Alternative Pathways of T‐Cell Activation and Positive Clonal Selection

476

360

Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones. Although expression of this gene has been demonstrated in multiple tissues, its regulation is not well understood. Our previous studies on the roles of the retinoblastoma protein (pRB) and p300͞CBP in the regulation of osteoblast differentiation revealed a link between osteopontin induction and the synthesis of alkaline phosphatase. In this paper, we describe results specifically linking induction of osteopontin to the enzymatic activity of alkaline phosphatase in the medium, which results in the generation of free phosphate. This elevation of free phosphate in the medium is sufficient to signal induction of osteopontin RNA and protein. The strong and specific induction of osteopontin in direct response to increased phosphate levels provides a mechanism to explain how expression of this product is normally regulated in bone and suggests how it may become up-regulated in damaged tissue. A s cells undergo terminal differentiation, various markers are induced in an ordered and sequential manner, but required steps in the induction process are not always clear because individual events in the sequence are not easily separable. The biology of the DNA tumor virus oncogene, adenovirus E1A, points to the cellular E1A targets, retinoblastoma protein (pRB) and p300͞CBP, as important regulators of gene expression during terminal differentiation (1-4). We have taken advantage of E1A genetics to explore the roles of the pRB and p300͞CBP protein families in expression of early and late markers during osteoblast differentiation. The MC3T3-E1 cell line is derived from newborn mice calvaria (5). It is an established cell line, but the cells maintain much of the tightly linked controls between proliferation and differentiation that usually are seen only in primary cells (6). Treatment with ascorbic acid stimulates these cells to differentiate along the osteoblast line (6-8). Induced cells deposit a collagenous extracellular matrix, accompanied by the activation of specific genes associated with the osteoblast phenotype, such as alkaline phosphatase, osteocalcin, and osteopontin. If a source of organic phosphate such as ␤-glycerol phosphate is present, a discrete zone of hydroxyapatitecontaining mineral is formed within the collagen fibrils. The sequence from induction to mineralization proceeds in a tightly regulated order over a span of 2 to 3 weeks (see schematic in Fig. 1), which permits a detailed analysis of the order of events.We previously have characterized two MC3T3-E1 cell lines stably express...

Section: Discussionmentioning

confidence: 97%

Phosphate is a specific signal for induction of osteopontin gene expression

Beck

Zerler

Morán

2000

476

360

“…Genes identified with this strategy then were studied in human cells exposed to M. tuberculosis and in tissues from patients with tuberculosis. One gene identified repeatedly in this screen was particularly interesting because its protein product, osteopontin, is a cytokine and macrophage chemoattractant (20,21). Osteopontin gene expression was induced by mycobacterial infection of human macrophages, and osteopontin protein was found in human tissue specimens from patients with clinical tuberculosis.…”

mentioning

confidence: 98%

A chemoattractant cytokine associated with granulomas in tuberculosis and silicosis

Nau

Guilfoile

Chupp

et al. 1997

183

145

Chronic inflammation and granuloma formation are associated with mononuclear cell infiltrates and are characteristic pathologic responses in tuberculosis. To identify host cell genes involved in tuberculous pathology, we screened macrophage cDNA libraries for genes induced by mycobacterial infection. One gene isolated in this screen, osteopontin (also known as early T lymphocyte activation protein 1 or Eta-1), was of particular interest because it is a cytokine and macrophage chemoattractant. Further study revealed that Mycobacterium tuberculosis infection of primary human alveolar macrophages causes a substantial increase in osteopontin gene expression. Osteopontin protein was identified by immunohistochemistry in macrophages, lymphocytes, and the extracellular matrix of pathologic tissue sections of patients with tuberculosis. Increased osteopontin expression also was found to be associated with silicosis, another granulomatous disease. The association of osteopontin with granulomatous pathology, together with the known properties of the protein, suggest that osteopontin may participate in granuloma formation. The strategy of identifying host genes whose expression is altered by infection thus can provide valuable clues to disease mechanisms and will be increasingly valuable as additional human genome sequences become available.Mycobacterial infections are among the most numerous in the world, with Mycobacterium tuberculosis believed to have infected one-third of the world's population (1). Aggravating the worldwide pandemic have been the emergence of resistant organisms and the concurrent HIV epidemic. These events have renewed interest in understanding the fundamental biology of the interactions between pathogenic mycobacteria and their host.Exposure to M. tuberculosis can lead to a pulmonary infection characterized by macrophage recruitment to the site of infection, followed in many cases by granuloma formation (2-4). M. tuberculosis is a facultative intracellular pathogen whose cellular habitat is the macrophage (5-9). Thus, the M. tuberculosis-macrophage relationship is of considerable interest. Previous investigations have revealed that interleukin 1, tumor necrosis factor-␣, interleukin 6, transforming growth factor-␤, and interleukin 8 production by macrophages is increased by exposure to mycobacteria (10)(11)(12)(13)(14). Negative effects of mycobacteria on macrophage antigen presentation (15) and responsiveness to cytokines (16, 17) also have been documented. However, little is known about the molecular mechanisms involved in granuloma formation.To gain insight into the host response to tuberculosis, we used differential screening of cDNA libraries to compare mRNAs of infected and uninfected macrophages. We reasoned that the identification of macrophage genes whose expression is altered after phagocytosis of M. tuberculosis might provide clues to pathogenesis. Using a model system that used a macrophage cell line (18, 19), we surveyed the population of macrophage mRNAs for those altered by my...

“…T cells that express the CD4-8+ phenotype (CD8 cells) recognize antigen associated with class I MHC products and mediate killer activity (reviewed in ref. 1).…”

mentioning

confidence: 99%

Thymocyte development in major histocompatibility complex-deficient mice: evidence for stochastic commitment to the CD4 and CD8 lineages.

Crump

Grusby

Glimcher

et al. 1993

The mechanism resulting in commitment of precursor cells in the thymus to either the CD4 or CD8 lineage remains poorly understood. In principle, this may reflect a stochastic process or may reflect instructional signals from host major histocompatiblity complex (MHC) molecules. We have examined the role of MHC products in subset commitment by using mice deficient in class I or class II MHC products. Normal numbers of committed CD4 intermediates (CD4+ CD8I0) develop in the thymus in the absence of class II molecules. Similarly, CD8 transitional cells (CD4IOCD8+) are present in the thymus of mice lacking class I products. These findings suggest that commitment of CD4+8+ precursor cells to either lineage is a stochastic process that does not depend on instructive signals from MHC molecules (i.e., expression of alternative differentiative options by uncommitted precursor cells is independent of this environmental signal). These studies also suggest that an interaction between the T-cell antigen receptor (TCR) and MHC molecules that is independent of CD4/CD8 coreceptor engagement enhances stochastic coreceptor downregulation substantially and leads to upregulation of TCR expression as a prelude to selective events that require joint coreceptor/TCR engagement. We suggest that this initial interaction molds the TCR repertoire of stochastically generated T-cell subsets toward recognition of self-MHC products.The thymus gives rise to two lineages of T lymphocytes that regulate immune responses. T cells that express the CD4+8-surface phenotype (CD4 cells) recognize antigen associated with class II major histocompatibility complex (MHC) products and mediate helper activity. T cells that express the CD4-8+ phenotype (CD8 cells) recognize antigen associated with class I MHC products and mediate killer activity (reviewed in ref. 1).The developmental steps resulting in the formation of these independent T-cell lineages remain poorly understood. Precursor cells in the thymus that expresses the CD4+8+ surface phenotype [termed double-positive (DP) thymocytes (2, 3)] downregulate either CD4 or CD8 to generate lineagecommitted intermediaries expressing the CD4+CD8l0 or CD41OCD8+ phenotype (4, 5). Development of mature singlepositive (SP) CD4 or CD8 progeny depends upon positive selection by intrathymic MHC molecules (6-9). The timing and regulation of this process have been studied primarily by using mice that express transgenic T-cell antigen receptors (TCRs) and/or coreceptors (CD4 and CD8) (10-13). Although this experimental model has contributed substantially to our understanding of thymocyte development, interpretation of these studies may be complicated by abnormally highThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.or premature expression of these transgenic products on thymocytes.Two general models have been proposed to explain the mechanism by which a si...