Cell Surface Thiols Research Articles

Redox regulation of the signaling pathways leading to eNOS phosphorylation

Oxidative stress mediates positive and negative effects on physiological processes. Recent reports show that H 2O 2 induces phosphorylation and activation of endothelial nitric oxide synthase (eNOS) through an Akt-phosphorylation-dependent pathway. In this study, we assessed activation of eNOS and Akt by determining their phosphorylation status. Whereas moderate levels of H 2O 2 (100 μM) activated the Akt/eNOS pathway, higher levels (500 μM) did not, suggesting differential effects by differing levels of oxidative stress. We then found that two pro-oxidants with activity on sulfhydryl groups, 1-chloro-2,4-dinitrobenzene (CDNB) and diethyl maleate (DEM), blocked the phosphorylation events induced by 100 μM H 2O 2. GSH was not a target thiol in this system because buthionine sulfoximine did not inhibit this phosphorylation. However, down-regulation of cell membrane surface and intracellular free thiols was associated with the inhibition of phosphorylation, suggesting that oxidation of non-GSH thiols inhibits the H 2O 2-induced phosphorylation of eNOS and Akt. DTT reversed the inhibitory effects of CDNB and DEM on Akt phosphorylation and concomitantly restored cell surface thiol levels more efficiently than it restored intracellular thiols, suggesting a more prominent role for the former. Similarly, DEM and CDNB inhibited TNF-α-induced Akt and eNOS phosphorylation, suggesting that thiol modification is involved in eNOS inductive pathways. Our findings suggest that eNOS activation is exquisitely sensitive to regulation by redox and that cell surface thiols, other than glutathione, regulate signal transduction leading to phosphorylation of Akt and eNOS.

Lymphocyte surface thiol levels.

Recent studies have implicated reduced thiols (cysteine -SH) in the function of individual cell surface proteins. Studies presented here demonstrate that the overall level of reduced thiols on cell surface molecules differs on individual subsets of peripheral blood mononuclear cells and that these levels can be manipulated in vitro by altering the level of intracellular glutathione (iGSH). To quantitate cell surface thiols, we have developed a Hi-D (11-color) fluorescence-activated cell sorter method in which we covalently couple a fluorescent molecule, Alexa-maleimide, to free (reduced) -SH groups on proteins or other molecules exposed on the cell surface (exofacial membrane). In addition, to reveal changes in cell surface thiol levels in response to various in vitro treatments, we used a pair of fluorescent Alexa dyes with distinct excitation and emission spectra to stain the cells before and after treatments. These in vitro studies demonstrate that decreasing iGSH, by specifically inhibiting its synthesis, decreases cell surface molecule thiols (csm-SH) and that preventing loss of iGSH also prevents loss of csm-SH. However, examination of peripheral blood mononuclear cell subsets tested immediately after isolation from healthy or HIV-infected subjects failed to reveal a similar relationship between internal iGSH and csm-SH. Although there is a relatively wide variation between individuals in both csm-SH and iGSH, there is no correlation between median iGSH and csm-SH compared for 22 healthy and 36 HIV-infected subjects. Collectively, our findings indicate that local environment plays a greater role in determining the redox status of cell surface molecules than the internal redox status of the cells.

Thiol-inducing and immunoregulatory effects of flavonoids in peripheral blood mononuclear cells from patients with end-stage diabetic nephropathy.

The cellular thiol status and its relationship to T-cell activation and cytokine synthesis of mononuclear cells was investigated in patients with end-stage diabetic nephropathy (ESDN) undergoing dialysis treatment. The functional effects of thiol repair by in vitro and in vivo treatment with flavonoids were elucidated. The thiol status of peripheral blood lymphocytes from 30 ESDN patients on hemodialysis and healthy controls was determined by flow cytometry. T-cell activation in response to pokeweed mitogen was analyzed by CD69 expression; cytokines were determined in cell culture supernatants. In result, compared to age-matched healthy subjects, a significant thiol deficiency in ESDN patients was obvious. The lowered total intracellular thiol levels correlated directly to a significant diminished T-cell activation and an elevated synthesis of TNF-alpha in the patient group. The treatment with flavonoids led to a restoration of the thiol status within 72 h in vitro and in vivo. This effect showed a biphasic kinetic that first utilized cell surface thiols and secondly intracellular thiols. In parallel, the T-cell activation was improved substantially along with a significant decrease in TNF-alpha release. These data provide the rational for clinical trials using flavonoids in ESDN to normalize immunoregulatory defects via restoration of the cellular thiol status.

Impaired suppressor cell activity due to surface sulphydryl oxidation in rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disorder with considerable evidence of impaired regulation of the immune response, including defective suppressor cell function, especially in the synovial membrane. We have investigated whether oxidation of cell surface thiols might be responsible for these defects and whether such cell function may be modulated towards normal by treatment with a sulphydryl-reactive drug, D-penicillamine. Using healthy mononuclear cells treated with an impermeant thiol blocker, induction of suppressor activity by incubation with the lectin Con A was not dependent on surface sulphydryl groups but suppressor activity was abolished by thiol blockade after Con A stimulation. Peripheral blood mononuclear cells from patients with active rheumatoid disease showed impaired Con A-induced suppressor activity which was enhanced to near-normal levels by incubating the rheumatoid cells with a sulphydryl reducing agent, 2-mercaptoethanol, or D-penicillamine. Con A-stimulation of cells from patients treated with intramuscular gold or D-penicillamine generated more active suppression than those from patients receiving non-steroidal drugs only. Mononuclear cells from patients with other chronic inflammatory joint diseases showed normal Con A-induced suppressor activity. These data support the conclusion that surface thiols on mononuclear cells in rheumatoid arthritis are reversibly oxidized by the disease process. This gives rise to aberrant cell function including impaired suppressor activity. Such a mechanism may be at least partly responsible for the defective immunoregulation seen in rheumatoid patients and thus be a relevant target for thiol containing antirheumatic drugs.

Role of thiols in human peripheral blood Natural Killer and Killer lymphocyte activities

The thiol reagents, dithiothreitol, diethyldithiocarbamate and reduced glutathione were each found to inhibit Natural Killer and Killer lymphocyte-mediated cytotoxicities. A biphasic aspect to the inhibition with increasing concentration was observed with diethyldithiocarbamate and reduced glutathione. The inhibition observed in response to reduced glutathione, a non-permeant compound, suggests that cell surface thiols may be critical functional groups in the processes of NK and K lymphocyte-mediated cytotoxicities.

Accessibility of cell surface thiols in human lymphocytes is altered by ionophores or OKT-3 antibody

The accessibility of cell surface sulfhydryl groups in human peripheral lymphocytes was investigated with 5,5′-dithiobis-(2-nitrobenzoic acid) in the presence and absence of ionophore antibiotics and the monoclonal antibody, OKT-3. Only a few accessible protein thiols have been found on the cells as demonstrated by labeling with a fluorescent non-penetrating thiol-marker, monobromotrimethyl-ammoniobimane and the subsequent gel electrophoretic analysis of the protein pattern. Difference spectrophotometric measurement of thiol-DTNB reaction revealed that ionophores altering the transmembrane potential induced an enhanced cell surface thiol-exposure on the minute time scale. The effect showed a dependence on the external concentration of the cations. The binding of monoclonal antibody, OKT-3, directed against T3 complexes, resulted in a similar, concentration-dependent increase of thiol-accessibility. These data are interpreted as early membrane-effects of ionophores and the specific antibody including changes in the conformational equilibrium or vertical displacements of certain membrane proteins. These events are likely to be coupled to the changes in the transmembrane potential of the lymphocytes.

Cell surface thiols, but not intracellular glutathione, are essential for cytolysis by a cloned murine natural killer cell line.

Cell surface thiols are required for a line of cloned murine natural killer lymphocytes to bind to and lyse tumor target cells. These lymphocytes neither bound to nor killed YAC-1 or G1Tc cells when the effector lymphocyte cell surface thiols were covalently coupled with the non-penetrating reagent, monobromotrimethylammoniobimane (qBBr). A limited number of thiol-bearing proteins were identified by gel electrophoresis on the cell surface using the fluorescence of the group that remains associated with the sulfur molecule. These results indicate that either one or more of these reactive proteins or different cell surface thiol-bearing molecules present at low frequencies are crucial to lymphocyte binding and killing. In contrast, we found little evidence that intracellular thiols are required for natural killer cell activity. Killing was relatively unimpaired when over 90% of lymphocyte glutathione was depleted with DL buthionine-S,R-Sulfoximine (BSO). Blocking the intracellular or the extracellular thiols of tumor targets had no effect on their ability to be lysed. Based on these data, we suggest that infrequently expressed extracellular thiols are required either for the conformation or for the disulfide crosslinking of proteins that participate in lymphocyte-mediated cytolysis.

Cell surface thiols, methylation, and complement-like components are involved in the early events of CML whereas proteases participate in the later, Ca++-dependent events.

The long term goals of our work are to define the chemical and metabolic functions required for cell-mediated lysis, to identify the molecules that participate in the lytic process, and ultimately, to characterize how the molecules mediate the required functions. We have used both murine cytotoxic T lymphocytes (CTL) and human natural killer (NK) cells as model systems. The data to be summarized here were obtained principally with the murine CTL system.

The mechanism of cell-mediated cytotoxicity. I. Killing by murine cytotoxic T lymphocytes requires cell surface thiols and activated proteases.

The mechanism of cell-mediated cytotoxicity. I. Killing by murine cytotoxic T lymphocytes requires cell surface thiols and activated proteases.