Effect of inflammatory cytokines on human endothelial cell proliferation.

Neovascularization, a common occurrence in chronic inflammatory lesions, requires endothelial cell (EC) proliferation. Because this form of inflammation is often mediated by immunologically generated cytokines, the effects of such cytokines on human umbilical vein EC proliferation in vitro were investigated. Low concentrations of recombinant interferon gamma (rIFN-gamma) (10-100 U/ml), but not a higher concentration (1,000 U/ml), enhanced both basal and endothelial cell growth factor (ECGF)-stimulated EC proliferation. Recombinant interleukin 1 (rIL-1) and recombinant tumor necrosis factor-alpha (rTNF) had minor effects on basal EC proliferation, but significant inhibition was observed in the presence of ECGF. A combination of rIFN-gamma and rTNF induced marked suppression of EC proliferation, which appeared to be due to a cytotoxic effect on the EC, as demonstrated by 51Cr release. In contrast, the combination of rIFN-gamma and rIL-1 had only an additive effect on EC proliferation, with no evidence of cytotoxicity. These results suggest that cytokines have important regulatory roles in local vascular proliferation. These effects varied not only with the individual cytokine, but also with the combination of cytokines used. The most striking effects were 1) the stimulation of proliferation by IFN-gamma at a low concentration and 2) the inhibition by both rIL-1 and rTNF of ECGF-stimulated proliferation.     

Induction of the synthesis of tumor necrosis factor receptors by interferon-gamma

Human HT-29 colon carcinoma and HeLa D98/AH2 and SK-MEL-109 melanoma cells were sensitive to synergistic growth inhibition by concentrations of recombinant human tumor necrosis factor (rTNF) and interferon-gamma (rIFN-gamma) which individually were only slightly inhibitory. We investigated whether this synergism could be explained by the presence of an increased number of TNF receptors in cells treated with rIFN-gamma. These receptors were measured by incubating cells resuspended from monolayers with 125I-rTNF. HT-29 cells treated for a few hours with rIFN-gamma could bind more 125I-rTNF than control untreated cells, but this binding returned to the level of control cells after 24 hr. The treatment with rIFN-gamma did not change the binding affinity of TNF receptors, but increased their number to 1800 per cell from a basal level of about 800 per cell. Inhibitors of RNA synthesis prevented this increase. HT-29 cells were significantly more growth-inhibited when treated first for 6 to 12 hr with rIFN-gamma and then with rTNF, than when treated first with rTNF and then with rIFN-gamma. Untreated HeLa D98/AH2 and SK-MEL-109 cells had 2400 and 9000 receptors per cell, with a KD similar to that of HT-29 cells (approximately 2 X 10(-10)M). A significant increase in TNF receptors after treatment with rIFN-gamma was observed in HeLa D98/AH2, but not in SK-MEL-109 cells. No increase in TNF receptors was detected in cells treated with rIFN-alpha 2. These results indicate that the synergism between rTNF and rIFN-gamma may be due, at least in part, to a transient induction of the synthesis of TNF receptors by rIFN-gamma in cells with a relatively low number of these receptors.     

Characterization of the receptor for tumor necrosis factor (TNF) and lymphotoxin (LT) on human T lymphocytes. TNF and LT differ in their receptor binding properties and the induction of MHC class I proteins on a human CD4+ T cell hybridoma

TNF-alpha and lymphotoxin (LT or TNF-beta) are structurally related cytokines that share several proinflammatory and immunomodulatory activities. The shared biologic activities of TNF and LT have been attributed to their binding to a common cell surface receptor(s). We observed that rTNF enhanced the expression of MHC class I proteins on the human T cell hybridoma, II-23.D7, however LT was largely unable to regulate MHC expression. To determine the molecular basis of this disparity between LT and TNF the receptor binding characteristics of rTNF and rLT were investigated by direct and competitive radioligand assays on the II-23.D7 T hybridoma, and for comparison, anti-CD3 activated human T lymphocytes. Specific 125I-rTNF binding to the II-23.D7 line revealed a single class of sites with a Kd = 175 pM and 3000 sites/cell; anti-CD3 activated T cells exhibited specific TNF binding with similar properties. The relationship of receptor occupancy to the induction of MHC class I Ag yielded a hyperbolic curve indicating a complex relationship between rTNF binding and biologic response. LT appeared to function like a partial agonist in that rLT was 10- to 20-fold less effective than rTNF in competitively inhibiting 125I-rTNF binding on the II-23.D7 line. Scatchard type analysis revealed a single class of low affinity binding sites for 125I-rLT. No differences in the competitive binding activity of rTNF and rLT were observed on the anti-CD3-activated T cells. Receptors for rTNF and rLT were immunoprecipitated from the II-23.D7 and activated T cells with anticytokine antibodies after cross-linking of radioiodinated rTNF or rLT to intact cells by using chemical cross-linking reagents. Analysis of the cross-linked adducts by SDS-PAGE and autoradiography indicated a major adduct of 92 kDa for rTNF and 104 kDa for rLT. Enzymatic digestion with neuraminidase or V8 protease revealed a unique structure to these adducts consistent with the cross-linking of a single chain of cytokine to a cell surface glycoprotein. rTNF inhibited the formation of the 104-kDa adduct formed with 125I-rLT on the II-23.D7 line, indicating these two cytokines bind to the same receptor of approximately 80 kDa. These results suggest that the disparate activities of LT and TNF to induce MHC class I proteins on the II-23.D7 cells are, in part, associated with a modified state of a common receptor.     

Induction of colony stimulating factor in vivo by recombinant interleukin 1 alpha and recombinant tumor necrosis factor alpha 1

In response to a potent inflammatory challenge, such as Gram-negative endotoxin, a number of cytokines are induced that, in turn, mediate many of the pathophysiologic alterations associated with endotoxicity. In this study, we have observed two endotoxin-associated monokines, recombinant interleukin-1 alpha (rIL 1 alpha) and recombinant tumor necrosis factor alpha (rTNF alpha), to induce colony stimulating factor (CSF) in vivo. The CSF activities produced in response to rIL 1 alpha or rTNF alpha gave rise to a mixture of granulocyte-macrophage colonies and were induced in a dose- and time-dependent fashion, peaking within 3 hr of cytokine injection (preceding peak CSF induction by endotoxin by several hours). Combined injection of suboptimal concentrations of rIL 1 alpha and rTNF alpha were additive, and simultaneous injection of optimal concentrations of each failed to increase CSF levels over that observed with either cytokine alone. Unlike endotoxin, neither cytokine induced interferon in vivo. These findings extend our understanding of the cytokine cascade that is operative in an inflammatory response and may account for many of the observed hematopoietic alterations that accompany inflammation.     

Anti-proliferative effect of IFN-gamma in immune regulation. II. IFN-gamma inhibits the proliferation of murine bone marrow cells stimulated with IL-3, IL-4, or granulocyte-macrophage colony-stimulating factor

A biphasic dose response curve was observed when the bone marrow-derived cell line FDCP1, used as an indicator line for IL-3 bioassays, was exposed to supernatants from some activated T cell clones but not others. The active component which inhibited proliferation at the higher supernatant concentrations appeared to be IFN-gamma, based on the following observations. 1) Only those culture supernatants which contained IFN-gamma gave a biphasic dose response curve; 2) with these supernatants, an anti-IFN-gamma mAb augmented the proliferation of FDCP1 cells at the higher supernatant concentrations; and 3) rIFN-gamma profoundly inhibited the proliferation of FDCP1 cells stimulated with rIL-3 or rIL-4. rTNF-alpha inhibited FDCP1 proliferation only to a modest extent, yet the combination of rTNF-alpha + rIFN-gamma provided greater inhibition than each agent alone. The proliferation of a second bone marrow-derived cell line, DA1, was not inhibited by rIFN-gamma or rIFN-gamma + rTNF-alpha when stimulated with rIL-3 or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Fresh bone marrow cells also showed a suboptimal proliferative response when stimulated with T cell supernatants containing IFN-gamma, and this response was augmented considerably upon the addition of anti-IFN-gamma mAb. Bone marrow cell proliferation was observed upon exposure to rIL-3, rIL-4, or rGM-CSF, and these responses were inhibited by rIFN-gamma; rTNF-alpha also produced a synergistic effect with these cells. Bone marrow cell colony formation stimulated by rIL-3 or rGM-CSF also was inhibited by rIFN-gamma. Colony formation in bone marrow cell cultures was not observed in response to rIL-4. Collectively, these results suggest that Th1 cells, which in addition to IL-3 and GM-CSF also produce IFN-gamma, may regulate hemopoietic cell proliferation and colony formation differently from the way Th2 cells do, which do not produce IFN-gamma.     

Recombinant granulocyte-macrophage colony-stimulating factor down-regulates expression of IL-2 receptor on human mononuclear phagocytes by induction of prostaglandin E

Recent studies have shown that normal human alveolar macrophages and blood monocytes, as well as HL-60 and U937 monocyte cell lines, newly express IL-2R after stimulation with rIFN-gamma or LPS. In addition, macrophages transiently express IL-2R in vivo during immunologically mediated diseases such as pulmonary sarcoidosis and allograft rejection. We therefore investigated in vitro factors that modulate macrophage expression of IL-2R. IL-2R were induced on normal alveolar macrophages, blood monocytes, and HL-60 cells using rIFN-gamma (24 to 48 h at 240 U/ml), and cells were cultured for an additional 12 to 24 h with rIL-2 (100 U/ml), recombinant granulocyte-macrophage CSF (rGM-CSF, 1000 U/ml), rGM-CSF plus indomethacin (2 X 10(-6) M), PGE2 (0.1 to 10 ng/ml), 1 X 10(-6) M levels of caffeine, theophylline, and dibutyryl cyclic AMP, or medium alone. IL-2R expression was quantitated by cell ELISA (HL-60 cells) or determined by immunoperoxidase staining (alveolar macrophages, blood monocytes, and HL-60 cells), using anti-Tac and other CD25 mAb. PGE production was assayed by RIA. We found greater than 95% of alveolar macrophages, monocytes, and HL-60 cells expressed IL-2R after rIFN-gamma treatment and remained IL-2R+ in the presence of IL-2R or medium alone. By comparison, greater than 95% of cells induced to express IL-2R became IL-2R- after addition of rGM-CSF, and the culture supernatants from GM-CSF-treated cells contained increased levels of PGE. This inhibition of macrophage IL-2R expression by rGM-CSF was blocked by indomethacin, and IL-2R+ macrophages became IL-2R- after addition of PGE2 alone. These findings indicate GM-CSF down-regulates IL-2R expression by human macrophages via induction of PGE synthesis. Moreover, a similar down-regulation of IL-2R expression was seen after stimulation with caffeine, theophylline, or dibutyryl cyclic AMP. Hence, GM-CSF, PGE, and other pharmacologic agents that act to increase intracellular levels of cAMP may play a modulatory role, antagonistic to that of IFN-gamma on cellular expression of IL-2R by human inflammatory macrophages in vivo.     

Studies on the mechanism of natural killer cell-mediated cytotoxicity. VII. functional comparison of human natural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor

The present study was undertaken to evaluate the possible contribution of other cytokines to the lytic activity of NKCF-containing supernatants. We compared some of the functional properties of human NKCF and purified recombinant human rLT and rTNF. It was found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF. The target specificity of rTNF was found to be related to that of NKCF with the exception of one NK-resistant cell line that was lysed by rTNF in a 20-hr 51Cr-release assay. However, rTNF was not toxic to any of the target cells tested as assessed by trypan blue exclusion in a 20-hr assay unless the targets were labeled with 51Cr. In contrast, NKCF did kill target cells as detected by trypan blue exclusion that were not labeled with 51Cr. Further analysis of this mechanistic difference in the lytic activity of rTNF and NKCF revealed that rTNF in combination with either cycloheximide or mitomycin C but not IFN-gamma could lyse unlabeled U937 target cells. In addition, pretreatment of U937 target cells with nonradioactive Na2CrO4 at concentrations equivalent to that used to 51Cr-labeled cells resulted in their susceptibility to lysis by rTNF as assessed by trypan blue exclusion. These findings suggest that lysis of several susceptible target cells in 20 hr by rTNF requires the presence of additional agents that may be sublethally toxic and/or inhibitory to macromolecular synthesis. Antibody inhibition studies revealed that anti-TNF mediated from partial to complete inhibition of lysis of U937 by unfractionated supernatants containing NKCF. However, fractionation of such supernatants on chromatofocusing columns yielded two distinct peaks of activity eluting in the pH range of 5 to 6 and 7 to 8. Anti-TNF could inhibit the acidic form of NKCF but not the neutral form. It is concluded that NKCF activity is mediated in part by TNF or an antigenically related molecule as well as some other distinct factor(s). The lack of consistent inhibition of NK CMC by anti-TNF suggests that TNF alone is not sufficient to mediate NK activity, or else it is inaccessible to the added antibody.     

Effects of recombinant murine granulocyte-macrophage colony-stimulating factor in cyclophosphamide-treated mice

To evaluate the efficacy of recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) in attenuating the myelosuppression associated with chemotherapy, the effects of 100 and 300 ng rGM-CSF, administered twice daily by intraperitoneal injection for 6 consecutive days to mice 24 hours after a dose of 200 mg/kg cyclophosphamide, were measured. Six days after the initial injection of rGM-CSF, a significant increase occurred in the absolute myeloid count compared to that of vehicle-treated animals. The difference was most pronounced on day 7, attaining levels of 327% and 428% of the control; these increases slowly declined to that of the control level by day 19. No significant effect was produced by rGM-CSF on the packed red cell volume or on the platelet count. Furthermore, the administration of rGM-CSF did not alter bone marrow cellularity or increase the number of marrow-derived hematopoietic stem cells. In contrast, a significant splenomegaly occurred, starting on day 6 and continuing until day 17. This was characterized by a pronounced increase in splenic-derived granulocyte (CFU-G), granulocyte-macrophage (CFU-GM), macrophage (CFU-M), megakaryocyte (CFU-MK), and erythroid (BFU-E, CFU-E) stem cells. The increases occurred between days 6 and 9 following the initial administration of rGM-CSF. These findings indicated that the administration of rGM-CSF to cyclophosphamide-treated animals causes an absolute increase in circulating myeloid cells and that these increases are derived from the spleen. The use of recombinant hematopoietic growth factors may permit the administration of more intensive chemotherapy through amelioration of chemically induced leukopenia.     

Effect of tumor necrosis factor and granulocyte/macrophage colony-stimulating factor on neutrophil degranulation

Both TNF and and granulocyte/macrophage CSF (GM-CSF) can activate neutrophils. The aim of this work was to determine the effect of these cytokines on neutrophil degranulation. The secretion of lactoferrin of secondary granules and myeloperoxidase (MPO) of primary granules from single adherent human neutrophils was assayed by use of a reverse hemolytic plaque assay. Both rTNF and rGM-CSF caused secretion of lactoferrin in a dose-dependent manner. Both agents also caused secretion of MPO, but only in the presence of cytochalasin B. Preincubation with pertussis toxin inhibited rGM-CSF-induced secretion of both lactoferrin and MPO. rTNF-induced MPO secretion was also blocked by pertussis toxin, whereas lactoferrin secretion was only slightly affected. Neither rTNF nor rGM-CSF caused any detectable changes in the concentration of cytoplasmic free Ca2+ in fura-2-loaded cells. However, when neutrophils were loaded with increasing concentrations of quin-2 to buffer any local, not detectable, changes in the concentration of cytoplasmic Ca2+, both rTNF- and rGM-CSF-induced secretion of lactoferrin and MPO were almost totally abolished at a relatively low quin-2 concentration. These results suggest a role of a regulatory G-protein and minute local changes in the concentration of cytoplasmic Ca2+ in TNF- and GM-CSF-induced neutrophil degranulation.     

Analysis of cytostatic/cytotoxic lymphokines: relationship of natural killer cytotoxic factor to recombinant lymphotoxin, recombinant tumor necrosis factor, and leukoregulin

Previous results that were obtained by using supernatants from the co-culture of human peripheral blood lymphocytes and the natural killer susceptible cell line K562 strongly inhibited the growth of various tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by natural killer cells. Rather the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin (LRG), a recently described lymphokine. Because of the recent availability of recombinant tumor necrosis factor (TNF) and lymphotoxin (LT), we compare the target selectivity and mechanism of action of these (TNF, LT, LRG) factors with natural killer cytotoxic factor (NKCF). The pattern of target cell susceptibility to growth inhibition or cytolysis by the factors were quite distinct from the pattern observed when cells were exposed to NKCF. Furthermore, antibodies to rLT or rTNF had no effect on LRG cytostasis or NKCF lysis, arguing against a requirement for or synergistic interaction with low levels of LT or TNF. Some of the targets susceptible to LRG were growth inhibited but were not lysed, thereby distinguishing it from NKCF. Furthermore, LRG cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which block natural killer cytotoxic factor. Therefore, LRG appears to be a cytostatic factor produced by large granular lymphocytes in response to K562 that is distinct from NKCF, TNF, and LT. In addition, NKCF, rLT, rTNF, and LRG, although having cytotoxic/cytostatic activity, are distinct functional factors and may represent a family of lytic factors.     

Infusion of tumor necrosis factor (TNF) causes an increase in circulating TNF-binding protein in humans

Serum samples from cancer patients receiving intravenous infusions of recombinant tumor necrosis factor (rTNF) and recombinant interferon-gamma (rIFN-gamma) were analyzed for TNF and the TNF-binding protein (TNF-BP). TNF-BP is a soluble fragment of the transmembrane TNF receptor with antagonistic effects to TNF and is released by proteolytic cleavage of the receptor. During a 60-min infusion of rTNF, peak serum levels of rTNF were observed after 30 to 60 min and a transient increase of circulating TNF-BP was observed with peak levels between 30 and 120 min. Injection of IFN-gamma alone did not affect the levels of TNF and TNF-BP. Thus administration of rTNF leads to release into the circulation of TNF-BP, which may modulate both systemic and local effects of TNF and influence its therapeutic efficacy.     

Regulation of tumor necrosis factor gene expression and protein synthesis in murine macrophages treated with recombinant tumor necrosis factor

The effect of murine rTNF-alpha on c-fos and TNF mRNA accumulation and protein synthesis was investigated in bone marrow-derived macrophages to examine the mechanism(s) by which TNF modulates macrophage activity. A rapid and transient expression of the c-fos gene was induced by murine rTNF. This was blocked by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, suggesting that the murine rTNF stimulated a protein kinase C-dependent signal transduction pathway. Although LPS induced the accumulation of one TNF mRNA species, murine rTNF induced the synthesis of two distinct TNF mRNA species. Both LPS- and murine rTNF-induced TNF mRNA accumulation was equally enhanced by pretreatment with mouse rIFN-gamma. In contrast, cycloheximide pretreatment had little effect on murine rTNF-induced TNF mRNA accumulation, whereas this treatment increased LPS-induced TNF mRNA by sevenfold. These results argue that TNF mRNA accumulation can be modulated in macrophages by distinct mechanisms. As assessed by Western blot and immunoprecipitation analysis, LPS stimulated the synthesis of both cell-associated and secreted forms of TNF protein. In comparison, newly synthesized TNF protein was not detected when macrophages were treated with murine rTNF alone or in combination with murine rIFN-gamma. This demonstrates that although murine rTNF stimulated the synthesis of two distinct TNF mRNA species, additional signal(s) are necessary for their translation into protein and that such signals are present after LPS stimulation.     

Tumor necrosis factor, alone or in combination with IL-2, but not IFN-gamma, is associated with macrophage killing of Mycobacterium avium complex

Organisms belonging to the Mycobacterium avium complex (MAC) are the most common bacterial pathogens in patients with AIDS but factors associated with the activation of cellular defense mechanisms against this atypical mycobacterium have not been defined. Peritoneal macrophages harvested from a chronic MAC infection in C57 black mice are able to kill approximately 86% of intracellular MAC in contrast to 0 to 20% killing by unstimulated human and mouse macrophages in vitro. The availability of human rTNF-alpha, rIFN-gamma, and rIL-2 permitted evaluation of the role of each of these lymphokines/monokines, alone or in combination, in activating macrophages in vitro to kill MAC. Human monocyte-derived macrophages were cultured in vitro, stimulated with rIL-2, rIFN-gamma, or rTNF, and then infected with MAC (serovars 1 and 8). Mouse peritoneal macrophages were harvested, cultured in vitro, and stimulated with rIFN-gamma. rTNF (10(4) U/ml) was associated with a modest increase of intracellular killing of MAC (58 +/- 5%) even when utilized 24 or 48 h after macrophage infection or when administered for 5 consecutive days after infection (78.1 +/- 4%). Both human and murine IFN-gamma were associated with increased intracellular growth of MAC (32 +/- 4% for murine and 38 +/- 3% for human macrophages). However, intracellular killing (53 +/- 6% compared with control) was observed after 6 days of treatment with IFN-gamma. This latter effect was fully blocked by anti-TNF antibody, whereas rIL-2 alone did not augment the intracellular killing of MAC by human macrophages. rTNF plus either rIFN-gamma or rIL-2 triggered significant increases in superoxide anion production, but subsequent MAC killing was no greater than with rTNF alone. Treatment of macrophages with 10 U/ml of rTNF followed by rIL-2 (200 U/ml) was associated with 68% of intracellular killing. TNF seems to be an important monokine, promoting activation of mycobactericidal mechanisms in human macrophages.     

Activity of recombinant tumor necrosis factor on Toxoplasma gondii and Trypanosoma cruzi

Activated macrophages produce tumor necrosis factor (TNF), a cytokine with anti-tumor and anti-plasmodia activities. This study revealed that recombinant TNF (rTNF) inhibits intracellular multiplication of blood trypomastigotes of Trypanosoma cruzi in murine peritoneal macrophages. rTNF did not have any apparent direct effect on the survival of extracellular T. cruzi or on its ability to infect mammalian cells. The degree of inhibition of the intracellular multiplication of T. cruzi was found to be a function of the time of exposure of the infected cells to rTNF. rTNF induced a comparable effect when different strains of the parasite were used. In contrast to its activity on T. cruzi, rTNF did not affect intracellular multiplication of Toxoplasma gondii tachyzoites or bradyzoites in normal murine peritoneal macrophages or in human fibroblasts. Killing of Toxoplasma tachyzoites by activated macrophages was not enhanced by rTNF.     

A recombinant human G-CSF/GM-CSF fusion protein from E. coli showing colony stimulating activity on human bone marrow cells

Granulocyte-Macrophage colony stimulating factor (GM-CSF) and Granulocyte colony stimulating factor (G-CSF) are cytokines involved in the differentiation of bone marrow progenitor cells into myeloid cells. They also activate mature myeloid cells to mediate a variety of antimicrobial activities and inflammatory responses. Recombinant GM-CSF and G-CSF proteins have been used to treat various diseases including cancer and hematopoietic diseases and to isolate peripheral blood progenitor cells for bone marrow transplantation. A plasmid construct expressing recombinant human G-CSF/GM-CSF fusion protein has now been prepared by linking the human G-CSF and GM-CSF coding regions and the recombinant fusion protein has been successfully expressed in E. coli. The recombinant human G-CSF/GM-CSF fusion protein was extracted and purified from the cellular inclusion and refolded into the biologically active form to show colony stimulating activity. The recombinant fusion protein exhibited colony stimulating activity on human bone marrow cell cultures, indicating that the linkage of GM-CSF and G-CSF by a linker peptide may not interrupt activities of the cytokines in the fusion protein. The colony forming unit of the fusion protein was also higher than those of the cultures treated with the same molar numbers of the recombinant human GM-CSF and G-CSF separately, which suggests that the fusion protein presumably retains both G-CSF and GM-CSF activities.     

Resistance to the cytolytic action of lymphotoxin and tumor necrosis factor coincides with the presence of gap junctions uniting target cells

The resistance of target cells to the cytolytic action of lymphotoxin (LT) and recombinant tumor necrosis factor (rTNF) has been investigated by using clonally derived cell lines with defined gap junction-mediated, intercellular communication properties. Gap junction-competent Chinese hamster ovary cells are normally insensitive to the action of LT/TNF. However, treatment with 12-o-tetradecanoylphorbol-13-acetate, which promotes the loss of gap junctions, or culturing at low cell density to reduce intercellular contacts, significantly increased their sensitivity to LT/TNF. The LT/TNF-sensitive murine CL-1D and L929 cell lines, which in normal culture conditions are unable to form gap junctions, were not changed in their susceptibility to LT/TNF after treatment with phorbol ester or low culture density. However, the formation of gap junctions by CL-1D can be promoted by treatment with 8-bromo-cyclic adenosine monophosphate (1 mM), and this treatment completely suppressed the ability of LT and rTNF to kill CL-1D. Additionally, the LA25-normal rat kidney cell line, which is infected with a temperature-sensitive mutant of Rous sarcoma virus (LA25), is gap junction-competent and resistant to the effects of LT at the restrictive temperature (39 degrees C). However, when shifted to the permissive temperature (33 degrees C), LA25-normal rat kidney cells express the pp60v-src viral gene product, lose their ability to form gap junctions, and become sensitive to the lytic activity of LT. The results demonstrate that the expression of the retroviral pp60v-src, a tyrosine protein kinase, is sufficient to render cells susceptible to the lytic effects of LT and rTNF. Collectively, these experiments demonstrate a strong correlation between the resistance of target cells to the action of LT/TNF and their ability to cooperate metabolically through gap junctions. The results do not completely exclude the possibility that other mechanisms, such as LT receptor modulation, are also occurring under these experimental conditions. These data also suggest that a possible physiologic function of the stable cytotoxic lymphokines is to induce cytolysis/cytostasis of cells that have lost gap junctional contact, such as those in the process of mitosis or metastasis that have separated from the main tissue mass.     

Effect of interleukin 2, interferon-gamma, and mitogens on the production of tumor necrosis factors alpha and beta

Human peripheral blood mononuclear cells (PBMC) were induced by recombinant interleukin 2 and mitogens to secrete two distinct cytotoxic polypeptides, tumor necrosis factor-alpha (TNF-alpha) and tumor necrosis factor-beta (TNF-beta), previously called lymphotoxin. Treatment of PBMC with recombinant human interleukin 2 (rIL 2) or mitogens in combination with recombinant human interferon-gamma (rIFN-gamma) resulted in augmented production of both TNF-alpha and TNF-beta. rIFN-gamma alone had no effect on production of either cytotoxic polypeptide. TNF-alpha was produced within 2 to 3 hr after induction and was the major cytotoxin produced by PBMC during the first 48 hr of culture, after which time TNF-beta became the predominant species. TNF-beta was first secreted into the media after 8 hr of induction. Enhanced levels of both TNF-alpha and TNF-beta were seen when the PBMC were separated into adherent and nonadherent cells. Both TNF-alpha and TNF-beta were induced in different tumor cell lines of hematopoietic origin. The results demonstrate that the production of TNF-alpha and TNF-beta can be enhanced by two lymphokines, IL 2 and IFN-gamma.     

Tumor necrosis factor-alpha enhances cytolytic activity of human natural killer cells

The effect of recombinant tumor necrosis factor-alpha (rTNF alpha) on human natural killer (NK) function was examined. Lysis of both the NK-sensitive K562 erythroleukemia line and the relatively insensitive renal carcinoma line Cur by nonadherent peripheral blood lymphocytes was significantly enhanced as a result of an 18-hr preincubation with either rTNF alpha or recombinant interleukin 2 (rIL 2). When cells were preincubated with rTNF alpha and low doses of rIL 2 (1 to 10 U/ml), marked additional augmentation of lysis of both targets was noted which was greater than that caused by either cytokine alone. Similar results were observed when responses of CD16+ large granular lymphocytes selected with the fluorescence-activated cell sorter after staining with the NK-specific monoclonal antibody Leu-11 were examined, indicating that the action of the cytokines was directly on the cytotoxic cells. Augmentation of tumor cell lysis could not be ascribed to a cytolytic activity of rTNF alpha on the targets, because no combination of rIL 2, rTNF alpha, or interferon-gamma caused lysis of K562 or Cur. By flow cytometric analysis, it was found that expression of IL 2 receptors was induced on purified CD16+ large granular lymphocytes by rTNF alpha alone and to an even greater degree by the combination of rTNF alpha and rIL 2. Additional analysis of the expression of surface antigens and blocking studies with monoclonal antibodies showed that enhanced tumor cell lysis was not caused by the augmentation of leukocyte function-associated antigen-1-mediated effector/target interactions. These data indicate that rTNF alpha alone, or in combination with rIL 2, directly augments NK cytotoxic activity.