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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.
Mononuclear phagocytes and lymphocytes can both synthesize cytotoxins which kill certain tumor cell lines and recent evidence suggests that these cytotoxins may be identical. In this study, human monocyte cytotoxin and lymphoblastoid lymphotoxin have been critically compared in terms of specificity, physicochemical characteristics and antigenicity. Despite their similar pattern of reactivity with a panel of cell lines, lymphotoxin was less readily adsorbed than monocyte cytotoxin by tumor cells. The cytotoxins could also be distinguished by their behavior on high performance liquid chromatography and by the failure of antibody to monocyte cytotoxin to react with lymphotoxin.
In this study we report that cloned Thy-1+, L3T4-, Lyt-1-, Lyt-2+, H-Y-specific and H-2Db-restricted cytotoxic T cell lines (CTLL) when induced by lectin or antigen secrete a soluble mediator(s) (SF) that inhibits proliferation and generation of cytotoxic lymphocytes (CTL) in mixed lymphocyte cultures (MLC). The biological activity was separable by gel filtration and appeared as a broad peak in the molecular mass range between 10 000 and 50 000 kDa. It was found that the suppressive activity released by CTLL neither strictly correlates with their cytotoxic potential nor with their ability to produce immune interferon or lymphotoxin. SF was shown to elicit its activity in an antigen-nonspecific manner in that it suppressed the maturation of T lymphocytes responding to both, the appropriate H-Y antigen as well as to unrelated H-2d alloantigens or to the hapten 2,4,6-trinitrophenyl (TNP). The effect of SF on CTL responses was most pronounced in early phases of primary or secondary MLC. When analyzed for its inhibitory activity on precursor cells in populations selected for either Lyt-2- or L3T4- lymphocytes, it was found that SF interfered with the maturation of both subsets. The inhibition of CTL responses elicited by SF could not be reversed by the addition of exogenous interleukin 2. The finding that SF also inhibited the proliferation of some but not all antigen-dependent cloned T cells with helper or cytotoxic potential provides evidence that the factor also may regulate effector lymphocytes. In addition, the results support the assumption that SF exerts its effect directly on the responder rather than the stimulator population, and demonstrate that the development of CTL from their precursor cells is controlled at least in part by the cytotoxic effector cells themselves via a soluble factor(s) that interferes with distinct stages of T cell maturation. These findings again emphasize the expression of multiple functions by CTL and indicate their possible role during the course of an immune response by their capability to eliminate target cells and to secrete a soluble product(s) that mediates feedback control.
T cell clones derived from patients with autoimmune diseases were found to be capable of producing tumor necrosis factor (TNF). This was demonstrated by stimulating the clones, in the absence of accessory cells, with antibodies against the Ti/T3 complex and with recombinant interleukin 2 (IL2). Analysis of RNA extracted from these clones showed that TNF mRNA was more abundant than lymphotoxin (LT) mRNA. We also found that TNF protein in the supernatants of these clones was generally more abundant than LT as assessed by using the murine L929 cell assay. TNF production was not limited to T cells from autoimmune individuals, since the T cell tumor HUT78 and T cells purified from the peripheral blood of healthy individuals also made TNF. Unlike the T cell clones, HUT78 produced greater amounts of LT mRNA than TNF mRNA. Induction of TNF mRNA in T cells from healthy individuals displayed a two-signal requirement (phorbol myristate 13-acetate and phytohemagglutinin or OKT3 and phorbol myristate 13-acetate), similar to that described for the induction of the T cell lymphokines IL 2 and interferon-gamma (IFN-gamma). Additionally we found that IL2 alone was sufficient to induce TNF in these cells when they had been precultured with phytohemagglutinin for 7 days to express IL 2 receptors. The cloned T cells we have characterized also produce IFN-gamma which was detected in the supernatants of the clones using a radioimmunoassay. The evidence suggests that T cells can produce TNF and have the potential to deliver by themselves the dual and synergistic signals of TNF/LT and IFN-gamma to target cells, a process which may be of importance in the pathogenesis of human autoimmunity.
Protein antigens with both allergenic and immunoprotective properties represent appropriate molecules to study IgE and IgG regulation. We have established a panel of T cell clones specific to bee venom phospholipase A2 (PLA) from human individuals allergic, hyposensitized or immune (protected) to bee sting. All clones obtained were CD3+, CD4+ and expressed alpha, beta T cell receptor. Depending on the T cell clone, maximal stimulation required 1 to 100 micrograms/ml of PLA, and the addition of interleukin (IL)-2 and/or IL-4 increased their antigen-dependent proliferation. Following antigen stimulation, the clones produced IL-4, interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor. Most clones also produced tumor necrosis factor alpha (TNF-alpha) and tumor necrosis factor beta (TNF-beta), and some produced IL-5 and/or IL-2. Both absolute and relative amounts of secreted cytokines depended on the antigen concentration. At low antigen doses, IL-4 was produced but little or not IFN-gamma, whereas at higher PLA concentrations significant amounts of both IL-4 and IFN-gamma were obtained. Thus, these PLA-specific T cell clones could be classified according to the changes in the ratio of IL-4/IFN-gamma production in response to increasing antigen concentrations. Clones derived from allergic and hyposensitized individuals required higher critical amounts of antigen for IFN-gamma induction, and expressed increasing IL-4/IFN-gamma ratios with increasing concentrations of PLA. Modulation of cytokine patterns by the dose of the antigen may be a driving force for IgE or IgG formation resulting in allergy or immunoprotection.
Human lymphokine-activated killer (LAK) cells expressed a membrane-associated lymphotoxin-related molecule (mLT) which was detected by flow cytometric analysis with anti-lymphotoxin antibody. Upon removal of exogenous interleukin-2 from LAK cell culture medium and another 24 h cultivation, the expression of mLT was decreased. Corresponding to the decrease of mLT expression, the killing activity of LAK cells towards L929 cells was remarkably reduced and killing of MIA PaCa-2 and U937 cells was moderately reduced, whereas killing of Daudi and K562 cells was fully restored. The supernatant of mLT-expressing LAK cells had no cytotoxic activity towards L929 cells in the absence of actinomycin D. Moreover, not only the killing of L929 cells but also that of human tumor cell lines (MIA PaCa-2, U937) by mLT-expressing LAK cells was partially inhibited in the presence of anti-lymphotoxin antibody. These results suggest an involvement of mLT in the killing of some tumor target cells by LAK cells.
(TGF)-beta is a pluripotent cytokine exerting differential effects on distinct components of the immune response. The present report, based on lymphokine determination in culture supernatants and Northern blot analysis of lymphokine mRNA, demonstrates that TGF-beta 2 markedly inhibits interleukin (IL)-4 and IL-5 synthesis by polyclonally activated human T cells in the absence of any significant effect on (IFN)-gamma, lymphotoxin or IL-2, suggesting a modulatory effect of TGF-beta 2 on the interferon Th1/Th2) balance of immune responses. The inhibitory effect of TGF-beta on IFN-gamma production by unfractionated peripheral blood mononuclear cells is likely to reflect the blunting of natural killer cell activation by TGF-beta.
Conditioned media from cultured Kupffer and mononuclear macrophagic cells obtained 48 hr after CCl4 administration to rats contains chemotactic factors for human skin fibroblasts and human monocytes. The chemotactic mediator for fibroblasts was approximately 17 kD and was more prominent at early stages of culture. It induced a dose-dependent chemotactic response in fibroblasts. Although the conditioned medium from cultured Kupffer cells of normal rats also contained detectable biological activity, it was significantly less than that in conditioned medium from cultured Kupffer cells from CCl4-treated rats. The activity obtained after purification by high-performance liquid chromatography was completely ablated by incubation with tumor necrosis factor-alpha antibody. Transforming growth factor-beta antibody diminished biological activity by 20%. Human recombinant tumor necrosis factor-alpha and transforming growth factor-beta used in the assay as control showed significant chemotactic activity. The chemotactic activity present in whole normal conditioned medium was only present after 24 and 48 hr of culture. Furthermore, this activity was not neutralized by human recombinant tumor necrosis factor-alpha or transforming growth factor-beta antibodies. Incubation of whole 6-hr conditioned medium with human recombinant tumor necrosis factor-alpha and transforming growth factor-beta antibodies demonstrated and confirmed that tumor necrosis factor-alpha plays a major role in inducing the chemotactic response. On acidification of this supernatant, we found a notable increase in the biological response that could be neutralized by transforming growth factor-beta antibody. Thus tumor necrosis factor-alpha and transforming growth factor-beta may sequentially provide important signals for fibroblast and monocyte recruitment in vivo at initial stages of liver injury.
Syrian-hamster-derived lymphotoxin inhibits benzo(a)pyrene and ultraviolet carcinogen-induced in vitro morphologic transformation of diploid secondary passage hamster embryo cells. When lymphotoxin is added up to 4 days post carcinogen, the reduction in the frequency of morphologic transformation 6 days after carcinogen treatment varies directly with lymphotoxin concentration. The frequency of transformation does not change following re-feeding with lymphotoxin-free medium and incubation for 6 additional days. Furthermore, transformation by ultraviolet is prevented when cells are pulsed with lymphotoxin for as few as 6 hours after irradiation. Thus, lymphotoxin has the potential to irreversibly inhibit the first recognizable morphologic step of carcinogenesis.
Treatment of neoplastic guinea-pig cells for 1-14 days with 300 units of guinea-pig lymphotoxin/ml medium enhanced 2- to 5-fold their susceptibility to natural killer (NK) cell-mediated cytotoxicity as measured by 4 h 51Cr and 72 h [3H]-thymidine-release assays. The NK susceptibility of human neoplastic and guinea-pig non-neoplastic cells refractory to guinea-pig lymphotoxin growth-inhibitory activity was unchanged after lymphotoxin treatment. Lymphotoxin preparations were free of detectable interferon and the enhancing activity copurified with lymphotoxin during diafiltration and isoelectric focusing. Treatment of NK cells with lymphotoxin preparations also augmented NK-mediated lysis but that activity did not copurify with lymphotoxin. As with neoplastic cells, lymphotoxin-sensitive preneoplastic cells previously treated with ultraviolet light, a chemical carcinogen, or carcinogen and the tumor-promoting agent tetradecanoylphorbol-acetate also exhibited lymphotoxin enhancement to NK-cell destruction. Thus, the immunologic hormone lymphotoxin enhances the susceptibility of lymphotoxin-sensitive cells to NK-cell destruction, indicating that these two natural immune mediators, lymphotoxin and NK cells, have the potential in concert to regulate carcinogenesis and neoplastic growth.
Phytohemagglutinin (PHA) or either of its isolectins, erythroagglutinin or leukoagglutinin, causes a dose-dependent decrease in 12-0-tetradecanoylphorbol 13-acetate (TPA)-promoted transformation of Syrian hamster embryo cells, but has no effect on transformation induced by ultraviolet irradiation. The ineffectiveness of concanavalin A indicates that not every lectin inhibits TPA. Galactose, a dominant sugar in receptors for PHA binding, reverses the inhibition of TPA promotion caused by PHA but galactose does not inhibit TPA promotion itself. Therefore, the TPA and PHA binding sites are functionally discrete. The PHA inhibition of TPA-promoted transformation is reversible because PHA is only effective if present with TPA, whereas lymphotoxin, an immunologic hormone, has a persistent anti-carcinogenic effect, regardless of whether it is added before or after TPA. PHA in conjunction with lymphotoxin causes additional inhibition of TPA-promoted transformation. PHA and lymphotoxin affect the biological activity of TPA by diverse mechanisms. Lymphotoxin alters the physiological state of the cell, causing a change in the cellular response to TPA. PHA may affect either the binding of TPA to a critical cellular receptor for promotion or a later step in promotion.
Lymphotoxin augments the susceptibility of tumorigenic guinea-pig cells to natural killer (NK) cell cytolysis in vitro but does not directly stimulate either NK cell or macrophage cytolytic action. The question whether lymphotoxin enhances the susceptibility of tumorigenic guinea-pig cells to cytolysis or other means of growth inhibition in vivo by syngeneic NK cells or macrophages was, therefore, examined using a modified tumor cell neutralization (Winn) assay. Mineral oil-, thioglycollate- or casein-induced peritoneal leukocytes, but not the macrophages isolated from the elicited leukocytes obtained from nonimmunized strain 2/N guinea-pigs, effected enhanced cytolysis of lymphotoxin-treated guinea-pig benzo (a)pyrene-induced 104CI tumor cells in vitro. Neither guinea-pig splenic NK cells nor oil-induced peritoneal macrophages alone inhibited the growth of 104CI cells as tumors in vivo when admixed with 104CI cells and injected into guinea-pigs. However, when the 104CI cells were treated with lymphotoxin before addition of effector cells, NK cells but not macrophages significantly reduced tumor growth in vivo. Therefore, the ability of lymphotoxin to increase the sensitivity of tumor cells to destruction mediated by natural leukocytes is specific for NK cells as compared to macrophages. This form of lymphokine amplification of natural leukocyte cytotoxicity may be one mechanism by which natural and acquired immunity serves or fails to prevent cancer and should be an important consideration in therapeutic approaches to eradicate or control tumor growth.
Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross-reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL-derived TNF did not bind to Concanavalin A-Sepharose or to several other agglutinin-Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS-PAGE revealed a single sharp peak of 16,500 +/- 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LF neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay.
The normal myeloid hematopoietic regulatory proteins include 4 different growth-inducing proteins (IL-3, MGI-1GM = GM-CSF, MGI-1G = G-CSF, and MGI-1M = M-CSF = CSF-1). There is also another type of normal myeloid regulatory protein (MGI-2) with no MGI-1 (CSF or IL-3) activity, which can induce differentiation of normal myeloid precursors and certain clones of myeloid leukemic cells. Studies on the binding of MGI-2 to differentiation-competent (D+) and differentiation-defective (D-) clones of mouse myeloid leukemic cells and to normal cells indicate that: (1) D+ clones of myeloid leukemic cells had about 2,500 high-affinity surface receptors per cell, like mature normal myeloid cells, and the bound MGI-2 was rapidly internalized with its cell-surface receptors at 37 degrees C causing down-regulation of MGI-2 receptors in both the normal and leukemic cells; (2) in some D- clones, the number and internalization of MGI-2 receptors were similar to those of D+ clones whereas other D- clones had only 0-100 MGI-2 receptors per cell; (3) normal thymus and lymph-node lymphocytes and T lymphoma cells did not show detectable MGI-2 receptors; (4) there was an independent expression of receptors for MGI-2 and for the 4 myeloid growth-inducing proteins on different clones of myeloid leukemic cells; and (5) none of the 4 myeloid growth-inducing proteins IL-3, MGI-1GM, MGI-1G, or MGI-1M, inhibited binding of MGI-2 to its receptors. The cytotoxic proteins lymphotoxin and tumor necrosis factor did not induce differentiation of the mouse myeloid leukemic cells and also did not inhibit binding of MGI-2 to its receptors. These results show that the myeloid differentiation-inducing protein MGI-2 binds to cell-surface receptors that are different from the receptors for the 4 myeloid growth-inducing proteins and these cytotoxic proteins.
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 control of haematopoietic colony-stimulating factors (CSF) gene expression by interleukin 1 (IL-1) and tumour necrosis factor alpha (TNF-alpha) in cultured endothelial cells was studied by RNA hybridization and nuclear gene transcription. Both IL-1 and TNF-alpha induced, with somewhat different kinetics, a slow but marked accumulation of granulocyte-macrophage (GM)- and granulocyte (G)-CSF mRNAs in endothelial cells; macrophage (M)-CSF mRNA increased more rapidly but more moderately. Simultaneous treatment with maximally stimulating concentrations of both IL-1 and TNF-alpha had an additive effect on the accumulation of the three mRNAs, suggesting that both mediators act via independent pathways. The mechanism of CSF mRNA accumulation in endothelial cells was explored by nuclear run-on experiments, which showed that both IL-1 and TNF-alpha increase GM-CSF, G-CSF and M-CSF gene transcription to varying degrees.
The plasma of a patient being prepared for allogeneic bone marrow transplantation was found to be severely lymphocytotoxic to donor cells. Three isovolaemic plasma exchanges carried out using a continuous-flow technique were required to reduce this titre to undetectable levels. Neither these procedures nor pretransplantation conditioning of the patient with 60 mg/kg or cyclophosphamide on two consecutive days resulted in overt haemolysis. However, a single identical plasmapheresis 12 hours after completing the second dose of the alkylating agent resulted in profound intravascular haemolysis requiring red cell transfusion. Subsequent studies in five patients receiving the same cyclophosphamide regimen demonstrated that red cell life span, when measured with radiochromium, was reduced from a normal of 25-35 days to a median of 11.5 days. Further studies in the same five patients carried out simultaneously demonstrated that red cell fragility, when assessed in vitro with osmotic or mechanical stress, was marginally increased but became clearly abnormal in only one individual. These doses of cyclophosphamide may result in accelerated but subclinical red cell destruction, and plasma exchange in this period may be associated with rapid intravascular haemolysis. This complication should now be added to the list of those that can occur when plasmapheresis is required in this specific clinical situation.
We have isolated, sequenced, and determined the chromosomal localization of the gene encoding human lymphotoxin (LT). The single copy gene was isolated from a human genomic library using a 32P-labeled 116 bp synthetic DNA fragment whose sequence was based on the NH2-terminal amino acid sequence of LT. The gene spans 3 kb of DNA and is interrupted by three intervening sequences. The LT gene is located on human chromosome 6, as determined by Southern blot analysis of human-murine hybrid DNA. Putative transcriptional control regions and areas of homology with the promoters of interferon and other genes are identified.