Adhesion regulation of stromal cell-derived factor-1 activation of ERK in lymphocytes by phosphatases.

We have investigated whether chemokine signaling to the extracellular-signal-regulated kinase (ERK) was regulated by beta 1-integrin-mediated adhesion in B- and T-cell lines. Activation of ERK by the chemokine SDF-1 can be regulated by adhesion to beta 1-integrin substrates in the T-cell lines MOLT-3, Jurkat, and H9 and in the Daudi B-cell line. In Jurkat T-cells, adhesion to the immobilized alpha 4 beta 1-integrin ligand VCAM-1 or to the alpha 5 beta 1-integrin ligand fibronectin regulated stromal-cell derived factor-1 (SDF-1) activation of ERK. Adhesion control of SDF-1 signaling was a rapid event, occurring as early as 10 min after adhesion, and loss of signaling occurred within 10 min of deadhesion. In contrast, SDF-1 activation of the ERK kinase MEK was independent of adhesion. Partial restoration of signaling to ERK in suspension was accomplished by pretreatment with pharmacological inhibitors of serine/threonine or protein-tyrosine phosphatases. In addition, we used a non-radioactive phosphatase assay using phosphorylated ERK as the substrate to determine relative ERK dephosphorylation in whole cell extracts. These results showed greater relative ERK dephosphorylation in extracts from Jurkat cells treated in suspension, as compared with adherent cells. Therefore, these data suggest that adhesion influences SDF-1 activation of ERK by regulating the activity of ERK phosphatases. This identifies a novel locus of adhesion regulation of the ERK cascade.

Versatility of merocyanine 540 for the flow cytometric detection of apoptosis in human and murine cells.

Since apoptosis plays many roles in development, immune function, and disease, there is an ongoing need to identify inexpensive and reliable fluorochromes for the quantitation of apoptosis. Merocyanine 540 (MC540) binds to the outer membrane of cells and readily fluoresces in the highly disordered membranes of apoptotic cells making them readily detectable by flow cytometry. Protocols for the effective labeling and gating of MC540br apoptotic cells are provided. For example, MC540br cells from dexamethasone (Dex) treated thymocytes were found to be equivalent in proportion to apoptotic cells noted in the propidium iodide (PI) stained and annexin-V stained populations. Sorting of the MC540br cells followed by counterstaining with PI demonstrated that these cells resided in the low DNA fluorescent or sub-G1 region and were small in size based on light scatter. Dexamethasone, etoposide, irradiation, and a calcium ionophore were used to induce cell death with equivalent numbers of apoptotic cells obtained with MC540 and PI. Moreover, apoptotic human bone marrow (BM) B cells, neutrophils, Jurkat T cells, and testicular cells could readily be identified with MC540. The latter is particularly noteworthy since some of the standard methods for identifying cell death have not worked well with human cells. The versatility of this dye is such that it was also possible to phenotypically label cells stained with MC540 to analyze apoptosis in heterogenous populations of cells. Finally, the rate of detection of apoptotic cells after treatment of thymocytes with dexamethasone at 2, 4, 6, and 8 h with MC540 was shown to be equivalent to PI and annexin-V. Taken together, the data demonstrate that when proper precautions are taken, MC540 is a reliable, versatile, and inexpensive fluorochrome that can be used to identify apoptotic cells of human or murine origin even in heterogenous populations that require multicolor labeling.

Rapid changes in the lymphopoietic and granulopoietic compartments of the marrow caused by stress levels of corticosterone.

Exposure to concentrations of glucocorticoids analogous to those produced during stress, trauma and malnutrition had rapid but varying effects on the major classes of cells within the marrow. Corticosterone (CS) was given as a subdermal implant in young mice and generated 60-95 microg CS/dl of blood compared to 5-15 microg CS/dl for sham controls over a period of 36 hr. Within 24 hr CS had caused losses of 30-70% among the early pro-B, pre-B and immature B cells. The pre-B cells were virtually eliminated by 36 hr and the capacity of surviving pro- and pre-B cells to cycle was reduced by 70-80%. Interestingly, the earliest of B cells, the prepro-B cells, showed considerable resistance to CS, being reduced by only 20% at 36 hr. Thus, the pattern of survival within the B-cell compartment paralleled the expression of Bcl-2. At the 36-hr time-point there were no changes in the proportion of progenitor cells, erythroid or monocytic cells, or number of nucleated cells in the marrow. By contrast, 36 hr after exposure to CS there was an increase of 30% in the proportion and absolute number of cells in the granulocytic compartment. Chronic production of CS appears to reprogramme lymphopoiesis and myelopoiesis, perhaps to preserve the first line of immune defence at the expense of the lymphoid branch. Resistance to apoptosis and modifications in the activity of the glucocorticoid receptor and cytokines produced by stromal cells are postulated as targets for CS-driven changes.

IL-7-mediated protection of pro and pre-B cells from the adverse effects of corticosterone.

The studies herein demonstrate that Interleukin-7 (IL-7) promotes survival of murine pro- and pre-B cells against stress levels of corticosterone (Cs). In short-term, 16-h, bone marrow cultures IL-7 abrogated Cs-induced apoptosis and cell cycle arrest in pro-B cells by decreasing apoptosis 60% and completely restoring the cell cycle. IL-7 also reduced Cs-induced apoptosis by 36% in pre-B cells and 24% in IgM(+) B cells, but did not restore deficits in the cell cycle. Among pro- and pre- B cells, substantial protection against high, pharmacological, levels of Cs was also provided by IL-7. Interestingly, stem cell factor, while reducing spontaneous apoptosis in pro-B cells, did not protect against Cs-induced death, either alone or with IL-7. In conclusion, IL-7 has potential immunotherapeutic value since it provides substantial protection to pro- and pre-B cells against the adverse effects of Cs.