TNF-R55-specific form of human tumor necrosis factor-alpha induces collagenase gene expression by human skin fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is a potent inhibitor of connective tissue formation. The cellular effects of TNF-alpha are mediated by two distinct cell-surface receptors, TNF-R55 and TNF-R75, both present on various types of cells, including fibroblasts. In this study we wanted to elucidate the role of TNF-R55 as a mediator of the connective tissue effects of TNF-alpha by using a mutant, TNF-R55-specific form of human TNF-alpha. This mutant TNF-alpha markedly induced collagenase and stromelysin-1 gene expression in dermal fibroblasts, the maximal activation (up to 42-fold) being 65%-89% of that noted with wild-type human TNF-alpha. In addition, TNF-R55-specific TNF-alpha suppressed type I collagen mRNA levels as potently as wild-type TNF-alpha (by 60%). The enhancement of collagenase gene expression by TNF-R55-specific TNF-alpha was augmented by simultaneous treatment of normal and scleroderma skin fibroblasts with interferon-gamma, indicating specific enhancement of TNF-R55 signaling pathway by interferon-gamma. These results show that stimulation of the TNF-R55 signaling pathway is sufficient for the inhibitory effects of TNF-alpha on extracellular matrix formation by dermal fibroblasts. It is conceivable that due to reduced systemic toxicity, TNF-R55-specific forms of human TNF-alpha may prove to be feasible in the therapy of fibrotic disorders.     

Modulation of melanocyte-stimulating hormone receptor expression on normal human melanocytes: evidence for a regulatory role of ultraviolet B, interleukin-1alpha, interleukin-1beta, endothelin-1 and tumour necrosis factor-alpha

In 1894, Halsted originated the standard radical mastectomy consisting of en bloc resection of the breast and axillary lymph nodes as a treatment for breast cancer. His approach was based on the theory that breast cancer was an essentially localized disease and progressed gradually through the lymphatic chains to the distant organs. His theory had been considered as a standard not only in the field of breast cancer, but also for most of the solid malignancies for a long time. Since the 70 s, the increase of early stage breast cancer and the trend to Quality-of-Life oriented treatment have led us to evaluate the modification of the extent of surgery. The results of several prospective randomized trials proved that breast conserving treatment was safe and popularized the idea that breast cancer was essentially a systemic disease. Based on the new theory, all effective treatments, including surgery, radiotherapy and medical treatment, are now used for patients with not only early stage but advanced or recurrent breast cancer. Nowadays surgeons should change the idea that they are leaders in cancer treatment to that of members of cancer treating teams. In this situation, there are many problems and much confusion. This paper was presented at the 8th Conference of the Japanese Clinical Oncology Meeting and concerned role of surgical treatment in multidisciplinary therapeutic strategy from the standpoint of the breast surgeon.     

Inhibitory effects of streptozotocin, tumor necrosis factor-alpha, and interleukin-1beta on glucokinase activity in pancreatic islets and gene expression of GLUT2 and glucokinase

Treatment of streptozotocin (ST), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) resulted in destroying insulin-secreting beta-cells of pancreatic islets and impairment of islet glucose oxidation and glucose-induced insulin secretion. IL-1beta and TNF-alpha inhibited insulin release and glucose utilization and oxidation. It was shown that the inhibitory effects of ST, IL-1beta, and TNF-alpha were due to impaired glucokinase activity. Glucokinase activity was severely impaired by ST, IL-1beta, and TNF-alpha treatments, as confirmed by assaying enzymes and nucleotides associated with glycolysis and glucose oxidation. On the other hand, nitric oxide was a factor of the deleterious effects of IL-1beta, TNF-alpha, and ST on pancreatic islets. Incubation of mouse pancreatic islets with ST at various concentrations of impairing insulin secretion resulted in generation of nitrite, stimulation of islet guanylyl cyclase and accumulation of cGMP, and inhibition of pancreatic islet mitochondrial aconitase activity to degree similar to those raised by IL-1beta and TNF-alpha. When the effects of IL-1beta and TNF-alpha on the gene expression of pancreatic GLUT2 and glucokinase were examined, the level of GLUT2 and glucokinase mRNA in pancreatic islets was significantly decreased. This suggested that IL-1beta and TNF-alpha downregulate gene expression of GLUT2 and glucokinase in pancreatic beta-cells.     

Regulation of cyclooxygenase-2 expression in bovine chondrocytes in culture by interleukin 1alpha, tumor necrosis factor-alpha, glucocorticoids, and 17beta-estradiol

OBJECTIVE: To determine the effects of interleukin 1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), dexamethasone, and 17beta-estradiol on the expression of cyclooxygenase-1 (COX-1) and COX-2 in bovine chondrocytes. METHODS: Northern blot analysis was used to quantify COX-1 and COX-2 mRNA expression in primary cultures of bovine chondrocytes and prostaglandin production to evaluate COX activity. RESULTS: IL-1alpha and TNF-alpha increased the expression of COX-2. This effect was independent of de novo protein synthesis and dependent on increased mRNA stability in the case of IL-1alpha. Dexamethasone inhibited the effects of both cytokines. 17beta-estradiol inhibited COX-2 mRNA expression in basal conditions, but had no effect on COX-2 expression induced by cytokines. The specific COX-2 inhibitor compound NS 398 prevented the increase in prostaglandin E2 (PGE2) production induced by the cytokines. COX-1 levels remained stable with all treatments. CONCLUSION: Increase in mRNA stability is a mechanism implicated in the induction of COX-2 by some cytokines. The effects of IL-1alpha and TNF-alpha on PGE2 production are mainly due to an increase in COX-2 activity as shown by the effect of compound NS 398. 17beta-estradiol inhibits COX-2 mRNA expression in basal conditions, suggesting that estrogens could be implicated in the control of cartilage metabolism.     

A modified human alpha 2-macroglobulin derivative that binds tumor necrosis factor-alpha and interleukin-1 beta with high affinity in vitro and reverses lipopolysaccharide toxicity in vivo in mice

The plasma protein alpha 2-macroglobulin (alpha 2M) has been reported to bind the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta), which play a central role in the pathogenesis of chronic inflammatory disorders, including Crohn's disease and rheumatoid arthritis. In this study, we chemically modified alpha 2M to stabilize a conformation of the protein (termed MAC, Macroglobulin Activated for Cytokine binding) with greatly increased TNF-alpha- and IL-1 beta-binding activity. The equilibrium dissociation constant (KD) for the binding of TNF-alpha to MAC was 80 +/- 20 nM, reflecting a 100-fold increase in affinity compared with native alpha 2M. To test the ability of MAC to neutralize proinflammatory cytokines in vivo, we treated mice with lipopolysaccharide (LPS) by intravenous injection. When MAC (2.5 mg) was administered by intraperitoneal injection 1 hour before the LPS, 12 of 12 mice survived and were without signs of toxicity at 5 days. None of the mice survived in the untreated control group (0/26) or in the group treated with 2.5 mg of unmodified alpha 2M (0/4). MAC also prevented the large increase in expression of inducible nitric oxide synthase in the liver, kidneys, and heart of LPS-treated mice. A novel property of MAC, compared with previously studied anticytokine agents, was its ability to reverse LPS toxicity in 12 of 24 mice when administered after the plasma level of TNF-alpha was elevated. These studies demonstrate that a naturally occurring protein, alpha 2M, can be modified so that it acquires the properties of clinically active monoclonal antibodies. Thus, MAC may have therapeutic potential in the control of chronic inflammatory disorders.     

Opposite effects of tumour necrosis factor-alpha on type I and III collagen gene expression by human dermal fibroblasts in monolayer and three-dimensional cultures

An HIV-1 infected immunosuppressed patient (CD4+ cell counts: 382 cells/microL; viral load 94,000 copies/mL) with recurrent perianal herpes simplex virus type 2 (HSV-2) infections is described, showing an unusual exophytic tumour resembling a squamous cell carcinoma in the lateral part of the tongue. He also had persistent facial herpes infection, oral candidosis, oral hairy leukoplakia and lymphadenopathy. The presence of HSV-2 was detected by polymerase chain reaction both in smears and in a tissue biopsy taken from the involved tongue area. Treatment with brivudin, a new oral virustatic drug, led to rapid regression of the tumour.     

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.     

Production and regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: roles of tumor necrosis factor alpha, interleukin-1, and interleukin-8

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.     

Inflammatory cytokine (interleukins 1, 6 and 8 and tumor necrosis factor-alpha) release from cultured human fetal membranes in response to endotoxic lipopolysaccharide mirrors amniotic fluid concentrations

OBJECTIVE: This study was conducted to quantitate and compare the amount of cytokines released from human fetal membranes in response to treatment with bacterial lipopolysaccharide and to compare this with amniotic fluid levels. STUDY DESIGN: Amniochorionic membranes were collected from women undergoing elective repeat cesarean section and showing no signs of infection- or pregnancy-related complications. Membranes were maintained in an organ explant system and stimulated with bacterial lipopolysaccharide for 24 hours. Media samples were collected and stored at -20 degrees C until cytokine levels were assayed by enzyme-linked immunosorbent assay. RESULTS: Enzyme-linked immunosorbent assay results demonstrated that lipopolysaccharide stimulated production of interleukins 1, 6 and 8 and tumor necrosis factor-alpha by the fetal membranes in comparison with the control cultures. A greater release of interleukin-6 and interleukin-8 compared with interleukin-1 and tumor necrosis factor-alpha was noticed. The relationships between cytokine concentrations observed in culture mirror those seen in amniotic fluid. CONCLUSION: Amniochorionic membranes can respond to an infectious process with increased secretion of interleukins 1, 6 and 8 and tumor necrosis factor-alpha. Cytokines produced from both amnion and chorion (interleukin-6 and interleukin-8) are released in greater quantities than those cytokines produced from chorion or amnion alone (interleukin-1 and tumor necrosis factor-alpha). These studies support a major role for amnion in infection-induced preterm labor.     

Plasma levels and gene expression of granulocyte colony-stimulating factor, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-8, and soluble intercellular adhesion molecule-1 in neonatal early onset sepsis

We have previously reported that T lymphocytes proliferating in vitro to the hapten trinitrochlorobenzene (TNCB) exhibit a very restricted V beta gene usage and response to TNCB is limited to T-cell receptors (TCR) composed of V beta 8.2 in combination with V alpha 3.2, V alpha 8 and V alpha 10. This paper investigates the role played by T lymphocytes expressing the V beta 8.2 gene segment in the contact sensitivity (CS) reaction to TNCB in the intact mouse and in its passive transfer into naive recipient mice. Mice injected with monoclonal antibodies to V beta 8 are unable to develop CS upon immunization with TNCB and 4-day TNCB-immune lymph node cells from mice that had been depleted in vivo or in vitro of V beta 8+ T lymphocytes fail to transfer CS. However, when separated V beta 8+ and V beta 8- cells were used for passive transfer, it was found that V beta 8+ T lymphocytes failed to transfer CS when given alone to recipient mice and a V beta 8- population was absolutely required. Further analysis revealed that within the V beta 8- population, T lymphocytes expressing the gamma delta TCR were fundamental to allow transfer of the CS reaction. These gamma delta cells were found to be antigen non-specific, genetically unrestricted and to rearrange the V gamma 3 gene segment. This indicates that transfer of the CS reaction requires cross-talk between V beta 8+ and gamma delta+ T lymphocytes, thus confirming our previous results obtained using TNCB-specific T-cell lines. Time-course experiments showed that V beta 8+ lymphocytes taken 4-24 days after immunization with TNCB were able to proliferate and produce interleukin-2 (IL-2) in response to the specific antigen in vitro. Similar time-course experiments were then undertaken using the passive transfer of the CS reaction system. The results obtained confirm that TNCB-specific V beta 8+ T lymphocytes are present in the lymph nodes of immunized mice from day 4 to day 24, and reveal that gamma delta+ T lymphocytes are active for a very short period of time, i.e. days 4 and 5 after immunization. In fact, TNCB-specific V beta 8+ cells are able to transfer CS when taken 4-24 days after immunization, providing the accompanying V beta 8- or gamma delta+ T lymphocyte are obtained 4 days after immunization. In contrast, injection of V beta 8+ T lymphocytes together with V beta 8- or gamma delta+ T lymphocytes that had been taken 2 or 6 days after immunization, failed to transfer significant CS into recipient mice. Taken together, our results confirm that cross-talk between V beta 8+ and gamma delta+ T lymphocytes is necessary for full development of the CS reaction and may explain why the CS reaction in the intact mouse lasts up to 21 days after immunization while the ability of immune lymph node cells to transfer CS is limited to days 4 and 5 after immunization.     

Modulation of collagen gene expression by cytokines: stimulatory effect of transforming growth factor-beta1, with divergent effects of epidermal growth factor and tumor necrosis factor-alpha on collagen type I and collagen type IV

Transforming growth factor-beta1 (TGF-beta1) is well recognized as a potent mediator of both fibrillar (collagen type I) and basement membrane (collagen type IV) production. However, tissue injury is characterized by the concomitant expression of many cytokines and/or growth factors in addition to TGF-beta1, and the ultimate extent of extracellular-matrix (ECM) deposition may reflect the interacting effects of TGF-beta1 and these other cytokines and/or growth factors. We, therefore, sought to determine whether other cytokines and/or growth factors, known to be produced after tissue injury, are capable either alone or in combination with TGF-beta1 of modulating collagen gene expression. Collagen type I and collagen type IV gene expression was assessed in NIH-3T3 cells, a murine fibroblast-like cell line that responds to TGF-beta1, with increases in both collagen type I and collagen type IV production. TGF-beta1 coordinately induced production of collagen type IV messenger ribonucleic acid (mRNA) to a level 3.8-fold above its baseline value (p < 0.001) and collagen type I mRNA to a level 2.6-fold above its baseline value (p < 0.001). Of the other cytokines and/or growth factors tested, only epidermal growth factor (EGF) had significant effects on collagen mRNA expression. We report the novel observation that EGF significantly induced collagen type IV mRNA (3.0-fold; p < 0.001) but did not alter collagen type I mRNA expression. Platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and insulin-like growth factor-1 (IGF-1) did not alter the expression of mRNA for collagen type IV or collagen type I. Addition of TGF-beta1 to cytokine- and/or growth factor-treated cells increased both collagen type IV and collagen type I mRNA levels. However, collagen type IV mRNA levels were similar in cultures given TGF-beta1 alone and cultures given TGF-beta1 with other cytokines and/or growth factors; there were no additive, synergistic, or antagonistic effects after coadministration of TGF-beta1 and other cytokines and/or growth factors. With regard to collagen type I mRNA expression, all cytokines and/or growth factors tested, with the exception of TNF-alpha, had no effect on collagen type I mRNA levels in TGF-beta1-treated cultures. Importantly, TNF-alpha antagonized the stimulatory effect of TGF-beta1 on collagen type I mRNA levels. These observations support a dominant role for TGF-beta1 in stimulating coordinate expression of collagen type I and collagen type IV mRNAs by NIH-3T3 cells; EGF and TNF-alpha are capable of inducing divergent expression of the genes for these two types of collagen.