Inflammatory bone diseases like rheumatoid arthritis and periodontal disease lead to increased bone loss in the inflamed areas. The multinucleated bone resorbing cells, the osteoclasts, present in these diseases are larger than normal, and these larger osteoclasts (10+ nuclei) resorb more bone and more often than smaller osteoclasts (2-5 nuclei). Thus, the focus of this thesis was to determine if there are differences in mechanisms of osteoclast activation between large and small osteoclasts. Experiments using authentic rabbit osteoclasts and RAW 264.7-derived osteoclasts revealed differences in the expression of a number of activating factors; with large osteoclasts expressing higher levels of activating receptors (RANK, IL-1RI, TNFR1 and integrins αv and β3), as well as enzymes involved in cellular resorption, while small osteoclasts expressed higher levels of an alleged fusion receptor and the inhibitory receptor, IL-1RII. Further studies revealed that large osteoclasts more readily responded to stimulation by IL-1 compared to small osteoclasts and at lower concentrations suggesting this is a result of their higher expression of activating receptors. Differences in responses to the IL-1 isoforms, IL-1α and IL-1β, were also seen in large osteoclasts: IL-1α generated more large osteoclasts over the course of differentiation, while IL-1β induced changes in cell morphology and in the induction of integrin β3 phosphorylation. These observations suggested that differences in osteoclast responses are induced by IL-1α and IL-1β and it led to the hypothesis that there are differences in signaling between large and small osteoclasts. To elucidate differences in signaling mechanisms a signaling pathway microarray was used which revealed higher expression of Vegfa in large compared to small osteoclasts. Osteoclast differentiation with RANKL increased Vegfa gene expression in a time-dependent manner and VEGF-A secretion was elevated in populations enriched for large osteoclasts. Furthermore, mechanistic studies with inhibitors of transcription factors involved in differentiation revealed that RANKL-mediated Vegfa expression in large osteoclasts was regulated by the NF-κB pathway via induction of Hif1α. These results support the hypothesis that signaling differences exist between large and small osteoclasts and implicates VEGF-A in osteoclast hyperactivity in inflammatory conditions.

Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (Ph. D. in Microbiology and Immunology)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

The pro-inflammatory cytokine interleukin 1 (IL-1) is involved in numerous physiological and pathological processes. It contributes to thermoregulation, sleep, feeding behaviour and notably to the exacerbation of non-communicable disorders such as cancer, heart disease, stroke and epilepsy, which are the greatest cause of mortality worldwide. Given this important role, IL-1 is tightly regulated, with regulation mechanisms present at the level of its synthesis, activation and receptor engagement. However, when studying IL-1 in vitro, little notice is taken of the disease microenvironment in which it acts. Acidosis is a hallmark of disease, often due to poor perfusion resulting in a shift to anaerobic respiration, a build-up of lactic acid and poor clearance of CO2. Additionally, highly active infiltrating immune cells favour anaerobic respiration and can contribute to this local acidosis. This thesis utilised primary cell cultures, cell lines and reporter cells to explore the mechanisms of IL-1 signalling under disease-relevant acidic conditions. Subsequently, a murine seizure model was developed to further explore IL-1 signalling in disease conditions in vivo. This work demonstrated that acidic pH itself did not induce IL-1β release, however, it did promote release of minimally active 20 kDa IL-1β in response to damage associated molecular patterns (DAMPs) such as ATP, monosodium urate crystals or calcium pyrophosphate dihydrate crystals. The cleavage of pro-IL-1β into 20 kDa IL-1β was mediated by cathepsin D and was also induced on addition of lactic acid to the culture media. This 20 kDa IL-1β was not further cleaved to the active mature 17 kDa IL-1β thus its production limits the spread of inflammation. The intranasal administration of kainic acid induces seizures in C57Bl/6J mice, however, IL-1β was not observed acutely in this model thus the presence of 20 kDa IL-1β in vivo was not confirmed. In recent years, the contribution of IL-1 to disease has become well established. However, despite successes in the development of novel therapeutics targeted at blocking IL-1 activity, such as anakinra, canakinumab or rilonacept to treat cryopyrin associated periodic syndromes, a number of studies have demonstrated poor efficacy and only minor improvements in patients when targeting IL-1. Thus further knowledge of the mechanisms of IL-1 signalling in disease is required to understand this system and develop improved novel therapeutics.

With increasing age, aspects of the innate and adaptive immune systems show functional decline. In the skin this is associated with an increased incidence of epidermal malignancies and infections, a decreased incidence of contact allergy, and the development of autoimmunity. The mechanisms underlying these clinical effects in aged skin are poorly understood. Langerhans’ cells (LCs), which are members of the wider family of dendritic cells (DCs), reside in the epidermis where they act as sentinels of the immune system by processing and presenting antigen and inducing T cell responses. Previous investigations have suggested that the number of epidermal LCs is reduced, and that the motility of LCs is impaired in aged skin. A series of investigations was performed to characterise the mechanistic basis for the reduced frequency and restricted mobility of epidermal LCs in the skin of the elderly. Initially LC-like cells were cultured from circulating monocyte precursors and characterised using flow cytometry. The ability of precursors to differentiate into LC-like cells was not impaired in the aged; furthermore there were no age-associated differences in expression of markers of LC activation at baseline or upon stimulation. The phenotype of epidermal LCs was assessed using flow cytometric analysis of epidermal cell suspensions and did not appear altered in aged individuals. In addition, using the same techniques with dermal cell suspensions the dermal DC population was not altered with age. Langerhans’ cell migration from epidermal explants prepared from the skin of aged individuals was impaired but could be restored with exogenous interleukin (IL)-1β. There was no age-related reduction in the epidermal levels of IL-1β or caspase-1 (IL-1β converting enzyme which converts pro-IL-1β to the active form) or the expression of the IL-1 receptor I (IL-1RI), to account for this observation. However, the amount of IL-1 receptor antagonist was reduced in aged skin suggesting a change in the overall local cytokine balance. Based on previous reports that topical retinoic acid (RA) can increase cutaneous IL-1 production, a 4-day patch test assay was performed using 0.025% all-trans RA cream to explore whether this could restore LC migration in the aged. There was no effect on LC migration from epidermal explants prepared after treatment with RA in the aged.These data demonstrate that changes in LC function in the elderly may not be associated with changes in systemic DC biology. Age related changes in the cutaneous microenvironment are likely to be more relevant.

Neuroinflammation is an important contributor to the pathogenesis of many neurological diseases. A key component of the innate immune response in the central nervous system is the migration of neutrophils into the brain parenchyma, where they exacerbate neuronal injury and worsen clinical outcome. A greater understanding of the mechanisms underlying neutrophil influx into the brain may aid the development of novel therapeutic interventions for the variety of diseases to which neutrophils contribute, notably including stroke and epilepsy. In vitro evidence implicates the pro- inflammatory cytokine, interleukin-1α (IL-1α), derived from platelets as a key mediator of cerebrovascular inflammation and neutrophil migration across brain endothelial cells.The aim of the work in this thesis was to test if this mechanism is important in vivo.We investigated the contribution of platelets and IL-1 in a murine model of neutrophil migration into the peritoneal cavity in response to injection of lipopolysaccharide (LPS). Depletion of platelets abrogated the migration of neutrophils in response to LPS- induced peritonitis, indicating an important role for platelets in the process. Genetic knockout of IL-1 had no effect on neutrophil influx, demonstrating that migration in the peritoneum occurs independently of IL-1.The discovery that neutrophil migration in LPS-induced peritonitis was independent of IL-1 contrasted with the finding that platelet-derived IL-1 was a mediator of neutrophil influx across mouse brain endothelial cells in vitro. The question arose as to whether IL-1 was required as a mediator of neutrophil migration in extra-cerebral tissues. Hence, we tested the contribution of platelets and IL-1 in two further in vivo models of neutrophil migration: LPS injection into a subcutaneous air pouch, and acute lung injury induced by LPS inhalation. Platelet depletion significantly reduced neutrophil migration into the air pouch in response to LPS, yet had no effect in acute lung injury. This indicated that neutrophil migration into the air pouch was dependent on platelets, and that migration into the lungs was platelet-independent. LPS induced the same degree of neutrophil migration in wild-type and IL-1 knockout mice, demonstrating that IL-1 was not required for neutrophil migration in either model.To determine the contribution of platelets and IL-1 to neutrophil migration in response to cerebrovascular inflammation, we injected LPS into the mouse striatum. In this model, neutrophil influx to the brain parenchyma in response to LPS was reduced by depletion of circulating platelets, and inhibition of the platelet adhesion molecule, GpIb. Genetic knockout of IL-1α significantly reduced the number of invading neutrophils induced by LPS. These data confirmed that both platelets and IL-1α were important contributors to cerebral neutrophil migration in vivo. To determine whether platelets in systemic circulation may be the source of IL-1α, we treated mice with IL-1 receptor antagonist or anti-IL-1 antibodies to block systemic IL-1 action. Neither intervention affected cerebral neutrophil migration in response to LPS, suggesting that the IL-1α that mediates neutrophil migration may originate in the brain.Overall, these data demonstrate that IL-1α and platelets make an important contribution to neutrophil migration to the brain, yet independently of each other. Our data also suggest there may be specific mechanisms driving innate immune responses in vivo even in response to the same inflammatory stimulus.

Interleukin-1 (IL-1), originally defined as a product of activated macrophages, has since been found to be produced by many cell types including keratinocytes. The nature of this IL-1 activity in keratinocytes, originally known as epidermal cell-derived thymocyte activating factor (ETAF) has been the subject of many studies. In the course of this work it was found that the human keratinocyte cell line COLO 16 contains mRNA homologous to human monocyte-derived IL-1B. A 1.2 kbp cDNA was selected with a human IL-B probe from a lgt11 library constructed from COLO 16 mRNA. Sequence analysis revealed that this eDNA was nearly identical to the 3' 1.2 kb of human monocyte IL-1B. In addition, a partial cDNA (F8) was isolated from COLO 16 cells which has a distinct sequence from either IL-1a or B. There is evidence to suggest that the F8 message may be derived from differential splicing of a region of the human genome which also gives rise to the cGMP-gated ion channel in rod photoreceptor cells. The F8 cDNA hybridized on Northern blots of COLO 16 mRNA to a 1.6 kb message of low abundance. Antisera generated against a synthetic peptide based on inferred protein sequence from the cDNA reacted with a 20 and 30 kDa species in both COLO 16 cells and PMA-stimulated normal human keratinocytes. Expression of the partial cDNA in COS-1 cells resulted in activity in the thymocyte co-stimulation and D10.G4.1 T-cell stimulation assays, suggesting that ETAF activity may be due to a combination of IL-1 and F8. / Thesis / Master of Science (MS)

Osteoarthritis (OA) is a debilitating disease of joints that afflicts horses of all ages and breeds and can result in lameness, suboptimal performance, and decreased quality of life. The pro-inflammatory cytokine interleukin-1 (IL-1) has been associated with the initiation and pathogenesis of joint disease. In part, this occurs by induction of proteases and oxidative pathways that contribute to the degradation of structural components of the articular cartilage extracellular matrix. Elucidating the complex macromolecular and molecular effects of IL-1 on articular tissues may further our understanding of the roles of IL-1 and inflammation in OA pathobiology. Full-length gene sequences encoding three recombinant equine interleukin-1 proteins (EqIL-1a, EqIL-1b, and EqIL-1 receptor antagonist), were previously cloned and expressed in-vitro. The objectives of this dissertation were to 1) establish EqIL-1 induced experimental models of equine OA, and 2) to investigate specific IL-1-induced immuno-inflammatory responses. Effects of EqIL-1 on articular cartilage explant proteoglycan metabolism and synthesis of a downstream inflammatory product, prostaglandin E2, established culturing conditions and furthered the rationale to use EqIL-1 in the in-vitro modeling of early joint disease. A customized cDNA array was used to profile changes in mRNA levels resulting from EqIL-1 treatments of cultured articular cartilage chondrocytes. EqIL-1a induced elevated mRNA levels corresponding to six genes after 1 hour relative to media control chondrocytes (p<0.05). EqIL-1b increased transcript levels of seven genes after 6 hours (p<0.0004); 102 additional transcripts were elevated > 2-fold over controls. A subset of the array-generated data was verified using optimized reverse transcriptase-PCR amplification. Results of principal component analysis indicate co-regulation of EqIL-1 induced transcript levels to relate to chondrocyte differentiation and cell-cycle processes. Subtractive hybridization-PCR identified 148 differentially expressed cDNAs in synovium resulting from a 6-hour intra-articular EqIL-1b injection. Combined results demonstrate the potent bioactivity of our equine IL-1 proteins and support the argument for crucial roles of IL-1 in pro-inflammatory processes and cytokine imbalances underlying early OA pathogenesis. These results add to the current knowledge of IL-1 modulated transcription that may precede ECM catabolic processes characteristic of OA. The culture systems, assays, and techniques for gene expression analysis may be useful for future studies attempting to elucidate macromolecular and transcriptional events underlying inflammatory-associated joint disease processes in horses. Reported information may further efforts toward improved diagnostic and preventive strategies and development of anti-IL-1 directed therapies. / Ph. D.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 180-198). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 5 uppsatser.