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.     

Synthesis and release of platelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin 1 alpha

Human endothelial cells synthesize large amounts of platelet-activating factor (PAF) after 30-min treatment with recombinant tumor necrosis factor (TNF). Synthesis of PAF peaks at 4-6 h, whereas in endothelial cells treated with interleukin 1 alpha (IL-1) it peaks at 8-12 h. More than twice as much PAF is synthesized in response to optimal concentrations of TNF than in response to IL-1. However, PAF synthesis is stimulated by lower molar concentrations of IL-1 than TNF. About 30% of PAF produced in response to either TNF or IL-1 is released into the medium, whereas approximately 70% remains cell-associated. Experiments with labeled precursors show that PAF is synthesized de novo in response to TNF. This activity of TNF is inhibited by treating endothelial cells with the inhibitors of protein or RNA synthesis cycloheximide or actinomycin D. This finding may be explained by the observation that TNF induces in endothelial cells an acetyltransferase required for PAF synthesis. The induction of this enzymatic activity precedes the peak of PAF synthesis in TNF-treated cells. After prolonged incubation with either TNF or IL-1, endothelial cells no longer respond to the same monokine, but are still capable of producing PAF when treated with the other monokine. The finding that these monokines do not show reciprocal tachyphylaxis in endothelial cells may be explained by their binding to different receptors. In cells treated simultaneously with different concentrations of TNF and IL-1, PAF synthesis is stimulated in an additive rather than synergistic way. This suggests that PAF is synthesized by the same pathway in response to TNF or IL-1.     

Endotoxemia and release of tumor necrosis factor and interleukin 1 alpha in acute heatstroke

To determine whether endotoxemia and release of tumor necrosis factor (TNF-alpha) and/or interleukin 1 alpha (IL-1 alpha) are involved in the pathogenesis of heatstroke, 17 adult patients with a mean rectal temperature of 42.1 +/- 0.2 degrees C were studied. Blood samples were taken on admission and after cooling was completed. TNF-alpha and IL-1 alpha levels were measured by enzyme-linked immunosorbent assay, and lipopolysaccharide (LPS) content was measured by the chromogenic substrate modification of the Limulus amebocyte lysate. TNF-alpha, IL-1 alpha, and LPS were elevated in all patients [199 +/- 25 (SE) pg/ml, 480.5 +/- 68.3 pg/ml, and 8.60 +/- 1.19 ng/ml, respectively, compared with normal control values of 31.4 +/- 8.4 pg/ml, 53.7 +/- 5.32 pg/ml, and less than 9 pg/ml]. There was no significant correlation between temperature and the circulating concentration of TNF-alpha, IL-1 alpha, and LPS. Postcooling TNF-alpha, IL-1 alpha, and LPS concentrations were significantly decreased but still above normal control values. The findings suggest that these mediators may have a role in the pathogenesis of heatstroke that could change the strategy of management.     

A urine inhibitor of interleukin 1 activity affects both interleukin 1 alpha and 1 beta but not tumor necrosis factor alpha

Urine from monocytic leukemia and other febrile patients contains an inhibitor of interleukin 1 (IL-1), as measured by prostaglandin E2 and collagenase production by human fibroblasts and synovial cells. With the use of recombinant IL-1, the IL-1 inhibitor was partially purified by using ammonium sulfate precipitation, anion-exchange, and gel filtration chromatographies. IL-1 inhibitory activity elutes with an 18,000 to 25,000 apparent molecular size. The same fractions also inhibit IL-1 assayed by the proliferation of murine thymocytes and human fibroblasts. Both forms of human recombinant IL-1, IL-1 alpha and IL-1 beta, which show only 26% homology, but nevertheless bind to the same receptor, are affected by this natural inhibitor to the same extent. In contrast, human recombinant tumor necrosis factor, which shares some of the biologic activities of IL-1, is not inhibited by the urinary IL-1 inhibitor. This study shows that the various biologic activities of both forms of human recombinant IL-1 are inhibited by a partially purified natural urine-derived factor.     

Eicosanoid pathway expression in bovine endometrial epithelial and stromal cells in response to lipopolysaccharide,interleukin 1 beta,and tumor necrosis factor alpha

During endometrial inflammation, bovine endometrium responds by increasing the production of pro-inflammatory mediators, such as interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα), and eicosanoids. The purpose of this study was to establish and characterize an in vitro model of endometrial inflammation using bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. We evaluated the effects of the infectious agent (bacterial lipopolysaccharide; LPS) and pro-inflammatory mediators (IL-1β and TNFα) on eicosanoid biosynthesis pathway gene expression and production by bEEL and bCSC cells. Based on concentration-response experiments, the optimal concentrations for responses were 1?μg/mL LPS, 10?ng/mL IL-1β and 50?ng/mL TNFα. Real-time PCR results show that there was an upregulation of relative mRNA expression of PTGS2 when bEEL and bCSC were treated with LPS, IL-1β and TNFα. An increase in PTGES3 expression was observed when bEEL cells were treated with LPS and IL-1β and PTGES2 when treated with IL-1β. In bCSC cells, FAAH relative mRNA was decreased upon treatments. Rate of production of PGE₂, PGF_2α, PGE₂-EA and PGF_2α-EA were also determined using liquid chromatography tandem mass spectrometry. Our results show that eicosanoid production was increased in both cell lines in response to LPS, IL-1β, and TNFα. We suggest that the characteristics of bEEL and bCSC cell lines mimic the physiological responses found in mammals with endometrial infection, making them excellent in vitro models for intrauterine environment studies.     

Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon

We have used the quantitative binding of murine monoclonal antibodies to the surface of cultured human umbilical vein endothelial (HUVE) cells to study the responses of HUVE cells to three different immune mediators: interleukin 1 (IL 1), tumor necrosis factor (TNF), and immune interferon (IFN-gamma). Antibody H4/18, reactive with an endothelial cell-specific activation antigen, does not bind to unstimulated HUVE cells but shows rapidly and transiently inducible binding (peak 4 to 6 hr) to cells stimulated by IL 1 or TNF that declines to basal levels by 24 hr, even in the continued presence of mediator. Binding of H4/18 is unaffected by IFN-gamma. Antibody RR1/1, reactive with intercellular adhesion molecule 1, binds to unstimulated HUVE cells, but binding is rapidly increased (plateau 24 hr) after stimulation by IL 1 or TNF and slowly increased (over several days) by IFN-gamma. In contrast to H4/18 binding, the increase in RR1/1 binding is sustained in the continued presence of mediator. Antibody W6/32, reactive with HLA-A,B antigens, binds to unstimulated HUVE cells and shows gradually progressive increases (over several days) in binding upon treatment with IFN-gamma or TNF. These observations demonstrate that HUVE cells show distinct but overlapping patterns of antigenic modulation in response to three different lymphokines, and suggest that the "activation" of endothelial cells observed in situ may represent a complex integration of several lymphokine-mediated signals.     

Neutrophil leukocyte emigration induced by endotoxin. Mediator roles of interleukin 1 and tumor necrosis factor alpha 1

The hypothesis that cytokines mediate neutrophil emigration induced by endotoxin (LPS) was studied by examining the potency, the kinetics of neutrophil emigration, and the tachyphylaxis of intradermal sites with IL-1, TNF-alpha and LPS. Human rIL-1 alpha and IL-1 beta, synthetic lipid A, and LPS were several orders of magnitude more potent than human rTNF. The kinetic profiles of neutrophil emigration induced by IL-1 alpha, TNF, and LPS were characterized by minimal emigration in the first 30 min, followed by rapid and transient emigration. After the injection of LPS, the onset and the time at which the rate of emigration was maximal consistently appeared 30 min later than IL-alpha or TNF, suggesting that neutrophil emigration in response to LPS was mediated by a locally generated cytokine. IL-1 and TNF were then examined as potential secondary mediators of LPS-induced emigration by comparing the patterns of tachyphylaxis between LPS and IL-1 alpha or TNF; i.e., the magnitude of neutrophil emigration into inflammatory sites was compared with sites injected 6 h previously (desensitizing injections) with a cytokine or with LPS. Tachyphylaxis was dose dependent with each and also between the IL-1 species; therefore, when tachyphylaxis between the cytokines and LPS was examined, relatively higher doses were selected for the desensitizing injections than for the test injections. With this approach, desensitizing injections of IL-1 alpha diminished the neutrophil accumulation after LPS, and LPS also desensitized sites to IL-1 alpha. However, tachyphylaxis was not observed between TNF and LPS, or between TNF and IL-1 alpha. These data suggest that IL-1, but not TNF, is a potential mediator of LPS-induced neutrophil emigration.     

Restoration of NF-kappaB activation by tumor necrosis factor alpha receptor complex-targeted MEKK3 in receptor-interacting protein-deficient cells

Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation. However, the mechanism by which RIP mediates TNF-alpha-induced signal transduction is not fully understood. In this study, we reconstituted RIP-deficient Jurkat T cells with a fusion protein composed of full-length MEKK3 and the death domain of RIP (MEKK3-DD). In these cells, MEKK3-DD substitutes for RIP and directly associates with TRADD in TNF receptor complexes following TNF-alpha stimulation. We found that TNF-alpha-induced NF-kappaB activation was fully restored by MEKK3-DD in these cells. In contrast, expression of a fusion protein composed of NEMO, a component of the IkappaB kinase complex, and the death domain of RIP (NEMO-DD) cannot restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells. These results indicate that the role of RIP is to specifically recruit MEKK3 to the TNF-alpha receptor complex, whereas the forced recruitment of NEMO to the TNF-alpha receptor complex is insufficient for TNF-alpha-induced NF-kappaB activation. Although MEKK2 has a high degree of homology with MEKK3, MEKK2-DD, unlike MEKK3-DD, also fails to restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells, indicating that RIP-dependent recruitment of MEKK3 plays a specific role in TNF-alpha signaling.     

Synergistic induction of interleukin 2 receptor (TAC) expression on YT cells by interleukin 1 or tumor necrosis factor alpha in combination with cAMP inducing agents

This study demonstrates synergistic effects on Tac expression by interleukin 1 (IL-1) or tumor necrosis factor alpha (TNF alpha) in combination with the adenylate cyclase stimulator, forskolin (FK), as well as by IL-1 with TNF alpha in the human NK-like leukemic cell line YT. The maximal expression level (greater than 80% positive cells) obtained with FK plus IL-1 or FK plus TNF alpha could not be obtained by increasing the concentration of either agent alone. Furthermore, we demonstrate that Tac protein expression is correlated with increased steady-state Tac mRNA levels. Other agents that increase intracellular cAMP, such as prostaglandin E (PGE) or isobutyl-methylxanthine (IBMX), also synergized with IL-1 or TNF alpha (but not with FK). The findings suggest that cAMP plays a role in regulating Tac expression in YT cells, and that IL-1, TNF, and FK use distinct signal transduction mechanisms, all resulting in the same end point effect, namely, induction of Tac mRNA and cell surface protein expression.     

E1A sensitizes cells to tumor necrosis factor alpha by downregulating c-FLIP S

Tumor necrosis factor alpha (TNF-alpha) activates both apoptosis and NF-kappaB-dependent survival pathways, the former of which requires inhibition of gene expression to be manifested. c-FLIP is a TNF-alpha-induced gene that inhibits caspase-8 activation during TNF-alpha signaling. Adenovirus infection and E1A expression sensitize cells to TNF-alpha by allowing apoptosis in the absence of inhibitors of gene expression, suggesting that it may be disabling a survival signaling pathway. E1A promoted TNF-alpha-mediated activation of caspase-8, suggesting that sensitivity was occurring at the level of the death-inducing signaling complex. Furthermore, E1A expression downregulated c-FLIP(S) expression and prevented its induction by TNF-alpha. c-FLIP(S) and viral FLIP expression rescued E1A-mediated sensitization to TNF-alpha by restoring the resistance of caspase-8 to activation, thereby preventing cell death. E1A inhibited TNF-alpha-dependent induction of c-FLIP(S) mRNA and stimulated ubiquitination- and proteasome-dependent degradation of c-FLIP(S) protein. Since elevated c-FLIP levels confer resistance to apoptosis and promote tumorigenicity, interference with its induction by NF-kappaB and stimulation of its destruction in the proteasome may provide novel therapeutic approaches for facilitating the elimination of apoptosis-refractory tumor cells.     

Transforming growth factor beta enhances respiratory syncytial virus replication and tumor necrosis factor alpha induction in human epithelial cells

Asthma is characterized as a chronic inflammatory disease associated with significant tissue remodeling. Patients with asthma are more susceptible to virus-induced exacerbation, which subsequently can lead to increased rates of hospitalization and mortality. While the most common cause of asthma-related deaths is respiratory viral infections, the underlying factors in the lung environment which render asthmatic subjects more susceptible to viral exacerbation are not yet identified. Since transforming growth factor beta (TGF-beta) is a critical cytokine for lung tissue remodeling and asthma phenotype, we have focused on the effects of TGF-beta on viral replication and virus-induced inflammation. Treatment of human epithelial cells with TGF-beta increased respiratory syncytial virus (RSV) replication by approximately fourfold. Tumor necrosis factor alpha (TNF-alpha) mRNA and protein expression were also significantly increased above levels with RSV infection alone. The increase in RSV replication and TNF-alpha expression after TGF-beta treatment was concomitant with an increase in virus-induced p38 mitogen-activated protein kinase activation. Our data reveal a novel effect for TGF-beta on RSV replication and provide a potential mechanism for the exaggerated inflammatory response observed in asthmatic subjects during respiratory viral infections.     

Epstein-Barr virus BHRF1 protein protects intestine 407 epithelial cells from apoptosis induced by tumor necrosis factor alpha and anti-Fas antibody.

Tumor necrosis factor (TNF) and cytotoxic T lymphocytes, which utilize Fas to induce apoptosis in target cells, are known to play a critical role in the host defense against viral infection. In this study, the Epstein-Barr virus BHRF1 protein was stably expressed in intestine 407 cells which were susceptible to cell death mediated through both the TNF receptor and Fas. WST-1 conversion assays and acridine orange staining showed that vector-transfected control cells were killed by TNF-alpha or anti-Fas antibody in a dose-dependent manner, whereas BHRF1-expressing cells were resistant to apoptosis induced by these mediators. DNA fragmentation, a characteristic of apoptosis induced by TNF-alpha and the anti-Fas antibody, was suppressed in BHRF1-expressing cells. These results indicate that the BHRF1 protein protects cells from apoptosis mediated by the TNF receptor and Fas. The role of BHRF1 as an inhibitor of cytokine-induced apoptosis during the Epstein-Barr virus lytic cycle in vivo is discussed.     

Synergistic stimulation of fibroblast prostaglandin production by recombinant interleukin 1 and tumor necrosis factor

Mononuclear cell production of cytokines that stimulate fibroblast prostaglandin (PG) elaboration is an important mechanism by which mononuclear cells regulate fibroblast function. However, the soluble factors mediating these PG-stimulatory effects are incompletely understood. We characterized the effects on PG production by confluent normal lung fibroblasts of recombinant interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta) and tumor necrosis factor (TNF), alone and in combination. All three cytokines stimulated fibroblast PG production with both IL 1 peptides being significantly more potent than TNF. In addition, TNF interacted in a synergistic fashion with both IL 1 peptides to augment fibroblast PGE elaboration further. The stimulatory effects of the cytokines were almost entirely caused by an increase in PGE2 production and were reversed when the cytokine(s) were removed. These changes in PG production could not be explained by alterations in cell number and were completely negated by specific anticytokine antibodies. Recombinant gamma interferon, although synergizing with TNF in regulating other cellular functions, did not interact with TNF to augment fibroblast PGE elaboration. In addition, the synergistic interaction of IL 1 and TNF did not extend to all biologic effects of IL 1 since TNF did not augment the ability of IL 1 to stimulate thymocyte proliferation.     

Human cytomegalovirus attenuates interleukin-1beta and tumor necrosis factor alpha proinflammatory signaling by inhibition of NF-kappaB activation

Viral infection is associated with a vigorous inflammatory response characterized by cellular infiltration and release of the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). In the present study, we identified a novel function of human cytomegalovirus (HCMV) that results in inhibition of IL-1 and TNF-alpha signaling pathways. The effect on these pathways was limited to cells infected with the virus, occurred at late times of infection, and was independent of cell type or virus strain. IL-1 and TNF-alpha signaling pathways converge at a point upstream of NF-kappaB activation and involve phosphorylation and degradation of the NF-kappaB inhibitory molecule IkappaBalpha. The HCMV inhibition of IL-1 and TNF-alpha pathways corresponded to a suppression of NF-kappaB activation. Analysis of IkappaBalpha phosphorylation and degradation suggested that HCMV induced two independent blocks in NF-kappaB activation, which occurred upstream from the point of convergence of the IL-1 and TNF-alpha pathways. We believe that the ability of HCMV to inhibit these two major proinflammatory pathways reveals a critical aspect of HCMV biology, with possible importance for immune evasion, as well as establishment of infection in cell types persistently infected by this virus.     

Proteomic analysis of human eosinophil activation mediated by mast cells,granulocyte macrophage colony stimulating factor and tumor necrosis factor alpha

We assessed mast cell influence on eosinophils, the prominent cells in late and chronic allergic reactions, by comparing the proteomic pattern of eosinophils incubated with mast cells, tumor necrosis factor alpha (TNF-alpha) or granulocyte macrophage colony stimulating factor (GM-CSF). Eosinophils were incubated with the human mast cell line HMC-1 cellular sonicate and their survival and GM-CSF production were evaluated. For proteomic studies, eosinophils were cultured with HMC-1 sonicate, TNF-alpha or GM-CSF in the presence of [(35)S]methionine, solubilized and submitted to isolelectric focusing separation and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the ISODALT system, followed by radiofluorography and computer image analysis. HMC-1-incubated eosinophils displayed increased survival partly mediated by mast cell-associated TNF-alpha, and produced GM-CSF. Metabolically labeled eosinophils incubated with either HMC-1, TNF-alpha or GM-CSF released eosinophil peroxidase. Comparison of two-dimensional gel spots from the eosinophils revealed that each of the three activating signals yielded a distinctly different proteomic pattern of labeled polypeptides. GM-CSF provided the strongest signal and the highest rate of protein synthesis (1,018 spots) followed by TNF-alpha (747 spots) and HMC-1 sonicate (611 spots). A portion of spots differed both in terms of quality and quantity. Although each stimulus induced similar functional effects, the resulting biosynthetic programs of the eosinophils greatly differed. The presented proteomic analysis is the first step in the exploration of molecular mechanisms involved in eosinophil activation.