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JOURNAL OF INTERFERON & CYTOKINE RESEARCHVolume 29, Number 7, 2009© Mary Ann Liebert, Inc.DOI: 10.1089/jir.2009.2907.diss

Dissertation Summaries

A Novel Role for IL-1 Cytokines and TNF-α in IFN-γ Production, Which is Mediated by IκBζ

Raquel Marie Raices Rodriguez, PhDOhio State University, 2008

The human immune response to pathogen infection involves a complex network of immune cells, sensor molecules, and signal-ing molecules that fi ght the invading organism and can lead to its elimination. Lipopolysaccharide (LPS) is an important com-ponent of the outer membrane of Gram-negative bacteria that elicits potent host immune responses leading to infl ammation. Monocytes and macrophages are some of the main responders to LPS via a surface-expressed sensor molecule known as Toll-like receptor 4 (TLR4). LPS recognition via TLR4 leads to signaling events that allow the cell to rearrange its gene expression pro-fi le and produce infl ammatory molecules, such as proinfl ammatory cytokines, which have a wide range of immune functions.

The fi rst part of this dissertation describes the early infl ammatory response, in terms of cytokine release, of human primary monocytes to LPS. Specifi cally, we detected an IFN-γ-inducing activity in the conditioned media from LPS-stimulated mono-cytes, which was not due to the prototypical IFN-γ-inducing cytokine, interleukin-18 (IL-18). Via neutralization, immunodeple-tion, and size-fractionation experiments, we uncovered that the IFN-γ-inducing activity was due to the synergistic action of the monocyte-derived cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Moreover, our data suggested that the prototypical IFN-γ-inducing cytokine, IL-18, might have been bound to an inhibitory protein, released in suboptimal amounts, or both. Hence, the lack of IL-18 mediated IFN-γ production in the monocyte-conditioned media. However, when the target IFN-γ-producing KG-1 cell line was directly stimulated with the recombinant forms of IL-18, IL-1β, and TNF-α, we found that IL-1β and IL-18 both could synergize with TNF-α for IFN-γ production. Since IL-1β and IL-18 belong to the same family of cytokines (the IL-1 family) and share similar signaling pathways, we hypothesized that common signaling events downstream of the IL-1β and IL-18 receptors (IL-1R and IL-18R, respectively) are crucial for the observed synergy between IL-1 cytokines and TNF-α.

In the second part of this dissertation, we tested the role of the transcriptional regulator, IκBζ, in IFN-γ production in re-sponse to IL-1 and TNF-α combined stimuli. IκBζ is known to be induced downstream of the IL-1R. We showed for the fi rst time that it can also be induced downstream of the IL-18R. Importantly, by silencing IκBζ expression, we uncovered that IκBζ has a crucial role in IL-1/TNF-α-induced IFN-γ production. IκBζ is known to regulate transcription by acting as a cofactor for the transcription factor, NFκB. Our fi ndings indicated that IL-1/TNF-α combined stimuli induces strong NFκB binding to two of three putative NFκB-binding sites present in the IFN-γ promoter and fi rst intron. This data suggested that IFN-γ produc-tion in response to IL-1/TNF-α combined stimuli may be due to IκBζ/NFκB-mediated transcription of the IFN-γ gene.

In summary, our studies revealed a novel synergistic role for IL-1 cytokines and TNF-α in IFN-γ production that is me-diated by the transcriptional regulator, IκBζ. Moreover, given the primary response nature of IL-1 cytokines and TNF-α, our fi ndings may have important implications in understanding the mechanisms leading to IFN-γ production during the initial stages of innate immune responses.

Cellular Proteins That Interact With the Hepatitis C Virus F Protein

Kamile Yuksek, PhDUniversity of Southern California, 2008

Hepatitis C virus (HCV) F protein of unknown function is produced from the core protein coding region by ribosomal frameshift. It is a short-lived protein with a half-life of slightly >10 minutes. It has been reported to affect transcription of the cellular genes such as p21, p53, and c-myc, and modulate cytokine expression. Moreover, it has been shown to associate with several cellular proteins including PFD2 and MM-1. These fi ndings suggest that the F protein may play important roles in persistent HCV infection. To identify the cellular cofactors associated with the F protein, a human liver cDNA library was

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DISSERTATION SUMMARIES416

screened using the F protein as a bait in yeast two hybrid system. Several cellular proteins were identifi ed to be associated with the HCV F protein including proteasome subunit alpha3 ([white square]3), heterogeneous nuclear ribonucleoprotein L (hnRNP L), phospholipid scramblase 1 (PLSCR1), apolipoprotein(a) (apo(a)), LIM domain containing protein 1 (LIMD1), com-plement protein 6 (C6), and fi bulin3. The interaction between [white square]3 and the F protein, as well as hnRNP L and the F protein, was confi rmed by GST pull-down assay, co-immunoprecipitation, and immnofl uorescence staining. Amino acids 40–60 of the F protein was identifi ed as the [white square]3-binding domain that together with its upstream sequence could signifi cantly destabilize the green fl uorescence protein (GFP), an otherwise stable protein. Expression of [white square]3 reduces the F protein level in cells in a dose-dependent manner. The F protein was degraded by 20S proteasome through ubiquitin-independent pathway. And this degradation pattern might be important for its regulatory activities. Although the biological signifi cance of PLSCR1, apo(a), LIMD1, C6, and fi bulin3 is unknown, F protein inhibits the IRES-mediated HCV translation in a dose-dependent manner and addition of exogenous hnRNP L relieves the suppression effect of the F protein. These results suggest that the F protein may regulate cellular function through interaction with cellular proteins, and thus contribute to pathogenesis of HCV.

Characterization of Host Responses Following Marek’s Disease Virus Infection or Vaccination Against Marek’s Disease

Mohamed Faizal Abdul Careem, PhDUniversity of Guelph, Canada, 2008

This thesis comprises a series of studies conducted to elucidate host responses to Marek’s disease virus (MDV) in two body compartments; spleen, which is a major secondary lymphoid organ, involved in elicitation of immune response and feathers, which are the site of virus shedding in MDV-infected chickens.

To establish a reliable method for quantifi cation of MDV genome, a real-time PCR technique was developed. Overall, this PCR technique was proven to be reproducible and to have a low inter- and intra-assay variation. The technique was then validated quantifying viral genome load in feathers of chickens infected experimentally with MDV. This technique was used in subsequent studies presented in this thesis for quantifi cation of MDV as well as related vaccine strains in feathers and spleen.

In a study aimed at identifying immunological mediators that are involved in immunity conferred by Marek’s disease (MD) vaccines, it was demonstrated that lower expression of cytokine genes such as interleukin (IL)-6, IL-10, and IL-18 in spleen was associated with protection in vaccinated chickens. Further, the study indicated that although vaccines can confer protection against disease and reduce MDV load in some tissues such as spleen, they are not able to reduce virus replication and shedding from feathers.

Considering the importance of feathers in MDV shedding, two separate experiments were conducted. In the fi rst study, in response to MDV, signifi cant increase in viral genome load and transcripts was associated with an increase in the ex-pression of major histocompatibility complex (MHC) class I gene, cytokine genes such as IL-6, IL-10, and interferon (IFN-γ) and the infi ltration of CD4+ and CD8+ T cells. In the second study, both CVI988 and herpesvirus of turkeys (HVT) vaccine viruses replicated in feathers and stimulated local host responses, characterized by the expression of cytokine genes such as IFN-γ and infi ltration of CD8+ T cells.

In conclusion, host responses are initiated in response to MDV infection and vaccination against MD both in spleen and feathers. There is a difference in elicited host responses in these body compartments; in spleen it is effective in curtailing MDV replication, whereas in feathers host responses are associated with the persistence of MDV.

Cross Talk Between Innate and Adaptive Immunity in Viral and Intracellular Bacterial Infection

Yumi Nakayama, PhDUniversity of Wisconsin, Madison, 2008

Typically, host immune responses are classifi ed into two arms: innate and adaptive immunity. Innate immunity induces a wide variety of mechanisms including complement activation and interferon (IFN) signaling. Adaptive immunity depends on specialized population of antigen-specifi c lymphocytes, the B and T lymphocytes. In this thesis proposal, I have investi-gated the role of complement and double-stranded RNA-dependent interferon-inducible protein kinase, PKR, in regulating antigen-specifi c T-cell responses to acute viral and intracellular bacterial infections.

In specifi c aim 1, I examined the role of complement component 3 (C3) in regulating CD8+ and CD4+ T-cell activation during infection of mice with an intracellular bacteria, Listeria monocytogenes (LM). My studies showed that C3 promotes

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DISSERTATION SUMMARIES 417

activation and expansion of CD4+ and CD8+ T cells in LM-infected mice. Additionally, I present evidence that C3-dependent expansion of T cells does not require C5a, a downstream effector of complement.

In specifi c aim 2, I examined the role of several possible mechanisms of complement-dependent CD8+ T-cell regulation including complement receptor 3 (CR3), antibodies/B cells, IL-4, and C5a in lymphocytic choriomeningitis virus (LCMV) infection. My studies indicated that C3 promotes T-cell responses to LCMV by mechanisms independent of CR3, C5a, B cells, or IL-4.

In LCMV infection, type I IFNs (IFN-I) play a critical role in controlling early viral replication and enhancing the primary virus-specifi c CD8+ T-cell response to LCMV. IFNs induce the antiviral effects through transcriptional up-regulation of IFN-stimulated genes (ISGs), which include PKR. I determined the role of PKR and IFN-I in regulating CD8+ T cells and viral con-trol during primary and secondary LCMV infections (specifi c aim 3). My studies provided strong evidence that IFN-I and PKR are required for optimal viral control during primary and secondary T-cell responses to LCMV, regardless of the magnitude of the virus-specifi c CD8+ T-cell response. Moreover, we found that the requirement for PKR is pathogen-dependent, because IFN-I but not PKR is required for control of vaccinia virus (VV) during a secondary response. These fi ndings have implications not only in understanding viral pathogenesis but also in development of effective vaccines against viral infections.

Functional Analysis of the Salivary Protein, Salp15

Ignacio J. Juncadella, PhDUniversity of Massachusetts, Amherst, 2008

The interaction between Ixodid ticks and their mammalian hosts is a complex relationship. While the mammalian host tries to avoid the completion of the feeding process, the tick has devised strategies to counteract these attempts. Tick saliva con-tains a vast array of pharmacological activities that presumably aid the tick to evade host responses, including anti-comple-ment, oxidative and innate and adaptive immune responses. The characterization of these activities has gained momentum in the last several years. One of the best-studied activities present in tick saliva corresponds to the antigen known as Salp15, which inhibits TCR-mediated CD4+ T-cell activation and IL-2 production.

This study identifi es CD4 as the specifi c receptor for Salp15 on T cells. A direct association occurs between CD4 and the C-terminal amino acids of Salp15, which allows Salp15 to act at the very beginning of TCR ligation-induced signaling cas-cades. Salp15 prevents the activation of Lck upon TCR engagement and the formation of lipid rafts and actin polymerization. Salp15 also affects tyrosine phosphorylation of several early signal components downstream of Lck, including Zap70, Lat, and PLCg1. This study also demonstrates that the peptide that mediates the interaction of Salp15 with CD4, P11, is able to recapitulate the immunosuppressive activity of the whole saliva protein. These results clarify the molecular mechanisms of action of Salp15 on T cells and suggest that binding to CD4 is suffi cient to elicit its immunosuppressive activity.

The differentiation of Th17 cells requires the concerted action of IL-6 and TGFb. However, the exact contribution of each cytokine has not been elucidated. This study also provides evidence indicating that the role of TGFb during the differenti-ation of CD4+ T cells is exclusively the repression of IL-2 production, which has been shown to counteract the generation of these effector cells. The inhibition of IL-2 during the differentiation of CD4+ T cells mediated by Salp15 and the specifi c IP 3 receptor inhibitor 2-APB resulted in Th17 differentiation in vitro. Furthermore, the treatment of PLP 139-151-immu-nized mice with Salp15 also resulted in increased differentiation of Th17 cells in the central nervous system and augmented pathology. Our results demonstrate that the role played by TFGb is circumscribed to the inhibition of IL-2 during the dif-ferentiation of CD4+ T cells.

Finally, this study shows that Salp15 inhibits the interaction between HIV-1 gp120 and CD4. Furthermore, Salp15 prevents the formation of syncitia of HL2/3 (a stable HeLa cell line expressing the envelop protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interaction with the glycoprotein. A phage display library screen provided the interacting residues in the C1 domain of gp120. These results provide a potential basis to defi ne exposed gp120 epitopes for the generation of neutralizing vaccines against HIV.

Innate and Adaptive Antiviral Responses in Mice and Ferrets to Wild Type and P/V Mutants of the Paramyxovirus Simian Virus 5

Gerald Anthony Capraro, Jr., PhDWake Forest University, The Bowman Gray School of Medicine, North Carolina, 2008

P/V gene substitutions convert the noncytopathic paramyxovirus Simian Virus 5 (SV5), which is a poor inducer of host cell responses in human tissue culture cells, into a mutant (P/V-CPI-) that induces high levels of apoptosis, interferon (IFN)-β, and proinfl ammatory cytokines. However, the effect of SV5-P/V gene mutations on virus growth and adaptive immune

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DISSERTATION SUMMARIES418

responses in animals has not been determined. Here, two distinct animal model systems were used to test the hypothesis that SV5-P/V mutants which are more potent activators of innate responses in tissue culture will also elicit higher antiviral antibody responses. In mouse cells, in vitro studies identifi ed a panel of SV5-P/V mutants that ranged in their ability to limit IFN responses. Intranasal infection of mice with these WT and P/V mutant viruses elicited equivalent anti-SV5 IgG responses at all doses tested, and viral titers recovered from the respiratory tract were indistinguishable. In primary cul-tures of ferret lung fi broblasts, WT and P/V-CPI-viruses had phenotypes similar to those established in human cell lines, including differential induction of IFN secretion, IFN signaling, and apoptosis. Intranasal infection of ferrets with a low dose of WT rSV5 elicited ~500-fold higher anti-SV5 serum IgG responses compared to the P/V-CPI- mutant, and this corre-lated with overall higher viral titers for the WT virus in tracheal tissues. There was a dose-dependent increase in antibody response to infection of ferrets with P/V-CPI-, but not with WT. Together these data indicate that WT rSV5 and P/V mutants elicit distinct innate and adaptive immunity phenotypes in the ferret animal model system, but not in the mouse system. The results presented here demonstrate that the humoral immune response to SV5 is directly related to the level of virus replication.

Lipid Raft-Mediated Lyn Kinase Activation Regulates Host Innate Immune Response Against Pseudomonas aeruginosa Lung Infection

Shibichakravarthy Kannan, PhDUniversity of North Dakota, 2008

Pseudomonas aeruginosa (PA) is a common opportunistic pathogen that invades the susceptible host. Alveolar macrophages (AM) play a pivotal role in defending against invading pathogens in the lungs. The effectiveness of AM in combating PA infection has not been studied in detail. The goal was to identify the key components involved in mounting an effective innate immune response using the murine lung infection as a model to understand the host–pathogen interactions. Our most important fi nding was the involvement of Lyn tyrosine kinase, a member of Src family tyrosine kinases, in orchestrat-ing the host cell response to PA invasion.

Preliminary fi ndings showed that Lyn is constitutively expressed in an epithelial cell type (A549, lung carcinoma derived) and associated with PA infection. PA infection of AEC II cells activates Lyn through innate immune receptor clustering lead-ing to aggregation of membrane lipid raft microdomains. Similarly in macrophage cell type raft-mediated Lyn activation plays a crucial role in regulating phagocytosis and respiratory burst activity.

Depletion of cholesterol was found to affect raft function and Lyn inhibition by multiple approaches signifi cantly reduced the phagocytosis of PA. The hypothesis proposes that AEC II and AM communicate through secreted factors and raft-mediated Lyn activation may play a role in regulating the cell–cell communication. Among the cytokines released by AEC II cells, monocyte chemoattractant protein (MCP-1) plays a major role in recruiting AM to the site of infection. Depleting cholesterol by mevastatin (cholesterol synthesis inhibitor) treatment in macrophages inhibits the raft-associated functions and down-regulates Lyn activity. Direct Lyn inhibition using siRNA-mediated gene silencing had very similar effects and signifi cantly decreased PA phagocytosis.

In conclusion, (1) PA infection of AEC II cells induces Lyn kinase activity by lipid raft-mediated mechanism; (2) infected AEC II cells secrete infl ammatory cytokines and predominantly MCP-1 that recruits and activates monocytes like alveolar macrophages; (3) effective phagocytosis of PA by activated AM depends on Lyn function and its downstream signaling mediators. The identifi cation of a novel lipid raft-based signaling mechanism that regulates host innate immunity has been demonstrated and holds promise for future therapeutic intervention to combat PA infection.

Mechanisms of Maternal–Fetal Immune Tolerance in Nonhuman Primates

Jessica G. Drenzek, PhDUniversity of Wisconsin, Madison, 2008

The mechanisms of maternal–fetal immune tolerance remain a paradox in the fi eld of classical immunology. Multiple mech-anisms regulate how the maternal immune system maintains a state of immunotolerance toward the fetal semiallograft. HLA-G, a nonclassical MHC class I molecule, has been proposed to play a key immunoregulatory role during pregnancy due to its restricted tissue distribution and limited polymorphism. Furthermore, Mamu-AG is expressed by the rhesus

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DISSERTATION SUMMARIES 419

monkey and is thought to be the functional homolog of HLA-G in the humans. Mamu-AG is found mainly in the placenta where it interacts with maternal leukocytes. Decidual natural killer (NK) cells make up 80% of the lymphocyte population in the decidua. Therefore, the interaction between NK cells and Mamu-AG was further investigated. NK cells were hypoth-esized to produce Th1 cytokines, in particular IFN-γ, in response to Mamu-AG that regulate blood vessel development and CG secretion. In order to investigate this proposal, NK cells were cocultured with classical and nonclassical MHC class I transfectants and trophoblasts. The cytokine profi les were analyzed to determine the effect that Mamu-AG had on NK cell cytokine production. The cytokines affected by the presence of Mamu-AG were IFN-γ, TNF, IL-1β, and GM-CSF. It was also determined that NK cells are most likely not a source of IL-4, IL-10, IL-12, IL-15, and IL-18.

Another mechanism of maternal–fetal immune tolerance is the role that indoleamine 2,3-dioxygenase (IDO) has in regu-lating maternal T cells during pregnancy. IDO catalyzes the rate-limiting step of tryptophan degradation along the kynure-nine pathway and is hypothesized to limit tryptophan availability and prevent maternal T-cell activation. Alternatively, metabolites of this reaction may be regulating T cells or serving as antioxidants to protect vascular endothelium from dam-age caused by oxidative stress. IDO was localized in the common marmoset and rhesus monkey placenta, uterus, spleen, lymph node, and small intestine by immunohistochemistry. In addition, the enzymatic activity of IDO was highest in the placenta when compared with these tissues.

The results from these experiments demonstrate that Mamu-AG is regulating NK cells and that IDO is present at the maternal–fetal interface during pregnancy.

Modulation of Macrophage Cytokine Responses and Antigen Presentation During African Trypanosomiasis

Bailey E. Freeman, PhDUniversity of Wisconsin, Madison, 2008

This thesis examines aspects of macrophage function during infection with the African trypanosome Trypanosoma brucei rhodesiense, the causative agent of fatal African sleeping sickness in humans. Relative resistance to this parasite is dependent on the development of a polarized type 1 immune response. Such immune activation occurs during early infection, but later stages of disease are characterized by chronic immunosuppression. Given the key role of macrophages in the regulation of all aspects of the immune response through cytokine production and antigen presentation, we sought to determine whether macrophage function is compromised during disease.

Trypanosome infection is characterized by repeated waves of parasitemia due to antigenic variation of the variant surface glycoprotein (VSG) molecules of the parasite’s surface coat, and parasite clearance by host antibody responses. Because of this repeated exposure of the host to antigen, we hypothesized that macrophages become tolerized to infl ammatory signals during infection, leading to a less robust immune response to later antigenic variants. Our results demonstrate that mac-rophages from infected mice display altered cytokine and signaling responses to microbial stimuli that are consistent with tolerance. Instead of producing high levels of proinfl ammatory cytokines such as IL-12, macrophages from infected mice produce IL-10 upon stimulation, which may contribute to the immunosuppressive environment during late stage disease.

Processing and presentation of foreign antigens is another way in which macrophages control the immune response. Based on previous evidence that macrophages from infected mice cannot present the model antigen hen egg lysozyme, we hypothesized that antigen-presenting cells are unable to present a newly encountered VSG that is distinct from the VSG of the infecting trypanosome population. Our results show that presentation of new VSG is indeed impaired, and mechanistic studies revealed that intracellular events leading to the formation of mature peptide-MHC II complexes are compromised in T. b. rhodesiense-infected mice. Thus, modulation of macrophage cytokine responses and antigen presentation contribute to the ability of T. b. rhodesiense to evade host immune responses.

Novel Strategies to Combat Ebolavirus

Peter J. Halfmann, PhDUniversity of Wisconsin, Madison, 2008

Ebolavirus is the causative agent of Ebola hemorrhagic fever, a viral disease with case fatality rates as high as 90%. Because there are therapeutic treatments against the virus, it is classifi ed as a biosafety level 4 (BSL4) agent. This high containment

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requirement impedes the characterization of the viral determinants of pathogenicity and the identifi cation of cellular pro-teins that are critical for the Ebolavirus life cycle.

To overcome the requirement for BSL-4 containment, a biologically contained Ebolavirus was generated. The essential viral VP30 gene, a transcription activator protein, was deleted. The resulting virus (EbolaDVP30) is replication-incompe-tent and therefore biologically safe. To maintain EbolaDVP30 virus, a cell line expressing the VP30 protein was created. EbolaDVP30 virus was morphologically similar to wild-type virus and grew in VP30-expressing cells to titers compa-rable to those obtained for wild-type virus, 108 viral particles/mL. The biologically contained viruses were genetically stable over multiple passages in cell culture. Collectively, these data show that the EbolaDVP30 system is suitable for basic research, therapeutic development, and the screening of siRNA libraries to identify cellular proteins that are critical for the Ebolavirus life.

The viral determinants of Ebolavirus pathogenicity are poorly understood. Cells infected with Ebolavirus are resistant to the transcriptional up-regulation of antiviral genes by interferon. Using an interferon-dependent luciferase reporter plas-mid, we and others identifi ed VP24 as an interferon-signaling antagonist. A number of viral interferon antagonists inhibit the JAK-STAT pathway. Treatment of cells with interferon leads to the phosphorylation and nuclear translocation of STAT-1. We and others showed that in interferon-treated cells expressing VP24 protein, the nuclear translocation of phosphorylated STAT-1 was inhibited. We also identifi ed a dileucine motif in the C-terminus of VP24 that is critical for this effect.

In conclusion, the generation of biologically contained Ebolaviruses has opened the door to a wide variety of applica-tions that will give us a better and broader understanding of fi loviruses. Together with basic research on the functions of viral proteins (such as VP24), we will gain a more detailed understanding of Ebolavirus pathogenicity, which will aid in the development of preventative and therapeutic treatments.

Regulation of Airway Smooth Muscle Proliferation: Cytokine-, Glucocorticoid-, and PKA-Dependent Mechanisms

Anna Maria Misior, PhDWake Forest University, The Bowman Gray School of Medicine, North Carolina, 2008

Increased mass of airway smooth muscle (ASM) is an important pathogenic mechanism in the obstructive airway disease asthma. Increased volume of ASM has been attributed to both smooth muscle hyperplasia and hypertrophy, and is thought to be driven by increased levels of mitogens and infl ammatory mediators in the airway. The effects of polypeptide growth factors and G protein-coupled receptor (GPCR) ligands on ASM proliferation have been extensively explored; however, reg-ulation of growth by the proinfl ammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α has not been well characterized. Both IL-1β and TNF-α, alone or in combination, have been shown to decrease proliferation of human ASM in vitro stimulated by a variety of growth factors or GPCR agonists and the effect is associated with the ability of the cytok-ines to induce cyclooxygenase (COX)-2 expression, production of prostaglandin E 2 (PGE 2), and increase in cAMP levels. Because cAMP-dependent protein kinase (PKA) is the primary cAMP effector, it has been assumed to mediate the antimi-togenic effects of IL-1β and TNF-α. Even though the role for COX-2 and PGE2 in the antimitogenic effects of the cytokines is assumed based on association, no evidence directly implicating PKA exists due to lack of effective and specifi c inhibitors of PKA in intact cells.

Studies described in this dissertation provide direct evidence for the critical role of PKA in regulating the mitogenic effects of IL-1β and TNF-α in ASM. Inhibition of PKA activity by heterologous expression of two different proteins is veri-fi ed and demonstrated to render IL-1β and TNF-α strong mitogens and enhancers of growth factor-stimulated ASM prolif-eration. Indirect inhibition of PKA activation, due to suppression of COX-2 induction or PGE 2 production, is the mechanism mediating the promitogenic effects of glucocorticoids (GC) and COX inhibitors on ASM. The PKA-dependent mechanisms in ASM involve regulation of the phosphoinositide-3 kinase and p42/p44 extracellular regulated kinases activation, cell cycle regulatory protein expression and activation, and gene expression. Additionally, our analyses characterize differential gene expression in ASM regulated by growth factors, cytokines, PKA, and GCs and provide novel insight into the effects of mainstream asthma therapies on ASM cell growth.

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