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G M Kammer

Publications and source records attributed to G M Kammer.

At least 19 recordsLinked to original sources
PubMed1992

Human T lymphocyte cAMP-dependent protein kinase: subcellular distributions and activity ranges of type I and type II isozymes.

The role of the type I and type II protein kinase A isozymes in the regulation of human T lymphocyte immune effector functions has not been ascertained. To approach this question, we first characterized the distribution and enzyme activities of the type I and type II protein kinase A (PKA) isozymes in normal, human T lymphocytes. T cells possess both type I and type II isozymes with an activity ratio of 5.0:1 +/- 0.71 (mean +/- SD). The type I isozyme associates predominately with the plasma membrane whereas the type II isozyme localizes primarily to the cytosol. Analyses of isozyme activities demonstrated that T cells from approximately one-third of 16 healthy donors exhibited significantly higher type II isozyme activities (higher type II, type IIH) than the remaining donors (lower type II, type IIL) (mean = 605 +/- 75 pmol.min-1.mg protein-1, P less than 0.001). Scatchard analyses of [3H]cAMP binding in the cytosolic fraction demonstrated similar Kd values (type IIH, 1.1 x 10(-7) M; type IIL, 9.0 x 10(-8) M); however, the Bmax (maximal binding) of the type IIH was 400 fmol/mg protein compared to the Bmax of the type IIL of 126 fmol/mg protein. Scatchard analysis of [3H]cAMP binding to the type I isozyme associated with membrane fragments had a Kd of 5.6 x 10(-8) M and a Bmax of 283 fmol/mg protein. Eadie-Hofstee plots of type IIH and type IIL gave a Km and Vmax of 2.3 mg/ml and 1.5 nmol.mg-1.min-1, and 2.1 mg/ml and 1.6 nmol.mg-1.min-1, respectively. The 3.2-fold higher maximal binding of the type II isozyme in one-third of healthy donors may reflect a greater amount of isozyme protein. The compartmentalization of type I PKA isozyme to the plasma membrane and type II PKA isozyme to the cytosol may serve to localize the isozymes to their respective substrates in T lymphocytes.

Adult↗
PubMed1990

Defective cAMP-dependent phosphorylation of intact T lymphocytes in active systemic lupus erythematosus.

The present study was undertaken to establish whether cAMP-dependent phosphorylation of endogenous substrates is impaired in T lymphocytes from subjects with active systemic lupus erythematosus (SLE). In normal human T lymphocytes, the cell-permeable cAMP analog, N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, induced phosphorylation of substrates with molecular masses of 17.5, 23/25, 33.5 kDa on one-dimensional SDS/PAGE. Maximal phosphorylation occurred at 60 min. In contrast to healthy T cells, the extent of substrate phosphorylation achieved in active SLE T cells (n = 8) was only 15% at 60 min in the 17.5-kDa substrate, 21% in the 23/25-kDa substrate, and 9% in the 33.5-kDa substrate. The rheumatic disease controls (rheumatoid arthritis; primary Sjögren syndrome; n = 8) exhibited a mean 72%, 124%, and 85%, respectively, of phosphorylation observed in healthy T cells. Because the only known mechanism by which cAMP acts is via cAMP-dependent protein kinase (protein kinase A), these data raise the possibility of a defect at the level of this kinase in SLE T lymphocytes.

Adult↗
PubMed1990

UVB radiation and human monocyte accessory function: differential effects on pre-mitotic events in T-cell activation.

Purified T lymphocytes fail to proliferate in response to antigenic and mitogenic stimuli when cultured in the presence of accessory cells that have been exposed in vitro to sublethal doses of UVB radiation. Because proliferation represents a final stage in the T-cell activation process, the present study was conducted to determine whether T cells were able to progress through any of the pre-mitotic stages when UVB-irradiated monocytes were used as model accessory cells. In these experiments, monoclonal anti-CD3 antibodies were employed as the mitogenic stimulus. Culture of T cells with UVB-irradiated monocytes did allow the T cells to undergo an increase in intracellular free calcium, which is one of the first steps in the activation sequence. The T cells expressed interleukin-2 receptors, although at a reduced level. However, T cells failed to produce interleukin-2 above background levels when they were placed in culture with monocytes exposed to UVB doses as low as 50 J/m2. Incubation of T cells with UVB-irradiated monocytes did not affect the subsequent capacity of T cells to proliferate, since they developed a normal proliferative response in secondary culture when restimulated with anti-CD3 antibodies and unirradiated monocytes. These studies indicate that T lymphocytes become partially activated when cultured with UVB-irradiated monocytes and mitogenic anti-CD3 monoclonal antibodies. In addition, they suggest that interleukin-2 production is the T-cell activation step most sensitive to inhibition when UVB-irradiated monocytes are employed as accessory cells.

Antigens, CD↗
PubMed1988

Association of cytoskeletal re-organization with capping of the complement decay-accelerating factor on T lymphocytes.

Recent studies have identified cell-associated proteins that are membrane anchored by glycosyl-inositol-phospholipid structures but the biologic implications of this mode of membrane attachment are incompletely understood. Among proteins anchored in this way is the decay-accelerating factor (DAF), a complement (C) regulatory factor that functions on blood cell surfaces to prevent autologous C attack. As one approach to investigate the functional consequences of glycosyl-inositol-phospholipid-anchoring of DAF in T lymphocytes, the effects of crosslinking surface DAF molecules were compared to those of crosslinking conventionally by anchored cluster of differentiation (CD) proteins. Upon incubation with anti-DAF mAb and anti-murine IgG, DAF re-distributed to a pole of the cell with a t1/2 at 37 degrees C of 4.4 min as compared to t1/2 of 3.5 to 7 min for CD3, CD4, and CD8. Re-distribution of DAF occurred independently of CD2, CD3, CD4, or CD8. Anti-DAF immunoprecipitates of membrane extracts of cells chemically cross-linked with dithiobis(succinimidylpropionate) contained only monomeric DAF. Immunofluorescent staining demonstrated clustered actin, tubulin, and vimentin beneath the capped DAF protein. Pre-treatment of cells with colchicine or 8-azidoadenosine 3',5'-cyclic phosphate, but not lumicolchicine, resulted in reduction of the t1/2 for DAF to 1 to 2.6 min. Conversely, treatment of cells with cytochalasins B or D completely blocked DAF capping. The results indicate that, upon cross-linking, glycosyl-inositol-phospholipid-anchored DAF molecules undergo capping similar to conventionally anchored CD molecules and that DAF capping is associated with cytoskeletal reorganization.

Adult↗
PubMed1988

Characterization of the human T lymphocyte adenosine receptor: comparison of normal and systemic lupus erythematosus cells.

To determine whether a defect in the T cell response to adenosine exists at the level of the adenosine receptor in systemic lupus erythematosus (SLE) we measured the binding affinity and maximum binding of T cell membranes from both normal and SLE T cells by utilizing radiolabeled adenosine ligands. Normal T lymphocyte membranes possess a single class of [3H]5-N-ethylcarboxamide adenosine binding sites with a Kd of 0.61 microM, a Bmax of 23.5 pmol/mg protein, and a Hill coefficient of 0.98, which indicates the presence of noncooperative sites. In contrast, T cell membranes do not bind significant amounts of either [3H]cyclohexyladenosine or [3H]phenylisopropyladenosine. These data indicate that T lymphocyte membranes have only A2, and not A1, adenosine receptors. Similarly, T cells from both active and inactive SLE subjects also express only A2 receptors with a Kd of 0.93 microM, a Bmax of 20.4 pmol/mg protein, and a Hill coefficient of 0.85, which is consistent with the presence of noncooperative sites. There is no difference in the on-rate, affinity, or density of T cell A2 receptors from active SLE patients, inactive SLE patients, or healthy controls. We conclude that T lymphocytes from both healthy and SLE subjects express A2, but not A1, receptors. Thus, the inability of SLE T cells to respond to adenosine does not reflect a decreased density of A2 (stimulatory) receptors, diminished A2 receptor binding, or an increased affinity or number of A1 (inhibitory) adenosine receptors. These observations support the conclusion that the defect in the T cell cAMP-dependent pathway may occur at a point distal to the adenosine receptor.

Adenosine↗
PubMed1988

Impaired mobility of human T lymphocyte surface molecules during inactive systemic lupus erythematosus. Relationship to a defective cAMP pathway.

The T lymphocytes of patients with active systemic lupus erythematosus (SLE) exhibit impaired capping of the surface molecules CD3, CD4, and CD8 and a defective cAMP-dependent pathway. Since the mobility of these molecules is regulated in part by cAMP, we sought to determine whether there is a specific defect(s) along the T cell cAMP pathway that contributes to the persistent capping disorder observed during inactive SLE. The data suggest that a defect may exist at the level of cAMP-dependent protein kinase activation or at a point distally. We propose that a disorder of cAMP-dependent protein kinase activity might account for the defect of capping observed in both the CD3, CD4 (helper/inducer) and CD3, CD8 (suppressor) subsets observed in SLE.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1988

Control of human T-lymphocyte interleukin-2 production by a cAMP-dependent pathway.

Cyclic AMP has long been proposed to be the intracellular second messenger that conveys the inhibitory signal for T-cell activation and clonal T-cell proliferation. The present study further explores the mechanism by which the cAMP pathway regulates human T-lymphocyte interleukin-2 (IL-2) production and T-cell blastogenesis. Activation of adenylate cyclase, inhibition of cAMP-dependent phosphodiesterase, or the direct addition of the cell-permeable cAMP analog, 8-N3-cAMP, increased occupancy of intracellular cAMP receptors, inhibited IL-2 production, and reduced T-cell proliferation. However, inhibition of cAMP-dependent protein phosphorylation by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a cell-permeable inhibitor of cyclic nucleotide-dependent protein kinase, partially restored IL-2 production. Our data support the conclusion that the cAMP pathway conveys an inhibitory signal for IL-2 production and T-cell proliferation via an integral protein phosphorylation step.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1988

Mobility of the human T lymphocyte surface molecules CD3, CD4, and CD8: regulation by a cAMP-dependent pathway.

The present study was undertaken to determine whether a cAMP pathway mediates the mobility of CD3, CD4, and CD8 within the membrane. Crosslinking CD3, CD4, and CD8 with monoclonal antibody and anti-antibody induced rapid accumulation of intracellular cAMP, occupancy of cAMP receptors, and was temporally associated with the mobilization and directed movement of these molecules to a pole of the cell. This capping process could be partially inhibited in a dose-dependent manner by treatment of T cells with 2',5'-dideoxyadenosine, a ribose-modified adenosine analogue that binds to the P site of the catalytic subunit of adenylate cyclase and reduces adenylate cyclase activity. Furthermore, inhibition of cAMP-dependent endogenous phosphorylation of 17.5-kDa, 23/25-kDa, and 33.5-kDa bands in intact T cells by N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide, a cell-permeable inhibitor of cyclic nucleotide-dependent protein kinase, blocked the capping event. Data support the conclusion that crosslinking of CD3, CD4, and CD8 activates a cAMP-dependent pathway that mediates the mobilization and directed movement of these molecules. cAMP-dependent protein phosphorylation is an integral step leading to the capping process.

Adenylyl Cyclase Inhibitors↗
PubMed1987

Intact proteoglycan is a polyclonal activator of murine B-lymphocytes.

Destruction of articular cartilage is the hallmark of both inflammatory and degenerative arthritides. Since degradation of cartilage results in the release of proteoglycan (PG) monomers and fragments into the synovial fluid, the present study was initiated to determine whether hyaline cartilage PG can induce a cellular immune response. Nonimmune spleen cells obtained from A/St and C57Bl/6 mice were cultured with a crude extract of bovine nasal cartilage, a PG aggregate fraction, PG monomer or degraded PG monomer for varying time periods. Only intact PG monomer induced a proliferative response which peaked at day 2. The responding cell was a B-lymphocyte which did not require T-helper cell activity. Our results suggest that intact PG monomer is a polyclonal activator of B-lymphocytes.

Animals↗
PubMed1986

Adenosine.

Explore the source record for details and available documents.

Adenosine↗
PubMed1986

Effect of anti-T cell autoantibodies from systemic lupus erythematosus sera upon T lymphocyte functions.

This study was undertaken to establish whether IgM and IgG anti-T cell autoantibodies obtained from sera of patients with active systemic lupus erythematosus, impair normal T lymphocyte functions. Two in vitro models of T cell function were examined: (a) the capacity of cells to cap, endocytose, and regenerate the T3, T4, and T8 surface antigens; and (b) the adenosine-induced T4----T8 phenotype switch. The results demonstrated that autoantibody neither impaired the capping process, nor impeded the phenotypic switch. Thus, bound anti-T cell autoantibodies do not appear to interfere with these specific T lymphocyte functions and cannot directly account for either the impaired T cell capping mechanism or the block in adenosine-induced phenotype switch observed during active systemic lupus erythematosus.

Autoantibodies↗
PubMed1986

Role of adenylate cyclase in human T-lymphocyte surface antigen capping.

Our recent studies indicated that capping of T3, T4 and T8 surface antigens on human T lymphocytes is augmented by interaction of adenosine with a purinergic receptor. We suggested that the T-cell capping process was mediated by an adenylate cyclase-coupled purinergic receptor that resulted in the generation of cAMP and occupancy of cAMP receptors. The present study was undertaken to examine whether activation of adenylate cyclase in the absence of purinergic stimulation is sufficient to regulate surface antigen capping. Treatment of T lymphocytes with forskolin or cholera toxin caused activation of adenylate cyclase and occupancy of intracellular types I and II regulatory subunits of protein kinase by cAMP, as demonstrated by photoaffinity labeling with [8-3H]N3-cAMP. Such treatment augmented the rate of capping of the T3, T4, and T8 antigens, which resulted in a significant decrement in the elapsed time to half-maximal capping of each antigen. These observations support the proposition that the normal T-lymphocyte capping mechanism of both T3+, T4+ (inducer/helper) and T3+, T8+ (suppressor) subsets can be augmented by activation of adenylate cyclase.

Adenosine↗
PubMed1985

Secretion of an articular cartilage proteoglycan-degrading enzyme activity by murine T lymphocytes in vitro.

Destruction of articular cartilage is the hallmark of inflammatory arthritides. Enzymes elaborated by mononuclear cells infiltrating the synovium mediate, in part, the degradation of the cartilage extracellular matrix. Since mononuclear cells are the dominant cell type found in chronic inflammatory synovitis, we investigated whether interaction of immune mononuclear cells with antigen initiated the synthesis and secretion of a proteoglycan-degrading enzyme activity. Proteoglycan-degrading enzyme activity was monitored by the capacity of murine spleen cell conditioned medium to release [3H]serine/35SO4 incorporated into rabbit cartilage proteoglycan monomer fraction (A1D1), and by the relative change in specific viscosity of bovine nasal cartilage proteoglycan monomer. The results demonstrated that both virgin and immune mononuclear cells spontaneously generated proteoglycan-degrading enzyme activity and that cellular activation and proliferation induced by the antigen keyhole limpet hemocyanin or the mitogen phytohemagglutinin was not required. Kinetic studies demonstrated stable release of the enzyme activity over 72 h. Cell separation studies showed that T lymphocytes, a thymoma line, and macrophages separately produced proteoglycan-degrading enzyme activity. The enzyme activity has been partially characterized and appears to belong to a class of neutral pH metal-dependent proteinases. These observations, the first to demonstrate that T lymphocytes secrete an enzyme capable of degrading cartilage proteoglycan, raise the possibility that this enzyme activity contributes to cartilage extracellular matrix destruction in vivo. Moreover, these data support the conclusion that production of this enzyme by T lymphocytes is independent of an antigen-specific stimulus.

Animals↗