Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells.

Dithiocarbamates and iron chelators were recently considered for the treatment of AIDS and neurodegenerative diseases. In this study, we show that dithiocarbamates and metal chelators can potently block the activation of nuclear factor kappa B (NF-kappa B), a transcription factor involved in human immunodeficiency virus type 1 (HIV-1) expression, signaling, and immediate early gene activation during inflammatory processes. Using cell cultures, the pyrrolidine derivative of dithiocarbamate (PDTC) was investigated in detail. Micromolar amounts of PDTC reversibly suppressed the release of the inhibitory subunit I kappa B from the latent cytoplasmic form of NF-kappa B in cells treated with phorbol ester, interleukin 1, and tumor necrosis factor alpha. Other DNA binding activities and the induction of AP-1 by phorbol ester were not affected. The antioxidant PDTC also blocked the activation of NF-kappa B by bacterial lipopolysaccharide (LPS), suggesting a role of oxygen radicals in the intracellular signaling of LPS. This idea was supported by demonstrating that treatment of pre-B and B cells with LPS induced the production of O2- and H2O2. PDTC prevented specifically the kappa B-dependent transactivation of reporter genes under the control of the HIV-1 long terminal repeat and simian virus 40 enhancer. The results from this study lend further support to the idea that oxygen radicals play an important role in the activation of NF-kappa B and HIV-1.

Enhancement of experimental metastasis by tumor necrosis factor.

The influence of endogenous and exogenous tumor necrosis factor (TNF) on metastasis was investigated in an experimental fibrosarcoma metastasis model. A single intraperitoneal injection of recombinant human (rh) TNF or recombinant mouse (rm) TNF into mice 5 h before intravenous inoculation of methylcholanthrene-induced fibrosarcoma cells (CFS1) induced a significant enhancement of the number of metastases in the lung. Dose responses of rmTNF and rhTNF demonstrated a stronger metastasis-augmenting effect by rmTNF compared with rhTNF. This effect was time dependent, as administration of rmTNF 5 h before or 1 h but not 24 h after tumor cell inoculation caused an increase of tumor cell colony formation on the lung surface, suggesting an influence of TNF on the vascular adhesion and diapedesis of tumor cells. Since tumor-bearing mice showed an enhanced ability to produce TNF after endotoxin injection compared to control mice, tumor-bearing mice were treated with anti-mTNF antibodies. Neutralization of endogenous tumor-induced TNF led to a significant decrease of the number of pulmonary metastases. Histological analysis of micrometastases in the lung on day 5 by silver staining of proteins associated with nucleolar organizer regions revealed more metastatic foci and augmented proliferative activity of the tumor cells after rmTNF pretreatment of mice. However, no direct effect of rmTNF on the proliferation rate of tumor cells was seen in vitro. These findings suggest that low doses of endogenous TNF or administered TNF during cytokine therapy might enhance the metastatic potential of circulating tumor cells.

The c-kit ligand, stem cell factor, can enhance innate immunity through effects on mast cells.

Mast cells are thought to contribute significantly to the pathology and mortality associated with anaphylaxis and other allergic disorders. However, studies using genetically mast cell-deficient WBB6F1-KitW/KitW-v and congenic wild-type (WBB6F1-+/+) mice indicate that mast cells can also promote health, by participating in natural immune responses to bacterial infection. We previously reported that repetitive administration of the c-kit ligand, stem cell factor (SCF), can increase mast cell numbers in normal mice in vivo. In vitro studies have indicated that SCF can also modulate mast cell effector function. We now report that treatment with SCF can significantly improve the survival of normal C57BL/6 mice in a model of acute bacterial peritonitis, cecal ligation and puncture (CLP). Experiments in mast cell-reconstituted WBB6F1-KitW/KitW-v mice indicate that this effect of SCF treatment reflects, at least in part, the actions of SCF on mast cells. Repetitive administration of SCF also can enhance survival in mice that genetically lack tumor necrosis factor (TNF)-alpha, demonstrating that the ability of SCF treatment to improve survival after CLP does not solely reflect effects of SCF on mast cell- dependent (or -independent) production of TNF-alpha. These findings identify c-kit and mast cells as potential therapeutic targets for enhancing innate immune responses.

Upregulation of TNF receptor type 2 in human and experimental renal allograft rejection.

An important role of TNF interacting with TNFR2 has been shown in different models of ischemic, nephrotoxic and immune-mediated renal injury. To systematically evaluate the expression of TNFR2 in renal allograft rejection, we investigated human renal allograft biopsies and, in addition, established an experimental transplantation model in rats to verify the human data under standardized conditions. The expression of TNFR2 was analyzed in 96 human renal allograft biopsies with different disease entities. In a 6-day and a 28-day experimental protocol, TNFR2 was examined in kidney specimens and in the urine of control, uni-nephrectomized and transplanted rats +/- cyclosporine treatment (n = 114). In human biopsies and in rat allografts on day 6 with acute allograft rejection, significantly elevated expression of TNFR2 was observed in tubular epithelial cells, podocytes, B cells and monocytes/macrophages. The expression level was associated with renal function. The TNFR2 expression level at day 28 was significantly lower compared to day 6. TNFR2 is markedly upregulated both in human and experimental acute renal allograft rejection. Our data are robust and consistent between different species, suggesting a role for TNFR2 in the early course of rejection.

Murine models of anaemia of inflammation: extramedullary haematopoiesis represents a species specific difference to human anaemia of inflammation that can be eliminated by splenectomy.

In contrast to humans, mice physiologically exhibit extramedullary haematopoiesis in the spleen. In spite of this crucial species specific difference not much is known about the contribution of extramedullary haematopoiesis to overall erythropoiesis in models of anaemia of inflammation (AI). The objective of this study is to characterize murine AI with respect to extramedullary haematopoiesis and to develop a model more closely resembling human AI. Three different models of AI [caecal ligation and puncture (CLP), collagen induced arthritis (CIA) and DSS induced chronic colitis (DSSC)] were characterized with respect to red blood parameters, iron metabolism and extramedullary haematopoiesis. Arthritic animals were splenectomised to prevent extramedullary haematopoiesis. Anaemia caused by systemic inflammation was found in all three models. Splenic extramedullary haematopoiesis was markedly increased as reflected by increment in spleen weights and increase of the red pulp resulting in increased reticulocyte counts. Splenectomised arthritic animals did not show increased reticulocyte counts indicating that most of the reticulocytes were produced in the spleen. Our results demonstrate that murine AI differs from human AI with respect to increased splenic extramedullary haematopoiesis. Our data demonstrate that induction of AI in splenectomised mice represents a good way to model human AI.

Lysis of tumor cells by natural killer cells in mice is impeded by platelets.

Natural killer (NK) cells provide effective antitumoral activity in the blood stream of mice, leading to reduced metastasis. There are, however, tumor cells that metastasize despite the presence of an intact NK system. The capability of tumor cells to induce platelet aggregation, on the other hand, correlates with their enhanced metastatic potential. A counteractive role of platelets for the NK function in metastasis has never been conceived. Here we demonstrate for the first time that platelets directly protect tumor cells from NK lysis in vitro as well as in vivo. Using three different tumor cell lines in a mouse model of experimental metastasis, tumor seeding in the target organs was reduced when the host was platelet depleted, but only if the tumor cells were NK sensitive. Aggregation of platelets around tumor cells also inhibited in vitro NK tumorilytic activity. This protection of tumor cells by platelets was mouse strain independent and was equally observed with platelets from beta2-microglobulin-deficient mice, excluding a NK inhibitory function of MHC class I on platelets. Thus, even if tumor cells are NK susceptible and cytotoxic NK cells threaten their survival in the blood, platelets are capable of protecting them from cytolysis, thereby promoting metastasis. Surface shielding by platelet aggregates seems to be the main mechanism of this protection.

Antimetastatic effect of CpG DNA mediated by type I IFN.

The mechanisms involved in the antimetastatic effect of CpG-containing DNA were investigated in a mouse model of experimental metastasis. Tumor cell colony formation in lungs or livers of mice after i.v. inoculation with syngeneic fibrosarcoma or thymoma cells was determined. The i.v. injection of plasmid DNA or synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs before tumor cell application strongly inhibited metastasis. Because synthetic CpG-ODN was not directly tumor cytotoxic, the target cells for this CpG-ODN effect were determined. The cytotoxic activity on standard natural killer (NK) targets as well as on fibrosarcoma cells of splenic NK cells and NKT cell-containing liver mononuclear cells derived from CpG-ODN-treated mice was strongly enhanced. Participation of NK/NKT cells in the CpG-induced antimetastatic effect was demonstrated by reduction of the antimetastatic effect in mice depleted of NK/NKT cells and beta2-microglobulin-deficient mice. Neutralization of interleukin 12, interleukin 18, or IFN-gamma did not interfere with the CpG-induced antimetastatic effect. However, in sera of CpG-ODN-treated mice, high levels of IFN-alpha were detected, and in IFN-alpha/beta receptor-deficient mice, the CpG-ODN-induced antimetastatic effect was strongly reduced. These data indicate that CpG-ODNs activate NK/NKT cells for antimetastatic activity indirectly via IFN-alpha/beta receptor activation. The exploitation of the stimulatory activity of CpG-ODN for the innate immune system might be a useful strategy for antimetastatic therapy.

Long-term effects of 2'-deoxycoformycin treatment on cytokine production in patients with hairy cell leukemia.

Deoxycoformycin (DCF) has been reported to cause immediate reduction and dysfunction of T lymphocytes, but the long-term effects on immune functions are still not known. As cytokine production is regulated by T helper-inducer lymphocytes and might represent a parameter for functional integrity of immunocompetent cells, we have measured the production of interleukin-2 (IL-2), tumor necrosis factor (TNF), and interferons (IFN) by peripheral mononuclear cells (PMNC) from 10 patients with hairy cell leukemia 11-24 months after end of therapy with DCF. The patients were in continuous remission at the time of study. Despite an absolute reduction in CD3+ and CD4+ lymphocytes. there were no significant differences in IL-2 or TNF release between patients and controls. Except for a significant reduction in IFN-alpha release stimulated by Newcastle disease virus (NDV), IFN productions induced by other mitogens (phytohemagglutinin, PHA; Concanavalin A, ConA; pokeweed mitogen, PWM) and viral antigens were within normal range. There was also a decrease in proliferative responsiveness to PHA, but responses to ConA, PWM, and other viral antigens were normal. In five of the patients, we have monitored closely the changes in IL-2, TNF, and IFN before, during, and after treatment and could demonstrate a rapid normalization of initially decreased IL-2 release in all cases and also of TNF if the initial production was reduced. This study shows that, even though the absolute number of T lymphocytes and helper cells are reduced in the long-term observation after DCF treatment, the capacity to produce IL-2, TNF, and IFN-gamma was within normal range. Parallel to this observation, no opportunistic infections or frequency of infectious complications occurred in these patients.

Recombinant, soluble LIGHT (HVEM ligand) induces increased IL-8 secretion and growth arrest in A375 melanoma cells.

The heterotrimeric lymphotoxin alpha(1)beta(2) (LTalpha(1)beta(2)) complex and LIGHT, a new member of the tumor necrosis factor (TNF) superfamily, have been identified as membrane-anchored ligands for the LTbeta receptor (LTbetaR), a member of the TNF receptor (TNFR) superfamily. Although some of the biologic activities of this receptor have been described using either soluble LTalpha(1)beta(2) as a ligand or agonistic monoclonal antibodies (mAb), very little is known about the signaling of LIGHT via the LTbetaR. To gain more insight into the biologic functions of LIGHT, we generated a recombinant soluble form of human LIGHT (rsHuLIGHT). We demonstrate here that this rsHuLIGHT is capable of binding to the LTbetaR. Interestingly, receptor-mediated ligand precipitation analysis revealed that rsHuLIGHT bound only to human LTbetaR but not to mouse LTbetaR, indicating a species-specific receptor ligand interaction. Activation of A375 human melanoma cells by rsHuLIGHT induced an increased secretion of interleukin-8 (IL-8). Furthermore, rsHuLIGHT caused growth arrest of A375 cells even in the absence of interferon-gamma (IFN-gamma).

Hypoxic upregulation of TNF receptor type 2 expression involves NF-IL-6 and is independent of HIF-1 or HIF-2.

Tumor necrosis factor (TNF) exerts its biologic activity via two distinct membrane receptors, TNF receptor type 1 (p55TNFR) and TNF receptor type 2 (p75TNFR). Whereas the p55TNFR gene is rather constitutively expressed, transcription of p75TNFR is strongly modulated by a number of stimulatory agents. Experimental evidence suggested the involvement of p75TNFR in endothelial cell activation. Therefore, we have tested the transcriptional activity of p75TNFR under conditions of hypoxia and reoxygenation. Northern blot analysis revealed that p75TNFR mRNA is upregulated in NIH3T3 cells under hypoxia and reoxygenation. This observation directly originates from transcriptional activation of the p75TNFR gene, as shown by reporter gene analysis. Cotransfection experiments clearly showed that the transcriptional induction of the p75TNFR gene is independent of the hypoxia-induced factors, HIF-1alpha and HIF-2alpha. Using deletion mutants of the 5'-flanking region of the p75TNFR gene, we were able to identify a putative DNA binding site for the transcription factor nuclear factor-interleukin-6 (NF-IL-6) to be responsible for the transcriptional upregulation of the p75TNFR gene under conditions of hypoxia and reoxygenation.

Tumor necrosis factor effects on ascites formation in an experimental tumor model.

In humans, treatment of malignant ascites with bolus TNF leads to resolution of the ascites. In an experimental model NMRI nude mice were inoculated intraperitoneally with human NIH-OVCAR3 adenocarcinoma cells, resulting in production of ascites and intraperitoneal tumor growth. Ascites formation and tumor growth after IP injection of recombinant human TNF was determined. Depending on the treatment schedule, a dual effect of TNF on the development of ascites was seen. Doses of TNF (1-10 micrograms/g) given once per week completely prevented ascites production, whereas the same doses of TNF given on a daily schedule induced enhanced ascites formation in an inverse TNF dose relationship. The area of tumor cell-covered peritoneal lining corresponded to these findings, indicating a correlation of tumor mass with ascites production. In an attempt to prevent renewal of ascites after drainage, neither inhibition nor enhancement in ascites production was seen when TNF was given five times per week. However, doses of 10 micrograms/g of TNF once per week led to almost complete inhibition of ascites reappearance. Histological examination of animals that received repeated TNF treatment demonstrated chronic peritonitis with strong stromal proliferation, angiogenesis, and increased adhesion of tumor cells to the peritoneum.

VEGF/Flk-1 interaction, a requirement for malignant ascites recurrence.

Vascular endothelial growth factor (VEGF) plays an important role in the production of ascitic fluid associated with malignant tumor growth. In an experimental model for malignant ascites formation, mice were inoculated intraperitoneally with syngeneic mouse sarcoma tumor cells. Ascites development was not prevented by administering tumor necrosis factor (TNF) simultaneously with the tumor cell inoculation. When the malignant ascites was first drained and renewal of ascites was monitored, however, a TNF dose-dependent inhibition of ascitic fluid accumulation was observed. Northern blot analyses indicated transient downregulation by TNF on the expression of VEGF mRNA in tumor cells. Monoclonal antibody, (mAb) DC101 generated against the mouse VEGF receptor Flk-1 prevented the recurrence of malignant ascites in mice similar to TNF inhibition. In addition, exogenous soluble human Flt-1 used as an inhibitor of endogenous VEGF binding also inhibited ascites recurrence. These data demonstrate that the observed inhibitory effect of TNF on reestablishment of malignant ascites can be achieved equally by inhibition of the interaction of VEGF with its receptor Flk-1.

A new assay for interleukin-1 in the presence of interleukin-2.

A simple and reliable assay for interleukin-1 (IL-1) is described which has the advantage over other assays that it is independent of interleukin-2 (IL-2) production by the test cells. The assay makes possible the detection of IL-1 in the supernatants of T cell populations. The ability of IL-1 to induce IL-2 receptor expression in the absence of T cell mitogen is the basis of this assay. Thus, proliferation of mouse thymocytes incubated in the presence of saturating concentrations of IL-2 (10 U/ml) was directly dependent on the concentration of IL-1. The sensitivity of the assay is comparable to the sensitivity of the classical thymocyte co-stimulator assay. Natural and recombinant human and murine IL-1 were measured in this test system with comparable sensitivity.

Inhibition of murine cytotoxic T cell responses by progesterone.

In the present study we evaluated the effect of sex hormone on the generation of murine cytotoxic T cell responses. We show that the in vitro CTL response was strongly inhibited by progesterone but not by E1, E2 or testosterone. Our experiments attempting to understand the mechanism of the hormone action on CTL development have revealed that the ability of the cells to generate helper signals was not affected. This was demonstrated by the fact that neither IL-2 production nor IL-2 receptor expression was altered by the hormone. Rather, it appears that the capacity of the cells to respond to the signals and to become cytotoxic was modified. Furthermore, we show that the hormone mediated an inhibition of CTL development in thymocyte cultures externally supplemented with all the required helper factors. These results strongly suggest that progesterone has a direct effect on the differentiation of cytotoxic effector cells.

Biological aspects of tumor necrosis factor.

Tumor necrosis factor (TNF) has been determined as an endogenous mediator for endotoxin-induced tumor necrosis. This macrophage product has been biochemically characterized and its protein structure defined by molecular cloning of the TNF gene. Experiments with antibodies to TNF demonstrated that TNF acts as an effector molecule of activated cytotoxic macrophages involved in tumor destruction. Purified TNF has been shown to exert direct necrotic activity against tumors in vivo. In addition, a number of similar effects in vivo and in vitro of TNF, endotoxin, and interleukin 1 (IL 1) have been observed. For example, in vivo the thermoregulatory activity of TNF is similar to IL 1. Since TNF was found to mediate other effects of endotoxin in modulating immune responses in addition to the tumor necrotic activity, it can be considered a true immunoregulator produced by macrophages after endotoxin stimulation.

A combination of soluble helper factors bypasses the requirement for stimulator cells and induces nonspecific cytotoxic T cell responses.

The specificity of cytotoxic T lymphocyte (CTL) responses generated in the presence of lymphokines was studied. Thymic responder cells were activated in the presence of stimulator cells that differed in their metabolic activity. After 5 days of culture, the cytotoxic response was estimated in a 4-h 51Cr-release test. Coculture of thymic responders with irradiated splenic stimulator cells in the presence of interleukin 2(IL 2) led to preferential cytolysis of target cells that expressed the same histocompatibility antigens as the cells used for sensitization. Addition of T cell cytotoxicity-inducing factor 1 (TCF1), however, to those cultures made the presence of stimulator cells unnecessary and induced cytotoxic responses against all target cells tested, including target cells syngeneic to the responder cells. This activation was neither due to contaminating mitogen nor to the effect of heterologous serum in the assay system. The conclusion of these findings was that either polyclonal activation of CTL was induced by TCF1 or that some specific CTL clones differentiated into unrestricted killer cells under the influence of TCF1.

Cellular and molecular mechanisms of TNF protection in septic peritonitis.

Requirement for endogenous TNF for survival of experimental septic peritonitis has been demonstrated in a mouse model of cecal ligation and puncture (CLP). Induction of endogenous TNF production before CLP or administration of TNF before or after CLP confered protection from death. Interaction of TNF with the p55TNF receptor, formation of fibrin deposits, and granulocyte function was necessary to survive CLP. The mast cell seems to be an important cell type to provide the TNF required for protection in this model.

Evidence for an intracellular activation loop in the IL-1 system.

The mechanism of action of the pleiotropic cytokine interleukin-1 (IL-1) is only incompletely understood. A unique feature among cytokines is its internalization and translocation to the nuclear area in nondegraded form, suggesting intracellular activities of the molecule. To define activities of that kind, a pair of IL-1 receptor type I (IL-1RI)-positive EL4 thymoma cells with differential receptor functionality was transfected with plasmids which caused intracellular expression of FLAGIL-1 alpha fusion peptides. Intracellular delivery of IL-1 costimulated expression of IL-2 mRNA and production of IL-2 protein. This effect was not mediated by the plasma membrane IL-1RI. The cells were permanently activated, and in cells with functional IL-1RI, appearance of membrane IL-1RI was abrogated. Thus, intracellularly delivered IL-1 can bypass and replace the plasma membrane IL-1RI, possibly via an as yet undefined intracellular receptor. This is a new modality of IL-1 action and suggests a role for the intracellular IL-1R antagonists (icIL-IRa).

Role of adhesion molecules and platelets in TNF-induced adhesion of tumor cells to endothelial cells: implications for experimental metastasis.

Mechanisms for TNF-enhanced adhesion of tumor cells to endothelial cells were investigated for their in vivo relevance in a model of experimental metastasis. Mouse fibrosarcoma and thymoma cells were used to analyze TNF-modified adherence to three different mouse endothelioma cell lines and the results were compared to the in vivo colonization behavior of the tumor cells. TNF enhanced tumor cell adhesion in vitro and extravasation in vivo with similar characteristics. The role of different adhesion molecules in these experimental systems was tested. Blocking of ICAM-1, LFA-1, VCAM-1, E-selectin, and P-selectin did not reduce TNF-enhanced metastasis even though tumor cell adhesion in vitro was reduced. However, the correlation between inhibition of integrin binding and inhibition of metastasis achieved with competing peptides indicated an important role for extracellular matrix components in tumor cell attachment. Platelets play a dual role: although in vitro platelets prevented tumor cell adhesion to endothelial cells, in vivo platelet-depletion of mice reduced metastasis.