Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 137-153).

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation eventually leading to the destruction of cartilage and bone. The aetiopathogenesis is not completely understood, but previous studies have shown that the disease is multifactorial with genetic, environmental and hormonal factors involved. Immune cells, e.g., T- and B-cells, and macrophages, migrate into the joints, with increased expression of numerous soluble factors such as cytokines, chemokines and adhesion molecules functionally active both locally and systemically. Analyses of blood samples from the Medical Biobank in Umeå from individuals before the onset of symptoms of joint disease showed that anti-citrullinated protein/peptide antibodies (ACPA) preceded the development of disease by years and this finding has been confirmed by other studies.                                         The aim of this thesis was to identify signs of activation of the immune system analysed as up-regulation of pro- and anti-inflammatory cytokines, sero-positivity for autoantibodies, and genetic factors identified as relevant for the development and disease progression of RA. The concentrations of 30 cytokines and chemokines were measured in blood samples from individuals before the onset of symptoms, and when diagnosed with RA, together with population-based matched controls using a multiplex system. The predictive value of different isotypes (IgG, IgA, and IgM) of ACPA and rheumatoid factor (RF) before onset of symptoms and different types of ACPA (e.g., mutated citrullinated vimentin, MCV) were analysed for disease development and progression in patients with early RA and controls from Northern Sweden. These factors were related to the genetic markers, HLA- shared epitope (SE) alleles and the 1858C/T polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene.                            In paper I, it was shown that in individuals who later developed RA (i.e., pre-patients) the levels of several cytokines and related factors that represent the adaptive immune system (Th1, Th2, and T regulatory cell related factors) were significantly elevated compared with controls, whereas, after the onset of disease the involvement of the immune system was more general and widespread. In paper II, the presence of different isotypes (IgM, IgA and IgG) of ACPA in pre-patients, patients and controls was evaluated showing that both the IgG and IgA isotype predicted the onset of RA by years with the IgG isotype having the highest predictive value. In paper III, the association of the 1858T variant of PTPN22 with RA was confirmed. Furthermore, the association was restricted to autoantibody positive disease and this variant was correlated with an earlier age for disease onset. In paper IV, anti-MCV antibodies were identified as being associated with a more severe disease course of RA, measured by disease activity score, erythrocyte sedimentation rate, and swollen joint count over time compared with anti-CCP2, anti-CCP3, and anti-CCP3.1 antibodies.                                                                                                In conclusion, individuals who later developed RA had increased concentrations of inflammatory markers reflecting an activation of the immune system years before the clinical symptoms of the disease developed. Also, the presence of ACPA of IgG and IgA isotype prior to disease onset predicted the development of RA. The PTPN22 1858T variant was associated with sero-positive RA and anti-MCV antibodies were associated with a higher inflammatory activity compared with anti-CCP2, -CCP3 and -CCP3.1 antibodies. These findings together present a possibility to better predict the development and progression of RA.

Type 1 diabetes (T1D) is due to a selective loss of the insulin producing β-cells. However, the process responsible for this loss is still unknown. There is accumulating evidence that enteroviruses (EVs) are involved in T1D. In addition to direct virus-induced cytolysis, EVs could facilitate β-cell destruction by inducing inflammatory cytokines. Induction of such genes has previously been shown in EV-infected islets in vitro. Modulation of inflammatory mediators expressed in the islets could be a possible strategy to reduce β-cell destruction. In the first paper we screened uninfected isolated human islets for genes with the potential to induce or modulate an immune response. We found that several of the genes expressed in the islets encode proteins with a powerful biological activity, such as IL-1β, IL-8, MIP-2α, MCP-1 and MIF. This indicates that the islets themselves can express several triggers of inflammation, and if expressed in vivo these mediators would probably contribute to β-cell destruction. The vitamin B3 derivate, nicotinamide (NA), has been shown to modulate expression of factors important for coagulation and inflammatory responses. Addition of NA into isolated islet cultures resulted in a reduced expression of the pro-inflammatory chemokine MCP-1 and the coagulation activator tissue factor, suggesting that NA may have implications for both inflammatory responses and the pro-coagulant activity of islets. We successfully isolated EVs from three newly diagnosed T1D patients. All isolates showed tropism for human islets and β-cells in vitro and clearly affected islet function. We also found that EV infection induced islet secretion of the chemokines IP-10 and MCP-1and that this induction could be blocked or reduced by addition of NA to the culture medium. Interestingly, NA also reduced viral replication and virus-induced islet destruction. To conclude, this thesis provides new information about expression and modulation of inflammatory mediators in infected and uninfected human islets that could trigger inflammatory reactions leading to β-cell destruction. Moreover, it further strengthens the causal relationship between EV and T1D.

Immune complexes (ICs) are produced during normal immune responses and facilitate clearance of foreign antigens. ICs not efficiently cleared from the circulation can cause tissue damage. This might happen if ICs are formed with autoantibodies and autoantigens. Well described effects of ICs are neutralization of antigen, classical complement activation or FcR-mediated phagocytosis, whereas cytokine inducing effects of ICs in human clinical settings are less well described. I have investigated cytokine-inducing properties in vitro of ICs from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and cryoglobulinemia in association with lymphoproliferative diseases. Cryoglobulin (CG)-induced cytokine production varied with changes in temperature and ionic strength in parallel to CG precipitation. IgG CG-induced cytokine production was also mediated via FcγIIa on monocytes. Blockade of the complement system, resembling the in vivo situation of complement consumption in CG-associated diseases, increased IgG CG induced IL-10 and decreased TNF-α production. This represents hitherto not described mechanisms for CG-associated inflammation. ICs from SLE patients induced IL-10 and IL-6 production from PBMC cultures via FcγRIIa. Occurrence of anti-SSA autoantibodies and signs of in vivo complement activation contributed to increased levels of circulating ICs in SLE patients, corresponding to increased amounts of IC-induced IL-10 in vitro. This represents a possible vicious cycle that might perpetuate antibody dependent pathology in SLE, and put anti-SSA in a new pathological context. RF-associated ICs from RA joints and ICs formed with antibodies against collagen type II from RA serum induced pro-inflammatory cytokine production from monocytes via FcγRIIa, showing how specific autoantibodies might induce or perpetuate joint inflammation in RA. I have described how ICs can induce significant amounts of pathophysiologically important monocyte-derived cytokines in three major IC-dependent diseases. Blockade of FcγRIIa and suppression of monocytes/macrophages might be a means of reducing pathogenic IC-induced cytokine production in these diseases.

Atopic asthma is characterized by excessive T helper 2 (Th2)-like immunity to allergens in the bronchial mucosa. The Th2-cytokine interleukin (IL)-4 induces IgE production, while the Th2-cytokine IL-5 promotes eosinophilic inflammation in the airways of asthmatics. Most asthmatics are atopic, but a subgroup is non-atopic. We hypothesize that allergen-induced Th2, particularly IL-5, responses can be observed in peripheral blood in both atopic and non-atopic asthmatic children but not in healthy control children. The aim of the present study was to determine IL-4, IL-5, IL-9, IL-10, IL-13 and IFN-γ secretion induced from peripheral blood mononuclear cells (PBMC) by a broad panel of inhalant allergens (timothy, cat, birch, dog and house dust mite) in asthmatic children with and without sensitization. The study included 13 atopic asthmatic, 5 non-atopic asthmatic, and 12 non-atopic non-asthmatic children. PBMC were stimulated with allergens and cytokine production was measured with enzyme-linked immunosorbent assay (ELISA). Higher levels of cat and dog antigen-induced IL-5 release were more commonly observed in both atopic and non-atopic asthmatics than in controls. Children with atopic, but not non-atopic, asthma produced higher levels of allergen-induced IL-4 and IL-9 than controls. Non-atopic asthmatics produced more IL-10 than atopic asthmatics after cat stimulation. High levels of eosinophilia-associated IL-5 responses are induced by cat and dog allergen in both atopic and non-atopic asthmatic children. The Th2 cytokines IL-4 and IL-9 were associated only with atopic asthma, probably due to their IgE-inducing properties.

The circadian system provides living organisms a means to adapt their internal physiology to constantly changing environmental conditions that exists on our rotating planet, Earth. Clocks in peripheral tissues are referred to as peripheral which may participate in tissue-specific functions. The first step to investigating the circadian regulation in the peripheral tissues of avians was to examine for the presence of avian orthologs of core components of the molecular clock using Quantitative real time (qRTPCR) assays. We investigated the avian spleen for daily and circadian control of core clock genes and regulation of the inflammatory response by the spleen clock. The core clock genes, bmal1, bmal2, per2, per3 and clock displayed both daily and circadian rhythms. Proinflammatory cytokines TNFα, IL-1β, IL-6 and IL-18 exhibited daily and circadian rhythmic oscillations. A differential expression of proinflammatory cytokine induction was observed in the spleen undergoing lipopolysaccharide (LPS)-induced acute inflammation. Exogenous melatonin administration during inflammation seems to enhance some and repress a few inflammatory cytokines, implying that melatonin is pleiotropic molecule. To compare and contrast the role of peripheral clocks in regulating energy balance and reproduction in layer vs. broiler chicken, the visceral adipose tissue (VAT), ovary and hypothalamus were examined for the presence of core clock genes were investigated in these two lines of poultry birds. Quantitative RT-PCR was employed to examine daily control of core clock genes in these three peripheral tissues over a 24hr period. The layer hens exhibit rhythmic oscillations in the mRNA abundance of the core clock genes in the VAT, ovary and the hypothalamus. The hypothalamus and VAT of the broiler hens exhibit rhythmic mRNA abundance of the core clock genes. However, the clock genes in the ovary of the broiler pullets exhibit marked reduction in their amplitude and rhythms over a 24hr period. The broiler hens are prone to poor energy balance, obesity and reproductive capacity. In summary, these data provide evidence for a functional link between the circadian clock and the ovary by determining clock gene regulation under conditions of disrupted or eliminated reproductive function vs. normal reproductive output.

<p>Normally, neutrophil pools are maintained by "steady-state" granulopoiesis. Infections and inflammation, however, trigger neutrophilias that are supported by a hematopoietic program of accelerated granulopoiesis known as "emergency" granulopoiesis. Steady-state and emergency granulopoiesis are thought to depend on distinct members of the CCAAT enhancer binding protein (C/EBP) family of transcription factors, yet the extracellular cues that determine these developmental pathways are unclear. I hypothesize that inflammation elicits IL-1 which acts directly on hematopoietic progenitor cells for the induction of emergency granulopoiesis. Indeed, IL-1RI<super>-/-</super> mice fail to mount reactive neutrophilias in response to adjuvant-induced inflammation. Analysis of this specific impairment revealed an unanticipated role for IL-1RI in supporting increased proliferation by granulocyte/macrophage progenitors (GMP) and, surprisingly, more primitive multipotent progenitors (MPP) and hematopoietic stem cells (HSC). Whereas IL-1 drives HSC proliferation directly <italic>in vitro</italic>, inflammation induces comparable rates of proliferation in IL-1RI deficient and -sufficient HSC, MPP, and GMP in mixed chimeric mice. Thus, IL-1RI signals play a necessary, but indirect role in the support of alum-induced neutrophilias by expanding both pluripotent and myeloid progenitor compartments to accelerate granulopoiesis.</p><p>The lack of alum-induced neutrophilia in IL-1RI<super>-/-</super> mice is due to defective mobilization of bone marrow (BM) neutrophils and impaired proliferation of hematopoietic stem and progenitor cells (HSPC). Coincident defects in neutrophil mobilization and HSPC proliferation suggest that the trigger for emergency granulopoiesis might be the exhaustion of neutrophil compartments rather than inflammatory inductions of growth factors. Consistent with this hypothesis, non-inflammatory reductions in BM neutrophil numbers elicit granulopoietic responses similar to those induced by adjuvant. Alum mobilizes BM neutrophils via G-CSF, but increased HSPC proliferation results from a density-dependent mechanism that is only partially dependent on G-CSF. Notably, C/EBP&beta;, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC, but plays a role in the terminal differentiation of neutrophils. These observations indicate that the draining of BM neutrophil pools is sufficient to activate a latent, homeostatic mechanism of accelerated granulopoiesis. I propose a common model for the regulation of neutrophil production that explains both steady-state and emergency granulopoiesis through negative feedback.</p> / Dissertation

The role of neutrophils in the pathogenesis of Salmonella typhimurium-induced ruminant and human enteritis and diarrhea remains incompletely understood. To address this question, the in vivo bovine ligated ileal loop model of non-typhoidal salmonellosis was used in calves with the naturallyoccurring Bovine Leukocyte Adhesion Deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from BLAD calves were compared to those from genetically normal calves negative for the BLAD mutation. Morphologic studies showed that the absence of significant tissue influx of neutrophils in intestine infected by S. typhimurium resulted in less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion compared to regular calves. Study of gene expression profile of cytokines by quantitative Real-Time PCR (qRTPCR) revealed that the massive tissue influx of neutrophils during acute infection is mainly driven by the CXC chemokine GRO- α especially in the last stages of acute infection and to a lesser extent, IL-8. In contrast, the pro-inflammatory cytokines IL-1 β and TNF- α were not significantly correlated with the presence or absence of tissue neutrophils. The precise in situ localization of gene expression of these major cytokines and chemokines was investigated by qRTCPR from specific groups of intestinal cells captured by Laser Capture Microdissection in S. typhimuriuminfected ileal loops from BLAD animals. Our data confirmed that gene expression of IL-8, GRO- α, and IL-1 β was predominantly localized to enterocytes of crypts with less expression in enterocytes of villi tips and cells that form the domed villi were not an important source of TNF- α gene expression. Microarray technology was used to determine the global transcriptional profile of bovine intestinal loops inoculated with S. typhimurium. The host samples were hybridized on a 13K bovine-specific oligoarray and microarray data was analyzed using a suite of gene expression analysis and modeling tools. Analysis of our data revealed that the tissue influx of neutrophils in ileal loops greatly influenced the host gene expression. Major differences in gene expression in relevant fields of Salmonella research including inflammation and immune response, Toll-like receptor signaling, cytokine profiles, apoptosis, and intracellular defense against infection are discussed.