Ann L. Moore

Activation of Potassium and Chloride Channels by Tumor Necrosis Factor Alpha: Role in Liver Cell Death

Despite abundant evidence for changes in mitochondrial membrane permeability in tumor necrosis factor (TNF)-mediated cell death, the role of plasma membrane ion channels in this process remains unclear. These studies examine the influence of TNF on ion channel opening and death in a model rat liver cell line (HTC). TNF (25 ng/ml) elicited a 2- and 5-fold increase in K+ and Cl− currents, respectively, in HTC cells. These increases occurred within 5–10 min after TNF exposure and were inhibited either by K+ or Cl−substitution or by K+ channel blockers (Ba2+, quinine, 0.1 mm each) or Cl− channel blockers (10 μm 5-nitro-2-(3-phenylpropylamino)benzoic acid and 0.1 mm N-phenylanthranilic acid), respectively. TNF-mediated increases in K+ and Cl− currents were each inhibited by intracellular Ca2+ chelation (5 mm EGTA), ATP depletion (4 units/ml apyrase), and the protein kinase C (PKC) inhibitors chelerythrine (10 μm) or PKC 19–36 peptide (1 μm). In contrast, currents were not attenuated by the calmodulin kinase II 281–309 peptide (10 μm), an inhibitor of calmodulin kinase II. In the presence of actinomycin D (1 μm), each of the above ion channel blockers significantly delayed the progression to TNF-mediated cell death. Collectively, these data suggest that activation of K+ and Cl− channels is an early response to TNF signaling and that channel opening is Ca2+- and PKC-dependent. Our findings further suggest that K+ and Cl− channels participate in pathways leading to TNF-mediated cell death and thus represent potential therapeutic targets to attenuate liver injury from TNF.

Amplification of antibody production by phosphorothioate oligodeoxynucleotides

A phosphorothioate oligodeoxynucleotide that is complementary (antisense) to the initiation region of the rev gene of HIV-1 causes hypergammaglobulinemia and splenomegaly in mice, and it induces B cell proliferation and differentiation in mouse spleen mononuclear cells (SMNCs) and human peripheral blood mononuclear cells in vitro. The current studies were performed to investigate the specificity of these immunomodulatory effects. Both the sense and antisense rev oligomers stimulated tritiated thymidine incorporation and secretion of immunoglobulin M (IgM) and immunoglobulin G (IgG) by mouse SMNCs in a concentration-dependent fashion, but the antisense oligomer produced greater immune effects. Studies comparing phosphorothioate oligomers (anti-rev, c-myc, and c-myb) either methylated or unmethylated at CpG dinucleotides showed that methylation effectively abrogated the proliferative effect and tended to reduce the immunoglobulin secretory activity, but the latter was not statistically significant except in the case of IgG in anti-rev oligomer-treated cultures. Mice were injected with the sense or antisense rev oligomers singly or in combination. The animals then were immunized with tetanus toxoid and received a booster 21 days later. Oligodeoxynucleotide-treated mice had significantly higher levels of IgM antibodies on days 28 and 35 and of IgG antibodies on days 14 and 35 as compared with mice that were immunized but received vehicle alone. There was no evidence for additive, synergistic, or antagonistic interactions of the sense and antisense rev oligomers. These results indicate that the unmethylated anti-rev oligomer is the most potent of the phosphorothioate oligomers tested at activating lymphocyte proliferation and differentiation and that a single intravenous injection of this oligodeoxynucleotide augments antibody production to a specific antigen as long as 35 days later.

Influenza and aging: Age-related changes and the effects of thymosin on the antibody response to influenza vaccine

Despite massive immunization programs, influenza remains a major cause of morbidity and mortality for elderly people. This may occur because immune senescent recipients may respond to vaccination with inadequate antibody production. We measured antibody response to the trivalent 1983–1984 influenza vaccine in young and elderly volunteers and found a significantly reduced response in the latter. The age-associated decreased antibody production was also observed in lymphocyte cultures in which specific antiinfluenza antibody synthesis was measured. In these cultures, however, the addition of a thymic hormone preparation (either thymosin fraction 5 or thymosin alpha 1) was shown to enhance specific antibody synthesis to a greater extent in the cultures established from the elderly volunteers. If thisin vitro observation of thymosin induced increased antibody production reflects what might occur in a clinical trial in which elderly subjects receive thymosin coincident with vaccine, greater protection against influenza infection may result.

Specific antibody synthesis in vitro. II. Age-associated thymosin enhancement of antitetanus antibody synthesis

A decline in T cell function accounts for many of the observed age-related deficient immune responses. Specific antibody response to many antigens requires T cell cooperation, and deficient antibody response to such antigens has been demonstrated with aging. In an effort to assess the potential reconstitutive capacity of Thymosin Fraction 5, in vitro antitetanus antibody production was measured in tetanus toxoid booster-immunized young and old volunteers. 22 young and 12 old volunteers were immunized with tetanus toxoid and plasma antitetanus antibody and in vitro lymphocyte production of antitetanus antibody was measured. Plasma antitetanus antibody response was significantly greater in the young. In vitro antitetanus antibody synthesis was negligible prior to immunization and peaked in cultures established 1 week after immunization from both young and old. When Thymosin Fraction 5 was added to the cultures, however, there was a dose-related enhancement of antibody synthesis in 7 of 10 from the group of elderly volunteers, but only 3 of 12 from the younger group. Our data indicate that specific antibody response is deficient in the elderly, but can be enhanced in vitro by thymosin. A future clinical trial of thymosin as an adjuvant to active immunization for the elderly is warranted.

Interleukin-2 and aging: Decreased interleukin-2 production in healthy older people does not correlate with reduced helper cell numbers or antibody response to influenza vaccine and is not corrected in vitro by Thymosin α1

The capacity of lymphocytes obtained from healthy young or old volunteers to produce interleukin-2 was measured and the results were compared with other measures of immune function. The in vitro effect of thymosin alpha 1 on interleukin-2 production was also measured. Interleukin-2 was lower in lymphocytes from the elderly, and individuals with low production also had lower proliferative responses in vitro to phytohemagglutinin. These individuals did not have a reduced helper T-cell number, abnormal ratio of helper to suppressor T-cells or reduced antibody production in response to vaccine. Thymosin alpha 1 did not have a consistent effect on interleukin-2 production.

Experimental tumors and aging: local factors that may account for the observed age advantage in the B16 murine melanoma model☆

In the B16 murine melanoma model tumor growth has been shown to be slower in animals of advanced age. One feature associated with this slower growth has been prominent fibrosis demonstrated in biopsies of the tumor in older animals. We have performed experiments to examine the fibrotic response in young and old mice. In non-tumor bearing animals the capacity to regain skin strength after surgical laceration and healing by primary intention was greater in old mice. Histologic preparations suggested a more prominent fibrosis at the wound site. The animals who were injected subcutaneously with B16 melanoma and treated with L 3,4-dehydroproline (an inhibitor of collagen synthesis) local tumor growth was significantly enhanced only for the old animals. Although this inhibition of collagen synthesis produced a differential growth enhancement, there remained a significant difference in tumor volume between young and old animals. We conclude that fibrogenesis is an important host defense for containing local tumor growth and that this mechanism is preserved if not enhanced in mice of advanced age. Nevertheless other factors are needed to account completely for the observed age-advantage in the B16 melanoma model.

Ca2+-activated IK1 Channels Associate with Lipid Rafts upon Cell Swelling and Mediate Volume Recovery

Restoration of cell volume in the continued presence of osmotic stimuli is essential, particularly in hepatocytes, which swell upon nutrient uptake. Responses to swelling involve the Ca2+-dependent activation of K+ channels, which promote fluid efflux to drive volume recovery; however, the channels involved in hepatocellular volume regulation have not been identified. We found that hypotonic exposure of HTC hepatoma cells evoked the opening of 50 pS K+-permeable channels, consistent with intermediate conductance (IK) channels. We isolated from rat liver and HTC cells a cDNA with sequence identity to the coding region of IK1. Swelling-activated currents were inhibited by transfection with a dominant interfering IK1 mutant. The IK channel blockers clotrimazole and TRAM-34 inhibited whole cell swelling-activated K+ currents and volume recovery. To determine whether IK1 underwent volume-sensitive localization, we expressed a green fluorescent protein fusion of IK1 in HTC cells. The localization of IK1 was suggestive of distribution in lipid rafts. Consistent with this, there was a time-dependent increase in colocalization between IK1 and the lipid raft ganglioside GM1 on the plasma membrane, which subsequently decreased with volume recovery. Pharmacological disruption of lipid rafts altered the plasma membrane distribution of IK1 and inhibited volume recovery after hypotonic exposure. Collectively, these findings support the hypothesis that IK1 regulates compensatory responses to hepatocellular swelling and suggest that regulation of cell volume involves coordination of signaling from lipid rafts with IK1 function.

Organ culture for thymus transplantation.

A method is presented for organ culture of postnatal thymus. Such tissue has been used for transplantation for nearly 20 years, but lasting benefit has been observed only in patients with the DiGeorge anomaly. Transplantation in other diseases has produced little or no results. Recently, improved methods for preparing the tissue as well as modifications of the culture media show marked improvement in quality and quantity of tissue suitable for transplant. In addition, using recently available monoclonal antibodies, preservation of vital stromal components can be monitored. The availability of reasonable amounts of high quality thymus tissue for transplantation may stimulate interest in further clinical trials where thymus transplantation may augment or restore T cell immunity.

Nitric Oxide Modulates Lymphocyte Proliferation But Not Secretion of IL-2

The objective of this study was to determine the effects of nitric oxide (NO) on lymphocyte proliferation and cytokine release. Bronchoalveolar lavage (BAL) cells served as the source of NO and were obtained from rats treated with a single, intratracheal dose of bleomycin (3.6 mg/kg). At the time of sacrifice, the spleens were removed and the lymphocytes separated. Co-cultures containing BAL cells, lymphocytes and concanavalin-A were established and incubated at 37 degrees C for 24 hours at which time proliferation, nitrite concentration and interleukin-2 (IL-2) production were measured. At ratios from 5:1 to 1:4 (BAL:lymphocyte) there was a significant reduction in lymphocyte proliferation. There was a significant, negative correlation between NO concentration and thymidine incorporation which was reversed when the NO synthase inhibitor NG-monomethyl-L-arginine (NMA) was added to the co-cultures. Despite marked inhibition of spleen lymphocyte proliferation by NO, released by BAL cells, there was no corresponding reduction in IL-2 production. These data demonstrate that macrophages, activated in vivo, produce NO which regulates lymphocyte growth but not necessarily functions such as the secretion of the cytokine IL-2. Further, the ability of IL-2-dependent CTLL-2 cells to proliferate in the presence of excess IL-2 was also inhibited by BAL cells, confirming that NO inhibits lymphocyte growth.

Immunosuppressive properties of chloroquinoxaline sulfonamide

Chloroquinoxaline sulfonamide (CQS), a sulfanilamide derivative with antitumor activity, was found to be toxic to lymphoid tissue during preclinical studies. The mechanism of this toxicity appears to involve profound inhibition of lymphocyte activation. Incubation of human peripheral blood mononuclear cells (PBMNCs) with CQS decreased cellular incorporation of thymidine and deoxyuridine in a dose-dependent manner. Analysis of cell cycle distribution by flow cytometry indicated that CQS blocked movement out of the G0/G1 phase. Drug-treated cells were smaller and expressed fewer receptors for interleukin-2 (IL-2) and transferrin than untreated mitogen-stimulated lymphocytes. These observations support the notion that CQS has cell cycle specificity in regulating lymphocyte proliferation. As little as 10 microM CQS markedly inhibited both human lymphocyte and murine CTLL cell replication in response to IL-2 containing growth factors. However, CQS did not block secretion of IL-2 into culture supernatant fractions by human PBMNCs. Finally, CQS inhibited in vitro production of immunoglobulins G and M by mitogen-stimulated lymphocytes, primarily by causing cytotoxicity. In all of these drug effects, CQS was approximately one to two logs more potent than the parent compound, sulfaquinoxaline (SQ). These studies indicate that CQS inhibits essential basic processes in human lymphocytes. This agent may find use as an immunosuppressive drug.