Cytokine expression by inflammatory cells obtained from the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis induced by inoculation with myelin basic protein and adjuvants.

Inflammatory cells were obtained from the spinal cords of rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the expression of mRNA for interleukin-2 (IL-2), IL-4, IL-10 and interferon-gamma (IFN-gamma) by cells from groups of rats studied 10-21 days after inoculation. On all days of study, the inflammatory cells, which were predominantly lymphocytes, expressed mRNA for IL-2, IL-4, IL-10 and IFN-gamma. In the mRNA from normal rat spinal cord tissue, there was little expression of cytokine mRNA. Cells from a short-term MBP-reactive T cell line expressed all the cytokines. Densitometry was used to measure the products of PCR, to assess the expression of each cytokine relative to that of beta-actin. IL-2 mRNA was expressed throughout the course of disease and reached a peak on day 18, during late clinical recovery. IFN-gamma was expressed throughout the course of the disease and was also high during late recovery. IL-4 mRNA was present in the spinal cord throughout the course of the disease, with a slight rise during late recovery. Relative expression of IL-10 rose to a peak on days 17-19, during late recovery from clinical disease. This study indicates that IL-2, IL-4, IL-10 and IFN-gamma are expressed by inflammatory cells in the spinal cord in EAE, with the relative expression of all cytokines being high during late clinical recovery.

A rapid method for the measurement of the oxoreductase activity of 11beta-hydroxysteroid dehydrogenase in granulosa-lutein cells from patients undergoing in-vitro fertilization.

The presence of 11beta-hydroxysteroid dehydrogenase (11beta-HSD; EC1.1.1.146), the enzyme responsible for the interconversion of cortisol and cortisone, in granulosa-lutein (GL) cells is associated with a poor outcome in in-vitro fertilization (IVF). We have developed a simple method of assessing the reductase component of 11beta-HSD in these cells which is sufficiently rapid to provide data on the enzyme's activity prior to embryo replacement. Cells were pooled from follicular aspirates and challenged with cortisone within 2 h of aspiration. Cortisol secretion was then measured by radioimmunoassay. Conversion of cortisone to cortisol was linear for up to 3 h and was completely inhibited by glycyrrhetinic acid, a specific 11beta-HSD inhibitor. Initial velocity rates were determined for eight cortisone concentrations (range 0.1-8 micromol/l), and the apparent Km calculated (1.6 +/- 0.4 micromol/l). There was no evidence of substrate/product inhibition and conversion of cortisone to cortisol was <2% in all experiments. In subsequent work, cells were challenged with cortisone (6 micromol/l) for 2 h. Cells challenged for 2 h immediately following purification from follicular aspirates produced varying amounts of cortisol (range 25-150 nmol/pooled follicles from each patient, n = 10 patients), while basal outputs were <6 nmol/l. Enzyme activity was also examined in cells on a per follicle basis from individual patients and found to vary considerably (e.g. 19, 53 and 36 nmol/l cortisol/1000 cells, three follicles). Having established the method for assessing 11beta-reductase activity within GL cells, we performed a small prospective study on a series of 20 patients examining the enzyme activity within 110 individual follicles. 11Beta-reductase activity varied greatly from patient to patient and from follicle to follicle ranging from <0.024-0.57 nmol cortisol/microg DNA but at present low patient numbers preclude a meaningful correlation between enzyme activity and pregnancy rate. In summary, we have developed a simple, rapid (<8 h) assay for detecting the reductase activity of 11beta-HSD in GL cells isolated from pooled or individual follicles. This procedure is sufficiently quick to aid in the choice of embryo for replacement.

Corticosteroid treatment of experimental autoimmune encephalomyelitis in the Lewis rat results in loss of V beta 8.2+ and myelin basic protein-reactive cells from the spinal cord, with increased total T-cell apoptosis but reduced apoptosis of V beta 8.2+ cells.

We have studied the effects of corticosteroid treatment on the numbers of lymphocytes obtained from the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Flow cytometric studies showed that treatment with dexamethasone (4 mg/kg) 8-12 h prior to study on day 14 after inoculation resulted in a reduction in the numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells in the spinal cord. Limiting dilution analysis indicated that dexamethasone treatment 12 h prior to study on day 12 after inoculation reduced the frequencies of MBP-reactive and interleukin-2-responsive lymphocytes in the spinal cord to low levels, but reduced the frequency of concanavalin-A-responsive lymphocytes to a lesser extent. Using propidium iodide staining of nuclear chromatin we also studied lymphocyte apoptosis. Greater numbers of apoptotic cells were found in the cells extracted from the spinal cords of rats, examined on day 14, that had been treated 1-12 h previously with dexamethasone, than in saline-treated controls. This increased level of apoptosis was observed in the CD5+ and TCR alpha beta + cell populations. At 1-4 h after dexamethasone treatment there was a reduction in the selective apoptosis of V beta 8.2+ cells that normally occurs during spontaneous recovery from EAE. Therefore apoptosis of V beta 8.2+ cells cannot explain the reduction in the numbers of V beta 8.2+ cells and MBP-reactive cells in the CNS after dexamethasone treatment. By 8-12 h after dexamethasone treatment the selectivity of the apoptotic process was restored. These studies suggest that a reduction in the number of T-lymphocytes in the central nervous system contributes to the beneficial effects of corticosteroids in EAE.

Apoptosis of V beta 8.2+ T lymphocytes in the spinal cord during recovery from experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein.

To study T cell apoptosis during spontaneous recovery from experimental autoimmune encephalomyelitis (EAE), we extracted lymphocytes from the spinal cords of Lewis rats with EAE induced by inoculation with myelin basic protein (MBP) and adjuvants. Using flow cytometry we assessed the numbers of CD5+ and TCR alpha beta + lymphocytes, as well as V beta 8.2+ lymphocytes, which constitute the predominant encephalitogenic MBP-reactive cells in Lewis rats. Rats developed neurological signs of disease 10-12 days after inoculation. The peak of disease was on day 14 after inoculation and was followed by clinical recovery. The numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells obtained from the spinal cord were greatest on day 13. During spontaneous clinical recovery, there was a decline in the numbers of all the cells studied, with a selective loss of V beta 8.2+ cells from the CD5+ and TCR alpha beta + populations. To determine whether the decline in lymphocyte numbers was due to apoptosis, we used simultaneous surface labelling and propidium iodide staining of the DNA of the cells extracted from the spinal cord. From day 14 onwards, there was selective enrichment of V beta 8.2+ cells in the apoptotic population, and the percentage of V beta 8.2+ cells undergoing apoptosis was greater than the percentages of CD5+ and TCR alpha beta + cells undergoing apoptosis. These findings indicate that recovery from acute EAE is associated with the selective apoptosis, in the central nervous system, of these disease-relevant cells. The findings in this study of actively induced EAE are similar to those of our previous study of EAE induced by transfer of encephalitogenic MBP-specific T cells (Z. Tabi et al., Eur. J. Immunol. 24: 2609-2617, 1994) and further support the hypothesis that selective apoptosis of autoreactive T cells in the central nervous system is of primary importance in spontaneous recovery from EAE.