Juan J. Muñoz

Expression and Function of the Eph A Receptors and Their Ligands Ephrins A in the Rat Thymus

Thymus development and function are dependent on the definition of different and graded microenvironments that provide the maturing T cell with the different signals that drive its maturation to a functional T lymphocyte. In these processes, cell-cell interactions, cell migration, and positioning are clues for the correct functioning of the organ. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, has been implicated in all these processes by regulating cytoskeleton and adhesion functioning, but a systemic analysis of their presence and possible functional role in thymus has not yet been conducted. In this regard, the current study combines different experimental approaches for analyzing the expression of four members of the Eph A family and their ligands, ephrins A, in the embryonic and adult rat thymus. The patterns of Eph and ephrin expression in the distinct thymic regions were different but overlapping. In general, the studied Eph A were expressed on thymic epithelial cells, whereas ephrins A seem to be more restricted to thymocytes, although Eph A1 and ephrin A1 are expressed on both cell types. Furthermore, the supply of either Eph A-Fc or ephrin A-Fc fusion proteins to fetal thymus organ cultures interferes with T cell development, suggesting an important role for this family of proteins in the cell mechanisms that drive intrathymic T cell development.

Thymic Alterations in EphA4-Deficient Mice

In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4+CD8+) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 −/− phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44−CD25+ triple-negative cells and, sometimes, of CD44+CD25− triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 −/− epithelial cells affects their development and finally results in altered T cell development.

Distinct Mechanisms Contribute to Generate and Change the CD4:CD8 Cell Ratio During Thymus Development: A Role for the Notch Ligand, Jagged1

In adult life, the high CD4:CD8 cell ratio observed in peripheral lymphoid organs originates in the thymus. Our results show that the low peripheral CD4:CD8 cell ratio seen during fetal life also has an intrathymic origin. This distinct production of CD4+CD8− and CD4−CD8+ thymocytes is regulated by the developmental age of the thymic stroma. The differential expression of Notch receptors and their ligands, especially Jagged1, throughout thymus development plays a key role in the generation of the different CD4:CD8 cell ratios. We also show that the intrathymic CD4:CD8 cell ratio sharply changes from fetal to adult values around birth. Differences in the proliferation and emigration rates of the mature thymocyte subsets contribute to this change.

EphrinB1-EphB signaling regulates thymocyte-epithelium interactions involved in functional T cell development

The Eph and ephrin families are involved in numerous developmental processes. Recently, an increasing body of evidence has related these families with some aspects of T cell development. In the present study, we show that the addition of either EphB2‐Fc or ephrinB1‐Fc fusion proteins to fetal thymus organ cultures established from 17‐day‐old fetal mice decreases the numbers of both double‐positive (CD4+CD8+) and single‐positive (both CD4+CD8– and CD4–CD8+) thymocytes, in correlation with increased apoptosis. By using reaggregate thymus organ cultures formed by fetal thymic epithelial cells (TEC) and CD4+CD8+ thymocytes, we have also demonstrated that ephrinB1‐Fc proteins are able to disorganize the three‐dimensional epithelial network that in vivo supports the T cell maturation, and to alter the thymocyte interactions. In addition, in an in vitro model, Eph/ephrinB‐Fc treatment also decreases the formation of cell conjugates by CD4+CD8+ thymocytes and TEC as well as the TCR‐dependent signaling between both cell types. Finally, immobilized EphB2‐Fc and ephrinB1‐Fc modulate the anti‐CD3 antibody‐induced apoptosis of CD4+CD8+ thymocytes in a process dependent on concentration. These results therefore support a role for Eph/ephrinB in the processes of development and selection of thymocytes as well as in the establishment of the three‐dimensional organization of TEC.

EphB2-mediated interactions are essential for proper migration of T cell progenitors during fetal thymus colonization

The ephrin‐Eph ligand receptor pair is known to control the repulsion/adhesion process in different tissues, including the immune system. Herein, we evaluated the role of EphB2 receptors in T cell progenitor migration during in vitro thymus colonization and to ECM or chemokine stimuli. EphB2 and their ligands, ephrin‐B1 and ephrin‐B2, are expressed in BM‐derived progenitors, and EphB2−/− cells had diminished thymus colonization capacity. Conversely, EphB2LacZ cells, which maintain a preserved ephrin‐binding domain, were capable of colonizing WT thymuses similarly to WT progenitors, highlighting the importance of reverse signals transmitted to normal fetal thymus. However, the EphB2 receptor expressed by microenvironmental cells also drives progenitor immigration, as recolonization of EphB2‐deficient fetal thymuses was compromised profoundly. Additionally, we observed lower depositions of ECM and chemokines on EphB2‐deficient thymuses but no changes in their receptor expression on BM‐derived progenitors and developing thymocytes. Migration of EphB2‐deficient progenitors and thymocytes was also reduced through ECM or chemokine stimuli. Furthermore, ephrin‐B1 costimulation also inhibited haptotaxis and chemotaxis of WT but not EphB2LacZ cells, demonstrating the specific involvement of EphB2 signaling on T cell progenitor migration. Our data suggest the relevance of a nonactivated EphB2 for regulating T cell progenitor migration and its modulation upon ephrin‐B engagement.

Oxidative polymorphism of debrisoquine in Parkinson's disease.

Oxidative phenotype and metabolic ratio (MR) of debrisoquine (DBQ) have been determined in 87 patients with Parkinsons disease and in 556 healthy control subjects. Three patients (3.45%) and 34 control subjects (6.12%), having an MR greater than 12.6, were classified as poor metabolisers (PM) of DBQ (ns). The distribution of MR values in the 84 Parkinsonian patients classified as extensive metabolisers (EM) showed a less efficient oxidative rate when compared with controls of the same phenotype (p less than 0.001). This difference may be due to enzymatic inhibition caused by drug treatment in 40 of these patients. As in patients not taking any drug known to inhibit the oxidation of DBQ, distribution of MR values was not different from that in controls. A negative correlation (r = -0.36, p less than 0.02) was found between MR of DBQ and age at onset of disease in patients free of drugs known to interact with DBQ metabolism. A higher rate of DBQ oxidation could be a genetic factor that delays the clinical onset of Parkinsons disease in predisposed people.

Alterations in the thymocyte phenotype of EphB‐deficient mice largely affect the double negative cell compartment

In the present study, we have analysed the phenotype of EphB2 and/or EphB3 deficient thymocytes confirming and extending previous studies on the role of this family of molecules in T‐cell differentiation. In all mutant thymuses statistically significant reduced cell contents were observed. This reduction of thymic cellularity correlated with increased proportions of apoptotic cells, largely both double negative (DN; CD4− CD8−) and double positive (CD4+ CD8+) cells, and decreased proportions of DN cycling cells. Adult deficient thymuses also showed increased proportions of DN cells but not significant variations in the percentages of other thymocyte subsets. In absolute terms, the thymocyte number decreased significantly in all thymocyte compartments from the DN3 (CD44− CD25+) cell stage onward, without variations in the numbers of both DN1 (CD44+ CD25−) and DN2 (CD44+ CD25+) cells. Remarkably, all these changes also occurred from the 15‐day fetal EphB2 and/or EphB3 deficient mice, suggesting that adult phenotype results from the gradual accumulations of defects appearing early in the thymus ontogeny. As a reflection of thymus condition, a reduction in the number of T lymphocytes occurred in the peripheral blood and mesenteric lymph nodes, but not in spleen, maintaining the proportions of T‐cell subsets defined by CD4/CD8 marker expression, in all cases.

Both tacrolimus and sirolimus decrease Th1/Th2 ratio, and increase regulatory T lymphocytes in the liver after ischemia/reperfusion

The protective effects of immunosuppressants against ischemia/reperfusion (I/R) injury have been attributed to their non-specific anti-inflammatory effect. However, these effects may also depend on their effect on T lymphocytes, which are increasingly considered to be key players in I/R. Here, we studied the effects of tacrolimus and sirolimus on lymphocyte subpopulations in an I/R rat model. The animals were treated with tacrolimus, sirolimus or vehicle, before undergoing a 60-min ischemia event of the right hepatic lobe, followed by excision of the remaining liver. After 2 h, I/R rats showed increased mortality, plasma lactate dehydrogenase (LDH) levels, hepatocyte apoptosis, liver histological injury and parenchymal infiltration by neutrophils, macrophages, NK cells and T lymphocytes. Most of the changes were antagonized by both immunosuppressants. Tacrolimus augmented the proportion of cycling cells in I/R rats, whereas sirolimus showed the opposite effect. The increased Th1/Th2 ratio found in I/R livers after 2 h was reverted by immunosuppressants, which also amplified the proportion of CD4+CD25+Foxp3+ regulatory T lymphocytes at 24 h. The protective effects of both tacrolimus and sirolimus correlated well with a decreased ratio of proinflammatory to anti-inflammatory T lymphocytes, and with an increase in the Treg proportion. This suggests a new mechanism to explain the known beneficial effect shown by immunosuppressants early after I/R.

Polymorphic Oxidation of Debrisoquine in Women with Breast Cancer

Oxidative polymorphism of debrisoquine (DBQ) was determined in 98 women with breast cancer (mean age 59.2 years, SD 10.7; 18 were premenopausal when diagnosed), and in 446 healthy control women (mean age 25.4 years, SD 9.15). All of them were free of drug interactions with oxidation of DBQ. Ten patients (10.2%), all of them postmenopausal when diagnosed, and 423 controls (5.2%), with values for metabolic ratio of DBQ greater than 12.6, were classified as poor metabolizers (PM) of DBQ (p = 0.006). Relative risk for developing breast cancer among women with PM phenotype was 2.09 (95% confidence limits 0.97-4.48). The PM status of DBQ might be a secondary risk factor for breast cancer in postmenopausal women.

CYP2D6 genotypes in Spanish women with breast cancer

Wild type and three mutated alleles of the polymorphic CYP2D6 gene were studied in genomic DNA samples from 187 women with breast carcinoma and 151 healthy women by a mutation-specific polymerase chain reaction. The prevalence of the enzyme-inactivating CYP2D6(B) allele was higher among patients (18.2%) than in controls (11.6%; OR = 1.7; 95% c.i. = 1.14-3.13; P = 0.018). This excess was more marked in postmenopausal patients (19.8%, P = 0.0086) and in patients with non-ductal infiltrating carcinomas (25.8%, P = 0.003). The percentage of carriers of only one active gene (heterozygote extensive metabolizers) was higher in patients (31% vs. 19.9%; OR = 1.81; 95% c.i. = 1.06-3.11; P = 0.02). The CYP2D6(B)-carrier state may be related to a greater risk of breast cancer in women.