Lymphotoxin Secretion Research Articles

A Novel MicroRNA-132-Surtuin-1 Axis Underlies Aberrant B-cell Cytokine Regulation in Patients with Relapsing-Remitting Multiple Sclerosis

Clinical trial results demonstrating that B-cell depletion substantially reduces new relapses in patients with multiple sclerosis (MS) have established that B cells play a role in the pathophysiology of MS relapses. The same treatment appears not to impact antibodies directed against the central nervous system, which underscores the contribution of antibody-independent functions of B cells to disease activity. One mechanism by which B cells are now thought to contribute to MS activity is by over-activating T cells, including through aberrant expression of B cell pro-inflammatory cytokines. However, the mechanisms underlying the observed B cell cytokine dysregulation in MS remain unknown. We hypothesized that aberrant expression of particular microRNAs might be involved in the dysregulated pro-inflammatory cytokine responses of B cells of patients with MS. Through screening candidate microRNAs in activated B cells of MS patients and matched healthy subjects, we discovered that abnormally increased secretion of lymphotoxin and tumor necrosis factor α by MS B cells is associated with abnormally increased expression of miR-132. Over-expression of miR-132 in normal B cells significantly enhanced their production of lymphotoxin and tumor necrosis factor α. The over-expression of miR-132 also suppressed the miR-132 target, sirtuin-1. We confirmed that pharmacological inhibition of sirtuin-1 in normal B cells induces exaggerated lymphotoxin and tumor necrosis factor α production, while the abnormal production of these cytokines by MS B cells can be normalized by resveratrol, a sirtuin-1 activator. These results define a novel miR-132-sirtuin-1 axis that controls pro-inflammatory cytokine secretion by human B cells, and demonstrate that a dysregulation of this axis underlies abnormal pro-inflammatory B cell cytokine responses in patients with MS.

Genetic control of multiple sclerosis: increased production of lymphotoxin and tumor necrosis factor-alpha by HLA-DR2+ T cells.

Lymphotoxin (LT) and tumor necrosis factor-alpha (TNF-alpha) play an important role in the pathogenesis of multiple sclerosis (MS). MS is associated with the HLA-DR2, Dw2, DQ6 HLA class II haplotype. Because both LT and TNF-alpha are encoded in the HLA region, the HLA association of MS may be related to the production of these cytokines. To test this hypothesis, we investigated the production of LT, TNF-alpha, and interferon-gamma (IFN-gamma) by CD4+ T-cell lines (TCLs) specific for myelin basic protein (MBP) or tetanus toxoid (TT) isolated from MS patients and normal controls. After stimulation with specific antigen but not mitogen, TCLs from HLA-DR2+ donors produced significantly more LT and TNF-alpha than TCLs from DR2- donors. In contrast, HLA-DR2+ and DR2- TCLs did not differ in the production of IFN-gamma, a cytokine also produced by T cells but not encoded in the HLA region. Increased secretion of LT and TNF-alpha was unrelated to the specificity (MBP vs TT), MHC restriction (HLA-DR2 vs other DR molecules), or source (MS vs normal) of the TCLs. There was no significant association of the cytokine production with individual LT or TNF-alpha alleles, indicating that the increased production of these cytokines may be linked to other polymorphic genes in this region. The results suggest that the association of MS with HLA-DR2 implies a genetically determined propensity of T cells to produce increased amounts of LT and TNF-alpha.

Lymphotoxin‐α/β heterodimer is expressed on leukemic hairy cells and activated human B lymphocytes

The expression of human lymphotoxin (LT) alpha/beta cell-surface complex was studied in human B-cell lines as well as in normal and neoplastic human B lymphocytes. In the absence of TNF receptors, only the human hairy-cell leukemia (HCL)-derived cell line JOK-I revealed constitutive cell-surface expression of LT but not TNF-alpha. Immunoprecipitation experiments with anti-LT monoclonal antibody (MAb) 9B9 from cell-surface radioiodinated JOK-I cells revealed that a cell-surface lymphotoxin molecule (25 kDa) is expressed in association with a 33-kDa molecule. Enzymatic digestion with F/N-glycosidase and O-glycosidase showed that both proteins contained N-linked carbohydrate residues, whereas only the 25-kDa molecule contained O-linked sugar residues. Analysis of mRNA expression revealed specific transcripts of LT-alpha and LT-beta in JOK-I cells. Resting tonsillar B cells did not express cell-surface LT. However, LT-beta mRNA was observable in unstimulated tonsillar B cells, whereas LT-alpha mRNA, cell-surface LT and LT secretion could only be detected upon in vitro activation. Thus LT-beta and alpha appear to be sequentially expressed in human B cells. Neoplastic B cells from chronic lymphocytic leukemia (BCLL), being devoid of constitutive cell-surface LT expression, could be induced to express surface LT by in vitro stimulation with Staphylococcus aureus Cowan I (SAC). Constitutive LT-beta transcripts, however, could also be detected in 4 out of 5 cases of BCLL. In contrast, human HCL cells displayed constitutive cell expression of lymphotoxin-alpha and beta. These findings demonstrate that cell-surface LT-alpha is expressed in association with LT-beta on activated normal B cells and neoplastic B cells representing an activated state.

Leishmania donovani-reactive Th1- and Th2-like T-cell clones from individuals who have recovered from visceral leishmaniasis.

Infections in humans by Leishmania donovani parasites can result in a fatal disease, visceral leishmaniasis (VL), or in a self-limiting asymptomatic infection. In murine models of the infection employing Leishmania major, the course of the disease can be directed into a VL-like syndrome by interleukin-4 (IL-4)-producing Th2 cells, or cure may result by Th1 cells secreting gamma interferon (IFN-gamma). The present study examined the potential of human T cells to generate Th1 or Th2 responses to L. donovani. The profiles of IFN-gamma, IL-4, and lymphotoxin secretion after antigen stimulation were analyzed in a panel of L. donovani-reactive CD4+ human T-cell clones generated from individuals who had recovered from VL after antimonial treatment. Two of the T-cell clones produced large amounts of IL-4 without production of IFN-gamma, seven clones produced both IFN-gamma and IL-4, and eight produced only IFN-gamma. This is the first report of a Th1- and Th2-type response in human leishmaniasis. These results suggest that in analogy with murine models, there is a dichotomy in the human T-cell response to L. donovani infections. Preferential activation of IL-4-producing Th2-like cells may be involved in the exacerbation of human VL, whereas activation of IFN-gamma-producing Th1 cells may protect the host from severe disease. Identification of leishmanial antigens activating one or the other type of T cells will be important in the development of vaccines against leishmaniasis.

Expression of surface lymphotoxin and tumor necrosis factor on activated T, B, and natural killer cells.

The expression of membrane-associated forms of lymphotoxin (LT) and TNF were examined on cell lines of T, B, and myeloid origin, IL-2 dependent T cell clones, and peripheral blood lymphocytes. Inducible and constitutive patterns of surface LT expression were found on T cells as exemplified by the II-23.D7, a CD4+T cell hybridoma, and HUT-78, a T cell lymphoma. Phorbol ester induced surface LT expression on Ramos, an EBV transformed B cell line, but at a slower rate of appearance when compared to the II-23.D7. Secretion of LT was rapidly inducible by phorbol ester in II-23.D7 and also in HUT-78 but with slower kinetics; surface LT expression continued in both lines after secretion had ceased. Low levels of membrane TNF were transiently induced on II-23.D7 and HUT-78, but none was observed on Ramos. Peripheral blood monocytes and some myeloid tumor lines did not express surface LT. Several T cell clones expressed surface LT after Ag-specific stimulation, and expression persisted several days. Stimulation through the TCR or by IL-2 rapidly induced surface LT on resting peripheral T cells and CD56+ NK cells; pokeweed mitogen activation induced expression on CD20+ B cells. Consistent with previous results, immunoprecipitation with anti-LT mAb showed that LT was complexed with a distinct 33 kDa glycoprotein (p33) on cells that expressed surface LT, whereas secreted LT was not associated with p33. Surface and secreted modes of LT expression by activated T, B, and NK cells suggests that LT can be utilized as either a localized or diffusible mediator in immune responses.

Protein kinase C regulates secretion of lymphotoxin (LT/TNFβ) and TNFα by human EBV-transformed B cells

Epstein-Barr virus (EBV)-transformed cell lines constitutively secrete lymphotoxin (LT/TNF beta) and not tumor necrosis factor-alpha (TNF alpha). To analyze the cellular processes that regulate LT and TNF alpha secretion by lymphoblastoid cell lines, we studied the role of two signal transduction pathways leading to either protein kinase C (PK-C) or PK-A activation. We demonstrate that PK-C activation, either after cross-linking of surface Ig or by direct activation with phorbolester, leads to increased production of both LT and TNF alpha, whereas no prominent role for PK-A was found. Interleukin (Il)-4 was found to synergize with PK-C activation in raising levels of secreted LT and TNF alpha. Increased levels of LT and TNF alpha did not correlate with augmented levels of immunoglobulin secreted by the cell lines nor with improved proliferation. These observations demonstrate that EBV B cells respond to B cell activation signals leading to PK-C activation with increased production of both LT and TNF alpha. It is, however, unlikely that these molecules serve as autostimulatory factors for EBV B cells, but in contrast might play a role in downregulation of biological functions in these cells.

Production of transmembrane and secreted forms of tumor necrosis factor (TNF)-alpha by HIV-1-specific CD4+ cytolytic T lymphocyte clones. Evidence for a TNF-alpha-independent cytolytic mechanism.

Candidate AIDS vaccines consisting of recombinant forms of the HIV-1 envelope glycoprotein induce, in seronegative human volunteers, an env-specific T cell response that includes CD4+, MHC class II-restricted CTL capable of lysing HIV-1-infected target cells. In this study, we have analyzed the production of the cytokines TNF-alpha and lymphotoxin (LT) by a set of env-specific CD4+ human CTL clones. TNF-alpha and LT are of interest because of their potential role in target cell destruction by CD4+ CTL. Our studies focused on the possibility that a cell surface form of TNF-alpha expressed by CTL after physiologic activation with target APC might participate in the cytolytic reactions mediated by these clones. We found that, upon interaction with target cells expressing env epitopes in the context of the appropriate MHC class II molecules, CD4+ CTL released TNF-alpha with kinetics that were rapid, compared with other cytokines, and that were generally similar to the kinetics of target cell destruction. LT secretion was not detected during the time course of the cytolytic reactions. A novel flow cytometric assay was used to show that physiologic activation of CD4+ CTL with target APC induced expression by the CTL of cell surface forms of TNF-alpha. Immunoprecipitations from activated, surface-iodinated CTL clones revealed two forms of surface TNF-alpha, a 26-kDa form, representing the transmembrane precursor of secreted TNF-alpha, as well as the 17-kDa secreted form bound to the cell surface. For a subset of CD4+ CTL, we found that treatment of CTL with cyclosporin A inhibited Ag-induced production of both transmembrane and secreted forms of TNF-alpha but had no effect on cytolysis. Thus, although transmembrane and secreted TNF-alpha produced by HIV-1-specific CD4+ CTL may have important effects in vivo, the rapid destruction of target APC by the set of CD4+ CTL clones described here occurs through a TNF-alpha-independent mechanism.

Tumor necrosis factor (TNF) receptor expression in T lymphocytes. Differential regulation of the type I TNF receptor during activation of resting and effector T cells.

The expression of TNF-alpha receptors (TNFR) was examined on a CD4+ T cell hybridoma, transformed T cell lines, CTL clones, and activated T cells from peripheral blood to determine the basis of the immunomodulatory activity of TNF on T cell function. Analyses by ligand cross-linking and competitive binding assays with mAb to the 80-kDa receptor (TNFR-I), demonstrated that the TNFR-I was the predominant receptor expressed on activated CD4+ and CD8+ T cell subsets. However, on T cell leukemic lines, a second, non-TNFR-I binding site was identified, most likely the 55-kDa form (TNFR-II). Additional subsets of T cells were readily distinguished by their expression of TNFR-I and related members of the TNFR gene family (CD40 and CD27). Expression of the TNFR-I was dependent upon the state of T cell activation. Signaling through the TCR for Ag or IL-2R was sufficient to induce TNFR mRNA and protein expression in resting T cells. Multiple sizes of TNFR-I transcripts were detected during T cell activation; however, biosynthetic studies showed these multiple species encode a single protein of 80 kDa. These results, combined with the known ability of TNF to induce IL-2R expression, indicate that TNF and IL-2 form a reciprocating receptor amplification circuit. In contrast, differentiated effector T cells triggered through the TCR or protein kinase C initiated a rapid down-regulation (transmodulation) of the TNFR-I that preceded TNF or lymphotoxin secretion. The mechanism of transmodulation involved proteolytic processing of the mature 80-kDa receptor releasing a soluble 40-kDa fragment. This indicates that a TNF autocrine loop is not likely to form during the response of an effector T cell. Collectively, these results suggest that transcriptional and post-translational modification of the TNFR-I are important control points regulating the expression of this receptor during T cell activation.

Effect of α-IFN on cytokine-induced antigen expression and secretion of TNF, LT and IgM in HCL

In order to investigate the possible mechanisms for the effect of alpha-interferon (α-IFN) in hairy cell leukaemia (HCL), blood cells from 4 cases were treated in vitro with α-IFN, tumour necrosis factor (TNF) and interleukin 2 (IL-2). Changes in the antigen expression, immunoglobulin (Ig) secretion and the production of TNF and lymphotoxin (LT) were investigated. TNF induced expression of CD4 and CD71, increased the intensity of HLA-DR, CD25, CD11c and CD13 expression and decreased both the intensity and frequency of sIg and cIg positivity. α-IFN decreased CD25 expression, the tartrate-resistant acid phosphatase activity (TRAP), reduced the TNF-induced CD4 and CD71 expression and antagonized the TNF effect on the Ig expression. Spontaneous TNF or LT production could not be detected in culture supernatants. However, TNF was found to induce LT production, an effect which α-IFN antagonized and IL-2 augmented. The reduction of CD25, TNF-induced CD71 and TRAP caused by α-IFN seems to represent a deactivation of the activated state of hairy cells (HCs). The failure of α-IFN to induce Ig secretion or CD38 expression in HCs speaks against a differentiation induction effect. The LT secretion induced by TNF suggests that other cytokines than TNF might be involved in the proliferation of HCs and that α-IFN by blocking the production of LT and perhaps other cytokines causes a growth arrest in HCs.

Tumor necrosis factor and lymphotoxin secretion by human natural killer cells leads to antiviral cytotoxicity.

Abstract NK cells mediate their cytotoxicity against tumor cells through abroad array of cytotoxic and cytostatic proteins. We investigated whether specific proteins could also be identified that contributed to NK cell-mediated antiviral immunity. Human CD16+/CD3- NK cells were obtained by using FACS and subsequently cloned by using limiting dilution. These NK cell lines, which were cytotoxic against NK-sensitive tumor targets and virally infected cells, also generated supernatants that selectively killed vesicular stomatitis virus-infected cells while sparing noninfected cells. This soluble antiviral activity was completely neutralized by antibodies specific for TNF and lymphotoxin. Purified human rTNF also duplicated this specific cytotoxicity against vesicular stomatitis virus-infected cells, as well as against CMV-, Theiler's murine encephalomyelitis virus-, and HSV-infected cells. The degree of cytotoxicity varied for the different viruses and depended on the cell type infected. These results suggest that NK cells can mediate selective and direct cytotoxicity against virally infected cells by the secretion of TNF and lymphotoxin.

Activation of cultured human endothelial cells by recombinant lymphotoxin: comparison with tumor necrosis factor and interleukin 1 species.

Recombinant human lymphotoxin (LT) was compared with recombinant human tumor necrosis factor (TNF) for direct actions on cultured human endothelial cells (HEC). At equivalent half-maximal concentrations (based on L929 cytotoxicity units) LT and TNF each caused rapid and transient induction (peak 4 to 6 hr) of an antigen associated with leukocyte adhesion (detected by monoclonal antibody H4/18), a rapid but sustained increased expression (plateau 24 hr) of a lymphocyte adhesion structure (ICAM-1), a gradual (plateau 4 to 6 days) increase in expression of HLA-A,B antigens, and gradual (4 to 6 days) conversion of HEC culture morphology from epithelioid to fibroblastoid, an effect enhanced by immune interferon (IFN-gamma). Induction of H4/18 binding by maximal concentrations of LT or TNF could not be augmented by addition of the other cytokine, and 24 hr pretreatment with LT or TNF produced hyporesponsiveness to both mediators for reinduction. H4/18 binding can be transiently induced by tumor-promoting phorbol esters. Pretreatment with either LT or TNF also fully inhibited induction of H4/18 binding by phorbol ester, whereas phorbol ester pretreatment only variably and partially inhibited reinduction by LT or TNF. These actions of LT on endothelium shared with TNF may serve in vivo to promote lymphocyte and inflammatory leukocyte adhesion and transendothelial migration. Recombinant human interleukin 1 species (IL 1 alpha and IL 1 beta) shared many of the actions of LT and TNF and were indistinguishable from each other. However, IL 1 species could be distinguished from LT/TNF by their relative inability to enhance HLA-A,B expression, by their ability to augment H4/18 binding caused by maximally effective concentrations of LT or TNF, and by their inability to inhibit reinduction of H4/18 binding by LT or TNF. In contrast to the actions of LT or TNF, pretreatment with IL 1 alpha or IL 1 beta only partially inhibited induction of H4/18 binding by phorbol ester, and phorbol ester pretreatment consistently, albeit partially, inhibited induction by IL 1 species. These studies suggest that activated T cells through the secretion of LT can in turn activate the local endothelial lining so as to promote homing and extravasation of inflammatory cells. Furthermore, these LT actions can be augmented or complemented by other locally produced mediators such as IFN-gamma or IL 1.

Normal levels of lymphotoxin secretion by freshly isolated and refrigerated human peripheral blood lymphocytes

Lymphocytes obtained by Ficoll-Hypaque density gradient centrifugation of leukocytes from 40 blood bank donors were incubated at 10 6 cells/ml for 24 h with 5 μg phytohemagglutinin/ml serum-free RPMI 1640 medium and secreted 34 ± 6 U lymphotoxin/ml and 52 ± 29 U interferon/ml. Addition of 5% fetal bovine serum (FBS) or acid-citrate-dextrose treated autologous plasma (AP) augmented lymphotoxin secretion 2-fold and interferon secretion 6–8-fold. Lymphotoxin alone was secreted in 39% of serum free cultures. Both lymphotoxin and interferon were secreted in the presence of FBS or AP. Lymphocytes refrigerated at 4°C for 18 h secreted upon serum-free culture similar levels of lymphotoxin as fresh cells but cultures secreting lymphotoxin alone increased from 39 to 77%. Lymphotoxin secretion by refrigerated lymphocytes was similar in the presence or absence of FBS or AP. Interferon secretion by refrigerated lymphocytes in the presence of FBS or AP, however, increased 30–40-fold approaching levels produced by freshly isolated cells and indicating that the modulatory effects of refrigeration, FBS, and AP affected interferon not lymphotoxin secretion. The levels of lymphotoxin and interferon secreted by lymphocyte populations producing both lymphokines, furthermore, varied independently of one another providing additional evidence for the dissociability of their secretion yet possible dependence of interferon upon lymphotoxin secretion. The ability to reliably quantitate lymphotoxin secretion using refrigerated lymphocytes permits transport of lymphocytes to laboratories performing quantitative bioassay of the lymphokine. This should facilitate further evaluation of the presence and role of lymphotoxin in immunopathological and other disease states.

Human T-cell hybridomas producing lymphokines: II. Enhancement of lymphotoxin secretion from human T-cell hybridomas by phorbol myristate acetate

Human lymphotoxin (LT)-producing T-cell hybridomas were constructed by fusing concanavalin A-activated human peripheral blood lymphocytes with emetine-actinomycin D-pretreated human acute lymphatic leukemia cells. LT secretion from these hybridomas was considerably enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA) and concanavalin A or PMA alone. A study using cloned hybrid lines revealed that PMA/Con A acted directly on the LT-producing clones. Furthermore, PMA/Con A stimulated A-B9-24, one of the cloned hybridomas, and secreted fourfold larger amounts of LT under serum-free conditions than under serum-containing conditions. However, MIF MAF and LT-producing cloned hybrid line E10–20 secreted rather decreased amounts of MIF MAF when stimulated with PMA, while the LT secretion from the same hybridoma was enhanced with PMA.

Comparative effectiveness of lymphotoxin anticarcinogenic and tumor cell growth-inhibitory activities

Prevention of ultraviolet radiation- or chemical carcinogen-induced morphologic transformation and inhibition of tumor-producing transformed cell growth by lymphotoxin and by normal spleen leukocytes were quantitatively compared to define the antineoplastic activity spectra of these natural immune mediators. When Syrian golden hamster embryo cells seeded for colony formation in culture dishes were treated simultaneously with carcinogen and lymphotoxin, the number of morphologically transformed cell colonies was irreversibly reduced by 50% in the presence of 6 units of lymphotoxin/ml. Lymphotoxin inhibition of tumor cell growth, however, was reversible and 50% reduction in tumor cell growth in three transformed lines required 124, 330, and 477 units/ml. Thus, the anticarcinogenic activity of lymphotoxin can be 20-fold or more greater than its tumor growth-inhibitory activity. Similarly spleen leukocytes also were more effective as an anticarcinogen than as an inhibitor of tumor cell growth, consistent with previous observations that naturally occurring spleen leukocyte antineoplastic activity may result from lymphotoxin secretion.

Lymphokines secreted from sodium periodate-treated lymphocytes

This study was designed to determine if sodium metaperiodate (NaIO 4)-treated lymphocytes secrete lymphokines and if these lymphokines are similar to those obtained from mitogen- or antigen-stimulated lymphocytes. A brief exposure of CBA spleen cells to NaIO 4 induced the secretion of significant amounts of migration inhibitory factor (MIF). This MIF had a molecular weight range between 30,000 and 58,000, and was stable when heated at 56 °C for 30 min, but unstable at 80 °C. These characteristics are similar to those previously reported for mitogen- and antigen-induced MIF. In addition, NaIO 4 induced the secretion of lymphotoxin (LT) from CBA and Balb/c spleen cells, as well as from guinea pig lymph node cells. NaIO 4 was compared to the other inducers in regard to the quantity of LT secreted. Supernatant derived from NaIO 4-treated mouse spleen cells contained less LT than supernatants derived from concanavalin A- or phytohemagglutinin-treated cells, but contained more activity than those supernatants derived from lipopolysaccharide-treated cells. CBA spleen cells secreted significantly more LT than Balb/c spleen cells after NaIO 4 stimulation. NaIO 4-stimulated CBA spleen cells secreted LT in cultures with or without serum, but stimulated Balb/c spleen cells secreted LT only in serum-containing cultures. The advantages of NaIO 4 as an inducer of lymphokines, as opposed to other mitogens or antigens, is the brief exposure of this agent to the cells after which the NaIO 4 is removed, and the lymphokines can be obtained free from the inducer.

Studies on the transformation of lymphocytes separated by suspension in dextran

Phytohemagglutinin (PHA)-stimulated cells were suspended in 10% dextran to prevent cellular aggregation. Cells suspended in dextran did not respond to PHA by any of the five parameters tested: [ 3H]leucine, [ 3H]uridine, and [ 3H]thymidine incorporation into acid-insoluble products, lymphotoxin synthesis, or morphologic transformation. Dextran itself was not cytotoxic to the lymphocytes. However, one major technical problem is attaining a proper dextran “control.” The difficulty in culturing lymphocytes in dextran without simultaneously causing some cell dispersion yielded dextran “control” values that showed less stimulation than cells without dextran. These results suggest that cell contact is necessary for PHA-induced transformation as judged by both DNA synthesis and lymphotoxin secretion. This technique now provides an experimental system for biochemically assessing the effects of antigens and mitogens without the attendant problems caused by cell clustering.

In vitro lymphocyte cytotoxicity : I. Evidence of multiple cytotoxic molecules secreted by mitogen activated human lymphoid cells in vitro

Multiple families of cytotoxic molecules [Lymphotoxin (LT)] have been identified in phytohemagglutinin (PHA-P) activated human lymphocyte supernatants and lymphocyte homogenates, using gel filtration chromatography on Sephadex G-150. These macromolecules have molecular weights of 80–90,000, 50,000, and 10–15,000 daltons and have been termed LT 2, LT 2 and LT 3, respectively. They are secreted by cells from a variety of lympboid tissues, i. e., tonsil, adenoid, and peripheral blood. The kinetics of appearance of the cytotoxins indicate that all three are present within 16 hr after lymphocyte activation. However, while LT 1 and LT 2 persist in these cultures through day 5, LT 3 is not detectable after day 3. These molecules can also be detected when either PHA or concanavalin A are employed as the stimulating agent. Moreover, the relative amounts of LT 1, LT 2 and LT 3 activity in a given supernatant vary dramatically from culture to culture. Extracellular levels of LT accumulate and peak by 4 to 5 days in culture, however, intracellular levels of LT reach a maximum on day 3 and decrease to very low levels on day 5. Mitogen-stimulated lymphocytes at 3 days contain intracellular levels of LT which are several logs higher than that detectable in unstimulated cells. This observation suggests that both the biosynthesis and secretion of lymphotoxin is governed by a regulatory control process(es).

The Role of Lymphotoxin in Target Cell Destruction Induced by Mitogen-Activated Human Lymphocytes in Vitro

Abstract The in vitro destruction of phytohemagglutinin (PHA) coated β L cells by non-immune human lymphocytes was resolved into two distinct phases—lymphocyte dependent and lymphocyte independent. The initial or lymphocyte-dependent phase occurred within the first 2 hr and proceeded equally well at 34 and 37°C. The amount of lymphotoxin (LT) secreted by PHA-activated human lymphocytes in vitro to PHA stimulation was the same at 34 and 37°C. Antiserum and complement inactivation of the aggressor lymphocytes at various intervals revealed that target cell lysis was lymphocyte independent. However, the latter phase was temperature dependent, i.e., proceeding at the permissive temperature of 37°C, but inhibited at the restrictive temperature of 34°C. Further experiments revealed that LT-induced destruction had the same temperature sensitivity as target cell cytolysis occurring during the lymphocyte-independent step. Trypsin treatment of target cells during an early period of the lymphocyte-independent phase protected the target cell from subsequent death, indicating the aggressor lymphocyte has deposited a cytotoxic effector material on its surface. These results suggest the lymphocyte-dependent stage involves the processes required for the induction of LT synthesis and secretion. The actual cytolysis occurring during the lymphocyte-independent stage may be caused by LT or LT-like material(s) deposited on the target cell surface by the mitogen-activated human lymphocyte.

THE EFFECT OF AGENTS WHICH MODULATE LEVELS OF THE CYCLIC NUCLEOTIDES ON HUMAN LYMPHOTOXIN SECRETION AND ACTIVITY IN VITRO

The ability of concanavalin-A (Con-A) to activate lymphocytes to secrete human lymphotoxin (LT) was tested in the presence of agents known to modify the intracellular levels of cyclic nucleotides (cAMP and cGMP). Lymphocytes were treated with these agents at different stages of activation: (1) during the first encounter with mitogens, and (2) after they had been fully activated and were restimulated. Two agents, Dibutyryl cAMP and theophylline, dramatically inhibit the amount of LT secreted during both "primary" and "secondary" activation by Con-A. In contrast; DL-isoproterenol had a weak effect during primary activation, but greatly reduced the level of LT secreted during secondary activation. Agents which affect the intracellular level of cGMP were also tested. Imidazole had no effect on LT secretion by either primary or secondary Con-A activated cells. In contrast, carbamyl choline greatly reduced LT secretion to a level comparable to Dibutyryl cAMP and theophylline. All agents tested protected, to some degree, the sensitive alpha-L cell against LT-induced destruction in vitro. Agents which affect the levels of cyclic nucleotides affect both the effectiveness of the aggressor cell and the sensitivity of the target cells in vitro.

Relationship of lymphotoxin secretion and DNA synthesis in the human mixed lymphocyte reaction in vitro

One-way mixed lymphocyte cultures employing human adenoid or peripheral blood lymphocytes activate lymphotoxin (LT)-secreting cells. Kinetic analysis of lymphocytes stimulated in mixed culture demonstrates that LT is secreted before the onset of DNA synthesis, but that maximum levels of LT secretion are reached simultaneously with maximum levels of DNA synthesis. Although the response of peripheral leukocytes is qualitatively similar to the response of adenoid-derived lymphocytes, unexplained high nonspecific background levels of toxic material(s) obscure early events in the former response. While cytochalasin B reversibly inhibits LT secretion, mitomycin C treated cultures are still capable of LT secretion. The results suggest that a population of cells exists, which does not require DNA synthesis to develop into effector cells. The requirement for DNA synthesis for the maximal development of effector cells may reside in a separate helper cell population as postulated by the two cell model of the mixed lymphocyte reaction.