Date of Award
Doctor of Philosophy (PhD)
Dr. Jack Gauldie
Cytokines are polypeptide hormones that act nonenzymatically to regulate host cell functions. These glycoproteins make up a fourth class of soluble intercellular signalling molecules that also include neurotransmitters, endocrine hormones and autacoids and are believed to play a central role in tissue remodelling in inflammation, infection, and wound repair. Numerous studies have now implicated cytokines to be of critical importance in host defense, and a more complete understanding of their molecular function is essential. What is also evident is that the majority of biological functions assigned to cytokines have been characterized by in vitro systems.
In vivo confirmation of these reported biological functions is required and has been attempted. To date, this has been difficult to attain with the available animal models. While studies in transgenic mice have revealed a number of biological activities, they probably do not reflect normal physiological responses, since tissues chronically exposed to a cytokine throughout development may undergo alterations in its effector phenotype. Administration of recombinant protein is also problematic as repeated injections with large doses of purified recombinant protein are usually required to maintain physiologic concentrations due to the short half life of most cytokines in the circulation.
To overcome these problems, we have developed an alternative approach to investigate cytokine function in vivo. This approach, which we have defined as a "pseudo transgenic" animal model, uses recombinant adenovirus vectors containing cytokine genes to deliver and transiently overexpress cytokines in vivo in a tissue-directed manner to normal adult animals. Using this vector approach, cytokine expression can be targeted to a tissue in a way that may mimic more normal physiologic responses. In this study, recombinant adenovirus type 5 vectors capable of expressing the murine cytokines interleukin-5 (IL-5), interleukin-6 (IL·6), and RANTES were constructed to investigate the in vivo effects of these cytokines on immune and inflammatory responses.
The first vector constructed, Ad5E3mlL6, contained the murine IL-6 gene incorporated into the E3 region of the viral genome and was used to characterize the capacity of recombinant adenovirus vectors for cytokine expression. This vector was very efficient for cytokine expression both in vitro and in vivo. In addition, using an adenovirus vector containing luciferase as a reporter gene, we demonstrated that expression could be targeted in a highly tissue-specific manner dependent upon the route of administration.
Since IL-6 was reported to be the major mediator of the acute phase response and intraperitoneal administration of adenovirus vector primarily targeted cytokine expression to the liver and spleen in Balb/c mice, our initial investigation involved intraperitoneal injection of the Ad5E3mlL6 vector into Balb/c mice. This study confirmed in vivo biological roles for IL-6 as a major mediator of the acute phase response and as a B and T cell proliferation factor.
We then analyzed the effects of expression of IL-5 and IL-6, alone and in combination, on humoral immune responses in the mucosal tissue of the lung. Both cytokines, produced by T helper type 2 lymphocytes, are critical to the development and differentiation of B lymphocytes and in particular to IgA antibody production in the mucosa-associated lymphoid tissue (MALT) and bronchus-associated lymphoid tissue (BALT). These studies, using Ad5E3mlL5 (a vector expressing murine IL-5 in the E3 region of the virus) in conjunction with Ad5E3mIL6, provided in vivo support for the roles of IL-5 and IL-6 in inducing lung mucosal immune responses. Co-administration of these two vectors in C57BI/6 mice synergistically induced up to a-fold increases in antigen-specific IgA antibody production in the lung.
In addition, we studied the in vivo effects of RANTES, a molecule reported to be chemotactic for monocytes, on lung inflammatory responses. A vector, Ad5E3mRANTES, was constructed which contained the murine RANTES cDNA in the E3 region of the virus. This vector, when intratracheally instilled into Sprague Dawley rats, targeted expression to the mucosal tissue and induced the recruitment of monocytes to the lung within 24 hours. These effects were transient and this expression did not result in detectable lasting pathologic changes to the organ. These in vivo findings on RANTES function are consistent with its proposed function as a potent monocyte chemotactic factor.
Finally, we applied this newly developed adenoviral technology to immune modulation in an animal model of breast cancer. Mammary tumor cells from transgenic mice expressing the polyoma middle T antigen under the control of the mouse mammary tumor virus promoter were transplanted into syngeneic mice to establish subcutaneous tumors. These tumors were directly injected with control virus or Ad5E1mlL6A + vector, a replication-deficient vector containing the murine IL-6 gene in the E1 region of the Ad5 genome. We found that localized vector-derived expression of IL-6 could attenuate tumor growth.
In conclusion, the results of this thesis study demonstrate the practical use for recombinant adenovirus vectors to aid in the investigation of cytokine function in vivo. This demonstration that vector-derived expression of cytokine is high and can be targeted to specific tissues suggests their use as potential therapeutic agents for the modulation of immune responses in the treatment of cancer and other diseases.
Braciak, Todd A., "Adenovirus Vectors for Cytokine Gene Expression" (1995). Open Access Dissertations and Theses. Paper 2264.