Activation of early enzyme production in small lymphocytes in response to high, nonmitogenic concentrations of concanavalin A

Lymphocyte mitogenesis is generally assessed by measuring the incorporation of [³H]thymidine into DNA. By this criterion, small lymphocytes, which are activated by relatively low doses of concanavalin A, are either unresponsive to or inhibited by higher concentrations. Because lymphocytes begin to synthesize DNA about 24 hr after addition of mitogen, the response is far removed temporally from the initial stimulus. We have chosen to use the induction of S-adenosylmethionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) to assess early activation events in bovine lymphocytes. Adenosylmethionine decarboxylase induction is bimodal, with an initial phase beginning 3 hr after addition of concanavalin A and a second wave coinciding with the onset of DNA synthesis. The initial accumulation of the decarboxylase (0-9 hr) in cultures treated with “nonmitogenic” levels of concanavalin A (108 μg/ml) was similar to that observed in cultures stimulated with optimally mitogenic doses (18 μg/ml). The early induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was also similar under these two culture conditions. However, the second phase of adenosylmethionine decarboxylase accumulation, the induction of thymidine kinase (ATP: thymidine 5′-phosphotransferase, EC 2.7.1.21), and DNA replication were blocked at the higher concentrations of concanavalin A. The inhibition of late events by high doses of concanavalin A was reversible. Cells treated with α-methyl-D-mannopyranoside 25 hr after addition of a high dose of lectin responded with a second period of adenosylmethionine decarboxylase accumulation, induction of thymidine kinase, and progression through S phase. These results suggest that initial lymphocyte activation occurs normally at high doses of concanavalin A, but that the cells are reversibly blocked prior to induction of “late” enzymes and progression through S phase.     

Depolarization of the Plasma Membrane of Neurospora During Active Transport of Glucose: Evidence for a Proton-Dependent Cotransport System

Intracellular microelectrodes were used to measure the effects of glucose transport on membrane voltage and membrane resistance in Neurospora crassa. Sudden activation of glucose uptake, via the high-affinity, derepressible system II, results in a very large depolarization of the plasma membrane. At saturating concentrations of glucose, the depolarization averages 120 mV; it is diphasic in time, with an initial shift at rates of 100-200 mV/sec followed by a slow, spontaneous, partial repolarization. Changes in intracellular ATP concentration are small and could account for only 10 mV of the initial depolarization, while the rest appears to depend upon the transport process itself. A plot of peak depolarization against the extracellular glucose concentration gives a saturation curve which is half-maximal at 42 μM, in good agreement with the K_1/2 reported for glucose transport via system II. The nonmetabolized analogue 3-O-methyl-D-glucose also causes depolarization, and in addition leads to a pulsed alkalinization of the medium occurring at approximately the same rate as 3-O-methyl-D-glucose uptake. The membrane resistance falls only slightly during glucose depolarization, a fact which requires the transport system itself to have a high internal resistance, or the membrane current-voltage relationship in glucose-starved cells to be quite nonlinear. All of the data support Mitchell's notion that sugar and hydrogen ions are contransported under the influence of the membrane potential, and lead to values for H⁺:glucose stoichiometry of 0.8 to 1.4.     

Selective blockers of voltage-gated K+ channels depolarize human T lymphocytes: mechanism of the antiproliferative effect of charybdotoxin.

Charybdotoxin (ChTX), a K+ channel blocker, depolarizes human peripheral T lymphocytes and renders them insensitive to activation by mitogen. We observed four types of K+ channels in human T cells: one voltage-activated, and three Ca(2+)-activated. To discern the mechanism by which ChTX depolarizes T cells, we examined the sensitivity of both the voltage-activated and Ca(2+)-activated K+ channels to ChTX and other peptide channel blockers. All four types were blocked by ChTX, whereas noxiustoxin and margatoxin blocked only the voltage-activated channels. All three toxins, however, produced equivalent depolarization in human T cells. We conclude that the membrane potential of resting T cells is set by voltage-activated channels and that blockade of these channels is sufficient to depolarize resting human T cells and prevent activation.     

Indole-3-acetic acid and fusicoccin cause cytosolic acidification of corn coleoptile cells

Microelectrodes were used to measure simultaneously the effects of indole-3-acetic acid (IAA) on membrane potential and cytosolic pH of corn coleoptile cells. IAA caused an initial depolarization followed by hyperpolarization, the latter displaying rhythmic oscillations. The extent of the changes in membrane potential was dependent on IAA concentration, and hyperpolarization, but not depolarization, could be detected with concentrations of IAA as low as 10 nM. Membrane hyperpolarization was preceded by a decrease in cytosolic pH. The decrease commenced ≈5 min after adding IAA and continued for 15-20 min before reaching a new steady state ≈0.1 pH unit lower than the original pH. The decrease in pH was readily detectable after treatment with 0.1 μM IAA. Fusicoccin and acetate, which, like IAA, induce elongation growth, caused a similar drop in cytosolic pH and subsequent membrane hyperpolarization, the decrease in pH commencing within seconds. The addition of fusicoccin to IAA-treated cells resulted in a further cytosolic acidification and membrane hyperpolarization. The two substances probably change cytosolic pH via different mechanisms. The results imply that one of the primary effects of auxins in coleoptiles is to lower cytosolic pH.     

Stimulation of canine lymphocyte subpopulations separated nonlytically by monoclonal anti-T and polyclonal Anti-B cell antibodies

Summary Canine blood lymphocytes were nonlytically separated on antibody-coated petri dishes into surface immunoglobulin-positive (SIg⁺) and -negative (SIg^–) populations. SIg^– cells were further separated into cells reactive or non-reactive with monoclonal antibody DT-2 recognizing canine T lymphocytes. The purity of the three enriched lymphocyte populations exceeded 90% as assessed by immunofluorescence.Mitogen stimulation showed a vigorous response of SIg⁺ cells to pokeweed mitogen and concanavalin A but only a weak response to phytohemagglutinin. In mixed lymphocyte cultures, SIg⁺ cells were poor responders but potent stimulators. DT-2^– and DT-2⁺ cells responded to phytohemagglutinin, concanavalin A and pokeweed mitogen, and both populations were good responders in mixed leukocyte culture. Only DT-2^– cells were potent stimulators; DT-2⁺ cells were not. Hence, canine blood T cells can be divided into two subsets, DT-2⁺ and DT-2^–, both of which are responsive to mitogens and alloantigens.Supported by Grants CA 31787, CA 15704, CA 28941, and CA 18221 awarded by the National Cancer Institute, DHHS     

Thymus-dependent membrane antigens in man: Inhibition of cell-mediated lympholysis by monoclonal antibodies to TH2 antigen

In prior studies a heteroantiserum to a surface membrane component termed T_H2 was used to define two subsets of human T cells (T_H2⁺ and T_H2^-), which were found to express distinct sets of activities in vitro. In the present studies we prepared monoclonal antibodies to surface determinants that are restricted to T cells belonging to each of these two subsets. Two antibodies, termed αLeu-2a and αLeu-2b, which seem to define the same surface antigen identified by the original T_H2 antiserum, reacted with 57-84% of thymocytes and 22-46% of the erythrocyte-rosette-forming cells (ERF-C) in peripheral blood. Two other monoclonal antibodies, termed αLeu-3a and αLeu-3b, reacted with the same subpopulation of thymocytes (78-89%) and peripheral blood ERF-C (47-78%) but, unlike αLeu-2a and αLeu-2b, did not exhibit cross-blocking; i.e., labeling cells with αLeu-3a did not inhibit the subsequent binding of αLeu-3b. T cells reactive with αLeu-2a were shown to be unreactive with αLeu-3a, indicating that two separate subpopulations of T cells, Leu-2 (formerly T_H2⁺) and Leu-3 (T_H2^-) T cells, were thereby defined. These two T cell subsets make up the subpopulation of ERF-C (80-95%) previously defined by a monoclonal antibody to a T cell membrane antigen (Leu-1) that has a thymus-dependent distribution on normal lymphocytes but is expressed by some surface-immunoglobulin-positive (sIg⁺) leukemic lymphocytes. None of the Leu antibodies reported here reacted with sIg⁺, Leu-1⁺ leukemic cells, nor did they react with normal hematopoietic cells or lymphoid cells that had surface markers characteristic of B cells. Studies of the blocking effects of Leu antibodies on killing in cell-mediated lympholysis by effector T cells were carried out in the absence of complement. These experiments established the following points: (i) αLeu-2a abolished the killing by cytotoxic T cells of allogeneic phytohemagglutinin-stimulated blasts, (ii) inhibition of killing by αLeu-2b was markedly less than inhibition by αLeu-2a, and (iii) other antibodies, including αLeu-1, αLeu-3a, and αLeu-3b, had little or no effect on killing in cell-mediated lympholysis. The relevance of these findings to prior studies done in the mouse and in man are discussed.     

Functional characteristics of human T-cell subpopulations distinguished by a monoclonal antibody

In animals and in man, diverse immunologic functions are mediated by specialized T-cell (thymus-derived lymphocyte) subsets that are distinguishable from one another on the basis of differences in cell surface determinants. Unfortunately, in humans, subset-specific antibodies have been difficult to generate. In this study, production of a murine monoclonal antibody specific for a subset of human T cells was achieved by fusing a sensitized B cell (bone marrow-derived cell) with a myeloma cell and isolating the antibody secreted by the resultant hybrid clone. This antibody binds 30-35% of peripheral T lymphocytes (T_a⁺ cells) but fails to bind remaining T lymphocytes (T_a^- cells), B lymphocytes, or monocytes. T_a⁺ and T_a^- subpopulations were separated with a fluorescence-activated cell sorter and their in vitro responses to various stimuli were assessed. T_a⁺ and T_a^- cells respond equally well to soluble antigens, allogeneic B cells, and autologous B cells, but only T_a⁺ cells respond to concanavalin A. T_a⁺ cells cultured in the presence of concanavalin A gradually lose the T_a marker, an effect not observed after stimulation with phytohemagglutinin, soluble antigens, or alloantigens. These results suggest that the functional subpopulation of T cells defined by T_a does not correspond to any previously described human T cell subset. Furthermore, somatic cell hybridization has been shown to be a feasible method for production of monoclonal antibodies specific for subpopulations of human lymphocytes.     

Emergence of Insulin Receptors on Human Lymphocytes During In Vitro Transformation

Essentially no specific binding sites for insulin are detected in small lymphocytes freshly isolated from human blood. Insulin-binding sites appear on the lymphocyte surface during transformation in vitro with concanavalin A, and the number of these receptors increases sharply to reach a maximum between 24 and 46 hr after exposure to the mitogen. The appearance of de novo binding sites for insulin coincides with the increase in [³H]thymidine uptake into nuclear DNA and clearly precedes the appearance of enlarged, morphologically transformed cells. No changes in insulin-binding are detected in unstimulated control cultures. A maximum of about 350 molecules of insulin can bind per transformed lymphocyte, while less than six insulin molecules bind to an untransformed cell. Circulating human leukemic lymphoblasts bind about as much insulin as the lymphocytes transformed in vitro. Giant, polynucleated, transformed lymphocytes cultured in the presence of cytochalasin B bind about 10 times more insulin than transformed lymphocytes, which is in harmony with a 10-fold increase in cell-surface area in these cells. Specific binding of insulin is a saturable process in transformed lymphocytes but not in the untransformed cells. In transformed cells, [¹²⁵I]-insulin is displaced by as little as 2 ng/ml of native insulin, while in untransformed cells no significant displacement is observed with native insulin. Digestion of transformed cells with phospholipase C (EC 3.1.4.3.) enhances the specific binding of [¹²⁵I]insulin 3-fold, but no effect occurs with untransformed cells. These observations indicate a possible functional role of insulin and of adenylate cyclase in cell growth and division.     

Effect of fatty acid composition of rat heart myocytes on their electrical activity

Rat heart myocytes were cultured for up to 16 days in medium containing 20% non-delipidated fetal calf and horse serum (control cells) and in medium containing lipid depleted serum supplemented with either palmitic or linoleic acid. The main change in fatty acid composition of cellular lipids consisted of an increase in the ratio of linoleic to oleic acid from 0.15 in palmitate supplemented cells and 0.64 in control cells to 2.6 in linoleate supplemented cells. Cells grown in the presence of linoleic acid showed enhanced repolarization compared to those grown with palmitic acid or to control cells. The maximal $\text{d}\text{V}\text{d}\text{t}$ during repolarization was 0.678 ± 0.113 V/s for cells grown with linoleic acid; 0.405 ± 0.105 V/s for cells grown with palmitic acid and 0.397 ± 0.007 V/s for the control cells. The duration of action potential and the value of $\text{d}\text{V}\text{d}\text{t}$, when compared at similar degrees of repolarization, showed significantly enhanced repolarization during the initial phase in cells grown with linoleic acid. Maximal diastolic membrane potential and the depolarization phase were similar in cells cultured with linoleic or palmitic acid.We explain these results by the hypothesis that the enrichment in linoleic acid of membrane lipids causes a rise in the flux of Cl^? and K⁺ through their respective channels leading to an augmented initial phase of repolarization.     

Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo

Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7^−/− erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119⁺/CD71^High stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.     

In vitro characterization of human intestinal intraepithelial lymphocytes

Enriched populations of human intestinal and colonic intraepithelial lymphocytes were isolated separate from lamina propria lymphocytes. These populations, and peripheral blood mononuclear cells, were characterized for membrane features, lymphocyte subsets, and proliferative potential to the nonspecific mitogenic lectins phytohemagglutinin, concanavalin A, and pokeweed mitogen. All three populations were predominantly T lymphocytes as measured by sheep red blood cell rosettes and monoclonal antibodies. Peripheral blood mononuclear cells had a T4:T8 ratio of 2.15 while intraepithelial lymphocytes were enriched for T8+ cells with a T4:T8 ratio of 0.06-0.14. Peripheral blood mononuclear cells and lamina propria lymphocytes registered vigorous proliferative responses to phytohemagglutinin, concanavalin A, and pokeweed mitogen. Intraepithelial lymphocytes, in contrast, failed to respond to the lectins. Control experiments with peripheral blood mononuclear cells indicated that the isolation reagents and procedures had no adverse effect on lectin-stimulated proliferation or on T-cell subset proportions. Viability of the intraepithelial lymphocytes after separation (88%) and after 3 days in culture with lectin (77%-85%) was confirmed by trypan blue dye exclusion, by light microscopy, by electron microscopy, and by ability to affect pokeweed mitogen-induced immunoglobulin synthesis when added to autologous and heterologous peripheral blood mononuclear cells. The failure of intraepithelial lymphocytes to respond to lectins was not due to failure of lectin binding, macrophage depletion, or absence of T-cell growth factor in culture. The possibility that the T-cell subset proportions (T4:T8 ratio) affect the proliferative response of isolated intraepithelial lymphocytes is discussed.     

Increased presence of effector lymphocytes during Helicobacter hepaticus-induced colitis

AIM: To identify and characterize drosophila mothers against decapentaplegic (SMAD)3-dependent changes in immune cell populations following infection with Helicobacter hepaticus (H. hepaticus).METHODS: SMAD3^-/- (n = 19) and colitis-resistant SMAD3^+/- (n = 24) mice (8-10 wk of age) were infected with H. hepaticus and changes in immune cell populations [T lymphocytes, natural killer (NK) cells, T regulatory cells] were measured in the spleen and mesenteric lymph nodes (MsLNs) at 0 d, 3 d, 7 d and 28 d post-infection using flow cytometry. Genotype-dependent changes in T lymphocytes and granzyme B⁺ cells were also assessed after 28 d in proximal colon tissue using immunohistochemistry.RESULTS: As previously observed, SMAD3^-/-, but not SMAD3^+/- mice, developed colitis, peaking at 4 wk post-infection. No significant changes in T cell subsets were observed in the spleen or in the MsLNs between genotypes at any time point. However, CD4⁺ and CD8⁺/CD62L^lo cells, an effector T lymphocyte population, as well as NK cells (NKp46/DX5⁺) were significantly higher in the MsLNs of SMAD3^-/- mice at 7 d and 28 d post-infection. In the colon, a higher number of CD3⁺ cells were present in SMAD3^-/- compared to SMAD3^+/– mice at baseline, which did not significantly change during infection. However, the number of granzyme B⁺ cells, a marker of cytolytic lymphocytes, significantly increased in SMAD3^-/- mice 28 d post-infection compared to both SMAD3^+/- mice and to baseline values. This was consistent with more severe colitis development in these animals.CONCLUSION: Data suggest that defects in SMAD3 signaling increase susceptibility to H. hepaticus-induced colitis through aberrant activation and/or dysregulation of effector lymphocytes.     

Sensitization-induced sodium influx in airway smooth muscle cells of guinea pigs

Airway smooth muscle (ASM) preparations isolated from Camm-Hartley male guinea pigs were passively in vitro sensitized using Austen's technique. During this sensitization, cells were frequently penetrated with glass microelectrodes (a resistance of 80-90 M omega) for a period of 120 min. Simultaneously, changes in the isometric force of ASM preparations were also monitored. It was found that: administration of serum (1:10 dilution) from sensitized animals caused a depolarization of ASM cells (-52.3 +/- 0.7 mV), achieving a peak at 4 min, followed by slow sustained hyperpolarization reaching a steady state after 15 min; (Resulting resting membrane potential of ASM cells was -68.8 +/- 0.5mV as compared to membrane potential of controls equal to -60.0 +/- 0.7 mV); in the presence of ouabain (10(-5)M), the development of hyperpolarization of ASM cells was completely inhibited; replacement of Na+ with choline chloride or lithium chloride partially inhibited the development of depolarization of ASM cells; in the presence of specific sodium entry blocker, amiloride the sensitization-caused depolarization was abolished; the presence of choline chloride, lithium chloride or amiloride in the experimental medium completely inhibited the contractile response of sensitized tissues to a specific antigen; response of ASM preparations to a specific antigen was absent during depolarization of ASM cells, but was always present during their hyperpolarization. It is concluded that in vitro passive sensitization leads to the interaction of serum specific antibodies with the membrane of airway smooth muscle cells so that permeability of membrane for Na+ is increased (transient depolarization) followed by the activation of electrogenic Na+-pump (sustained hyperpolarization). These events seem to determine the contractile response of sensitized ASM cells to a specific antigen.     

Electrical correlates of peristaltic and nonperistaltic contractions in the opossum smooth muscle esophagus

The electrical correlates of peristaltic and nonperistaltic esophageal contractions were characterized in anesthetized opossums using simultaneous suction electrode and intraluminal pressure recordings from sites 1 and 5 cm above the lower esophageal sphincter. Transient midesophageal balloon distention produced peristaltic contractions with faster propagation velocity than swallow-induced peristalsis. This was associated with prompt circular smooth muscle hyperpolarization at both sites, then depolarization, spike burst, and esophageal contraction. At the 5-cm site hyperpolarization was followed by rapid depolarization, whereas at the 1-cm site there was a much slower rate of depolarization after the initial hyperpolarization. Thus the longer latency to contraction at the 1-cm site correlated with the timing of the depolarization phase and not with the duration of the initial hyperpolarization. With prolonged balloon distention the inflation-related membrane hyperpolarization was followed by either partial or complete repolarization during sustained distention. Upon balloon deflation there was rapid membrane depolarization and spike burst at both sites such that the resultant contractions were usually nonperistaltic. In some instances balloon distention evoked contractions of slow peristaltic velocity. These correlated with membrane potential oscillations at the 1-cm site, with threshold depolarization being reached at the second depolarization in the hyperpolarization-depolarization sequences that followed balloon deflation. A similar pattern was seen with swallow-induced peristalsis. These studies demonstrate that differences in timing of the depolarization that follows initial hyperpolarization, and not differences in the duration of the initial hyperpolarization, are important in the genesis of peristaltic esophageal contractions.     

Changes in membrane potential of human granulocytes antecede the metabolic responses to surface stimulation

Human granulocytes (polymorphonuclear leukocytes) exposed to surface stimuli [e.g., immune complexes, concanavalin A (Con A)] generate O(2).(-), undergo a respiratory burst, and secrete lysosomal enzymes. To study the earliest reaction of ligands with surface receptors of granulocytes, purified cells were exposed to bovine serum albumin-anti-albumin complexes (Fc receptors) or Con A (glycoprotein receptors). The membrane potential (DeltaPsi) was measured by distribution of the lipophilic cation [(3)H]triphenylmethyl phosphonium ion. The Nernst equation yielded a resting DeltaPsi of -26.7 mV. Beginning within 10 sec after exposure to the antigen-antibody complex or to Con A, the cells responded with a rapid hyperpolarization --> depolarization --> slow hyperpolarization. Even when phagocytosis was inhibited by cytochalasin B, the triphasic response was obtained: evidence for surface interaction. The hyperpolarization response anteceded O(2).(-) generation (continuous recording) by at least 20-30 sec. O(2).(-) generation in response to immune complexes was stimulated by Ca(2+) whereas DeltaPsi remained unchanged; lack of Ca(2+) in the medium did not inhibit the DeltaPsi response. Dissociation of membrane hyperpolarization from subsequent metabolic responses (O(2).(-) generation) was also found in the presence of steroids (hydrocortisone, methylprednisolone), which inhibited O(2).(-) generation but did not inhibit the DeltaPsi response to antigen-antibody complex. Because O(2).(-) generation could be stimulated (Ca(2+)) or depressed (steroids) without affecting DeltaPsi, the data suggest that DeltaPsi is involved in primary triggering of phagocytic cells and that metabolic stimulation is a secondary consequence of ligand-receptor interactions.     

24-hour pattern of circulating prolactin and growth hormone levels and submaxillary lymph node immune responses in growing male rats subjected to social isolation

To assess the effect of social isolation of growing rats on 24-h rhythmicity of circulating prolactin and growth hormone (GH) levels and submaxillary lymph node immune responses, male Wistar rats were either individually caged or kept in groups (4–5 animals per cage) for 30 d starting on d 35 of life. Plasma prolactin and GH levels, and submaxillary lymph node lymphocyte subset populations, interferon (IFN)-γ release and mitogenic responses to concanavalin A (Con A) and lipopolysaccharide (LPS) were determined at six time intervals during the 24 h span. Social isolation brought about changes in mean values and 24-h pattern of plasma prolactin and GH levels and lymph node immune responses. After isolation, prolactin and GH mean values decreased, and lymph node T, B, non T-non B, CD8⁺, and CD4⁺-CD8⁺ cells augmented, whereas lymph node CD4⁺/CD8⁺ ratio, IFN-γ release and mitogenic responses decreased. Social isolation resulted in disruption of 24 h rhythmicity of every immune parameter tested. CD4⁺/CD8⁺ ratio, IFN-γ release and Concanavalin A (Con A) and lipopolysaccharide (LPS) responses correlated significantly with plasma prolactin or GH levels while T/B ratio correlated with plasma prolactin levels only. B, non T-non B, and CD4⁺-CD8⁺ cells correlated negatively with plasma prolactin. Modifications in mean value and 24-h rhythmicity of plasma prolactin and GH levels are presumably involved in the effect of social isolation on immune responsiveness.     

Activated rat hepatic stellate cells influence Th1/Th2 profile in vitro

AIM: To investigate the effects of activated rat hepatic stellate cells(HSCs) on rat Th1/Th2 profile in vitro.METHODS: Growth and survival of activated HSCs and CD4+ T lymphocytes cultured alone or together was assessed after 24 or 48 h. CD4+ T lymphocytes were then cultured with or without activated HSCs for 24 or 48 h and the proportion of Th1 [interferon(IFN)-γ+] and Th2 [interleukin(IL)-4+] cells was assessed by flow cytometry. Th1 and Th2 cell apoptosis was assessed after 24 h of co-culture using a caspase-3 staining procedure. Differentiation rates of Th1 and Th2 cells from CD4+ T lymphocytes that were positive for CD25 but did not express IFN-γ or IL-4 were also assessed after 48 h of co-culture with activated HSCs. Galectin-9 expression in HSCs was determined by immunofluorescence and Western blotting. ELISA was performed to assess galectin-9 secretion from activated HSCs.RESULTS: Co-culture of CD4+ T lymphocytes with activated rat HSCs for 48 h significantly reduced the proportion of Th1 cells compared to culture-alone conditions(-1.73% ± 0.71%; P 0.05), whereas the proportion of Th2 cells was not altered; the Th1/Th2 ratio was significantly decreased(-0.44 ± 0.13; P 0.05). In addition, the level of IFN-γ in Th1 cells wasdecreased(-65.71 ± 9.67; P 0.01), whereas the level of IL-4 in Th2 cells was increased(82.79 ± 25.12; P 0.05) by co-culturing, as measured by mean fluorescence intensity by flow cytometry. Apoptosis rates in Th1(12.27% ± 0.99%; P 0.01) and Th2(1.71% ± 0.185%; P 0.01) cells were increased 24 h after co-culturing with activated HSCs; the Th1 cell apoptosis rate was significantly higher than in Th2 cells(P 0.01). Galectin-9 protein expression was significantly decreased in HSCs only 24 h after coculturing(P 0.05) but not after 48 h. Co-culture for 48 h significantly increased the differentiation of Th1 and Th2 cells; however, the increase in the proportion of Th2 cells was significantly higher than that of Th1 cells(1.85% ± 0.48%; P 0.05).CONCLUSION: Activated rat HSCs lower the Th1/Th2 profile, inhibiting the Th1 response and enhancing the Th2 response, and this may be a novel pathway for liver fibrogenesis.     

Beat-to-beat interplay of heart rate, ventricular depolarization, and repolarization

To improve malignant arrhythmia risk stratification, the causal and random components of spatiotemporal dynamics of heart rate (RR distances), ventricular depolarization sequence, and repolarization disparity were studied based on body surface potential map records taken for 5 minutes, in resting, supine position on 14 healthy subjects (age range, 20-65 years) and on 6 arrhythmia patients (age range, 59-70 years). Beat-to-beat QRS and QRST integral maps, Karhunen-Loève (KL) coefficients, RR, and nondipolarity index time series were computed. Tight relationship was found between RR and QRS integrals in healthy subjects with less association in arrhythmia patients. Tight KL-domain multiple linear association (r² > 0.72) was found between the QRS and QRST integral dynamics (ie, depolarization sequence and repolarization disparity). Beat-to-beat probability of the generation of significant nondipolarity index spikes was proportional to the QRST KL-component standard deviations (SD_i) and inversely proportional with the mean dipolar KL components (M_i) of the average QRST integral map.     

Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes

The potassium channel Kv1.3 has recently been located to the inner mitochondrial membrane of lymphocytes. Here, we show that mouse and human cells that are genetically deficient in either Kv1.3 or transfected with siRNA to suppress Kv1.3-expression resisted apoptosis induced by several stimuli, including Bax over-expression. Retransfection of either Kv1.3 or a mitochondrial-targeted Kv1.3 restored cell death. Bax interacted with and functionally inhibited mitochondrial Kv1.3. Incubation of isolated Kv1.3-positive mitochondria with recombinant Bax, t-Bid, or toxins that bind to and inhibit Kv1.3 successively triggered hyperpolarization, formation of reactive oxygen species, release of cytochrome c, and marked depolarization. Kv1.3-deficient mitochondria were resistant to Bax, t-Bid, and the toxins. Mutation of Bax at K128, which corresponds to a conserved lysine in Kv1.3-inhibiting toxins, abrogated its effects on both Kv1.3 and mitochondria. These findings suggest that Bax mediates cytochrome c release and mitochondrial depolarization in lymphocytes, at least in part, via its interaction with mitochondrial Kv1.3.     

Ly-6C regulates endothelial adhesion and homing of CD8+ T cells by activating integrin-dependent adhesion pathways

Ly-6C belongs to the Ly-6 family of glycosyl phosphatidylinositol-anchored surface glycoproteins and is expressed on a subset of mature CD8⁺ T cells. Ly-6C ligation can mediate T cell activation and causes interleukin 2 secretion in cytolytic T cell clones. We characterize herein a new mAb 1G7.G10 against Ly-6C that recognizes an epitope involved in lymphocyte adhesion and in lymphocyte homing. Pretreatment of lymph node lymphocytes and of purified CD8⁺ T cells (but not of lymphocytes depleted of CD8⁺ T cells) with 1G7.G10 reduced their in vitro binding to lymph node high endothelial venules by 28% and 34%, respectively. This effect was bypassed by cross-linking Ly-6C molecules with 1G7.G10 and a second-step antibody. The in vivo homing of (donor) CD8⁺ T lymphocytes to lymph nodes was reduced by Ly-6C blocking with 1G7.G10 (whole antibody) or with its fragments [F(ab) or F(ab)₂] by 20% or by 32% and 48%, respectively. Cross-linking of Ly-6C in vitro induced very late antigen-4 and lymphocyte function-associated antigen 1-mediated aggregation of CD8⁺ T cells, suggesting that ligand binding to Ly-6C leads to activation of integrins. This activation may facilitate homing of Ly-6C⁺ CD8⁺ T cells in vivo.