Anatomical and developmental patterns of interleukin-2 gene expression in the mouse: analysis of IL-2 expressing cells and partial characterization of interactions involved in mediating IL-2 gene expression in vivo

Interleukin-2 (IL-2) is an important mediator in the vertebrate immune system. IL-2 is a potent growth factor that mature T lymphocytes use as a proliferation signal and the production of IL-2 is crucial for the clonal expansion of antigen-specific T cells in the primary immune response. IL-2 driven proliferation is dependent on the interaction of the lymphokine with its cognate multichain receptor. IL-2 expression is induced only upon stimulation and transcriptional activation of the IL-2 gene relies extensively on the coordinate interaction of numerous inducible and constitutive trans-acting factors. Over the past several years, thousands of papers have been published regarding molecular and cellular aspects of IL-2 gene expression and IL-2 function. The vast majority of these reports describe work that has been carried out in vitro. However, considerably less is known about control of IL-2 gene expression and IL-2 function in vivo.

To gain new insight into the regulation of IL-2 gene expression in vivo, anatomical and developmental patterns of IL-2 gene expression in the mouse were established by employing in situ hybridization and immunohistochemical staining methodologies to tissue sections generated from normal mice and mutant animals in which T -cell development was perturbed. Results from these studies revealed several interesting aspects of IL-2 gene expression, such as (1) induction of IL-2 gene expression and protein synthesis in the thymus, the primary site of T-cell development in the body, (2) cell-type specificity of IL-2 gene expression in vivo, (3) participation of IL-2 in the extrathymic expansion of mature T cells in particular tissues, independent of an acute immune response to foreign antigen, (4) involvement of IL-2 in maintaining immunologic balance in the mucosal immune system, and (5) potential function of IL-2 in early events associated with hematopoiesis.

Extensive analysis of IL-2 mRNA accumulation and protein production in the murine thymus at various stages of development established the existence of two classes of intrathymic IL-2 producing cells. One class of intrathymic IL-2 producers was found exclusively in the fetal thymus. Cells belonging to this subset were restricted to the outermost region of the thymus. IL-2 expression in the fetal thymus was highly transient; a dramatic peak ofiL-2 mRNA accumulation was identified at day 14.5 of gestation and maximal IL-2 protein production was observed 12 hours later, after which both IL-2 mRNA and protein levels rapidly decreased. Significantly, the presence of IL-2 expressing cells in the day 14-15 fetal thymus was not contingent on the generation of T-cell receptor (TcR) positive cells. The second class of IL-2 producing cells was also detectable in the fetal thymus (cells found in this class represented a minority subset of IL-2 producers in the fetal thymus) but persist in the thymus during later stages of development and after birth. Intrathymic IL-2 producers in postnatal animals were located in the subcapsular region and cortex, indicating that these cells reside in the same areas where immature T cells are consigned. The frequency of IL-2 expressing cells in the postnatal thymus was extremely low, indicating that induction of IL-2 expression and protein synthesis are indicative of a rare activation event. Unlike the fetal class of intrathymic IL-2 producers, the presence of IL-2 producing cells in the postnatal thymus was dependent on to the generation of TcR+ cells. Subsequent examination of intrathymic IL-2 production in mutant postnatal mice unable to produce either αβ or γδ T cells showed that postnatal IL-2 producers in the thymus belong to both αβ and γδ lineages. Additionally, further studies indicated that IL-2 synthesis by immature αβ -T cells depends on the expression of bonafide TcR αβ-heterodimers. Taken altogether, IL-2 production in the postnatal thymus relies on the generation of αβ or γδ-TcR^+ cells and induction of IL-2 protein synthesis can be linked to an activation event mediated via the TcR.

With regard to tissue specificity of IL-2 gene expression in vivo, analysis of whole body sections obtained from normal neonatal mouse pups by in situ hybridization demonstrated that IL-2 mRNA^+ cells were found in both lymphoid and nonlymphoid tissues with which T cells are associated, such as the thymus (as described above), dermis and gut. Tissues devoid of IL-2 mRNA^+ cells included brain, heart, lung, liver, stomach, spine, spinal cord, kidney, and bladder. Additional analysis of isolated tissues taken from older animals revealed that IL-2 expression was undetectable in bone marrow and in nonactivated spleen and lymph nodes. Thus, it appears that extrathymic IL-2 expressing cells in nonimmunologically challenged animals are relegated to particular epidermal and epithelial tissues in which characterized subsets of T cells reside and thatinduction of IL-2 gene expression associated with these tissues may be a result of T-cell activation therein.

Based on the neonatal in situ hybridization results, a detailed investigation into possible induction of IL-2 expression resulting in IL-2 protein synthesis in the skin and gut revealed that IL-2 expression is induced in the epidermis and intestine and IL-2 protein is available to drive cell proliferation of resident cells and/or participate in immune function in these tissues. Pertaining to IL-2 expression in the skin, maximal IL-2 mRNA accumulation and protein production were observed when resident Vγ_3^+ T-cell populations were expanding. At this age, both IL-2 mRNA^+ cells and IL-2 protein production were intimately associated with hair follicles. Likewise, at this age a significant number of CD3ε^+ cells were also found in association with follicles. The colocalization of IL-2 expression and CD3ε^+ cells suggests that IL-2 expression is induced when T cells are in contact with hair follicles. In contrast, neither IL-2 mRNA nor IL-2 protein were readily detected once T-cell density in the skin reached steady-state proportions. At this point, T cells were no longer found associated with hair follicles but were evenly distributed throughout the epidermis. In addition, IL-2 expression in the skin was contingent upon the presence of mature T cells therein and induction of IL-2 protein synthesis in the skin did not depend on the expression of a specific TcR on resident T cells. These newly disclosed properties of IL-2 expression in the skin indicate that IL-2 may play an additional role in controlling mature T-cell proliferation by participating in the extrathymic expansion of T cells, particularly those associated with the epidermis.

Finally, regarding IL-2 expression and protein synthesis in the gut, IL-2 producing cells were found associated with the lamina propria of neonatal animals and gut-associated IL-2 production persisted throughout life. In older animals, the frequency of IL-2 producing cells in the small intestine was not identical to that in the large intestine and this difference may reflect regional specialization of the mucosal immune system in response to enteric antigen. Similar to other instances of IL-2 gene expression in vivo, a failure to generate mature T cells also led to an abrogation of IL-2 protein production in the gut. The presence of IL-2 producing cells in the neonatal gut suggested that these cells may be generated during fetal development. Examination of the fetal gut to determine the distribution of IL-2 producing cells therein indicated that there was a tenfold increase in the number of gut-associated IL-2 producers at day 20 of gestation compared to that observed four days earlier and there was little difference between the frequency of IL-2 producing cells in prenatal versus neonatal gut. The origin of these fetally-derived IL-2 producing cells is unclear. Prior to the immigration of IL-2 inducible cells to the fetal gut and/or induction of IL-2 expression therein, IL-2 protein was observed in the fetal liver and fetal omentum, as well as the fetal thymus. Considering that induction of IL-2 protein synthesis may be an indication of future functional capability, detection of IL-2 producing cells in the fetal liver and fetal omentum raises the possibility that IL-2 producing cells in the fetal gut may be extrathymic in origin and IL-2 producing cells in these fetal tissues may not belong solely to the T lineage. Overall, these results provide increased understanding of the nature of IL-2 producing cells in the gut and how the absence of IL-2 production therein and in fetal hematopoietic tissues can result in the acute pathology observed in IL-2 deficient animals.

Constraining the interpretation of 2-methylhopanoids through genetic and phylogenetic methods

Hopanoids are a class of sterol-like lipids produced by select bacteria. Their preservation in the rock record for billions of years as fossilized hopanes lends them geological significance. Much of the structural diversity present in this class of molecules, which likely underpins important biological functions, is lost during fossilization. Yet, one type of modification that persists during preservation is methylation at C-2. The resulting 2-methylhopanoids are prominent molecular fossils and have an intriguing pattern over time, exhibiting increases in abundance associated with Ocean Anoxic Events during the Phanerozoic. This thesis uses diverse methods to address what the presence of 2-methylhopanes tells us about the microbial life and environmental conditions of their ancient depositional settings. Through an environmental survey of hpnP, the gene encoding the C-2 hopanoid methylase, we found that many different taxa are capable of producing 2-methylhopanoids in more diverse modern environments than expected. This study also revealed that hpnP is significantly overrepresented in organisms that are plant symbionts, in environments associated with plants, and with metabolisms that support plant-microbe interactions; collectively, these correlations provide a clue about the biological importance of 2-methylhopanoids. Phylogenetic reconstruction of the evolutionary history of hpnP revealed that 2-methylhopanoid production arose in the Alphaproteobacteria, indicating that the origin of these molecules is younger than originally thought. Additionally, we took genetic approach to understand the role of 2-methylhopanoids in Cyanobacteria using the filamentous symbiotic Nostoc punctiforme. We found that hopanoids likely aid in rigidifying the cell membrane but do not appear to provide resistance to osmotic or outer membrane stressors, as has been shown in other organisms. The work presented in this thesis supports previous findings that 2-methylhopanoids are not biomarkers for oxygenic photosynthesis and provides new insights by defining their distribution in modern environments, identifying their evolutionary origin, and investigating their role in Cyanobacteria. These efforts in modern settings aid the formation of a robust interpretation of 2-methylhopanes in the rock record.

Geology of the Pacoima Area, Los Angeles County, California

The Pacoima area is located on an isolated hill in the northeast section of the San Fernando, the northeast portion of the Pacoima Quadrangle, Los Angeles County, California. Within it are exposed more than 2300 feet of Tertiary rocks, which comprise three units of Middle Miocene (?) age, and approximately 950 feet of Jurassic (?) granite basement. The formations are characterized by their mode of occurrence, marine and terrestial origin, diverse lithology, and structural features.

The basement complex is composed of intrusive granite, small masses of granodiorite and a granodiorite gneiss with the development of schistosity in sections. During the long period of erosion of the metamorphics, the granitic rocks were exposed and may have provided clastic constituents for the overlying formations.

As a result of rapid sedimentation in a transitional environment, the Middle Miocene Twin Peaks formation was laid down unconformably on the granite. This formation is essentially a large thinning bed of gray to buff pebble and cobble conglomerate grading to coarse yellow sandstone. The contact of conglomerate and granite is characterized by its faulted and depositional nature.

Beds of extrusive andesite, basalt porphyry, compact vesicular amygdaloidal basalts, andesite breccia, interbedded feldspathic sands and clays of terrestial origin, and mudflow breccia comprise the Pacoima formation which overlies the Twin Peaks formation unconformably. A transgressing shallow sea accompanied settling of the region and initiated deposition of fine clastic sediments.

The marine Topanga (?) formation is composed of brown to gray coarse sandstone grading into interbedded buff sandstones and gray shales. Intrusions of rhyolitedacite and ash beds mark continued but sporatic volcanism during this period.

The area mapped represents an arch in the Tertiary sediments. Forces that produced the uplift of the granite structural high created stresses that were relieved by jointing and faulting. Vertical and horizontal movement along these faults has displaced beds, offset contacts and complicated their structure. Uplift and erosion have exposed the present sequence of beds which dip gently to the northeast. The isolated hill is believed to be in an early stage of maturity.