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Two isoforms of lymphotoxin beta (LTbeta) were isolated from mRNAs of a panel of human lymphoid cell lines and tumor tissues obtained from patients with non-Hodgkin's lymphoma (NHL). The truncated LTbeta mRNA variant lacked 46 base pairs complementary to the complete sequence of exon 2, suggesting that both isoforms are produced by an alternative splicing mechanism. Skipping out of exon 2 causes a reading frame shift and a premature stop codon in the LTbeta mRNA variant. The predictive translated polypeptide would correspond to a severely shortened LTbeta protein that would lack the majority of the extracellular domain of the native molecule, thus impairing its normal complex assembly with LTalpha. These observations provide new insights into the molecular heterogeneity and biological function of LTbeta within the tumor necrosis factor and LT ligand-receptor system., (Copyright 1997 Academic Press.)

The pterins neopterin and xanthopterin have been measured together with the cytokines IFN-gamma, TNF-alpha and TNF-beta in the supernatants of 20 allogeneic mixed lymphocyte reactions. We have calculated rank correlation coefficients between these analytes and the immune response. Our findings confirm that neopterin is a sensitive early marker of immune activation and show that another pterin, xanthopterin could be used likewise. Cytokine production was also proportional to immune activation but none could be used consistently as early markers of immune activation.

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.

It was previously reported that spleen cells of rabbits infected with Treponema pallidum produced anti-treponemal lymphotoxins (ATL). This ability was distinctly disturbed when circulating immune complexes (CIC) and autolymphocytotoxins (AL) were present in the sera of cell donors. ATL liberated from cells of donors without CIC and AL displayed a marked ability to immobilize treponemes. The percentage of immobilized treponemes varied according to the type of cells used for ATL liberation and their density. The most active was ATL from T cells (density 4 x 10(8) ml-1) and the weakest was the one from B lymphocytes. In the presence of CIC in sera of cell donors the weakest ATL was from macrophages and in the presence of AL from T lymphocytes. When both factors (CIC and AL) were present ATL from T lymphocytes did not immobilize treponemes. This seems to suggest that the impairment of the cells' ability to produce ATL may facilitate the survival of treponemes in the host despite the presence of immunologically competent cells.

Anti-CD3 antibody induced non-MHC-restricted cytolytic activity in murine spleen cells within 4 hr of incubation in vitro and within 8 hr in vivo after intraperitoneal injection. Interstitial lung leukocytes acquired the capacity to kill both NK-sensitive and -resistant targets within 24 hr after anti-CD3 injection. In vivo-stimulated spleen cells produced significantly more IL-2 and TNF than unstimulated cells. When these cells were restimulated with anti-CD3 in vitro, a potentiation of TNF production was seen. Interstitial lung leukocytes doubled their TNF production ex vivo after anti-CD3 injection, and the suppressed TNF production which was seen in lung cells from tumor-bearing animals was restored after anti-CD3 stimulation. The TNF production by alveolar macrophages was augmented 5-fold 48 hr after injection of antibody. Anti-CD3 antibody also induced significant accumulation of both TNF alpha and TNF beta mRNA in spleen and lung leukocytes. Pre-treatment with anti-TNF antibodies, both in vitro and in vivo, did not eliminate the cytolytic activation of lung and spleen cells. Our data indicate that anti-CD3 antibodies can induce rapid activation of both cytolytic activity and cytokine production in lung lymphocytes and macrophages.

The cytotoxin production by regional lymph node cells was examined in 25 patients with uterine cervical cancer and 10 patients with uterine myoma. The patients in stage I had significantly increased spontaneous release of cytotoxins compared with that in stages II, III, and IV. The spontaneous release in stages III and IV was markedly reduced. There was no difference in the release of cytotoxins from peripheral blood lymphocytes between cancer patients and patients with myoma or healthy controls. The cytotoxin production by lymph node cells was increased in stage III by stimulating with formalin-fixed QG-K cells derived from uterine cervical cancer, but not in stages I and II. Almost all of the cytotoxic activity of cytotoxin was abrogated by antilymphotoxin antibody. However, the cytotoxin activity was partially inhibited by anti-tumor necrosis factor antibody. These results suggest that cytotoxins released from the regional lymph node cells of uterine cancer patients are derived from, most of all, lymphotoxin.

Human lymphocytes were stimulated in vitro with mitogens, phytohemagglutinin and concanavalin A to secrete proliferation inhibitory factor (PIF), cloning inhibitory factor (CIF), and lymphotoxin (LT). These three activities were demonstrable in the same supernatant, moreover, the particular effect observed was shown to depend on the concentration of the medium and the type of target cell employed. In general, the medium effects on target cells were: a) cytotoxic at high concentrations, b) growth inhibitory at intermediate concentrations, and c) only temporarily growth inhibitory at low concentrations. The absolute concentration producing a certain effect varied, depending on the target cell type employed. In addition, the sensitivity of the target cells to LT parallels the sensitivity of the cell to each of the other activities, PIF and CIF, and no species specificity was observed.

The ability of various subsets of human mononuclear cells to produce human lymphotoxin (LT) was examined. Peripheral blood mononuclear cells were separated into OKT4+, OKT8+, and Leu-11a+ subpopulations by flow cytometry. Both OKT4+ and OKT8+ cells produced LT upon phytohemagglutinin (PHA) stimulation, but the OKT4+ T cells were the major source of LT. In contrast, Leu-11a+ cells failed to produce LT. The LT production and the proliferative response to PHA, of OKT4+ and OKT8+ cells, were inhibited by prostaglandin E2 and histamine. The LT, derived from PHA-stimulated peripheral blood lymphocytes, was purified by a procedure involving Blue-agarose, Con A-Sepharose chromatography, preparative gradient polyacrylamide gel electrophoresis and preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The LT activity was recovered from SDS gel with major activity peak in the Mr 76,000 region and the minor activity peak in the Mr 24,000 region. The LT derived from 1788 lymphoblastoid cell line also showed heterogeneity on SDS gel. The activity was recovered from two peaks in the region of Mr 70,000 and 20,000.

Two different lymphotoxins synthesized by human blood lymphocytes stimulated with phytohemagglutin (PHA) are described. The two toxins are called alpha-LT and beta-LT relative to their elution order on gel chromatography. Their m.w. are 75,000 and 45,000 daltons, respectively. Both toxins appear as early as 7 hr after the addition of PHA, with the amount of beta-toxin exceeding that of alpha-LT initially. Both toxins are differentiated from a third toxin (adherent cell toxin, ACT) made by plastic-adherent cells without requiring mitogen-stimulation. Depletion of macrophages or B cells does not affect the synthesis of either lymphotoxin. Monospecific antisera were elicited to alpha-LT. Antisera elicited to beta-LT also neutralized alpha-LT but to a significantly lesser degree. Alpha-LT appears to be the lymphotoxin described by most other workers. Beta-LT is unstable at 37 degrees C which may explain why the low m.w. lymphotoxin has not been described previously.

Spleen lymphocytes from C3H/He mice when treated with cisplatin show increased cytostasis and cytotoxicity against Dalton's lymphoma cells in vitro. Cisplatin treatment of splenocytes does not render them cytotoxic/or cytostatic against normal splenocytes. Splenocytes on treatment with cisplatin also produce tumor cell-specific cytotoxic/cytostatic factors (lymphotoxins) which have cytolytic and cytostatic effect on Dalton's lymphoma cells. The increased cytotoxicity of splenocytes on treatment with cisplatin is reversed by the calcium channel blocker nifedipine and the calmodulin antagonist chlorpromazine, suggesting a role of calcium in cisplatin-activated lymphocyte-mediated cytotoxicity.

The in vitro destruction of phytohemagglutinin (PHA) coated Beta 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 degrees C. The amount of lymphotoxin (LT) secreted by PHA-activated human lymphocytes in vitro to PHA stimulation was the same at 34 and 37 degrees 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 degrees C, but inhibited at the restrictive temperature of 34 degrees 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.

In lymphocyte cultures (LC) from 5 immune donors blast transformation occurred as a result of the stimulting action of adenovirus antigen; the supernatants obtained from these cultures produced a cytotoxic effect on L cells, thus suggesting the presence of lymphotoxin (LT). No blast transformation and no LT production were detected in nonstimulated LC used for control, as well as in LC from 9 nonimmune donors. LT production was shown to depend on the time elapsed after recovery from acute adenovirus infection. LT production was found to be most intensive during the first 2 months after recovery.

Rubella virus-stimulated lymphocytes from rubella-seropositive donors produced in the culture medium cytotoxic activity with preferential action against rubella-infected over uninfected target cells. The ability of lymphocytes to produce the cytotoxic activity upon stimulation by rubella virus correlated with the humoral rubella-immunity status, i.e. no such cytotoxic activity developed in the supernatants of lymphocyte cultures of rubella-seronegative donors. Stimulation of lymphocytes from seropositive donors by rubella virus was also detected by thymidine incorporation, but the correlation of lymphocyte responsiveness to the humoral rubella antibody status was not so clear as in the cytotoxicity assay. Conversion of lymphocytes from unresponsive to responsive to rubella virus following natural rubella infection and after rubella vaccination was demonstrated using both methods. Following vaccination rubella-specific cell-mediated immunity first became demonstrable at 14 days. The responsiveness of lymphocytes to phytohaemagglutinin (PHA) after rubella vaccination was followed by studying thymidine uptake and the ability of lymphocytes to produce lymphootoxin. By both tests marked suppression of PHA response occurred at days 3 and 7 after vaccination.

A mitogenic factor (MF) from guinea pig lymph node cells (LNC), which was produced by stimulating immune LNC with the specific antigen, ovalbumin, was partially purified by the sequential use of Sephadex G-200 gel filtration. CM-cellulose ion exchange chromatography, DEAE-cellulose ion exchange chromatography, and polyacrylamide disc gel electrophoresis. The product was purified at least 100-fold with regard to protein content. In addition, the purified MF was functionally pure with respect to the absence fo lymphotoxin (LT), another guinea pig lymphokine.

A total of 43 lymphocyte culture (LC) from 12 adults (aged 32--48 years) and 21 children 6--7 years of age were studied. In PPD-stimulated adults LC stimulation ratio ranged from 8 to 39%; their cytotoxic activity, i.e. lymphotoxin (LT) production, was determined by staining target L cells with crystal violet and measuring protein synthesis in surviving target cells by radioactive amino acid incorporation. In children blastogenic response occurred in 11 out of 21 PPD-stimulated LC studied, but only 8 supernatant culture fluids were toxic to L cells and 3 were not. These findings were confirmed by repeated tests 3 weeks later. Probable correlation of BTL and LT production with the functional state of specific immunity to tuberculosis is discussed.

Lymphotoxin was found to be present in supernatants from 22 human lymphocytes cultures stimulated with phytohemagglutinin in a dose of 5 and 10 microgram/ml. The lymphocytes were obtained from the peripheral blood of 6 apparently healthy persons. Lymphotoxin activity was determined by simple and objective method, i.e. by staining the target cells (mouse L-cells) monolayer with crystal violet, with the following determination of optic densities of the L-cells lysates at 570 nm in the spectrophotometer. As revealed, 1 : 5 dilutions of the supernatants from the lymphocyte cultures incubated for 48 hours inhibited the L-cells growth by from 40 to 60%. With further incubation of the cultures (up to 72 and 96 hours) the cytotoxicity of their supernatants for the target cells showed no increase, whereas the blasttransformation index reaches the maximal value by 72nd incubation hour. Supernatants from unstimulated lymphocyte cultures failed to produce any cytotoxic effect on L-cells.

Human blood monocytes and lymphocytes were separated by Percoll gradient fractionation. The synthesis of RNA was inhibited by actinomycin D (AcD) or alpha-amanitin (Amn). Monocytes were stimulated with LPS, lymphocytes were stimulated with phytohaemagglutinin (PHA). The activity of tumor necrosis factor (TNF-alpha) and lymphotoxin (LT) was measured by L-929 cell assay. It was shown that induction of TNF-alpha synthesis by LPS was not blocked by AcD and Amn. In contrast, the production of LT was blocked in cultures of lymphocytes treated by the inhibitors. Moreover, TNF-alpha synthesis was induced in resting monocyte cultures by AcD. Cycloheximide (Cy) inhibited the production of TNF-alpha. The data imply that TNF-alpha synthesis by human blood monocytes can be induced by posttranscriptional regulation of TNF-alpha mRNA presynthesized in vivo.

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 micrograms 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 degrees 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.

We present evidence that human peripheral blood lymphocytes, free of contaminating monocytes, rapidly produce high levels of tumor necrosis factor (TNF) when stimulated with phorbol diester and calcium ionophore, and lower but significant levels of TNF when stimulated with mitogens. These two types of inducers act preferentially on T cells, both CD4+ and CD8+. NK cells produce TNF only when stimulated with phorbol diester and calcium ionophore, and they do so at a much lower level than T cells. The procedures used in the purification of lymphocytes and the differential ability to respond to various inducers allow us to exclude that monocytes or basophils contaminating the lymphocyte preparation participate in the production of TNF. In particular, LPS, a potent inducer of TNF production from monocytes, is unable to induce significant levels of TNF in the lymphocyte preparations. The TNF produced by lymphocytes has antigenic, physicochemical, and biochemical characteristics identical to those of the TNF produced by myeloid cell lines or monocytes upon stimulation with LPS. LT is also produced by lymphocyte preparations. Production of TNF and LT proteins in response to the different inducers is paralleled by accumulation of cytoplasmic TNF and LT mRNA. Both at mRNA and at protein levels, stimulation of T lymphocytes with phorbol diester and calcium ionophore preferentially induces TNF, whereas mitogen stimulation preferentially induces LT. Our data suggest that the TNF and LT genes, two closely linked genes encoding two partially homologous proteins with almost identical biological functions, are independently regulated in lymphocytes.

A significantly increased spontaneous cell-mediated cytotoxicity (SCMC) has been reported in synovial fluid lymphocytes (SFL) as compared to peripheral blood lymphocytes (PBL) of patients with rheumatoid arthritis (RA) and that of normal controls [1-3]. To determine whether this increased SCMC activity is due to the production of a lymphokine and related to the production of a lymphotoxin(LT)-like mediator, PBL from normal controls and PBL and SFL from RA patients were incubated either with a human melanoma cell line (IGR 3) or with cell-free synovial fluid (SF) from RA patients. The SF and the cell-free supernatants of the different cultures were tested for LT activity by estimation of inhibition of DNA synthesis of HeLa cell monolayers and they were added to a SCMC assay system using normal PBL and IGR 3 as target. In the supernatants from cocultures of either PBL from controls or PBL and SFL from RA patients with IGR 3 cells, there was no significant difference in LT activity. An LT-like mediator was observed in the supernatants of all lymphocytes cocultured with SF, whereas SF alone and supernatants of lymphocytes alone exhibited little or no LT activity. In a control experiment, LT induction was not observed when normal lymphocytes were cultured with the serum of RA patients. Absorption of the culture supernatants with an insolubilised goat anti-human Ig did not remove LT activity. The demonstrated release of an LT-like mediator from lymphocytes incubated with SF might be one contributing mechanism to the inflammatory joint reaction in RA patients.

Treatment of tuberculin-sensitive guinea pig spleen or lymph node cells with a burro anti-T cell serum plus complement diminished markedly the number of functionally detectable T cells, but did not affect the amount of migration inhibitory factor (MIF) or lymphotoxin produced by the residual T cell-depleted populations.

Pretreatment of human peripheral blood lymphocytes with interferon significantly enhances the release of lymphotoxins (LTs) observed at subsequent incubation of the cells, for 3 hr, with phytohemagglutinin (PHA). Fractionation of the LTs by gel filtration shows that interferon (IFN) strongly increases the release of certain LTs produced by these cells, while it has little effect on the release of others. The release of LTs from the IFN-treated cells is dependent on stimulation by PHA, requires Ca2+ ions, and can be blocked by prostaglandin E1, but it is independent of protein synthesis.

The present investigation was initiated for the purpose of elucidating which cell type, T and/or B lymphocytes, were responsible for human lymphotoxin (LT) and human chemotactic factor (CF) production in vitro. T- and B-enriched subpopulations were tested for proliferative responses and lymphokine release in the presence of a human T mitogen, phytohaemagglutinin (PHA), and a human B-mitogen, Nocardia water-soluble mitogen (NWSM). We found that PHA-stimulated T cells produced LT and CF whereas NWSM-stimulated B-enriched subpopulation released LT and CF.

A study was made of the production of a blastogenic factor and lymphotoxin in the cultures of lymphocytes of the palatine tonsils removed from patients with chronic tonsillitis; the activity of this blastogenic factor and lymphotoxin was studied in the test-cultures of autologous and allogenic lymphocytes and the transplantable HeLa cells. The antigens of the pathogenic streptococcus and staphylococcus induced production of the blastogenic factor and lymphotoxin; as to the antigens of saprophytic bacteria-they produced no such action. The antigen-specific blastogenic factor intensified the immune response to the homologous antigen, whereas the factor obtained in stimulation of lymphocytes with PHA-to all the microbial antigens under study.

Human peripheral blood mononuclear cells (PBMC) were induced by recombinant human interleukin 2 (rIL 2) to secrete lymphotoxin (LT) and interferon-gamma (IFN-gamma). Induction of both LT and IFN-gamma by rIL 2 is regulated at the transcriptional level. Treatment of PBMC with rIL 2 in combination with recombinant human IFN-gamma (rIFN-gamma) resulted in an earlier appearance of LT mRNA and an augmented production of LT than did rIL 2 treatment alone. IFN-gamma alone had no effect on production of either LT or its mRNA. PBMC cultured in the presence of rIL 2 plus anti-rIFN-gamma resulted in decreased LT production. Only a 15-min incubation of PBMC with rIL 2 is needed to stimulate LT production, whereas 3 hr is necessary for IFN-gamma production. These results suggest that rIL 2, in addition to being a T cell growth factor, may exhibit other activities through induction of LT and IFN-gamma.

The tumor promoters mezerein and phorbol myristate acetate, and the phorbol diesters phorbol diacetate, phorbol dibenzoate, and phorbol didecanoate synergistically enhanced the production of lymphotoxin (LT) by phytohemagglutinin-stimulated human peripheral blood or tonsil and adenoid lymphocytes. LT production was elevated 2-20-fold, depending on such parameters as the nature of the promoter and dose, the lectin dose, and the lymphocyte source. The increased LT levels were primarily due to enhanced production of the alpha-light (alpha L) class of LT. The alpha L-class obtained from supernatants from promoter-synergized, lectin-stimulated lymphocyte cultures was compared with the alpha L from lectin-stimulated cultures. They were indistinguishable by molecular sieving on Ultrogel AcA44, were both composed primarily of the alpha 2-subclass as determined by ion-exchange chromatography on DEAE-Sepharose, and were immunologically cross-reactive. Lectin-affinity chromatography on concanavalin A-Sepharose and on lentil-lectin--Sepharose revealed that both alpha L preparations were dominated by components with affinity for these matrices. Affinity chromatography on alkyl sorbents also indicated very similar hydrophobicities. Chromatofocusing of the alpha L preparations demonstrated a comparable pattern of isoelectric points. Thus, the use of these drugs in lectin-stimulated human lymphocyte cultures provides an effective means for significantly increasing the yield of alpha L-LT suitable for biochemical purification and analysis, and biological testing in vitro.