peritoneum and dendritic cell development from bone-marrow (BM) cultures. I found that while the accumulation of inflammatory monocytes/macrophages appeared quantitatively similar in WT and D6 KO animals, D6 KO cells expressed greater levels of CD11c, suggesting preferential accumulation of DC-like cells in the inflamed peritoneum. D6 may influence the development and function of myeloid lineage cells. As D6 is expressed at high levels in the small and large intestine, I next investigated both tolerogenic and inflammatory intestinal responses in D6 KO animals. As detailed in Chapter 5 of this thesis, the induction of oral tolerance in response to a high dose feeding protocol was normal in D6 KO mice. However, D6 KO mice showed increased resistance to experimental colitis. As described in Chapter 6, various D6 KO populations displayed differential chemokine receptor profiles compared with their WT counterparts. The results suggest a role for D6 in the normal development of leukocytes populations, with absence of this atypical receptor leading to the dysregulated expression of other chemokine receptors. Taken together, my data suggest that the biological functions of D6 may be more complicated than previously appreciated. Indeed, I found no evidence for a decoy role of D6 in vivo, but D6-deficient animals were characterised by altered leukocyte development, aberrant chemokine receptor expression and increased resistance to experimental colitis induction.

Some mounted ill. / Bibliography: leaves 197-226 / xiv, 226, [137] leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1987

Bibliography: leaves 205-233 / xix, 234 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.S.)--University of Adelaide, 1985

Nociceptin/OrphaninFQ, N/OFQ, and its receptor NOP represent a non-opioid branch of the opioid family. There is growing interest in the involvement of this system during inflammation and sepsis as it is present in immune cells and modifies immunocyte function. Systemic N/OFQ increased mortality in an animal model of sepsis and there is limited evidence for increased plasma N/OFQ in patients with sepsis who died compared to those who survived. This thesis explores changes in the expression of NOP and ppN/OFQ-mRNA by polymorphs (PMN) and of N/OFQ peptide in plasma during inflammation and sepsis. A further aim was to investigate the relationship between the N/OFQ system with physiological and biochemical indicators of severity of disease. Forty patients undergoing cardiopulmonary bypass (CPB) and 49-patients with sepsis in the Intensive Care Unit (ICU) were recruited into 2-studies. In the CPB study we observed a 57% reduction of NOP-mRNA and a 95% reduction of ppN/OFQ-mRNA expression in PMN. Plasma N/OFQ concentrations increased by over 30%. Higher plasma N/OFQ was associated with lower NOP-mRNA. These changes were related to prolonged aortic cross clamp time. In patients with sepsis there was an 85% reduction of ppN/OFQ-mRNA expression compared to a sample taken after recovery from sepsis. Lower expression of ppN/OFQ-mRNA was associated with increased inotropic support and lactate concentrations on the first day of sepsis. Our data did not show any differences amongst survivors and non-survivors. During inflammation(CPB) and sepsis there was reduced expression of NOP and ppN/OFQ-mRNA with an inverse relationship between plasma N/OFQ(CPB study) and NOP-mRNA expression, suggestive of a possible feedback mechanism. Based on the current evidence (this thesis and literature) we suggest that N/OFQ could contribute to the complex pathophysiological process occurring during inflammation and sepsis and warrant further study.