Induction of lymphokine-activated killer activity in mice by prothymosin α

We have recently demonstrated that prothymosin (ProT) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor (TNF) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT in vivo. We demonstrate that i.p. injections of ProT enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8⁺ cells, NK cells and activated CD3⁺ cells. The ProT-induced effect persisted for 30 days after the end of the ProT treatment period and returned to normal levels 20 days later. SPlenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT in vitro. The ProT-induced NK an LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF and IL-2 were generated in response to ProT in LAK cultures. These findings suggest that ProT may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF in the spleen, peritoneal cavity and probably other lymphoid organs.This work was supported by a CEC grant to M. Papamichail     

Augmentative effect ofNocardia rubra cell-wall skeleton on the induction of human lymphokine-activated killer (LAK) cells by the production of LAK cell helper factor(s)

Summary Nocardia rubra cell wall skeleton (N-CWS) was found to synergistically augment lymphokine-activated killer (LAK) cell generation from human peripheral blood mononuclear cells (PBMC) in the presence of a suboptimal dose of recombinant interleukin-2 (rIL-2). N-CWS increased the number of PBMC expressing IL-2 receptor on their surfaces, and the presence of N-CWS at the early stage of the culture period was essential for the exertion of its augmentative activity on the LAK induction. The predominant phenotype of LAK precursor cells responding to N-CWS and rIL-2 was CD3^– CD16⁺. Culture supernatant from N-CWS-stimulated PBMC was found to act as a substitute for N-CWS in the induction of LAK generation in the presence of rIL-2, suggesting that these cells produced a factor capable of augmenting LAK cell induction (LAK helper factor, LHF). LHF was found to have a molecular mass of 29 kDa by gel filtration, and could also function as a killer helper factor to augment allo-antigen-specific cytotoxic T lymphocyte generation from human peripheral blood T cells as well as murine thymocytes. LHF showed no species specificity, indicating that it is different from IL-4. The enhancing activity of LHF was not neutralized with anti-TNF, anti-IL-1, or anti-IL-1 antibodies. Furthermore, no tumor necrosis factor- (TNF), TNF, IL-1, , IL-2, IL-5, IL-6 or interferon activity was detected in semi-purified LHF during enzyme-linked immunosorbant assay and biological assays. The present findings indicate that LHF produced from N-CWS-stimulated PBMC is a molecule distinct from TNF, TNF, interferon, IL-1, -2, -4, -5, and -6, and suggest that LHF might be a novel lymphokine involved in LAK generation.This work was supported by a Grant-in-aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan     

Phenotypic analyses of lymphokine-activated killer cells that release interferon γ and tumor necrosis factor α

Summary We recently reported that interleukin-2(IL-2)-activated peripheral blood lymphocytes and CD3⁺, lymphokine-activated killer (LAK) cell clones release tumor necrosis factor (TNF) and interferon (IFN) when stimulated with K562 erythroleukemia cells. We examined the phenotype of IL-2-activated peripheral blood leukocytes that secrete TNF and IFN when stimulated with K562 cells and demonstrated that TNF secretion is not due to the presence of contaminating mononuclear phagocytes. Further, we demonstrate that IL-2-activated natural killer (NK) cells release only IFN when stimulated with K562 cells while T lymphocytes exposed to monoclonal anti-CD3 and K562 cells secrete both TNF and IFN. However, T cells stimulated only with K562 cells did not release IFN or TNF while the admixture of these T cells with NK cells, when stimulated with K562 cells, released levels of TNF comparable to those produced by the unseparated cells. At present it is unclear whether only one or both effector cell types respond to K562 by releasing TNF or why the presence both cell types is needed.This work was supported by grants from the national Institutes of Health (CA 23074 and CA 17094) and the Arizona Disease Commission (8277-000000-1-0-YR-9301)     

The CySF-L2 factor from dialysable human leucocyte extract activates natural killer cytotoxicity by induction of interferon γ

Summary The mechanism of natural killer (NK) cytotoxicity activation of human peripheral blood mononuclear cells (PBMC) by CySF-L2 was elucidated. CySF-L2 is a cytotoxicity-stimulating factor isolated from dialysable human leucocyte extract, which activates NK cytotoxicity against NK-sensitive and insensitive tumour cells (K562; Daudi; Raji; MOLT4) when preincubated with effector cells for 72 h. CySF-L2-mediated activation was synergistic to interleukin-2(IL-2)-mediated activation of NK cytotoxicity. Induction of interferon (IFN) release was the crucial step during CySF-L2-mediated NK cytotoxicity activation since enhancement of NK activity was completely blocked when anti-IFN antibodies were present during treatment of PBMC. Anti-IFN, anti-TNF (tumour necrosis factor ) anti-IL-1 and anti-IL-2 antibodies showed no blocking effect. Analysis of the supernatant culture medium after 72 h incubation of PBMC and their highly purified subpopulations demonstrated that CySF-L2 induced release of IFN from CD3⁺T cells and CD56⁺CD3^– NK cells and of TNF and prostaglandin E₂ from monocytes. CySF-L2 was also capable of activating NK cytotoxicity of highly purified (98%) CD56⁺CD3^– NK cells as well as of monocytes (94% pure). Cell cooperation studies connected with analysis of cytokine release and enhancement of NK cytotoxicity indicated that CySF-L2 might play an essential role in the up and down regulation of NK cytotoxicity by the cytokine network.     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

AViscum album oligosaccharide activating human natural cytotoxicity is an interferon γ inducer

Summary CommercialViscum album extract Helixor-M contains a dialysable oligosaccharide (HM-BP) that activates natural killer (NK) cytotoxicity against K562 tumour cells when preincubated with human peripheral blood mononuclear cells (PBMC) for 72 h. The activated effector cells were exclusively found in the monocyte/macrophage subpopulation. However, when peripheral non-adherent cells (PNAC) were preincubated with HM-BP for 72 h the NK cytotoxicity of CD56⁺CD3^– NK cells was activated. This discrepancy was found to be due to the release of prostaglandin E₂ from activated monocytes/macrophages, which blocked activation of the cytotoxicity of NK cells. Analysis of the supernatant culture medium after 72 h preincubation demonstrated that HM-BP induced release of interferon (IFN) from T cells (preferentially from CD3⁺CD4⁺ cells) and of tumour necrosis factor (TNF) from monocytes/macrophages. Release of IFN was the crucial step for activation of NK cytotoxicity since enhancement of NK cytotoxicity during pretreatment of PBMC or PNAC with HM-BP was completely blocked in the presence of anti-IFN antibodies. Anti-interleukin-2, anti-TNF or anti-IFN antibodies had no effect on the HM-BP-induced enhancement of NK cytotoxicity. The activation of the NK cytotoxicity of nonadherent cells by interleukin-2 treatment was found to be synergistic to the enhancement of NK cytotoxicity by treatment with HM-BP.     

Endogeneous interferon α/β produced by murine Kupffer cells augments liver-associated natural killing activity

Summary Nonparenchymal liver cells from untreated C3HeB/FeJ mice, when incubated in medium containing-10% fetal bovine serum or portal serum, produced significant amounts of interferon alpha/beta (IFN/). In contrast, other cell populations (spleen, mononuclear blood cells and peritoneal cells) from C3HeB/FeJ mice or nonparenchymal liver cells from other strains of mice (C3H/HeJ, germ-free C3H/HeN and C57Bl/6J) produced little or no detectable IFN in fetal bovine serum under the same culture conditions. The cells in the nonparenchymal liver cell population responsible for IFN/ production were adherent, phagocytic, silica-sensitive, carbonyl-iron-sensitive, and Thy1.2^–, presumably Kupffer cells or resident liver macrophages. IFN/ production by cultured Kupffer cells was not observed if medium containing fetal bovine serum or portal serum was treated with polymyxin B or if Kupffer cells were cultured in serum-free medium. This suggested that small amounts of endotoxin in fetal bovine or portal serum stimulated Kupffer cells to produce IFN/. Possibly, Kupffer cells are in a different state of activation/maturation than peritoneal and splenic macrophages since the sensitivity of resident Kupffer cells from C3HeB/FeJ mice to the stimulatory effects of endotoxin. The endogenous production of IFN/ by Kupffer cells from C3HeB/FeJ mice can augment liver-associated natural killer (NK) activity against YAC-1 cells (4 h) and induce liver-associated cytotoxic activity, not restricted by the major histocompatibility complex, against NK resistant P815 mastocytoma cells (18 h).This work was supported by National Institutes of Health grant CA28835, VA Merit Grant and by the Margaret Duffy and Robert Cameron Troup Fund Abbreviations used: NPC, nonparenchymal liver cells; FBS, fetal bovine serum; IFN, interferon; -AsGm-1, anti-asialo-GM1; -Thy1.2, anti-Thy1.2; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; HBSS, Hanks' balanced salt solution; GBSS, Gey's balanced salt solution; SRBC, sheep red blood cells; Ab, mouse anti-SRBC; NK, natural killer; MHC, major histocompatibility complex; polyI·polyC, polyinosinec·polycytidylic acid     

Fibroblast growth factor increases TNFα-mediated prostaglandin E2 production and TNFα receptor expression in human fibroblasts

To examine the possible role of basic fibroblast growth factor (FGF) in regulating the effects of TNF, we tested the effect of FGF on TNF-mediated PGE₂ production and TNF receptor expression in human fibroblasts. We found that, while FGF alone had no effect on PGE₂ production, it enhanced the amount of PGE₂ produced in response to TNF between 3 and 11-fold. FGF stimulated TNF-induced PGE₂ production independent of potential TNF-mediated IL-1 production, as neither anti-IL-1 mAbs nor IL-1 receptor antagonist protein (IRAP) inhibited TNF induced-PGE₂ production or the stimulatory effect of FGF. A one minute exposure of cells to FGF prior to removal was sufficient to significantly enhance TNF-induced PGE₂ production; the maximal FGF effect was reached after a 6 h preincubation. We also found that FGF significantly enhanced TNF receptor expression. Untreated fibroblasts expressed 3,900 receptors/cell, while cells treated with FGF for 6h expressed 9,500 receptors/cell, a 2.4-fold increase in receptor number; there was no apparent change in affinity for TNF (K_d 3.8×10^–11 M). The FGF-mediated increase in TNF receptor expression and TNF-mediated PGE₂ production could be abolished by FGF mAbs, indicating a specific FGF effect. These results show that FGF increases TNF receptor expression and suggest that this may account, at least in part, for the ability of FGF to enhance TNF-mediated PGE₂ production in human fibroblasts.     

Free radicals,cytokines and nitric oxide in cardiac failure and myocardial infarction

Myocardial infarction is the most common cause of congestive cardiac failure. Free radicals, cytokines, nitric oxide (NO) and antioxidants play a major role both in atherosclerosis and myocardial damage and preservation. In the early stages of atherosclerosis, neutrophils and monocytes infiltrate the intima and generate free radicals which damage the endothelial cells. As a result, production of NO and prostacyclin by the endothelial cells declines, which have cardioprotective actions. This also has relevance to the beneficial action of aspirin since, it can modulate both prostanoid and l-arginine-NO systems and NF-kB translocation. In both acute myocardial infarction and chronic congestive cardiac failure, the plasma levels of various inflammatory mediators such as interleukins and tumour necrosis factor- (TNF) are elevated. TNF, produced by the inflammatory cells and the myocardium, can suppress myocardial contractility and induce the production of free radicals, which in turn can further damage the myocardium. Transforming growth factor (TGF), polyunsaturated fatty acids and the glucose-insulin-potassium regimen can antagonize the harmful actions of TNF and protect the myocardium. This explains why efforts made to reduce the levels of pro-inflammatory cytokines have beneficial action and preserve the myocardium.     

Establishment of a mutant from human monocytic leukaemia U937 that exhibitsa genetically dominant resistance to TNFα-induced apoptosis

Tumour necrosis factor- (TNF) is a cytokine that induces apoptosis in various cell systems by binding to a TNF receptor (TNFR). To study TNF-induced apoptosis, we isolated and characterized a novel TNF resistant variant, U937/TNF clone II-5, from human monocytic leukaemia U937 cells. The II-5 cells resist apoptosis by TNF and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and II-5 showed that the apoptosis resistance to TNF in II-5 was genetically dominant. This dominant mutation in II-5 cells blocks TNF-induced disruption of mitochondrial membrane potential and caspase-3 activation. Expression of TNFR, Fas and Bcl-2 family proteins were not changed in II-5 cells. These results suggest that the apoptosis-resistant II-5 cells could have a functional defect in apoptosis signalling from TNFR to mitochondria and caspase activation. The II-5 cells could be useful in studying the signa lling linkage between TNFR and mitochondria.     

Effects of cytokines on in vitro growth of tumor-infiltrating lymphocytes obtained from human primary and metastatic liver tumors

Summary Tumor-infiltrating lymphocytes (TIL) were isolated from 22 human primary and metastatic liver tumors, and expanded in vitro in the presence of either interleukin-2 (IL-2, 100 U/ml) plus tumor necrosis factor (TNF, 1000 U/ml), IL-2 (1000 U/ml) plus IL-4 (1000 U/ml) or IL-2 (1000 U/ml) alone. TIL proliferated in culture in 20/22 cases. Among different cytokine combinations, TNF and IL-2 were most effective in promoting the outgrowth of CD3⁺ CD8⁺ T lymphocytes (mean ± SEM: 90%±5) in the cultures of TIL from primary liver tumors. Cytotoxicity against autologous tumor cells was demonstrated in all early cultures of TIL from primary liver cancers in the presence of IL-2 plus TNF. In contrast, cultures of TIL derived from colon cancer metastatic to liver had significantly lower levels of autotumor cytotoxicity and proportions of CD3⁺ CD8⁺ cells (40%±13) than those of TIL from primary liver tumors. The addition on day 0 of interferons ( or ) to TIL cultured in the presence of TNF and IL-2, significantly augmented cytotoxicity against autologous tumor. In contrast, incubation of TIL in the presence of IL-4 and IL-2 did not result in increased autotumor responses in the cultures of TIL from primary liver tumors. The expansion (-fold) of TIL (day 30) cultured in the presence of IL-2 alone compared to that in the presence of TNF and IL-2 was significantly greater for hepatocellular carcinoma (median, 280 vs 260) than for autologous peripheral blood lymphocytes (36 vs 27), cholangiocarcinoma (42 vs 51) or TIL from metastatic colon cancer (39 vs 30). Outgrowth of TIL in IL-2 plus TNF offers an opportunity for in vitro enrichment in cells with autotumor cytotoxicity in primary liver tumors. However, this cytokine combination was unable to promote and sustain growth of autotumor effectors from TIL in metastatic liver cancer.Supported by ACS grants IM27 077 and IM588 A (TLW) and Organ Transplant Program Project 1P01-CA-4744501 AZ     

Phase I study of TNFα AutoVaccIne in Patients with metastatic cancer

We evaluated the safety and immunogencity of a novel vaccine directed against autologous TNF in a Phase I fixed dose escalation trial. The vaccine consisted of two recombinant TNF proteins, with specific peptides replaced by foreign immunodominant T cell epitopes from tetanus toxoid. The main objectives were to establish a safe dose and evaluate the vaccines ability to raise neutralising TNF antibodies. Secondary objectives were improvements in body weight and tumour response. Thirty-three patients were vaccinated with three doses (20, 100, or 400 g) of TNF vaccine at 2-weekly intervals adjuvanted with aluminium hydroxide. Anti-TNF antibody titres were measured by both a RIA, using soluble native TNF as the antigen, and by an ELISA using immobilized partly denatured TNF. Eleven patients (33%) had mild grade1/2 injection site reactions at the higher doses. In 10 of 20 patients, serum antibodies recognize denatured TNF in the ELISA, whereas, antibody titres against native TNF in the RIA were undetectable. This suggests that the production process had partly denatured the vaccine preventing the formation of cross-reacting antibodies to native TNF. In conclusion, TNF vaccine was able to elicit vaccine specific antibodies. However, since the antibodies were only able to cross-react with partly denatured TNF, evaluation of safety and tumour responses to the TNF vaccine was compromised.     

Common expression of a tumor necrosis factor resistance mechanism among gynecological malignancies

Summary The efficacy of tumor necrosis factor (TNF) as an anticancer agent is limited. This limitation might be related to the expression of a protein-synthesis-dependent resistance mechanism that prevents the lysis of tumor cells by TNF. To test this possibility eight randomly selected human cell lines, three derived from ovarian carcinomas and five derived from cervical carcinomas, were tested for their in vitro sensitivity to TNF-mediated lysis. The results of this analysis showed that all eight cell lines are normally resistant to lysis by TNF. However, in the presence of inhibitors of protein synthesis, seven of them showed a significant increase in TNF-mediated lysis. Measurement of protein synthesis showed that there is a linear correlation between the level of inhibition of protein synthesis and the level of TNF-mediated lysis. The fact that seven of eight randomly selected cell lines are resistant to TNF because they express a protein-synthesis-dependent resistance mechanism suggests that this mechanism of resistance may be common among gynecological cancers. The results also suggest that a therapy involving TNF and inhibitors of protein synthesis might be useful for the treatment of gynecological malignancies.     

Cytokines in Brain Ischemia—The Role of TNFα

1. The role of cytokines and other inflammatory mediators in the progression of ischemic brain injury is a new and exciting era of research. Evidence in support for a role for TNF in this respect is emerging as evidence on de novo upregulation of TNF following ischemia is now well established.2. TNF administered directly to the brain parenchyma elicits local microvascular injury in the form of pericapillary edema and leukocyte adhesion to cerebral capillaries.3. TNF administered into the cerebroventricular space prior to ischemia augment the extent of tissue damage and neurological deficits.4. Specific and potent inhibitors of TNF synthesis or TNF receptors must be developed and tried to prove firmly a role for TNF in ischemic brain injury.     

Interferon γ but not tumor necrosis factor α decreases susceptibility of human renal cell cancer cell lines to lymphokine-activated killer cells

Summary Human renal cell cancer (RCC) cell lines, ACHN and KRC/Y, with or without exposure to cytokines, were examined for their susceptibility to lymphokine-activated killer (LAK) cells. Flow-cytometric analysis demonstrated constitutional expression of class I antigen on both cell lines, which was enhanced by interferon (IFN), IFN and tumor necrosis factor (TNF). A 4-h⁵¹Cr-release cytotoxicity assay demonstrated that pretreatment of both cell lines with IFN or IFN, but not with TNF, decreased their susceptibility to LAK cells. IFN also decreased susceptibility to natural killer cells in a 16-h⁵¹Cr-release cytotoxicity assay. IFN treatment decreased the susceptibility of ACHN cells in a dose-dependent manner. Cold-target competition assay clearly showed that IFN- but not TNF-pretreated cells compete less effectively than do untreated target cells. Pretreatment with IFN, however, increased expression of intercellular adhesion molecule-1 (ICAM-1) to a degree comparable to that with TNF. Northern blot analyses using a 520-base-pair ICAM-1 cDNA as a probe demonstrated that more 3.3-kb mRNA is expressed in IFN- and TNF-pretreated cells. These results suggest that IFN-treated RCC cell lines may reduce their ability to be recognized by LAK cells, and that IFN-induced protection of RCC cell lines against LAK cells may depend upon a mechanism independent of the expression of class I antigens or ICAM-1 on tumor cells.     

Calpeptin suppresses tumor necrosis factor-α-induced death and accumulation of p53 in L929 mouse sarcoma cells

The cytokine tumor necrosis factor (TNF) induces caspase-dependent cell death in a subset of tumor cells. In this report, we show a novel suppressive effect of calpeptin, a calpain inhibitor, on TNF-induced cell death and accumulation of p53 in L929 mouse fibrosarcoma. Exposure to 10 ng/ml TNF induced cell death in >50% of L929 cells within 12 h and stimulated accumulation of p53 (8-fold). Preincubation of cells with calpeptin blocked both TNF-induced cell death and accumulation of p53 as examined with Western blot. TNF-induced accumulation of p53 was in part contributed by increase of p53 mRNA level (2.2-fold) in a calpeptin-insensitive manner. Interestingly, other calpain inhibitors tested did not show these effects like calpeptin and TNF treatment did not increase apparent calpain activity in L929 cells, suggesting that calpeptin may have another function besides targeting calpain. Expression of dominant negative mutant p53Val¹³⁵ reduced the incidence of TNF-mediated cell death. Taken together, our findings suggest that TNF induces calpeptin-dependent, but calpain-independent accumulation of p53 protein as a necessary step leading to death in L929 cells.     

Influence of TNFalpha on the sialylation of mucins produced by a transformed cell line MM-39 derived from human tracheal gland cells

In order to investigate the influence of inflammation on the peripheral glycosylation of airway mucins, a human respiratory glandular cell line (MM-39) was treated by TNF. The expression and the activity of sialyl- and fucosyl-transferases, involved in the biosynthesis of peripheral carbohydrate determinants like sialyl-Lewis x, were investigated by RT-PCR and by HPAEC respectively. The mRNA steady-state level of sialyl- (ST3Gal III) and of fucosyl- (FUT3) transferases was moderately up-regulated by TNF; a 52% increase of 2,3-sialyltransferase activity was also observed in TNF-stimulated MM-39 cells. After metabolic radio-labelling with [³H]glucosamine and [³H]fucose, the mucins released inthe culture supernatant were purified by Sepharose CL-4B, density-gradient centrifugation and treatment with glycosaminoglycans-degrading enzymes. The mucins, released in the culture supernatant from control MM-39 cells, were constituted by two populations of molecules having the same 1.39–1.44 mg/ml density but carrying either high or low amounts of sialic acid residues at their periphery. TNF was able to increase the sialylation of the weakly sialylated mucins. This effect and the enhancement of the 2,3-sialyltransferase activity by TNF argue in favour of a regulation of the mucin sialylation by this pro-inflammatory cytokine. Despite the moderate overexpression of FUT3, no fucosylation of mucins produced by MM-39 cells was induced by TNF. In conclusion, the influence of TNF on the sialylation of mucins could explain why the mucins from infected patients suffering either from cystic fibrosis or from chronic bronchitis are more sialylated.     

Combined effects of interferon α and interleukin 2 on the induction of a vascular leak syndrome in mice

Summary Immunotherapy with interleukin 2 (IL-2) alone or in combination with lymphokine-activated killer cells can mediate tumor regression in mice and in man. Further dose escalation of IL-2 along with lymphokine-activated killer cells has been prevented by the development of a vascular leak syndrome produced by IL-2. Because we have found that interferon (IFN-) or tumor necrosis factor (TNF-) has synergistic antitumor effects when administered together with IL-2, we have tested the vascular leakage induced by these lymphokine combinations. We used a murine model to quantify vascular leakage by measuring the extravasation of ¹²⁵I-albumin from the intravascular space as well as the wet and dry lung weights after treatment with different cytokines. Cytokines (or Hanks balanced salt solution) were administered to C57BL/6 mice and 4 h after the last injection the vascular leak was quantified. IFN- alone did not cause extravasation of radiolabel or increase in wet lung weights, though when given in combination with IL-2, significantly greater extravasation (P<0.01) as well as increase in lung water weights (P<0.05) was observed compared to the response in mice treated with IL-2 alone. IFN- in combination with IL-2 induced significant vascular leakage earlier than the response induced by IL-2 alone. For example treatment with IFN- and IL-2 induced accumulation of 14674±605 cpm in the lungs at day 1 while IL-2 alone induced 12340±251 cpm. The degree of vascular leakage was highly related to the dose of IFN- administered along with IL-2 and increased vascular leak syndrome was evident even at low doses (5000 units) of IFN-. Immunosuppression of mice by pretreatment irradiation (500 rad) markedly decreased the development of vascular leak syndrome induced by IL-2 and IFN-. Interestingly IFN- and TNF- did not induce vascular leakage in the lungs when given alone, and did not add or synergize with IL-2 in causing the syndrome. Thus the administration of IFN- in combination with IL-2 produces a dose-limiting vascular leakage that is more severe than that caused by IL-2 alone, and may be mediated, directly or indirectly by host radiosensitive cells. Abbreviations used: LAK, lymphokine-activated killer; IFN, interferon; TNF, tumor necrosis factor; IL-2, interleukin-2     

Tnf-α and IL-1α inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: Evidence for primary impairment of mitochondrial function

Cytokines such as tumor necrosis factor (TNF) and Interleukin-1 (IL1) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNF and IL1. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNF (1, 10, 100, 1000 I.U./ml) and IL1 (0.1, 1, 10, 100 I.U./ ml).TNF- and IL1- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNF and IL1 and a subsequent down regulation of PDH activity.     

Stimulation by hexose esters of lactate production by rat erythrocytes,insensitivity to 3-O-methyl-D-glucose and inhibition by 2-deoxy-D-glucose and its tetraacetic ester

Selected esters of D-glucose were recently proposed as tools to provide the sugar to cells, whilst bypassing the carrier system for hexose transport across the plasma membrane. In the present study, -D-glucose pentaacetate, -D-glucose pentaacetate, -D-mannose pentaacetate and, to a lesser extent, 6-O-acetyl-D-glucose, all tested at a 1.7 mM concentration, were found to increase lactate production above basal value in rat erythrocytes. Over 90 min incubation, the increment in lactate production ranged from about 1.2 (-D-glucose pentaacetate) to 0.6 (6-O-acetyl-D-glucose) mol/l of erythrocytes. Little or no change in lactate production was observed in cells exposed to -L-glucose pentaacetate, -D-glucose pentaethylsuccinate, -D-galactose pentaacetate or -D-galactose pentaacetate. The metabolic response to -D-glucose pentaacetate was resistant to 3-O-methyl-D-glucose (10-80 mM) which suppressed, however, that evoked by D-glucose. D-mannoheptulose (10 mM) virtually failed to affect the response to D-glucose and its pentaacetate ester. On the contrary, 2-deoxy-D-glucose (10.6 mM) inhibited to the same relative extent (55% decrease) lactate production in erythrocytes exposed to either unesterified D-glucose or -D-glucose pentaacetate. The tetraacetic ester of 2-deoxy-D-glucose was more efficient than unesterified 2-deoxy-D-glucose in inhibiting lactate production from -D-glucose pentaacetate. It is proposed that selected esters of saccharides represent useful tools to bypass defects in hexose transport, and to increase their nutritional or therapeutic efficiency.