The association of peripheral monocyte derived interleukin 1beta (IL-1beta), IL-1 receptor antagonist, and tumor necrosis factor-alpha with osteoarthritis in the elderly

OBJECTIVE: To examine the association of peripheral blood mononuclear cell (PBMC) derived interleukin 1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor alpha (TNF-alpha), and radiographic osteoarthritis (OA) in the elderly. METHODS: A total of 703 subjects (436 women, 267 men, mean age 78.5+/-4.5 yrs) had both knee and hand radiographs, and cytokines were measured during the 22nd biennial examination of the Framingham Cohort. PBMC derived IL-1beta , IL-1Ra, and TNF-alpha production was assessed using a non-cross reacting polyclonal radioimmunoassay. Knee OA was defined as a score of > 2 using a modified Kellgren and Lawrence scale. The presence of osteophytes and joint space narrowing were scored separately on a 0-3 scale, in which disease was defined a priori as a score > 0 for each feature. Sex-specific odds ratios were calculated for knee OA after adjusting for weight, history of knee injury, and use of estrogen and nonsteroidal antiinflammatory drugs. RESULT: No uniform associations were found for IL-1beta or IL-1Ra in men, or for TNF-alpha production and radiographic OA in either sex. We found possible associations for the highest levels of IL-1beta production and the presence of knee osteophytes [OR=2.0 (1.2-3.5)] and joint space narrowing [OR=1.7 (1.1-2.8)] in women. Our data suggested a possible protective effect for IL-1Ra production and hand OA in women [OR=0.6 (0.4-1.0)]. CONCLUSION: We found no consistent association of PBMC cytokine production and radiographic OA. However, women with the highest production of IL-1beta and IL-1Ra had respectively higher rates of knee OA and lower rates of hand OA than expected.     

Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells. IL-1 beta-mediated induction regulated by tumor necrosis factor-alpha and IFN-gamma

We investigated the effects of murine rTNF-alpha, human rIL-1 beta, and rat rIFN-gamma in various concentrations and/or combinations on inducible nitric oxide (NO) production in primary cultures of rat aortic endothelial cells. Northern blot analysis of total RNA from induced and control cultures using the cloned mouse macrophage gene of inducible NO synthase as probe as well as polymerase chain reaction using a specific primer sequence gave a positive signal for activated cells only. A RNA approximately 4.4 kb of length similar to the inducible form of NO synthase in macrophages was labeled. The concentration of nitrite as a stable reaction product of NO in culture supernatants was determined 24 h after incubation with the various cytokines. IL-1 beta alone (40 to 1000 U/ml) induced formation of increasing amounts of nitrite with increasing concentrations of IL-1 beta present. Neither TNF-alpha alone (10 to 2000 U/ml) nor IFN-gamma alone 25 to 500 U/ml) showed significant effects on nitrite production. Simultaneous incubation with low concentrations of TNF-alpha (< or = 100 U/ml) and IL-1 beta abrogated the induction effect of IL-1 beta. Conversely, addition of high concentrations of TNF-alpha (> or = 500 U/ml) led to near maximal levels of nitrite formation even at lowest IL-1 beta concentrations (40 U/ml). In addition, simultaneous incubation of endothelial cells with IFN-gamma plus IL-1 beta and/or TNF-alpha led to near maximal NO production of endothelial cells, even at lowest IFN-gamma concentrations (25 U/ml). We hypothesize that the regulating effect of TNF-alpha may in vivo help to prevent local inflammatory responses from spreading to intact sites.     

The epidermal growth factor receptor is not required for tumor necrosis factor-alpha action in normal mammary epithelial cells

Our laboratory has shown that tumor necrosis factor-alpha (TNF alpha) can regulate normal mammary epithelial cell (MEC) growth, morphogenesis, and, under certain circumstances, functional differentiation in a manner similar to epidermal growth factor (EGF). As TNF alpha has been shown to up-regulate EGF receptor (EGFR) expression and function in other systems, the present studies were undertaken to determine whether TNF alpha action in MEC was indirect through stimulation of the EGFR. An inhibitor of EGFR tyrosine kinase activity, PD158780, failed to block proliferation induced by 40 ng/ml TNF alpha and only partially inhibited growth in response to 2 ng/ml TNF alpha. PD158780 was also unable to suppress the extensive morphological development induced by either TNF alpha concentration. In contrast, the effects of TNF alpha and PD158780 on functional differentiation (i.e. casein accumulation) were time dependent. When measured on day 7 after 48 h of treatment, casein accumulation was unaffected by either concentration of TNF alpha or by PD158780. When assessed on day 21 after 16 days of treatment, however, casein levels were decreased by 40 ng/ml TNF alpha and increased by PD158780. Significantly, this PD158780-induced increase in casein was not observed in MEC that had been treated with both PD158780 and TNF alpha. These results thus suggest that EGFR tyrosine kinase activity is not necessary for TNF alpha action in normal MEC.     

The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor

Tumor necrosis factor (TNF) can induce a variety of cellular responses at low picomolar concentrations. This is in apparent conflict with the published dissociation constants for TNF binding to TNF receptors in the order of 100-500 pM. To elucidate the mechanisms underlying the outstanding cellular sensitivity to TNF, we determined the binding characteristics of TNF to both human TNF receptors at 37 degrees C. Calculation of the dissociation constant (Kd) from the association and dissociation rate constants determined at 37 degrees C revealed a remarkable high affinity for TNF binding to the 60-kDa TNF type 1 receptor (TNF-R1; Kd = 1.9 x 10(-11) M) and a significantly lower affinity for the 80-kDa TNF type 2 receptor (TNF-R2; Kd = 4.2 x 10(-10) M). The high affinity determined for TNF-R1 is mainly caused by the marked stability of ligand-receptor complexes in contrast to the transient interaction of soluble TNF with TNF-R2. These data can readily explain the predominant role of TNF-R1 in induction of cellular responses by soluble TNF and suggest the stability of the TNF-TNF receptor complexes as a rationale for their differential signaling capability. In accordance with this reasoning, the lower signaling capability of homotrimeric lymphotoxin, compared with TNF, correlates with a lower stability of the lymphotoxin-TNF-R1 complex at 37 degrees C.     

Tumor necrosis factor-alpha-induced apoptosis in a human keratinocyte cell line (HaCaT) is counteracted by transforming growth factor-alpha

Pulmonary surfactant is a complex mixture of lipids and proteins that functions to keep alveoli from collapsing at the end of expiration. Dipalmitoylphosphatidylcholine has been identified as the most important component for lowering surface tension at the air-liquid interface. Hydrophobic surfactant apoproteins, SP-B and SP-C, play essential roles in the biophysical functions of the surfactant phospholipids. Hydrophilic surfactant apoproteins (SP-A and SP-D) that are members of C-type lectin superfamily, interact with phospholipids and glycolipids and modulate host defense functions in the lung. SP-A also plays an important role in regulating phospholipid homeostasis in the alveolar spaces. Recent advances in genetics and molecular biology have clarified the structure-function relationship of surfactant apoproteins.     

Interferon-gamma potentiates interleukin (IL)-6 and tumor necrosis factor-alpha but not IL-1beta induced by endotoxin in the brain

Because interferon-gamma (IFN gamma) is present in the central nervous system during neurologic diseases associated with inflammation, its effect on endotoxin-induced cytokines was studied. Cerebrospinal fluid (CSF) and serum levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha (TNF alpha), their messenger RNA expression in brain areas (hypothalamus, hippocampus, and striatum) and in spleen were evaluated 2 and 8 h after endotoxin [lipopolysaccharide (LPS), 25 microg/rat i.c.v.], IFN gamma (2.5 microg/rat i.c.v.) or after their coadministration in rats. CSF and serum IL-1beta levels were increased by LPS alone and IFN gamma coadministration did not furtherly increase them. IFN gamma potentiated LPS effect on IL-6 and TNF alpha levels in both CSF and serum. LPS and IFN-gamma coadministration did not alter IL-1beta messenger RNA expression induced by LPS in brain areas and in spleen, but it potentiated that of IL-6 and TNF alpha. The present in vivo data show that i.c.v. coadministration of LPS and IFN gamma results in a potentiation of cytokine production (IL-6 and TNF alpha) which may trigger a cascade of events relevant to neurodegenerative processes. This action is independent of IL-1beta because the production of this cytokine is not altered by IFN gamma treatment.     

Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, induces endothelial proliferation in vitro and vascular permeability in vivo. The human transmembrane c-fms-like tyrosine kinase Flt-1 has recently been identified as a VEGF receptor. Flt-1 kinase has seven immunoglobulin-like extracellular domains and a kinase insert sequence, features shared by two other human gene-encoded proteins, kinase insert domain-containing receptor (KDR) and FLT-4. In this study we show that the mouse homologue of KDR, Flk-1, is a second functional VEGF receptor. Flk-1 binds VEGF with high affinity, undergoes autophosphorylation, and mediates VEGF-dependent Ca2+ efflux in Xenopus oocytes injected with Flk-1 mRNA. We also demonstrate by in situ hybridization that Flk-1 protein expression in the mouse embryo is restricted to the vascular endothelium and the umbilical cord stroma. VEGF and its receptors Flk-1/KDR and Flt-1 may play a role in vascular development and regulation of vascular permeability.     

Denopamine, a beta1-adrenergic agonist, prolongs survival in a murine model of congestive heart failure induced by viral myocarditis: suppression of tumor necrosis factor-alpha production in the heart

OBJECTIVES: This study was designed to examine the effects of denopamine, a selective beta1-adrenergic agonist, in a murine model of congestive heart failure (CHF) due to viral myocarditis. BACKGROUND: Positive inotropic agents are used to treat severe heart failure due to myocarditis. However, sympathomimetic agents have not been found beneficial in animal models of myocarditis. METHODS: In vitro: The effects of denopamine on lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha) production was studied in murine spleen cells. In vivo: Four-week-old DBA/2 mice were inoculated with the encephalomyocarditis virus (day 0). Denopamine (14 micromol/kg), denopamine (14 micromol/kg) with a selective beta1-blocker metoprolol (42 micromol/kg), or denopamine (14 micromol/kg) with metoprolol (84 micromol/kg) was given daily, and control mice received the vehicle only. Survival and myocardial histology on day 14 and TNF-alpha levels in the heart on day 6 were examined. RESULTS: In the in vitro study, TNF-alpha levels in treated cells were significantly lower than in controls (p < 0.05). In the in vivo study treatment with denopamine significantly improved the survival of the animals (14 of 25 (56%) treated, vs 5 of 25 (20%) control mice), attenuated myocardial lesions, and suppressed TNF-alpha production (66.5+/-7.5 pg/mg of heart in treated mice vs 113.5+/-15.1 pg/mg of heart in control mice, mean+/-SE). There was a strong linear relationship between mortality and TNF-alpha levels (r=0.98, n=4, p < 0.05). These in vitro and in vivo effects of denopamine were significantly inhibited by metoprolol. CONCLUSIONS: These results suggest that denopamine may exert its beneficial effects, in part, by suppressing the production of TNF-alpha via beta1-adrenoceptors.     

Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic beta cell line, INS-1

The ability of beta cells to endure assaults by various environmental agents, including toxins and viruses, may be relevant to the development of diabetes. We have examined the mode of cell death caused by streptozotocin (STZ) in a murine pancreatic beta cell line, INS-1. Apoptosis was identified by detection of initial endonuclease-mediated DNA strand breaks by DNA gel electrophoresis. Apoptosis and necrosis were distinguished morphologically by light and electron microscopy. Higher rates of apoptosis, as compared to necrosis, were observed when cells were exposed to 15 mM STZ for 1 hr followed by a 24 hrs recovery period. Higher doses of STZ (30 mM) caused the cells to undergo necrosis (22%) as well as apoptosis (17%). These results suggest that the cytotoxic effect of STZ, at low doses, on beta cells involves the activation of the apoptotic pathway, whereas, at high doses, the mode of beta cell death is predominantly necrosis.     

The neonatal Fc receptor is not required for mucosal infection by mouse mammary tumor virus

The milk-borne mouse mammary tumor virus (MMTV) infects newborn mice via the intestine. Infection is initially restricted to Peyer's patches and later spreads to the epithelial cells of the mammary gland. The receptor that mediates uptake and transport of MMTV across the intestinal barrier has not yet been identified, The neonatal Fc receptor (nFcR), which is expressed by enterocytes during the first two weeks of life, is downregulated at weaning, and its disappearance correlates with the onset of intestinal resistance to MMTV. To test whether the nFcR mediates transport and allows infection, we foster nursed on infected MMTV mothers beta2 microglobulin-deficient (beta2m-deficient) newborn mice that are unable to express the nFcR at the surface of their enterocytes. Exposure of beta2m-deficient mice to milk-borne virus resulted in the deletion of peripheral blood T cells reactive to the superantigen encoded by MMTV. Since beta2m-deficient newborn mice are susceptible to MMTV infection despite the lack of the nFcR, we conclude that the nFcR is not required for MMTV transport.     

Characterization of the release of cholecystokinin from a murine neuroendocrine tumor cell line, STC-1

Seven fatty acid derivatives containing one or two butenolide moieties along with ancepsenolide [1], a known bis-butenolide of marine origin, were isolated from the Caribbean gorgonian Pterogorgia citrina. The structures of the new metabolites were elucidated on the basis of spectroscopic data and were confirmed upon chemical conversion to either ancepsenolide or to its homolog, homoancepsenolide [4].     

Characterization and quantitation of NF-kappaB nuclear translocation induced by interleukin-1 and tumor necrosis factor-alpha. Development and use of a high capacity fluorescence cytometric system

A new quantitative cytometric technique, termed the ArrayScanTM, is described and used to measure NF-kappaB nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-alpha (TNFalpha). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-kappaB was found to move to the nucleus with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes, a rate in each case about 4-5 min slower than that of Ikappa Balpha degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNFalpha and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNFalpha stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNFalpha stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent Ikappa Balpha degradation and subsequent NF-kappaB translocation could also be discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor with an IC50 of 2 microM, while clastolactacystin beta-lactone was a complete inhibitor with an IC50 of 10 microM. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNFalpha stimulation in both cell types with an IC50 of 0.4 microM. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNFalpha concentration measured after 17 h of stimulation. These results suggest that the ArrayScanTM technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-kappaB.     

Acid sphingomyelinase is not essential for the IL-1 and tumor necrosis factor receptor signaling pathway leading to NFkB activation

A recent report has suggested that tumor necrosis factor (TNF) utilizes acid sphingomyelinase (SMase) pathway to activate NFkB (Schutze et al. 1992. Cell 71:765). To directly investigate the role of acid SMase in IL-1 and TNF receptor-mediated signal transduction, we examined the ability of Niemann-Pick disease (NPD) type A fibroblasts, which are deficient in acid SMase, to induce IL-8 gene expression through activating NFkB. Unexpectedly, IL-1 alpha and TNF-alpha efficiently induced IL-8 production and IL-8 mRNA in NPD type A fibroblasts as in normal fibroblasts. Furthermore, activation of NFkB was also induced in NPD type A fibroblasts in response to IL-1 alpha and TNF-alpha stimulation to a similar extent as in normal fibroblasts. These results provide evidence that acid SMase is not essential in IL-1 and TNF receptor signaling leading to NFkB activation as well as the cytokine gene activation which is regulated by NFkB.     

Differential effects of interleukin 1-alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) on motility of human melanoma cell lines on fibronectin

Interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) induce a motogenic response in a number of benign and malignant cells. We examined the chemokinetic effects of these cytokines on the cell migration of four melanoma cell lines on fibronectin using modified Boyden chambers and video-time lapse analysis. Flow cytometry analysis of IL-1 receptors, TNF receptors, and shifts in beta 1 integrin expression were correlated with the effects of these cytokines on cell migration on fibronectin. The four melanoma cell lines exhibited heterogeneous expression of types I and II IL-1 receptors as well as p60 TNF receptors. Scant p80 TNF receptor expression was detected on only one cell line. Three of four melanoma cell lines demonstrated type I IL-1 receptors by Western blotting. IL-1 alpha and TNF-alpha induced heterogeneous modulation of beta 1 integrin expression in the four melanoma cell lines tested; downward shift of the alpha 2, alpha 3, alpha 4, and beta 1 integrin subunits was detected among three of the melanoma cell lines as were upward shifts of the alpha 4, alpha 5, and alpha 6 integrin subunits among three of the melanoma cell lines. IL-1 alpha and TNF-alpha induced enhanced migration on fibronectin in one of the melanoma cell lines and were related to an upward shift in the alpha 4 and alpha 5 integrin subunit expression. Taken together, the findings indicate that expression of a particular receptor for IL-1 or TNF does not necessarily signal a motogenic response in melanoma cells, but induces heterogeneous shifts in beta 1 integrin expression. However, upregulation in alpha 4 and alpha 5 integrin subunits appears to relate to enhanced migration on fibronectin.     

Flt-1, a receptor for vascular endothelial growth factor, has transforming and morphogenic potentials

A paradox of Flt-1, a tyrosine kinase receptor for vascular endothelial growth factor (VEGF), is that the ligand cannot activate the receptor to stimulate growth of cells that exogenously overexpress the receptor. In order to find Flt-1 kinase-dependent biological systems, we obtained for the first time activated forms of the Flt-1 kinase in a ligand-independent manner. Replacement of the ABL sequences in the human leukemia oncoprotein BCR-ABL with the cytoplasmic domain of Flt-1 (BCR-FLT) followed by a retroviral random mutagenesis scheme gave constitutively active artificial chimera BCR-FLTm with mutations within the Flt-1 sequence. Like BCR-ABL it could, but not the original BCR-FLT, transform Rat1 fibroblasts, abrogate cytokine dependence in Ba/F3 cells, and induce neurite-like structures in neuronal PC12 cells. Interestingly, Rat1 cells transformed by BCR-FLTm formed tube-like structures in basement membrane matrix. BCR-FLTm retroviruses may be a very useful tool to investigate an as yet uncovered functions of the Flt-1 kinase.     

The role of complement, antibody, and tumor necrosis factor alpha in the killing of Plasmodium falciparum by the monocytic cell line THP-1

Killing of Plasmodium falciparum blood forms by the differentiated human myelomonocytic THP-1Mo cell line was studied by a radiometric assay. Results showed that parasite killing was promoted by complement, antimalarial antibody, and the cytokines tumor necrosis factor alpha and gamma interferon. Differentiated THP-1Mo appears to be a useful monocytic cell line for the study of mechanisms of immunity to Plasmodium.