Differential effects of interleukin-1 and formylmethionylleucylphenylalanine on chemotaxis and human endothelium adhesivity for A549 tumor cells

We investigated the effects of formylmethionylleucylphenylalanine (FMLP), interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) on tumor cell chemotaxis and tumor cell/endothelial cell adhesion. Chemotaxis of A549 human lung carcinoma cells was measured as the number of tumor cells which migrated across a nitrocellulose filter in a Boyden chamber. Tumor cell/endothelial cell adhesion was measured as the number of 125IUdR tumor cells adherent to monolayers of endothelial cells. Confluent monolayers of human umbilical endothelial cells were incubated from 10 to 240 minutes with FMLP, monocyte-derived interleukin-1, or recombinant IL1 alpha or IL1 beta. The endothelial cells were washed and then incubated with 125IUdR-tumor cells. Thirty minutes later the number of adherent tumor cells was assessed isotopically. Our results demonstrate that (a) interleukin-1 but not FMLP, has chemotactic activity for tumor cells, and (b) both FMLP and interleukin-1 enhance tumor cell adhesion to the endothelium independent of any chemotactic activity. Furthermore, we demonstrate that IL1 alpha and IL1 beta have different effects on tumor cell/endothelial cell adhesion, and raise the possibility that IL1 alpha but not IL1 beta is continuously synthesized and stored within the endothelium. We postulate that IL1 alpha and IL1 beta influence tumor cell/endothelial cell adhesion independent of chemotaxis through the expression of adhesive receptors on the endothelial cell surface.     

Role of interleukin 1 in mycoplasma mitogen-induced proliferation of human T cells

Recently, a mitogenic effect of the supernatant of cultured mycoplasma arthritidis (MAS) on human and murine lymphocytes has been described. Here, we studied the role of accessory cells (AC) in MAS-induced T cell proliferation in a system of human leukocytes. Nylon-wool purified T cells were non-responsive to MAS with regard to both proliferation and IFN-gamma production. The capacity of T lymphocytes to respond to MAS could be restored when viable AC were added. Treatment of AC with UV light resulted in a cell population which was incapable of reconstituting T cells. Addition of human recombinant interleukin 1 alpha (IL 1 alpha) or IL 1 beta again showed a reconstituting effect. However, only a partial reconstitution of the T cell response could be achieved by addition of recombinant IL 1 alpha or IL 1 beta. The optimal restoration was achieved by adding IL 1 at a concentration of 100 U/ml IL 1 alpha or 100 U/ml IL 1 beta. The results indicate that metabolically active AC were required for MAS-induced T cell proliferation to occur and that IL 1 was able to substitute for the role of AC. Since this restoration was only partial, it remains to be determined whether factors others than IL 1 are required to fully substitute the role of accessory cells.     

Autoregulation of interleukin 1 production

Interleukin 1 (IL 1) alpha and beta are distinct cytokines with a common receptor on target cells. Both have been implicated in immunity, inflammation and connective tissue metabolism. Their production is stimulated by microbial products and also by other cytokines derived from accessory cells and lymphocytes. Following reports that IL 1 can stimulate its own production, we have tested the effects of recombinant IL 1 on the synthesis and release of IL 1 alpha and beta by human blood mononuclear cells (MNC). We confirmed that autoinduction occurs but report also the novel finding that this effect is very concentration dependent. At some concentrations within the range found in vivo, recombinant IL 1 not only failed to stimulate further IL 1 production but also inhibited the background level of synthesis in 20-h MNC cultures. The negative feedback appears unrelated to prostaglandin E2 and interferon-gamma levels and could not be reproduced by adding transforming growth factor beta. This previously unrecognized autoregulation may be relevant to clinical diseases associated with pathogenic over-production of IL 1.     

Degenerative changes in the A375 melanoma line induced by transforming growth factor beta 1.

A375 human melanoma cell cultures grown in the presence of TGF beta contained greatly reduced cell numbers and exhibited drastic alterations in cell morphology compared to the control cultures. Preincubation of the cells with the cytokine for only 18 h was sufficient to induce these changes irreversibly. Examination of TGF beta-treated cells in the electron microscope revealed large numbers of lipid-filled vacuoles in the cytoplasm, greatly contracted nuclei and some loss of the otherwise abundant microvilli. Thus TGF beta may have a direct toxic effect on the A375 melanoma cells.     

Recombinant human tumor necrosis factor--I. Cytotoxic activity in vitro

Cytotoxic activity of recombinant human TNF (rHu-TNF) on various human cell lines was examined in vitro. rHu-TNF exerted a cytostatic effect on various types of human tumor cells such as carcinoma, sarcoma, leukemia, melanoma and other types. When the cytocidal effect was examined on the tumor cells which were cytostatically susceptible to rHu-TNF, the cytocidal effect of rHu-TNF was also noticed on many of these tumor cells. However, some tumor cells were affected cytostatically only. Human diploid cells were not affected cytostatically or cytocidally by rHu-TNF. WI-38 VA13 cells which are an SV-40-transformed derivative of WI-38 diploid cells, were affected both cytostatically and cytocidally by rHu-TNF. These results suggest that rHu-TNF exerts cytostatic and cytocidal effects against a broad spectrum of human tumor cells, and its cytotoxic activity is tumor-specific.     

Differential activity of interleukin 1 alpha and interleukin 1 beta in the stimulation of the immune response in vivo

The biological activities of human recombinant interleukin (IL) 1 alpha and IL 1 beta were compared in different biological systems. The two IL 1 forms were equally active in vitro in inducing proliferation of murine thymocytes and of the murine T helper clone D10.G4.1, and in triggering release of prostaglandin E2 from human skin fibroblasts. In vivo, IL 1 alpha and IL 1 beta were similarly pyrogenic both in rabbits and mice, and could equally increase the circulating levels of the acute phase protein serum amyloid A in mice. However, only IL 1 beta showed immunostimulatory activity in vivo, as it could enhance the number of specific antibody-producing cells in the spleen of mice immunized with either a T-dependent or a T-independent antigen. Although devoid of immunostimulatory activity, IL 1 alpha could efficiently compete immunostimulation induced by IL 1 beta, suggesting an effective interaction with the IL 1 receptor. Thus, IL 1 beta appears to have an important role in the positive regulation of immune responses, while IL 1 alpha may act as down-regulator of the IL 1 beta effect.     

Recombinant alpha2(IV)NC1 domain inhibits tumor cell-extracellular matrix interactions, induces cellular senescence, and inhibits tumor growth in vivo

Cellular interaction with the extracellular matrix is thought to be a critical event in controlling angiogenesis and tumor growth. In our previous studies, genetically distinct noncollagenous (NC) domains of type-IV collagen were shown to interact with integrin receptors expressed on the surface of endothelial cells. Moreover, these NC1 domains were shown to inhibit angiogenesis in vivo. Here, we provide evidence that a recombinant form of the alpha2(IV)NC1 domain of type-IV collagen could bind integrins alpha1beta1 and alphavbeta3 expressed on melanoma cells and inhibit tumor cell adhesion in a ligand-specific manner. Systemic administration of recombinant alpha2(IV)NC1 domain potently inhibited M21 melanoma tumor growth within full thickness human skin and exhibited a dose-dependent inhibition of tumor growth in nude mice. Interestingly, alpha2(IV)NC1 domain enhanced cellular senescence in tumor cells in vitro and in vivo. Taken together, these results suggest that recombinant alpha2(IV)NC1 domain is not only a potent anti-angiogenic reagent, but it also directly impacts tumor cell behavior. Thus, alpha2(IV)NC1 domain represents a potent inhibitor of tumor growth by impacting both endothelial and tumor cell compartments.     

Differences in the synthesis and kinetics of release of interleukin 1 alpha, interleukin 1 beta and tumor necrosis factor from human mononuclear cells

Cell-associated and secreted interleukin 1 alpha (IL 1 alpha), IL 1 beta and tumor necrosis factor alpha (TNF-alpha), produced by human mononuclear cells (MNC) in vitro in response to lipopolysaccharide, were measured by radioimmunoassay. After 18 h of incubation, total production of IL 1 alpha in medium containing 1% heat-inactivated serum was two-to-three times higher than IL 1 beta. However, in the presence of 1% serum and 5% fresh plasma, IL 1 alpha and IL 1 beta were produced in similar amounts. Independent of the culture conditions, 90% of the IL 1 alpha remained cell associated whereas 80% of IL 1 beta was extracellular. The kinetics of production and release of IL 1 alpha, beta and TNF-alpha were also studied. IL 1 alpha and TNF-alpha reached maximal levels within 6 h of stimulation, whereas IL 1 beta reached maximal levels between 12 and 16 h. IL 1 alpha remained primarily cell associated (80%) for the first 24 h. After 48 h, extracellular IL 1 alpha exceeded cell-associated levels. IL 1 beta was primarily secreted (80%), appearing in the extracellular fluid within 6 h. TNF-alpha appeared in the extracellular fluid within 1 h of incubation, with less than 10% cell associated at any time during the 48 h of incubation. Although the three cytokines share many biological activities, this study provides evidence that MNC IL 1 alpha is predominantly a cell-associated cytokine acting on a cell-cell basis, whereas IL 1 beta and TNF-alpha are secreted as paracrine mediators.     

Action of recombinant human interleukin 6, interleukin 1 beta and tumor necrosis factor alpha on the mRNA induction of acute-phase proteins

The rat hepatoma cell line Fao was used to study the role of three inflammatory mediators on the mRNA regulation of several acute-phase proteins. In the presence of 10(-6) M dexamethasone beta-fibrinogen mRNA levels increased 6-fold after addition of recombinant human IL 6 (rhIL 6). rhIL 1 beta or recombinant human tumor necrosis factor alpha (rhTNF alpha) had essentially no effect on beta-fibrinogen mRNA induction but led to a 20-fold increase in alpha 1-acid glycoprotein mRNA in the presence of dexamethasone. On the other hand, rhIL 6 was a much weaker stimulator of alpha 1-acid glycoprotein mRNA synthesis. All three mediators reduced albumin mRNA concentrations to about 30% of controls. Whereas the induction of beta-fibrinogen mRNA was potentiated by dexamethasone, the synthetic glucocorticoid analog was an absolute requirement for the stimulation of alpha 1-acid glycoprotein mRNA. The mRNA levels of the negative acute-phase protein albumin were induced 5-fold by dexamethasone alone. The beta-fibrinogen mRNA induction started immediately after addition of rhIL 6 and reached a maximum between 12 and 18 h. In contrast, the time-course for alpha 1-acid glycoprotein mRNA synthesis showed a lag phase of 8 h followed by an increase up to 20 h after rhIL 1 beta. rhTNF alpha led to an even more delayed increase in alpha 1-acid glycoprotein mRNA. Whereas in the case of beta-fibrinogen mRNA induction no synergistic effect was observed between various concentrations of the three mediators, the combination of rhIL 6/rhIL 1 beta as well as rhIL 6/rhTNF alpha or rhIL 1 beta/rhTNF alpha regulated synergistically alpha 1-acid glycoprotein and albumin mRNA. It is concluded that discrete acute-phase proteins are regulated differently by the inflammatory mediators IL 6, IL 1 beta and TNF alpha, indicating that the acute-phase response is more complex than previously assumed. The Fao cell line used in this study turned out to be an ideal model for acute-phase protein regulation, suitable for the discrimination between the inflammatory mediators IL 6 and IL 1/TNF alpha.     

Apoptin基因通过激活caspase-3诱导人黑色素瘤细胞A375凋亡

目的研究caspase-3在肿瘤特异性凋亡基因诱导人黑色素瘤细胞A375凋亡中的作用。方法用含有apoptin基因的真核表达载体瞬间转染体外培养的人黑色瘤细胞A375;采用RT-PCR、DNA凝胶电泳、流式细胞术检测A375细胞的凋亡;以比色法检测caspase-3的相对活性。结果Ap-optin基因瞬间转染的A375细胞可出现典型的细胞凋亡所具有的DNA梯状带;流式细胞术发现实验组细胞凋亡率明显高于其他各组(P<0.01);转染后24h实验组caspase-3的活性开始升高,72h达高峰,明显高于其他各组(P<0.01)。结论Apoptin基因可通过激活caspase-3诱导人黑色素瘤细胞A375凋亡。     

Interleukin 1 and tumor necrosis factor-alpha additively increase the levels of granulocyte-macrophage and granulocyte colony-stimulating factor (CSF) mRNA in human fibroblasts

Recombinant interleukin (IL) 1 beta and tumor necrosis factor/cachectin (TNF-alpha) induce, usually within 2 h, a dose-dependent increase in the levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF mRNA in cultured human fibroblasts. Maximal induction is reached at about 4-8 h and usually last for at least 48 h. IL 1 beta and TNF have additive effects on the levels of GM- and G-CSF mRNA, and on the secretion of G-CSF activity into the culture medium. IL 1 alpha has the same additive effect that IL 1 beta has with TNF, but no additive effect with IL 1 beta. In contrast, the high basic level of M-CSF (CSF-1) mRNA shows little or lower variations in response to IL 1, TNF-alpha or both IL 1 and TNF-alpha also induce, with similar kinetics, an increase in IL 1 beta but not mRNA level. In contrast to what is observed with macrophages and endothelial cells, E. coli lipopolysaccharide does not modify the fibroblast CSF mRNA level up to 48 h of culture.     

Recombinant tumor necrosis factor can induce interleukin 2 receptor expression and cytolytic activity in a rat x mouse T cell hybrid

Tumor necrosis factor (TNF), an endotoxin-induced macrophage monokine, is known for its cytotoxic and cytostatic effect on some tumor cell lines. Here we show that highly purified recombinant TNF, in combination with interleukin 2 (IL2), can induce IL2 receptor expression and cytolytic activity in a rat x mouse T cell hybrid (PC60). Previously, it was shown that IL1 had a similar effect on PC60 cells. The ability of TNF to co-induce IL2 receptor expression suggests that it may play a role in the activation of certain lymphoid effector cells. This observation augments the growing list of biological activities attributed to TNF.     

Regulation of interleukin 1 generation in immune-activated fibroblasts

In the present study we have demonstrated that fibroblasts can generate the inflammatory cytokine interleukin 1 (IL 1) under conditions similar to those abundant in cellular immune responses. Thus, induction of IL 1 requires a sequential two-step protocol which consists of preactivation of mouse embryo fibroblasts (MEF) with crude preparations of T cell or macrophage-derived conditioned media (CM; 72 h), followed by a challenge with lipopolysaccharide (LPS; 24 h). Unstimulated fibroblasts or such cells activated by either CM or LPS produced only low levels of IL 1, while a synergism between both signals was observed for obtaining maximal IL 1-like activity in MEF. Each of a series of individual recombinant lymphokines and cytokines (IL 2, granulocyte/macrophage-colony-stimulating factor, tumor necrosis factor, IL 1 beta and interferons-alpha, beta and gamma) was shown to serve as an efficient priming signal for the induction of IL 1. IL 1-like activity in fibroblasts was detected in cell lysates or associated with the producing-cell membrane but not in culture fluids. Immune-stimulated fibroblasts, activated under such experimental conditions, were shown to actively transcribe mRNA of both IL 1 genes (alpha and beta). For the expression of IL 1-specific mRNA in fibroblasts a single stimulus, provided by either LPS or a lymphokine/cytokine, was sufficient; however, a more intense signal was observed when both stimuli were applied. The IL 1-like biological activity of fibroblast origin was significantly reduced by anti-IL 1 alpha antibodies. Thus, fibroblasts, when activated by immune and bacterial products, generate IL 1 which in turn possibly amplifies cellular immune responses or inflammatory processes in connective tissues.     

Cysteine-Rich Domain of Human ADAM 12 (Meltrin α) Supports Tumor Cell Adhesion

The ADAMs (A disintegrin and metalloprotease) comprise a family of membrane-anchored cell surface proteins with a putative role in cell-cell and/or cell-matrix interactions. By immunostaining, ADAM 12 (meltrin alpha) was up-regulated in several human carcinomas and could be detected along the tumor cell membranes. Because of this intriguing staining pattern, we investigated whether human ADAM 12 supports tumor cell adhesion. Using an in vitro assay using recombinant polypeptides expressed in Escherichia coli, we examined the ability of individual domains of human ADAM 12 and ADAM 15 to support tumor cell adhesion. We found that the disintegrin-like domain of human ADAM 15 supported adhesion of alphavbeta3-expressing A375 melanoma cells. In the case of human ADAM 12, however, recombinant polypeptides of the cysteine-rich domain but not the disintegrin-like domain supported cell adhesion of a panel of carcinoma cell lines. On attachment to recombinant polypeptides from the cysteine-rich domain of human ADAM 12, most tumor cell lines, such as MDA-MB-231 breast carcinoma cells, were rounded and associated with numerous actin-containing filopodia and used a cell surface heparan sulfate proteoglycan to attach. Finally, we demonstrated that authentic full-length human ADAM 12 could bind to heparin Sepharose. Together these results suggest a novel role of the cysteine-rich domain of ADAM 12 -- that of supporting tumor cell adhesion.     

Role of beta 1 integrins in tumor cell adhesion to cultured human endothelial cells.

We investigated the effects of beta 1 integrins on tumor cell (TC) adhesion to unstimulated and interleukin-1 (IL-1) activated endothelial cells (EC). IL-1 treatment (20 units/ml for 6 hours) of cultured human umbilical vein EC significantly increased adhesion of seven human TC lines of different origin. A goat antiserum raised to purified alpha 5 beta 1 integrin abolished the IL-1 induced increment in adhesion of two osteosarcomas, one melanoma, one lung, and one kidney carcinoma, whereas it did not affect adhesion of two colon carcinoma cell lines. Further studies were performed on MG63 osteosarcoma cells. Adhesion of MG63 osteosarcoma cells to EC was dependent on time of EC treatment with IL-1: it was maximal at 12 hours and declined at 24 hours. alpha 5 beta 1 antiserum blocked IL-1 induced increase in MG63 adhesion at any time of EC treatment. This effect appears to be mainly directed to MG63 integrins since selective incubation of the antiserum with EC, but not with MG63, did not modify TC adhesion. Using a series of antibodies to different alpha and beta chains, we found that only monoclonal antibodies (mAb) to alpha 4, alpha 5, and beta 1 could inhibit MG63 adhesion to IL-1 activated EC, whereas alpha 2, alpha 6, and beta 3 antibodies were ineffective. Antibodies to fibronectin had very little activity on MG63 adhesion to EC matrix and did not significantly affect MG63 adhesion to control or IL-1 treated EC. Antibodies to alpha 4, alpha 5, and beta 1 were only partially effective in inhibiting MG63 adhesion to EC matrix. These data indicate that the capacity of alpha 4 beta 1 and alpha 5 beta 1 integrins to bind fibronectin contributed very little to MG63 adhesion to EC. The importance of beta 1 integrins in promoting a direct interaction between EC and MG63 was further shown by inhibition of rosette formation among these cells in suspension by the alpha 5 beta 1 antiserum. Only a VCAM-1/INCAM110 mAb, but not ELAM-1 or ICAM-1 mAbs, could inhibit MG63 adhesion to IL-1 activated EC. Overall these data indicate that at least two members of the beta 1 integrin subfamily (alpha 4 beta 1 and alpha 5 beta 1) are involved in MG63 adhesion to cytokine treated EC. This integrin function might be important at early stages of TC interaction with the vessel wall.     

Transcription, translation and secretion of interleukin 1 and tumor necrosis factor: effects of tebufelone, a dual cyclooxygenase/5-lipoxygenase inhibitor.

We examined the effect of tebufelone, a dual cyclooxygenase (CO)/5-lipoxygenase (LO) inhibitor, on the synthesis, secretion and gene expression of interleukin (IL) 1 beta and tumor necrosis factor (TNF)-alpha by human peripheral blood mononuclear cells (PBMC). Basal concentrations of immunoreactive IL 1 beta and TNF-alpha after 18-24 h, in the absence or presence of tebufelone (less than or equal to 12.5 microM), were near the limit of detection (100 pg/ml). By contrast, preincubation (1 h) of cells, in amounts of tebufelone which decrease the formation of leukotriene (LT) B4, markedly enhanced (up to 500%) the synthesis of IL 1 beta and TNF-alpha following lipopolysaccharide (LPS), heat-killed Staphylococcus epidermidis or concanavalin A stimulation. Moreover, a disproportionate amount of the overall increase in IL 1 (alpha and beta) was secreted in contrast to the amount which remained cell associated, an effect unrelated to cell damage or leakage as tebufelone had no effect on either lactate dehydrogenase release by PBMC, or mitochondrial dehydrogenases of adherent monocytes as detected by enzymatic cleavage of the substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. There was no inverse correlation between the changes in prostaglandin (PG)E2 levels and TNF-alpha or IL 1 beta synthesis, and when PG formation was maximally inhibited by preincubating the cells in indomethacin, tebufelone, added 1 h before the stimulus, continued to enhance the synthesis of IL 1 beta although not that of TNF-alpha. The addition of the CO/5-LO inhibitor 2 h after LPS stimulation, however, did not interfere with IL 1 beta synthesis, suggesting that tebufelone interacts with an early event(s) in the activation of PBMC. For IL 1 beta and TNF-alpha, basal and stimulated (4 h post LPS) mRNA levels were not increased by tebufelone, despite a concomitant increase in the synthesis of IL 1 beta. In conclusion, we have demonstrated that tebufelone enhances IL 1 (alpha and beta) and TNF-alpha synthesis at concentrations which suppress leukotriene formation. These findings argue against a role of 5-LO products as necessary intermediates of IL 1 (alpha and beta) and TNF-alpha synthesis.     

Recombinant interleukin 1 alpha and beta prime human monocyte superoxide production but have no effect on chemotaxis and oxidative burst response of neutrophils

We report that recombinant human IL 1 alpha and beta and the two synthetic short fragments 163-171 and 226-254 do not exhibit chemotactic activity for human peripheral blood neutrophils and monocytes. These products up to concentrations of 4 X 10(4) units/ml failed to show chemotactic activity. Furthermore, IL 1 alpha and beta failed to generate chemotactic factors from human serum. Recombinant IL 1 alpha, IL 1 beta, or IL 1 beta fragments 163-171 and 226-254 did not induce any superoxide response by monocytes or neutrophils. However, exposure of monocytes to recombinant IL 1 alpha or IL 1 beta resulted in enhanced generation of superoxide response following stimulation with PMA. No priming was observed in neutrophils. These results suggest that IL 1 alpha and beta are involved in regulation of monocyte oxidative burst response, but play no direct regulatory role on neutrophil function.     

Response of LFA-1-deficient B cells to interleukin 4 (BSF-1) and low molecular weight B cell growth factor (BCGFlow)

T cell-depleted B cells from a patient with LFA-1 deficiency were tested in costimulation assays for responsiveness to recombinant human IL4 (BSF-1) and purified low molecular weight B cell growth factor (BCGFlow). In both cases the response of LFA-1-deficient B cells was comparable with normal controls. Monoclonal antibodies to LFA-1 alpha (CD11a) and beta (CD18) chains were unable to mimic the action of IL4 on normal B cells in costimulation assays with anti-IgM, and did not inhibit normal B cell proliferation in response to IL4 and anti-IgM. Epstein-Barr virus-transformed lymphoblastoid B cell lines (LCL) from normal and LFA-1-deficient donors both responded in proliferation assays to BCGFlow but not IL4. Similarly, both normal and LFA-1-deficient LCL increased IgM secretion in response to BCDF, BCGFlow and, interestingly, IL4. The normal LCL also increased IgG secretion in response to these factors, but no IgG was detected in supernatants from the LFA-1-deficient LCL. These results show that LFA-1 expression is not essential for B cell responses to B cell growth and differentiation factors.