Absence Of TNF-alpha Research Articles

Polyamines found in the inflamed periodontium inhibit priming and apoptosis in human polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) are exposed to high concentrations of polyamines in the inflamed periodontium and possess a transport system for taking up these compounds. Previous studies suggest that polyamines are involved in priming of the PMN respiratory burst by tumor necrosis factor-alpha (TNF-alpha) and can stabilize DNA against degradation. The purpose of this study was to determine whether exogenous polyamines can modulate priming by TNF-alpha or delay nuclear changes associated with PMN apoptosis (programmed cell death). Isolated human PMNs were incubated with putrescine or spermidine in vitro. Superoxide generation was measured with a cytochrome C reduction assay, and apoptotic changes were assessed by fluorescence microscopy (after cell staining with acridine orange and ethidium bromide). Incubation with 1 mM putrescine for 1 hour inhibited superoxide production by TNF-primed PMNs by 20%, but enhanced the production of superoxide by unprimed cells by 38%. Both effects were dose dependent and statistically significant (P <0.03, repeated measures ANOVA and Dunnett's test). Spermidine had no significant effects on PMN oxidative function. With regard to apoptosis, 1 mM putrescine or spermidine produced a statistically significant reduction in the proportion of apoptotic PMNs within 6 to 9 hours (P <0.05). In cells incubated for 7 hours with 300 microM putrescine or spermidine, the proportion of apoptotic cells was approximately 30% lower than in untreated controls (P <0.05, Dunnett's test). The delay of apoptosis by spermidine was less profound than that produced by TNF-alpha and was not additive to the effects of this cytokine. Polyamines could potentially impair the priming of PMN oxidative function by TNF-alpha at sites where this cytokine is present. In the absence of TNF-alpha, polyamines could enhance PMN superoxide release and enhance the maintenance of PMN function in the periodontal pocket.

Induction of mdr1b mRNA and P-glycoprotein expression by tumor necrosis factor alpha in primary rat hepatocyte cultures.

Mammalian liver exhibits expression of members of the family of multidrug resistance (mdr) transporters (P-glycoproteins). P-glycoprotein isoforms encoded by mdr1 genes participate in extrusion of an array of xenobiotics into the bile. Induction of mdr1b mRNA expression has been shown to occur in rat hepatocytes in response to hepatotrophic growth factors. As the cytokine tumor necrosis factor alpha (TNF-alpha) is known to exert a direct mitogenic effect on hepatocytes, its influence on mdr1b expression was investigated. In primary rat hepatocytes cultured in the absence of TNF-alpha, a time-dependent increase in basal expression of mdr1b mRNA and in immunodetectable P-glycoprotein was observed. In cells treated with TNF-alpha (4,000 U/ml) for 3 days, expression of mdr1b mRNA and of immunodetectable P-glycoprotein was induced approximately twofold. Moreover, intracellular steady-state levels of the mdr1 substrate rhodamine 123 were decreased in cells pretreated with TNF-alpha in comparison to controls, indicating an increase in functional transporter(s) mediating dye extrusion. Treatment of hepatocytes with antioxidants (1 mM ascorbic acid and 2% dimethyl sulfoxide) for 3 days markedly suppressed mdr1b mRNA and P-glycoprotein expression both in cells cultured in the presence of TNF-alpha and in the absence of the cytokine, but did not fully abolish mdr1b mRNA induction by TNF-alpha, supporting the notion that reactive oxygen species participate in regulation of basal mdr1b gene expression during hepatocyte culture. In conclusion, the present data indicate that by inducing mdr1b expression in hepatocytes, TNF-alpha may affect the capacity of the liver for extrusion or detoxification of endogenous or xenobiotic mdr1 substrates.

Carbon monoxide contributes to the cytokine-induced inhibition of surfactant synthesis by human type II pneumocytes.

An increase in cyclic guanosine 3',5'-monophosphate (cGMP) due to nitric oxide generation is known to participate in the mediation of the tumor necrosis factor alpha (TNF-alpha) effect in type II cells. Because guanylyl cyclase can be activated also by carbon monoxide (CO), in this study we examined the ability of human type II pneumocytes to produce CO in the presence of cytokines and the relative contribution of this molecule to the TNF-alpha and interleukin 1 effects. Type II pneumocytes were isolated from cadaveric multiple-organ donors by enzymatic digestion, adherence separation of macrophages, and gradient purification. After preculture for 24 hours, cells were cultured for 24 hours in the presence or absence of TNF-alpha, interleukin 1, sodium nitroprusside, N(omega)-nitro-1-arginine, CO, hemin, zinc-protoporphyrin type IX, deferoxamine mesylate, S-adenosyl-L-methionine, alpha-tocopherol, methylene blue (a guanylyl cyclase inhibitor), 8-bromine-cGMP, and combinations of these reagents. Both CO (picomole per microgram of protein) and nitric oxide release to the medium and the cGMP (picomole per microgram of protein) content of the cells were measured. In a different set of experiments, D-glucose labeled with radioactive carbon (14C) was added to the medium, and the labeling of several lipid fractions was determined (picomole per microgram of protein). D-[14C]glucose incorporation into phosphatidylcholine, the main surfactant component, was selectively inhibited in the presence of cytokines, CO, sodium nitroprusside, or 8-bromine-cGMP. The inhibitory effect of TNF-alpha was partially reversed by N(omega)-nitro-L-arginine, deferoxamine, or alpha-tocopherol and totally reversed by methylene blue. Tumor necrosis factor alpha induced an increase in cGMP cell content and in the CO and nitric oxide release to the medium. Hemin increased CO and cGMP production and decreased phosphatidylcholine synthesis. Zinc-protoporphyrin type IX, an inhibitor of heme oxygenase, and all 3 antioxidants, which inhibited CO production, also antagonized the TNF-alpha effect on cGMP and phosphatidylcholine synthesis. Intracellular cGMP increase due to an endogenous generation of both CO and nitric oxide mediates the cytokine-induced inhibition of surfactant synthesis by type II pneumocytes. Both lipid peroxidation and heme oxygenase activity are sources for the observed CO production.

Tumor necrosis factor alpha regulates proliferation in a mouse intestinal cell line

BACKGROUND & AIMS: Tumor necrosis factor (TNF)-alpha is a prominent cytokine in the pathogenesis of inflammatory bowel disease, yet its effects on the intestinal epithelium remain poorly understood. This study was designed to investigate the action of TNF-alpha on intestinal cell proliferation. METHODS: Young adult mouse colon cells were studied under nontransformed conditions with epidermal growth factor, keratinocyte growth factor, insulin-like growth factor 1, or serum in the presence or absence of TNF-alpha and cell numbers determined. The expression and independent actions of the 55-kilodalton TNF-alpha R1 and 75-kilodalton TNF-alpha R2 receptors were studied by immunologic methods. RESULTS: TNF-alpha stimulated proliferation at 0.1 and 1 ng/mL and inhibited proliferation at 100 and 1000 ng/mL without altering cell viability. TNF-alpha inhibited the mitogenic effect of growth factors and epidermal growth factor receptor tyrosine phosphorylation. TNF- alpha R1 receptor agonist antibody inhibited proliferation, whereas a TNF-alpha R2 receptor-blocking antibody prevented the proliferative effect of low-dose TNF-alpha. CONCLUSIONS: TNF-alpha displays a novel influence on intestinal cell growth, stimulating proliferation at physiological concentrations and inhibiting proliferation at pathological concentrations. The regulation of intestinal cell mitogenesis by TNF-alpha seems to be mediated differentially by the two TNF-alpha receptors, with the TNF-alpha R1 receptor inhibiting proliferation and the TNF-alpha R2 receptor promoting proliferation. (Gastroenterology 1997 Apr;112(4):1231-40)

TGF-beta 1 promotes in vitro generation of dendritic cells by protecting progenitor cells from apoptosis.

Our previous studies demonstrated that TGF-beta 1 is required for efficient in vitro generation of dendritic cells (DC) from CD34+ progenitor cells under serum-free conditions. Here we show that TGF-beta 1 promotes the growth and differentiation of DC primarily by protecting the viability of DC precursors and not by enhancing their proliferative response. Addition of TGF-beta 1 to TNF-alpha, granulocyte-macrophage CSF, and stem cell factor-supplemented cultures had no significant effect on the proportions of cycling cells. Already at 72 h of culture, however, the proportion of apoptotic cells was reduced by more than 60% in the presence of TGF-beta 1. This early protective effect of TGF-beta 1 correlates with the outgrowth of higher numbers and proportions of CD1a+ DC at day 7 of culture. It also correlates with a significantly reduced Fas/APO-1 expression on TGF-beta 1 cultured cells. In contrast, granulomonocytic cells, also arising under these culture conditions, are not affected to such an extent. They are found at equal proportions, both in the presence and absence of TGF-beta 1. The striking DC growth-promoting effect of TGF-beta 1 could only be observed when both TGF-beta 1 and TNF-alpha were present in the cultures. TGF-beta 1, in the absence of TNF-alpha, rather inhibited than enhanced cell expansion. Thus, for optimal in vitro DC development to occur, all four cytokines must obviously act in concert and the combination of TGF-beta 1 with TNF-alpha seems to be particularly critical.

Absence of tumor necrosis factor alpha, interleukin-6 (IL-6), and granulocyte-macrophage colony-stimulating factor expression but presence of IL-1beta, IL-8, and IL-10 expression in human monocytes exposed to viable or killed Ehrlichia chaffeensis.

Ehrlichia chaffeensis is a recently isolated minute gram-negative obligatory intracellular bacterium of monocytes/macrophages and is the etiologic agent of human monocytic ehrlichiosis. It is not known how macrophages respond when they encounter ehrlichiae in terms of cytokine production. In this study, we examined cytokine mRNA expression by incubating E. chaffeensis with THP-1 cells and performing competitive reverse transcription-PCR (RT-PCR). At 2 h postinfection, the levels of interleukin-1beta (IL-1beta), IL-8, and IL-10 mRNAs were significant but lower than those following Escherichia coli lipopolysaccharide (LPS) stimulation. Unlike the situation with E. coli LPS stimulation, however, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha) mRNAs were not induced. Time course and dose-response studies confirmed the absence of IL-6, GM-CSF, and TNF-alpha mRNA induction with E. chaffeensis. Viable E. chaffeensis organisms were not required for IL-1beta IO, IL-8, and IL-10 mRNA induction, since heat-killed E. chaffeensis induced identical time course responses. IL-1beta, IL-8, and IL-10 mRNAs were detected for up to 21, 21, and 24 h postexposure with E. chaffeensis, respectively, which were shut off more rapidly than with LPS stimulation. Although heat treatment of E. chaffeensis had no effect, periodate treatment completely abolished the ability of E. chaffeensis to induce IL-1beta, IL-8, and IL-10 mRNAs. The capture enzyme-linked immunosorbent assay result corresponded with the RT-PCR results, showing that viable and heat-killed E. chaffeensis produced and secreted the same levels of IL-1beta and IL-8. IL-10 production was significantly reduced by heat treatment. Periodate-treated ehrlichiae did not induce production of any of the cytokines tested. Anti-CD14 monoclonal antibody and polymyxin B did not inhibit IL-1beta mRNA expression upon exposure to E. chaffeensis. The absence of TNF-alpha, IL-6, and GM-CSF mRNA induction may delay the development of a protective immune response, thereby allowing E. chaffeensis to set up residence in macrophages.

Alterations in TNF-alpha signal transduction in resistant human papillary thyroid carcinoma cells.

Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) inhibit the growth of the human papillary thyroid carcinoma (PTC) cell line, NP. Exposure of NP cells to TNF-alpha resulted in the development of several PTC cell lines (R30, R45, and R60) with graded loss to the TNF-alpha-induced antiproliferation, termed resistance. In contrast, the NP cells and the resistant cells were equally sensitive to the antiproliferative action of interferon-gamma. Utilizing TNF-alpha receptor-specific agonist monoclonal antibodies, we demonstrated that the TNF-alpha receptor p55 mediated the antiproliferative action of TNF-alpha, while the p75 receptor did not affect cell proliferation in the NP cell line. The resistant PTC cell lines, however, showed a graded loss of p55 receptor-mediated antiproliferation and a concomitant activation of a p75 receptor-mediated growth stimulation. Shedding of TNF receptors is an important mechanism of TNF-alpha receptor metabolism. The p55 receptor mediated the TNF-alpha-induced up-regulation of the shedding of the p75 TNF-alpha receptor. The p75 receptor mediated the TNF-alpha-induced down-regulation of the shedding of the p55 receptor. However, the shedding of the p75 receptor was decreased and the shedding of the p55 receptor was increased in the resistant R60 cell line compared with the NP cell line, in the presence and absence of TNF-alpha. In contrast, IFN-gamma increased shedding of both p55 and p75 TNF-alpha receptors in NP and R60 cell lines with equal potency. Furthermore, the resistant PTC cell lines have increased basal manganous superoxide dismutase (MnSOD) expression and blunted induction of MnSOD mRNA upon short-term. TNF-alpha treatment (less than 2 h of treatment). The results indicate that a decrease in signal transduction via the p55 TNF-alpha receptor and concomitant increase in signal transduction via the p75 TNF-alpha receptor are involved in the development of PTC cell resistance.

Influence of cryopreservation on the sensitivity of human islets to tumor necrosis factor.

Tumor necrosis factor (TNF alpha) has been shown to inhibit insulin release and it has been postulated to-be an important effector in islet rejection. We studied the effect of cryopreservation on glucose oxidation rate (GOR), lipid synthesis, hormone secretion (insulin, glucagon, somatostatin, thyrotropin-releasing hormone), and cyclic guanosine 3',5'-monophosphate (cGMP) content of human islets, in the presence or absence of TNF alpha, looking for changes that could explain a different susceptibility to rejection for cryopreserved islets. Islets were isolated from multiple organ donor pancreata by collagenase digestion. The islets were then cultured for 7 days, cryopreserved (-0.25 degrees C/min), and stored in liquid N2. After 24 h of culture, thawed islets were cultured for an other 24 h in the presence or absence of TNF alpha. Islets were then washed to remove the cytokine and incubated in Krebs-Ringer bicarbonate (5 or 20 mM glucose), and both the cGMP content of the islets and the hormone concentration in the medium were determined by radio-immunoassay. GOR was measured as the production of 14CO2 from 5 or 20 mM D-[U-14C]glucose, and de novo lipid synthesis was determined as D-[U-14C]glucose incorporation into different lipidic fractions. Cryopreservation did not significantly modify the hormone response to glucose but it partially reversed the TNF alpha-induced inhibitory effect on insulin release in the presence of 20 mM glucose. In addition, the inhibitory effect of TNF alpha on phosphatidylcholine labeling was attenuated in cryopreserved islets compared with noncryopreserved islets. TNF alpha significantly stimulated islet nitrite production and cGMP accumulation, both effects being of a similar magnitude in cryopreserved and noncryopreserved islets. Our results suggest that cryopreservation can modify the metabolic and hormone response of human islets to TNF alpha. This effect is not mediated by changes in the TNF alpha-induced islet nitric oxide production or cGMP accumulation.

A point mutation in the HIV-1 Tat responsive element is associated with postintegration latency.

Study of the mechanism of HIV-1 postintegration latency in the ACH2 cell line demonstrates that these cells failed to increase HIV-1 production following treatment with exogenous Tat. Reasoning that the defect in ACH2 cells involves the Tat response, we analyzed the sequence of tat cDNA and Tat responsive element (TAR) from the virus integrated in ACH2. Tat cDNA sequence is closely related to that of HIV LAI, and the encoded protein is fully functional in terms of long terminal repeat (LTR) transactivation. Cloning of a region corresponding to the 5'-LTR from ACH2, however, identified a point mutation (C37 -> T) in TAR. This mutation impaired Tat responsiveness of the LTR in transient transfection assays, and the measured defect was complemented in cells that had been treated with tetradecanoyl phorbol acetate or tumor necrosis factor type alpha (TNF-alpha). A compensatory mutation in TAR (G28 -> A), designed to reestablish base pairing in the TAR hairpin, restored wild-type Tat responsiveness. When the (C37 -> T) mutation was introduced in an infectious clone of HIV-1, no viral production was measured in the absence of TNF-alpha, whereas full complementation was observed when the infection was conducted in the presence of TNF-alpha or when a compensatory mutation (G28 -> A) was introduced into TAR. These experiments identify a novel mutation associated with HIV-1 latency and suggest that alterations in the Tat-TAR axis can be a crucial determinant of the latent phenotype in infected individuals.

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: effect of tumor necrosis factor-alpha.

Recent work by a number of investigators has demonstrated that the process of bone matrix formation and mineralization is under the influence of growth factors and cytokines present in the local environment. Utilizing primary and established osteoblast cell culture systems, these studies have examined the regulation of bone matrix protein synthesis and deposition into the extracellular matrix (ECM) and subsequent mineralization. In previous studies, we have utilized the human osteoblastic cell line, HOS TE85, to study the effects of Tumor Necrosis Factor-alpha (TNF-alpha) on the regulation of matrix proteins and proteolytic function in monolayer cultures as well as during the development and calcification of ECM formed by HOS TE85 cells during extended culture. Our studies demonstrate that TNF-alpha inhibited formation and mineralization of nodules. In the study reported here, we evaluated the ultrastructural morphology of the cell-matrix complex formed by HOS TE85 cells in the presence and absence of TNF-alpha at selected time points during the matrix development process utilizing both transmission electron microscopy and light microscopy. In the presence of TNF-alpha, the cell-matrix complex does not develop normally, with a lack of organization and mineralization, when compared to untreated cells. The lack of mineralization appears to result from the lack of normal collagen fibril deposition and formation of an appropriate ECM essential for the mineralization process. These results support our previous observations that TNF-alpha inhibits HOS TE85 cells from forming a mineralizing ECM by inhibiting incorporation of collagen into the ECM and inducing the synthesis of proteolytic enzymes capable of degrading collagen in the ECM.

Both prostaglandin E2 and nitric oxide sequentially mediate the tumor necrosis factor alpha-induced inhibition of surfactant synthesis by human type II pneumocytes.

Tumor necrosis factor alpha (TNF-alpha)-induced inhibition of surfactant synthesis seems to participate in the pathogenesis of the adult respiratory distress syndrome. To examine the ability of human type II pneumocytes to produce nitric oxide (NO) in the presence of TNF-alpha and, in addition, to explore the role of this radical in the transduction of the cytokine signal. Multiple organ donors were the source of lung tissue specimens. Type II pneumocytes were isolated by enzymatic digestion, adherence separation of macrophages, and gradient purification. After 24-hour preculture, cells were cultured for 24 hours in the presence or absence of TNF-alpha (100 ng/mL), sodium nitroprusside (100 mumol/L), N omega-nitro-L-arginine methyl ester (NAME) (1 mmol/L), methylene blue (10 mumol/L),8-bromo-3',5'-cyclic guanosine monophosphate (8-Br-cGMP) (1 mmol/L), prostaglandin E2 (PGE2) (0.1 mumol/L), indomethacin (30 mumol/L), and combinations. The NO release to the medium and cGMP and PGE2 contents of the cells were measured. The incorporation of 14C-labeled glucose (D-[U-14C]glucose) into phosphatidylcholine and phosphatidylglycerol was selectively inhibited either by 8-Br-cGMP or in the presence of TNF-alpha, PGE2, or nitroprusside, all of which caused an increase in the intracellular levels of cGMP. The inhibitory effect of TNF-alpha was partially reverted by indomethacin, NAME, N-monomethyl arginine, or methylene blue. The inhibitory effect of PGE2 was partially reverted by NAME, while that of nitroprusside was reverted by methylene blue, but not by indomethacin. Tumor necrosis factor alpha induced an increase in PGE2 (4.31 +/- 0.27 vs 1.65 +/- 0.17-pg/microgram protein, n = 10, P < .01) and cGMP (0.238 +/- 0.012 vs 0.109 +/- 0.014-pmol/microgram protein, n = 10, P < .01) cell content and in the NO release to the medium (3.10 +/- 0.14 vs 1.19 +/- 0.11-nmol/microgram protein, n = 10, P < .01). The basal NO release to the medium was also increased in the presence of PGE2. The NAME, which blocked NO generation and cGMP increase, did not affect PGE2 production in response to TNF-alpha. However, indomethacin, which blocked PGE2 production, also blunted NO generation and cGMP increase. The NO generation, secondary to PGE2 production, seems responsible for the TNF-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes. Nitric oxide seems to exert this effect through activation of guanylyl cyclase.

Enhancing effect of staurosporine on NO production in rat peritoneal macrophages via a protein kinase C-independent mechanism.

Staurosporine (3-100 nM), frequently used as a protein kinase C (PKC) inhibitor, increased accumulation of nitrite in the culture medium of rat peritoneal macrophages up to 6 times above the control level. Moreover, when used in combination with the stable analogue of cyclic AMP, dibutyrylcyclic AMP (db cyclic AMP; 0.1 mM), and/or a cytokine, tumour necrosis factor-alpha (TNF alpha; 100 u ml-1), staurosporine synergistically potentiated, up to 30 times, nitrite accumulation. On the other hand, the other PKC inhibitors, calphostin C and H-7 (10 nM-10 microM) were not effective under the same conditions. The staurosporine-induced nitrite accumulation, in both the presence and the absence of TNF alpha and/or db cyclic AMP was effectively inhibited by the protein synthesis inhibitor, cycloheximide, or by the nitric oxide (NO) synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA). Thus our data suggest that staurosporine may enhance NO production in macrophages via intracellular mechanisms unrelated to the PKC inhibition.

Nuclear c-Myc Plays an Important Role in the Cytotoxicity of Tumor Necrosis Factor Alpha in Tumor Cells

The phosphoprotein c-Myc has the potential to kill cells by apoptosis. To investigate whether c-Myc is involved in tumor necrosis factor alpha (TNF-alpha)-mediated cell killing, we have examined two HeLa cell lines (D98 and H21) which show dramatic differences in their susceptibilities to TNF-alpha cytotoxicity. Northern (RNA) blot analyses showed that there were no significant differences between these cell lines in basal or TNF-alpha-induced mRNA expression for a variety of proteins, including manganous superoxide dismutase, A20 zinc finger protein, plasminogen activator inhibitor type 2, and hsp70, all of which are known to influence the susceptibility of certain cells to TNF-alpha killing. On the other hand, there was a dramatic increase in c-Myc mRNA expression in TNF-alpha-sensitive D98 cells, but not in TNF-alpha-resistant H21 cells, which was only observed when the cells were treated with cycloheximide. Western blot (immunoblot) analyses revealed that even in the absence of TNF-alpha or cycloheximide, c-Myc was detectable only in nuclear extracts of TNF-alpha-sensitive D98 cells, implying a role for preexisting c-Myc in TNF-alpha killing. In support of this interpretation, a c-myc antisense oligonucleotide specifically inhibited the TNF-alpha killing of D98 cells, provided that the oligonucleotide was added 6 h prior to TNF-alpha treatment. Either dexamethasone treatment or transient expression of c-myc antisense cDNA fragments decreased nuclear c-Myc in D98 cells and rendered the cells more resistant to TNF-alpha cytotoxicity. Nuclear c-Myc was also detectable in a TNF-alpha-sensitive human HT-1080 fibrosarcoma cell line, but it was undetectable in a derivative of HT-1080 (SS-HT-1080) known to be resistant to TNF-alpha killing because of overexpression of plasminogen activator inhibitor type 2. HT-1080 cells transfected with antisense c-myc cDNA had significantly less nuclear c-Myc and were resistant to TNF-alpha cytotoxicity. Together, these data indicate that a nuclear transcription factor, c-Myc, plays an important role in sensitizing two different tumor cell types to TNF-alpha cytotoxicity.

Nuclear c-Myc plays an important role in the cytotoxicity of tumor necrosis factor alpha in tumor cells.

The phosphoprotein c-Myc has the potential to kill cells by apoptosis. To investigate whether c-Myc is involved in tumor necrosis factor alpha (TNF-alpha)-mediated cell killing, we have examined two HeLa cell lines (D98 and H21) which show dramatic differences in their susceptibilities to TNF-alpha cytotoxicity. Northern (RNA) blot analyses showed that there were no significant differences between these cell lines in basal or TNF-alpha-induced mRNA expression for a variety of proteins, including manganous superoxide dismutase, A20 zinc finger protein, plasminogen activator inhibitor type 2, and hsp70, all of which are known to influence the susceptibility of certain cells to TNF-alpha killing. On the other hand, there was a dramatic increase in c-Myc mRNA expression in TNF-alpha-sensitive D98 cells, but not in TNF-alpha-resistant H21 cells, which was only observed when the cells were treated with cycloheximide. Western blot (immunoblot) analyses revealed that even in the absence of TNF-alpha or cycloheximide, c-Myc was detectable only in nuclear extracts of TNF-alpha-sensitive D98 cells, implying a role for preexisting c-Myc in TNF-alpha killing. In support of this interpretation, a c-myc antisense oligonucleotide specifically inhibited the TNF-alpha killing of D98 cells, provided that the oligonucleotide was added 6 h prior to TNF-alpha treatment. Either dexamethasone treatment or transient expression of c-myc antisense cDNA fragments decreased nuclear c-Myc in D98 cells and rendered the cells more resistant to TNF-alpha cytotoxicity. Nuclear c-Myc was also detectable in a TNF-alpha-sensitive human HT-1080 fibrosarcoma cell line, but it was undetectable in a derivative of HT-1080 (SS-HT-1080) known to be resistant to TNF-alpha killing because of overexpression of plasminogen activator inhibitor type 2. HT-1080 cells transfected with antisense c-myc cDNA had significantly less nuclear c-Myc and were resistant to TNF-alpha cytotoxicity. Together, these data indicate that a nuclear transcription factor, c-Myc, plays an important role in sensitizing two different tumor cell types to TNF-alpha cytotoxicity.

Spontaneous abortion in immunodeficient SCID mice.

Immunodeficient SCID mice on the CB-17 have been used to test the role of "rejection" in a xenogeneic blastocyst transfer model of recurrent miscarriage, but interpretation of the data requires knowing syngeneic within-species matings have a high success rate and do not require immunotrophic factors expected only in immunocompetent non-T-cell deficient mice. Resorption rates were studied in a SCID CB-17 barrier facility that provided the mice used to test the role of immunology in the resorption model. Spontaneous resorption in syngeneically mated immunodeficient SCID mice on the CB-17 background occurred at an unexpectedly high rate and could not be prevented by treatment with anti-asialo GM1 antibody or GM-CSF, both of which are effective in ameliorating abortion in DBA/2J-mated CBA/J mice. Immunocompetent CB-17 +/+ mice showed an even higher rate of loss. The latter was also not affected by treatment with antiasialo GM1 antibody or by GM-CSF and was not prevented by tetracycline (which is effective in the DBA/2-CBA/J system) or progesterone treatment. Mating experiments showed a scid/+ x scid@+ cross gave the highest rate of loss, and it appeared that the presence of +/(+)-type embryos in the uterus could be augmenting abortion with selective discrimination against scid/scid embryos. High abortion rates were associated both with appearance of a coagulase-negative Staphylococcus sp. in feces and with loss of one component of the SPF flora. Decidual tissue from mated CB-17 +/+ mice showed premature release of TNF-alpha in absence of TGF-beta 2-related suppressor activity, and vascular lesions (fibrinoid necrosis), varying in extent, were associated with both scid/scid x scid/scid and +/+ x +/+ pregnancies. TNF-alpha also appeared prematurely in pregnant scid/scid mice, but the levels were lower (and areas of necrosis smaller than in +/+ x +/+ pregnancies). Outcrossing onto a C57B1/6 background dramatically reduced the abortion rate, indicating an important genetic effect on susceptibility with heterogeneity protecting against abortion. SCID mice on the CB-17 background do not have a high rate of successful syngeneic pregnancies, and a TNF-alpha induced vasculopathy may be responsible. Abortion was not caused by immunodeficiency leading to loss of immunotrophism because immunocompetent non-SCID CB-17 mice had a higher rate of loss. Factors augmenting the abortion rate included the presence of embryos of the +/+ genotype in the uterus and treatment with anti-asialo GM1 antibody. Abortion rates were not reduced by treatments effective in the DBA/2-mated CBA/J mouse model but were reduced by re-establishing a new colony with defined flora (a temporary effect) and by outcrossing mice with a different (C57B1/6) background. Together, the data suggest an infectious trigger (identity uncertain) of the vasculopathy and an important genetic influence on susceptibility with heterozygosity and a SCID mouse mutation providing against abortion a degree of protection.

Effect of pentoxifylline on the inhibition of surfactant synthesis induced by TNF-alpha in human type II pneumocytes.

Tumor necrosis factor alpha (TNF-alpha) seems to play an important role in the pathogenesis of the adult respiratory distress syndrome (ARDS). This study was designed to determine the effect of TNF-alpha and pentoxifylline (PTXF) on surfactant synthesis by isolated human type II pneumocytes. In order to isolate the pneumocytes, lungs obtained from both previously healthy multiple organ donors (n = 11) and patients who underwent surgical excision for lung cancer (n = 8) were used. Surfactant synthesis was measured by the incorporation of labeled glucose into the two most important phospholipid components of surfactant: phosphatidylcholine (PC) and phosphatidylglycerol (PGL). The pneumocytes of the donor group showed a greater degree of PC synthesis than those from the cancer group (3.44 +/- 0.19 versus 2.15 +/- 01.5 pmol/micrograms protein, p < 0.001). The synthesis of PC by pneumocytes in both the donor (1.13 +/- 0.19 versus 3.44 +/- 0.19 pmol/micrograms protein, p < 0.01) and cancer (0.99 +/- 0.11 versus 2.15 +/- 0.15 pmol/micrograms protein, p < 0.01) groups was decreased by TNF-alpha (100 ng/ml). This effect was blocked by PTXF (100 micrograms/ml), a substance that also increased PC production in the control-group pneumocytes from cancer patients, the final PC levels being similar to those of the donors in the absence of TNF-alpha. These results suggest that one of the mechanisms of TNF-alpha participation in the pathophysiology of ARDS is inhibition of surfactant synthesis, and support the hypothesis of in vivo production of TNF-alpha in lung-cancer patients, with subsequent chronic exposure of the lung epithelial cells to this cytokine.(ABSTRACT TRUNCATED AT 250 WORDS)

TNF-alpha induces spreading of B-CLL via the CD11c/CD18 molecule.

Activation of malignant B cells can lead to extensive morphological changes of these cells. A combination of PMA and TNF-alpha can induce adherence of purified B-CLL cells, which acquire a dendritic cell-like appearance. This phenomenon that we have termed spreading, is accompanied by upregulation of expression of both beta 1 and beta 2 integrin molecules. Spreading was inhibited by the addition of antibodies against CD18 or CD11c. In other B cell malignancies (HCL and NHL), morphological changes could be induced by PMA in the absence of TNF-alpha. Culturing in the presence of prednisolone resulted in an inhibition of spreading, most likely mediated via a down regulation of CD18 and CD11c expression. These data indicate that the CD11c/CD18 complex might be important for the adhesive properties of B cells.

Tumor necrosis factor alpha decreases 1,25-dihydroxyvitamin D3 receptors in osteoblastic ROS 17/2.8 cells.

Bone remodeling is a complex process regulated by systemic hormones, local cytokines, and growth factors. One cytokine, tumor necrosis factor alpha (TNF-alpha), is known to have potent inhibitory effects on osteoblast matrix protein production and to stimulate osteoclast recruitment. We have previously shown that TNF-alpha inhibits 1,25-(OH)2D3-stimulated synthesis of bone gla protein (BGP), an abundant and osteoblast-specific matrix constituent. We hypothesized that the mechanism of TNF-alpha action included inhibition of intracellular 1,25-(OH)2D3 receptor (VDR) number or function. To test this, the osteoblastic cell line ROS 17/2.8 was cultured in the presence or absence of TNF-alpha (100 ng/ml), and binding of [3H]1,25-(OH)2D3 to 0.3 M KCl extracts of cytosol was measured by equilibrium assay. Specific [3H]1,25-(OH)2D3 binding decreased 70%, 25 h after addition of TNF-alpha. The decrease in [3H]1,25-(OH)2D3 binding was seen by 18 h, was sustained throughout the 72 h culture period, and was greater in low-density cultures. Scatchard analysis confirmed that TNF-alpha (100 ng/ml for 24 h) caused a decrease in the number of binding sites without change in VDR affinity. Northern analysis with a VDR riboprobe revealed that the decrease in VDR occurred without a change in the 4.4 kb steady-state VDR mRNA [VDR/cyclophilin mRNA signal ratio: control, 2.25; TNF-alpha, 2.24 (24 h), 2.17 (40 h), n = 2 flasks/time point]. These results suggest that TNF-alpha action on osteoblastic cells includes an inhibitory effect on VDR number at a point distal to the synthesis of VDR mRNA.

Tumor necrosis factor-alpha modifies adhesion properties of rat islet B cells.

The characteristic three-dimensional cell type organization of islets of Langerhans is perturbed in animal models of diabetes, suggesting that it may be important for islet function. Rat islet cells in culture are able to form aggregates with an architecture similar to native islets (pseudoislets), thus providing a good model to study the molecular basis of islet architecture and its role in islet function. Sorted islet B cells and non-B cells were permanently labeled with two different fluorescent dyes (DiO and DiI), mixed, and allowed to form aggregates during a 5-d culture in the presence or absence of TNF-alpha (100 U/ml), a cytokine suggested to be implicated in the early physiological events leading to insulin-dependent diabetes mellitus. Confocal microscopy of aggregates revealed that TNF-alpha reversibly perturbs the typical segregation between B and non-B cells. Insulin secretion, was altered in the disorganized aggregates, and returned towards normal when pseudoislets had regained their typical architecture. The homotypic adhesion properties of sorted B and non-B cells cultured for 20 h in the presence or absence of TNF-alpha were studied in a short term aggregation assay. TNF-alpha induced a significant rise in Ca(2+)-independent adhesion of B cells (from 24 +/- 1.1% to 44.3 +/- 1.2%; n = 4, P < 0.001). These findings raise the possibility that the increased expression of Ca(2+)-independent adhesion molecules on B cells leads to altered islet architecture, which might be a factor in the perturbation of islet function induced by TNF-alpha.

Regulation of C4b-binding protein gene expression by the acute-phase mediators tumor necrosis factor-alpha, interleukin-6, and interleukin-1.

C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. During an acute-phase response, we have shown that hepatic levels of murine C4BP mRNA are elevated 2.5-fold while rat liver C4BP gene expression exhibits a 4-fold induction. Furthermore, a survey of different mouse tissues showed that during acute inflammation C4BP gene expression was confined to the liver. To gain a better understanding of the acute-phase regulation of C4BP gene expression we utilized the rat hepatoma cell line FAO in which tumor necrosis factor-alpha (TNF-alpha) produced a 2.7-fold induction of C4BP mRNA levels. In the absence of TNF-alpha, interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6) had little effect on C4BP gene expression but when all three cytokines were used together a synergistic 4-fold induction of C4BP mRNA levels was observed. In contrast the synthetic glucocorticoid dexamethasone inhibited TNF-alpha-induced C4BP gene expression. Cycloheximide-mediated inhibition of inducible C4BP gene expression demonstrated the requirement for ongoing protein synthesis. Rapid induction of C4BP mRNA levels by TNF-alpha and IL-6 (within 1 h) and the observation that stimulation was inhibited by actinomycin D provided evidence that regulation of C4BP gene expression during the acute-phase response is regulated at the transcriptional level. Isolation of a genomic clone extending into the 5' regulatory region of the rat C4BP gene enabled us to identify the major transcriptional start site and putative response elements through which TNF-alpha, IL-6, IL-1 alpha, and dexamethasone may exert their effects on C4BP gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)