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.     

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 that blocks ligand binding

Urine from febrile patients was found to contain a novel inhibitor of interleukin 1 (IL-1) bioactivity that blocked the specific binding of radioiodinated IL-1 to its receptor in a dose-dependent fashion. Strong inhibition of IL-1 binding was still obtained when cells were preincubated with the inhibitor and washed, thus suggesting that the inhibitor binds to a surface structure (possibly the IL-1 receptor itself). The inhibitor was distinct from IL-1 as determined by both physical parameters (size and antigenicity) and receptor-binding characteristics (apparent affinity and dissociation rate). These data provide evidence for a physiologic regulator of IL-1 activity that functions in vivo via direct interference with ligand binding.     

Serum levels of interleukin 1beta, interleukin 8 and tumour necrosis factor alpha as markers of gastric cancer.

Despite the efforts made, a serum marker reliable for the screening and follow-up of patients with gastric cancer has not yet been identified. The aim of this preliminary study was to test the role of pro-inflammatory cytokines interleukin 1beta, interleukin 8 and tumour necrosis factor alpha in patients with gastric cancer and in control groups. The statistical analysis of cytokines serum levels in the group with gastric cancer versus control groups has shown considerable differences (p < 0.001) in their mean rates. The results indicate that the cytokines interleukin 1beta, interleukin 8 and tumour necrosis factor alpha might perhaps act as diagnostic markers in patients with gastric cancer. Therefore, it is hypothesized that after more extended trials, their use in the screening and prognostic assessment of these patients could be a possibility.     

Overlapping polypeptide induction in human fibroblasts in response to treatment with interferon-alpha, interferon-gamma, interleukin 1 alpha, interleukin 1 beta, and tumor necrosis factor

Cytokine-induced polypeptides were identified in whole cell lysates of human fibroblasts by computer-based analysis of two-dimensional gels with the use of the PDQuest System. Treatment with interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) enhanced the synthesis of 12 and 28 polypeptides, respectively. Exposure to interleukin 1 alpha (IL-1 alpha) or interleukin 1 beta (IL-1 beta) resulted in the increased synthesis of seven identical polypeptides. Treatment with tumor necrosis factor (TNF) at 100 U/ml led to enhanced expression of seven polypeptides, whereas exposure to TNF at 1000 U/ml increased the levels of these seven plus two additional polypeptides. The antiviral and antiproliferative effects of these cytokines in strain 153 fibroblasts were also assessed. Both IFN-alpha and IFN-gamma exhibited antiviral activity, whereas both IL-1 and TNF stimulated fibroblast growth. IFN-gamma was alone in inhibiting proliferation. Thus, although these cytokines exhibit low degrees of structural homology, they share some common functions, and a number of polypeptides were induced in common by two or more of these agents. The greatest similarities in polypeptide induction occur between IFN-alpha and IFN-gamma and between the IL-1s and TNF. However, polypeptides were also induced in common by IFN-alpha and TNF, IFN-gamma and IL-1, and IFN-gamma and TNF. These similarities in polypeptide induction may reflect the overlapping functions of these cytokines and may be indicative of common biochemical pathways in their mechanisms of action.     

Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage

Introduction

The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.

Methods

We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1β) as well as tumor necrosis factor alpha (TNF-α) were tested with a modified Boyden chamber assay. The influence of IL-1β and TNF-α was additionally examined by scratch assays and outgrowth experiments.

Results

A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1β and TNF-α significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.

Conclusion

These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1β and TNF-α inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.     

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.     

Purification and biologic characterization of a specific tumor necrosis factor alpha inhibitor

The purpose of the present investigation was to purify a urine-derived tumor necrosis factor alpha inhibitor (TNF alpha INH) and to characterize its mechanism of action. For the purification procedure, urine was concentrated and TNF alpha INH purified by ion-exchange chromatographies, gel filtration, TNF alpha affinity column, and reverse-phase chromatography. The TNF alpha INH migrates with an apparent Mr of approximately 33,000 when estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under both reducing and nonreducing conditions. Elution of TNF alpha INH activity from the gel yields also a approximately 33,000-Da inhibitory fraction. Besides inhibiting TNF alpha-induced cytotoxicity in L929 cells in the presence of actinomycin D, the TNF alpha INH impeded in a dose-dependent manner prostaglandin E2 production and expression of cell-associated interleukin-1 by human dermal fibroblasts. Therefore, TNF alpha INH is active on both actinomycin D-treated and untreated cells. In contrast to TNF alpha, TNF beta-induced cytotoxicity was only slightly affected by the inhibitor. This specificity was confirmed by the fact that it affected neither interleukin-1 alpha nor interleukin-1 beta biologic activities. The mechanism of action of TNF alpha INH involves blocking of 125I-TNF alpha binding to the promonocytic cell line U937. Moreover, preincubation of 125I-TNF alpha with TNF alpha INH increased binding inhibition, suggesting an interaction between TNF alpha and the inhibitor.     

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.     

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.     

Cytostatic product(s) released by activated macrophages, unrelated to interleukin 1, tumor necrosis factor alpha, and interferon-alpha/beta

Murine peritoneal macrophages activated for cytotoxicity by trehalose dimycolate in vivo and lipopolysaccharide in vitro released cytostatic factor(s) against EMT6 target cells, in 8-hr conditioned medium (CM). The cytostatic factor(s) completely blocked DNA synthesis by EMT6 cells within 16 hr. Other cell lines are less sensitive (P815 and R-L929) or resistant (KB and HT29) to the cytostatic effect of CM. The anti-proliferative activity of CM had a MW greater than 10,000 Da, as judged by ultrafiltration. It was destroyed by proteases and strongly inhibited by P815 cell product(s). Conditioned media from nonactivated macrophages were not cytostatic against EMT6 cells. No relationship was found between cytostatic factor(s) in CM and interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and interferon-alpha/beta (IFN-alpha/beta): the growth of EMT6 cells was unaffected by Hu.r.IL-is and Hu.r.TNF-alpha and was only slightly inhibited by IFN-alpha/beta. Furthermore, cytostatic CM contained low levels of TNF and IFN activities. Finally, antibodies raised against murine IFN-alpha/beta had no effect on the cytostatic activity of CM.     

肿瘤坏死因子α和β对电离辐射诱导细胞凋亡的效应

为探讨肿瘤坏死因子（tumor necrosis foctor)α和β（TNFα和β）对电离辐射诱发细胞凋亡的效应及其机理,采用DNA琼脂糖凝胶电泳和FACS分析等方法,观察了人肿瘤坏死因子α（hTNFα)和β（hTNFβ)对^60Co-γ射线诱发细胞凋亡的形态学,生化学变化。结果显示：hTNFα或hTNFβ均可明显抑制^60Co-γ射线诱发正常人胚肺二倍体细胞（2BS）的凋亡,而相同剂量的hTNFα能促进^60Co-γ射线诱发的人体肺腺癌细胞系A549细胞凋亡,而对另一株人体肺癌SPC细胞的效应比A549降低1倍;hTNFβ能分别增强A549和SPC的细胞凋亡频率。由此认为,hTNFα和hTNFβ均可通过调节细胞的生理生化反应来改变细胞对电离辐射的敏感性,可保护正常细胞免受辐射损伤,而增加某些肿瘤细胞对辐射的敏感性。     

Interferon-gamma synergizes with tumor necrosis factor and with interleukin 1 and requires the presence of both monokines to induce antitumor cytotoxic activity in macrophages

Small concentrations of recombinant murine interferon-gamma (MuIFN-gamma), recombinant human interleukin 1 (HuIL-1), and recombinant murine tumor necrosis factor (MuTNF), added separately to cultures of thioglycolate-elicited peritoneal macrophages, produced no cytotoxic activity against L5178Y cells, a tumor cell line which is resistant to the direct toxic effects of these cytokines, either alone or in combination. However, small concentrations of MuIFN-gamma when combined with small concentrations of either HuIL-1 or MuTNF activated these macrophages to produce cytotoxic effects against L5178Y cells; small concentrations of HuIL-1 and MuTNF in combination had no macrophage activating activity. Specific antibody to MuTNF blocked the macrophage-activating synergistic effects of MuIFN-gamma + HuIL-1, and specific antibody to HuIL-1 blocked the macrophage-activating activity of MuIFN-gamma + MuTNF, indicating that MuTNF was induced in macrophage cultures treated with MuIFN-gamma + HuIL-1, and that murine IL-1 was induced in macrophage cultures treated with MuIFN-gamma + MuTNF. These results indicate that all three cytokines are required for induction of antitumor cytotoxic activation of macrophages. Experiments with a concentration of MuIFN-gamma which alone could activate macrophages revealed that both MuTNF and murine IL-1 were required for this activation. The demonstration that small concentrations of these three cytokines can act synergistically, but not separately, to activate macrophages indicates the importance of cytokine combinations in immunoregulation and in anti-tumor cell-mediated immune responses.     

Circulating integrins: alpha 5 beta 1, alpha 6 beta 4 and Mac-1, but not alpha 3 beta 1, alpha 4 beta 1 or LFA-1.

The alpha 5 beta 1, alpha 6 beta 4 and Mac-1 integrins all participate in the endocytotic cycle. By contrast, alpha 3 beta 1, alpha 4 beta 1 and LFA-1 do so much more slowly, or not at all, in the cell lines examined. This indicates that the alpha-chains appear to determine whether an integrin cycles or not, and that alpha 5 beta 1, alpha 6 beta 4 and Mac-1 can be brought to the leading edge of a moving cell by endocytosis and recycling.     

Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocyte-macrophage colony-stimulating factor

Interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are molecularly distinct cytokines acting on separate receptors. The release of these cytokines can be concomitantly induced by the same signal and from the same cellular source, suggesting that they may cooperate. Administered alone, human recombinant (hr)IL-1 alpha and hrTNF alpha protect lethally irradiated mice from death, whereas murine recombinant GM-CSF and hrG-CSF do not confer similar protection. On a dose basis, IL-1 alpha is a more efficient radioprotector than TNF alpha. At optimal doses, IL-1 alpha is a more radioprotective cytokine than TNF alpha in C57BL/6 and B6D2F1 mice and less effective than TNF alpha in C3H/HeN mice, suggesting that the relative effectiveness of TNF alpha and IL-1 alpha depends on the genetic makeup of the host. Administration of the two cytokines in combination results in additive radioprotection in all three strains. This suggests that the two cytokines act through different radioprotective pathways and argues against their apparent redundancy. Suboptimal, nonradioprotective doses of IL-1 alpha also synergize with GM-CSF or G-CSF to confer optimal radioprotection, suggesting that such an interaction may be necessary for radioprotection of hemopoietic progenitor cells.