Cls cDNA clones, which together contained the entire coding region of the protein, were isolated from two human-liver cDNA libraries. The initial Cls clones were identified using a synthetic oligonucleotide probe which corresponded to a region of low degeneracy near the C-terminus of the Cls catalytic chain. Fragments of the Cls cDNA were used to screen a cosmid library in an attempt to isolate the Cls gene, but this proved unsuccessful and no positive clones were isolated. The complete primary sequence of Cls revealed that the homology between the Cls and Clr catalytic chains also extends throughout their non-catalytic chains. Like Clr, Cls can be divided into six structurally independent domains of which the sixth represents the catalytic B chain. Domains I and III in the A chain of Cls are internally homologous, as are domains IV and V. The latter domains are homologous to the internally repeating 60-residue sequences found in Factor B, C2 and other proteins. Domain II of Cls is similar to the 40-residue repeat sequences found in epidermal growth factor precursor and many of the vitamin K-dependent proteins. The assignment of these domains to the different regions of Cls tertiary structure has still to be achieved, but studies in this area should be facilitated now that the complete primary sequence for the Cls zymogen is available.

In this study, the effect of aging on various cells of the immune system was investigated. The two experimental models used were normal young (1-2 months) and old (22-24 months) DBA/2 mice and autoimmune-susceptible young (1-2 months) and old (5-6 months) MRL-Ipr/Ipr (Ipr) mice. Autoreactive T cell clones isolated from DBA/2 mice were used to study the age-induced differential responses of syngeneic T cells and B cells. These cell interactions were found to be greatly diminished in old DBA/2 mice, and this appeared to be due to an intrinsic defect in the cells from old mice. A decreased syngeneic mixed lymphocyte reaction (SMLR) was also found to be associated with these defects in T-T and T-B interactions. The decreased SMLR was due to a reduction in the production of interleukin-1 by macrophages from old mice. In the Ipr mice, age-induced alterations in the cell surface characteristics of the abnormal T cells that accumulate in the lymph nodes were studied. The double-negative T cells from the lymph nodes of old Ipr mice were found to express a cell surface marker, J11d, that is normally present only on immature T cells in the thymus. Furthermore, the number of double-negative J11d⁺ T cells also increased in the thymus of old Ipr mice. Autoreactive T cell clones isolated from DBA/2 and /pr mice exhibited the properties of both T_H1 and T_H2 subsets as the clones secreted IL-2, IL-4 and IFN-γ, and activated both B cells and macrophages. The current study indicates that with increasing age, the autoreactive T cell-induced immunoregulation is disturbed, which may account for reduced immune responsiveness to foreign antigens and increased susceptibility to autoimmune diseases. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI) / Neuropathic pain is a serious health problem that greatly impairs quality of life. The International Association for the Study of Pain (IASP) defines neuropathic pain as ‘pain arising as a direct consequence of a lesion or disease affecting the nervous system’. It is important to note that with neuropathy the chronic pain is not a symptom of injury, but rather the pain is itself a disease process. Novel interactions between the nervous system and elements of the immune system may be key facets to a chronic disease state. One of particular note is the recent finding supporting an interaction between an immune response protein high mobility group box 1 (HMGB1) and Toll like receptor 4 (TLR4). HMGB1 is an endogenous ligand for TLR4 that influences the induction of cytokines in many non-neuronal cells. After tissue damage or injury, HMGB1 may function as a neuromodulatory cytokine and influence the production of pro-nociceptive mediators altering the state of sensory neurons. Very little is known about the HMGB1-TLR4 interaction in sensory neurons and whether chronic changes in endogenous HMGB1 signaling influence the establishment of neuropathic pain. This thesis aims to determine whether a physiologically relevant neuroimmune interaction involving endogenous HMGB1 and TLR4 in the dorsal root ganglia is altered following a tibial nerve injury model of neuropathic pain. I hypothesized that sensitization of sensory neurons following a peripheral nerve injury is dependent on endogenous HMGB1 and TLR4. The studies presented here demonstrate that HMGB1 undergoes subcellular redistribution from the nucleus to the cytoplasm in primary afferent neurons following peripheral nerve injury. Further, the presence of extracellular HMGB1 may directly contribute to peripheral sensitization and injury-induced tactile hyperalgesia. Though thought to be important as a pivotal receptor for HMGB1 activation, neuronal protein expression of TLR4 does not appear to influence the effects of HMGB1-dependent behavioral changes following peripheral nerve injury. Taken together, these findings suggest that extracellular HMGB1 may serve as an important endogenous cytokine that contributes to ongoing pain hypersensitivity in a rodent model of neuropathic pain.

Indiana University-Purdue University Indianapolis (IUPUI) / The immune system initiates tissue repair following injury. In response to sterile tissue injury, neutrophils infiltrate the tissue to remove tissue debris and subsequently undergo apoptosis. Proper clearance of apoptotic neutrophils in the tissue by recruited macrophages, in a process termed efferocytosis, is critical to facilitate the resolution of inflammation and tissue repair. However, the events leading to suppression of sterile inflammation following efferocytosis, and the contribution of other innate cell types are not clearly defined in an in vivo setting. Using a sterile mouse peritonitis model, we identified IL-4 production from efferocytosing macrophages in the peritoneum that activate invariant NKT cells to produce cytokines including IL-4 and IL-13. Importantly, IL-4 from macrophages functions in autocrine and paracrine circuits to promote alternative activation of peritoneal exudate macrophages and augment type-2 cytokine production from NKT cells to suppress inflammation. The increased peritonitis in mice deficient in IL-4, NKT cells, or IL-4Ra expression on myeloid cells suggested that each is a key component for resolution of sterile inflammation. The phagocyte NADPH oxidase, a multi-subunit enzyme complex we demonstrated to require a physical interaction between the Rac GTPase and the oxidase subunit gp91phox for generation of reactive oxygen species (ROS), is required for production of ROS within macrophage phagosomes containing ingested apoptotic cells. In mice with X-linked chronic granulomatous disease (X-CGD) that lack gp91phox, efferocytosing macrophages were unable to produce ROS and were defective in activating iNKT during sterile peritonitis, resulting in enhanced and prolonged inflammation. Thus, efferocytosis-induced IL-4 production and activation of IL-4-producing iNKT cells by macrophages are immunomodulatory events in an innate immune circuit required to resolve sterile inflammation and promote tissue repair.

Indiana University-Purdue University Indianapolis (IUPUI) / CD4 T helper cells control immunity to pathogens and the development of inflammatory disease by acquiring the ability to secrete effector cytokines. Cytokine responsiveness is a critical component of the ability of cells to respond to the extracellular milieu by activating Signal Transducer and Activator of Transcription factors that induce the expression of other transcription factors important for cytokine production. STAT4 is a critical regulator of Th1 differentiation and inflammatory disease that attenuates the gene-repressing activity of Dnmt3a. In the absence of STAT4, genetic loss of Dnmt3a results in de-repression of a subset of Th1 genes, and a partial increase in expression that is sufficient to observe a modest recovery of STAT4-dependent inflammatory disease. STAT4 also induces expression of the transcription factors Twist1 and Etv5. We demonstrate that Twist1 negatively regulates Th1 cell differentiation through several mechanisms including physical interaction with Runx3 and impairing STAT4 activation. Following induction by STAT3-activating cytokines including IL-6, Twist1 represses Th17 and Tfh differentiation by directly binding to, and suppressing expression of, the Il6ra locus, subsequently reducing STAT3 activation. In contrast, Etv5 contributes only modestly to Th1 development but promotes Th differentiation by directly activating cytokine production in Th9 and Th17 cells, and Bcl6 expression in Tfh cells. Thus, the transcription factors Twist1 and Etv5 provide unique regulation of T helper cell identity, ultimately impacting the development of cell-mediated and humoral immunity.