Biomedical subjects

G Gorgas

Publications and source records attributed to G Gorgas.

6 recordsLinked to original sources

PubMed1997

Diminished activation of the MAP kinase pathway in CD3-stimulated T lymphocytes from old mice.

Stimulation of the ERK family of protein kinases ('extracellular signal regulated kinases', also known as MAP kinases) plays an important role in the activation of many cell types, including T lymphocytes. ERKs are activated when they are phosphorylated by an upstream activator, the dual-specific protein kinase MEK. To see if aging leads to an impairment of MEK activation in mouse T cells, we used a mobility shift assay in which activation of MEK leads to phosphorylation and altered mobility of ERK-2 kinase. Similarly, we monitored mobility of pp90rsk, a known ERK substrate, as an indication of ERK function. We found an age-related decline in the ability of mouse T cells to activate both MEK and ERK function in response to stimulation by antibodies to the CD3 chain of the T cell receptor. Aging did not alter the kinetics of enzyme activation, but did diminish (by about 2-fold) the maximal level of substrate converted into the slower migrating form. Naive and memory CD4 T cells from young mice were equally able to convert ERK2 to its slower migrating form, suggesting that the decline in MEK function is not likely to be attributable to the shift, with age, from naive to memory T cell predominance. Our data suggest that age-dependent declines in gene activation, including genes for key cytokines like IL-2, may be due to declines in the upstream signals that lead to activation of the MEK/ERK protein kinase cascade.

Aging↗

PubMed1996

Reciprocal expression of P-glycoprotein and TAP1 accompanied by higher expression of MHC class I antigens in T cells of old mice.

We have shown previously that (a) aging leads to an increase in the proportion of murine splenic T cells that express high activity of P-glycoprotein (PGP), the ATP-dependent plasma membrane pump that mediates multiple drug resistance, and (b) PGPhi CD4 memory cells from mice of any age do not proliferate or secrete IL-4 after activation with anti-CD3 and IL2. We now report that the age-associated increase in expression of MHC Class I molecules is limited to the subset of T cells that overexpress PGP and thus extrude the fluorochrome R123 (the "R123lo" subset). Although H-2 levels increase on T cells of old mice, the levels of TAP1, a component of the polypeptide pump responsible for assembly and internal transport of Class I MHC molecules, decline, unexpectedly, by about fourfold in T cells from old donors. Thus, aging leads to reciprocal changes in the level of T-cell expression of PGP and TAP1, two closely related members of the ABC superfamily of peptide transport proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Extrusion of the P glycoprotein substrate rhodamine-123 distinguishes CD4 memory T cell subsets that differ in IL-2-driven IL-4 production.

Approximately 25% of T cells in young mice are able to extrude the fluorescent P glycoprotein substrate Rhodamine-123 (R123), and these R123low cells are present within the naive and memory cell populations of both CD4 and CD8 type. The proportion of R123low T cells increases with age to approximately 60% in mice older than 18 mo of age. CD4 memory T cells from young mice secrete more IL-4 compared with cells from old donors when activated by anti-CD3 and further cultured for 7 to 10 days in the presence of IL-2. To determine whether this age-related decline in IL-4 production was related to the parallel accumulation of R123low T cells within the CD4 memory subset, we compared IL-4 production in cell preparations enriched for R123high and R123low cells by electronic cell sorting. IL-2-driven IL-4 production by CD3-activated CD4 memory cells was found to be limited almost entirely to the R123high subset, i.e., the subset that declines with age. Proliferation under these culture conditions also declined with age and was also much more vigorous in R123high than in R123low cells. Not all R123low T cells, however, were resistant to IL-2-dependent proliferation and differentiation: limit dilution analyses showed nearly equal proportions of Con A-responsive proliferative and cytotoxic clones within the R123high and R123low subsets of CD8 naive cells. These data show that P glycoprotein-mediated extrusion of R123 delineates CD4 memory T cell subsets that differ in function.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Age-sensitive T cell phenotypes covary in genetically heterogeneous mice and predict early death from lymphoma.

We have assessed several age-sensitive indicators of immune status in young (i.e., 6 to 11-month-old) mice of a genetically heterogeneous population to see if these varied in parallel and to determine if one or more of the status indices predicted life span or cancer incidence. We report that the number of memory (i.e., CD44hi) T cells within the CD8 subset is correlated with number of memory cells in the CD4 population, and inversely correlated with the number of naive (i.e., CD45RBhi) CD4 cells at both 6 and 11 months of age, suggesting that the conversion of naive to memory cells may occur at similar rates in both T cell subsets. Mice that ranked high in the proportion of memory T cells (within the CD4 and CD8 pools) at 6 months of age tended to retain their ranking at 11 months, suggesting that the pace or extent of memory cell formation may be a consistent trait that distinguishes mice at least within a genetically heterogeneous population. Mice that at 6 months of age exhibited high levels of CD4 or CD8 memory T cells, low levels of naive CD4 cells, or low levels of T cells able to proliferate in response to Con A and IL-2 were found to be significantly more likely than their littermates to die within the first 18 months of life. Cases of follicular cell lymphoma, lymphocytic and lymphoblastic lymphoma, and hepatic hemangiosarcoma were seen within the group of mice dying at early ages.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1993

Myosin light chain phosphorylation does not increase during yeast phagocytosis by macrophages.

We have studied the role of myosin II light chain phosphorylation in yeast phagocytosis by J774 cells. J774 cells, which are mouse cells of monocyte/macrophage lineage, ingest opsonized yeast particles, and the rate of internalization is linear for 60 min at 37 degrees C. Immunoprecipitation of myosin II from cells labeled with 32P, using an affinity-purified antibody to myosin II purified from J774 cells, demonstrated phosphorylation of both the myosin heavy chain and the 20-kDa light chain (PMLC) prior to the addition of the opsonized yeast. However, the levels of heavy chain and PMLC phosphorylation did not change during the linear phase of yeast uptake by J774 cells. Other experiments demonstrated that the amount of myosin II associated with the cytoskeleton did not change during phagocytosis, further supporting the observation that PMLC phosphorylation does not increase during phagocytosis. In contrast, F-actin increased by 1.6-fold during the linear phase of phagocytosis. Two additional approaches were used to analyze in greater detail the role of myosin II phosphorylation in phagocytosis. First, antibodies to myosin light chain kinase (MLCK), the enzyme that phosphorylates PMLC, were electroinjected into J774 cells. These antibodies, which inhibit MLCK activity, inhibited chemotaxis as previously described but had no effect on phagocytosis. Second, quantitation of phagocytosis and chemotaxis following treatment with the phosphoprotein phosphatase inhibitor okadaic acid demonstrated that chemotaxis was much more sensitive than phagocytosis to okadaic acid treatment; at 0.3 microM okadaic acid, there is a substantial increase in myosin phosphorylation and chemotaxis is inhibited by 60%, whereas phagocytosis is unaffected. These data indicate that PMLC phosphorylation and, by implication, myosin II are not involved in yeast phagocytosis. They also suggest that PMLC phosphorylation displays a high degree of specificity with respect to mediating energy-dependent cellular processes in macrophages.

Animals↗

PubMed1991

An increase or a decrease in myosin II phosphorylation inhibits macrophage motility.

Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin-independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells.

Animals↗