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Lymphotoxin-α requires high expression of the p55-TNF receptor for efficient cell signaling
550 I Abstracts
CYTOKINE,
P3 October 2: InJammation
Vol. 6, No. 5 (September
1994: 539-582)
and Cytokines
A66 SELECTIVE INDUCTION OF VASCULAR CELL ADHESION MOLECULE-l AND AMPLIFICATION OF IL-6 PRODUCTION BY IL-13 IN ENDOTHELIAL (EC) AND MESOTHELIAL (MC) CELLS. M. Sironi, F.L. Sciacca, C. Matteucci, M. Conni , A. Vecchi, A. Minty, D. Caput, P. Ferrara, F. Colotta, A. Mantovani. 1st. Ric. Farmacol. “Mario Negri”, Milano, Italy. In vitro exposure to IL-13 of vascular EC induced surface expression of VCAM-1. At optimal concentrations (lo-50 rig/ml) and exposure times (24h), IL-13 was a 2 to 3-fold less effective inducer of VCAM-1 than IL-l, used as reference EC activator. When IL-13 was combined with IL-l, almost additive induction of VCAM was observed. Induction of VCAM-1 by IL-1 3 was selective in that E-selectin and ICAMwere unaffected. IL-13 caused a modest reduction of IL-l induction of E-selectin and ICAM-I. Surface expression of VCAM-1 on IL-13-treated cells was associated with mRNA induction, with predominance of transcripts encoding the 7 lg domain form of this molecule. IL-13 inhibited cytokine production in monocytes but was a weak inducer and amplifier (in concert with IL-l) of IL-6 expression in EC. MC were stimulated to express VCAM-1 and IL-6 by IL-13. Thus, IL-13 elicits a spectrum of responses in EC remarkably similar to that of IL-4.
LYMPHOTOXIN-a REQUIRES HIGH EXPRESSION OF THE ~55.TNF RECEPTOR FOR EFFICIENT CELL SIGNALING A. SUNDAN, A: MEDVEDEV AND T. ESPEVIK. Institute for Cancer Research, MTS, Univ. of Trondheim, N-7005 Trondheim, Norway
AfiS . ---
Tumor Necrosis Factor-u (TNF) and Lymphotoxin-a (LT) are pleiotropiccytokines which both mediatetheir effects by binding to the two known TNF receptors, -the ~55 and the ~75 TNFR. These receptors are almost ubiquitously expressed on cells, and TNF and LT bind the receptors with comparable affinities. However, the expression of the two receptor types are independentlyregulated,and the ratio of the two receptors expressed on cells varies. Here we present experiments demonstrating that whereas some human ceils,i.e. the FS4 and WHEI ~1.13,are equally sensitive to TNF and LT, other human cells such as the KYM-1 and Sii480 cells were much more resistant towards LT than TNF. Bindingexperiments with iodinatedLT and TNF indicated that both cytokines were bound to the these cell lines in a similar manner with comparable association constants and number of binding sites. However, binding experiments in the presence of antibodies which block the binding to one or the other TNFR indicated that the Fs4 and WEHI cells expressed a high proportion of p55 compared to ~75 receptors, whereas the KYM-1 and SW480 cells expressed high numbers of ~75 compared to p55 TNFR. Cells expressmg a high proportion of ~75 TNFR therfore appears to be resistant to LT. The relative expression of the two TNFR may therfore be a way for the cells to discriminate between suceptibility towards TNF and LT. We also present data indicatingthat whereas TNF have a relatively high on-off rate in the Interactionwith the ~75 TNFR, the rate of dissociation of LT is much lower. The resistance towards LT of some cell linesexpressing a high proportion of the p75 TNFR may therfore be due fo a lack of ability to pass LT on from the ~75 to the ~55 TNFR, wheras such a mechanism may operate in the case 01TNF.
A67
A70 SOLUBLE TNF RECEPTOR MEDIATED REDUCTION OF MIP-la EXPRESSION IN BLEOMYCIN-INDUCED LUNG INJURY. R. E. Smith, S.H. Phan, R.M. Stricter, and S.L. Kunkel(Sponsor). Depts. of Pathology and Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, 48109-0602. Bleomycin-induced lung injury, a model of interstitial lung disease, is a T-cell dependent pulmonary inflammatory response characterized by an increase in leukocyte infiltration, fibroblast proliferation and collagen synthesis. Cytokines such as TNF are believed to have a central role in the emanation of the inflammatory response to bleomycin. Previously, we have demonstrated that intratracheal challenge of CBA/J mice with bleomycin resulted in a significant time-dependent increase in MIP-la protein levels both in whole lung homogenates and broncho-alveolar lavage fluid (BALF) with detectable protein peaking at days 2 and 12 post-bleomycin instillation. In addition, anti-MIP-la antibodies significantly reduced inflammatory cell content and fibrosis. We now demonstrate that treatment of bleomycin challenged mice with soluble TNF receptor (sTNFr) reduces MIP-la production (measured by ELISA) in the mouse lung. TNF levels (measured by bioassay) in BALF fluid were significantly elevated, compared to controls, 2. 8, and 16 days postbleomycin injection. CBA/J mice challenged with 0.025 units of bleomycin were injected intraperitoneally with 100 ug of IgG or sTNPr either at time of bleomycin instillation or at 6 days post-instillation. Lung homogenates from these mice demonstrated a significant reduction in MI&-laprotein in lung homogenates fmm 611 f 166 pg/animal (mean f SEM) (IgG) to 302 f 139 pg/animal (sTNPr) at 2 days and a reduction from 956 f 150 pg/animaJ to 706 f 147 pg/animal at 10 days post-bleomycin instillation. These results suggest MP-la production is mediated by TNFa.
HUMAN C3a ENHANCES TUMOR NECROSIS FACTOR, INTERLEUKIN1, AND INTERLEUKIN-6 PRODUCTION IN ENDOTOXIN-STIMULATED HUMAN PBMC. T. Takabayashi, C. Dinarello, N. Margolis, E. Vannier, B. Clark, J. Burke and J. Gelfand New England Medical Center, Boston, MA 02111 and Massachusetts General Hospital, Boston, MA 02114 Anaphylatoxins and cytokines have been implicated in the pathophysiology of sepsis. We therefore investigated the effect of purified human C3a on protein synthesis and gene expression of TNF, IL-l, and IL-6 in human PBMC. C3a by itself did not induce protein synthesis and gene expression for these cytokines. C3a at 1 to 10 yg/ml, however, significantly increased LPS-induced TNF production by 285-361%, IL-1R by 457.576%, and IL-6 by 285542%) compared with stimulation with LPS alone. Moreover, C3a at 5 pglml enhanced and sustained TNF, IL-113, and IL-5 MRNA levels in LPS-stimulated PBMC. These results suggest that C3a may enhance the sustain inflammatory effects on host responses to Gram-negative infections by up-regulating TNF, IL-lB, and IL-6 production in PBMC.
RECEPTORS AND SIGNALING MECHANISMS OF MONOCYTE CHEMOTACTIC PROTEIN(MCP)-1, MCP-2 AND MCP-3 IN HUMAN MONOCYTES AND NK CELLS. S. Sozzani, D. Zhou, G. Bianchi, M. Rieppi, W. Luini, G. Iamorte, P. Allavena and A. Mantovani Istituto di Ricerche Fannacologiche ‘Mario Negri’, Milan, Italy MCP-1, MCP-2 and MCP-3 are closely related proteins (60.70% homology) part of the C-C branch of the chemokine family. At chemotactic concentrations (50-100 @ml), MCP-1 and MCP-3 induced a rapid accumulation of [3H]arachidonic acid which was dependent on extracellular [Ca2+]i and important for chemotaxis. Amchidonate release was increased by pretreatment with PAF and was associated with the activation and translocation of cPLAz to the membrane fraction. On the contrary, MCP-2 did not induce appreciable change in [Ca2+]i nor activated PLAz. Pertussis toxin (PTox) blocked calcium fluxes, actin polymerization, PLA? activation and chemotaxis by MCP-1 and MCP-3 but it was ineffective in inhibiting MCP2 action. These results together with receptor binding studies suggest that MCP-1 and MCP-3 share a common receptor on monocytes while MCP-2 uses a specific receptor. MCP-1, MCP-2 and MCP-3 also were active in inducing NK cell (CD3., CD14. CD16+, CD56+) migration. The migration was higher in IL-Zactivated cells, required a positive chemoattractant gradient and was blocked by PTox. Similarly to monocytes, MCP-1 induced a concentration-dependent rise in [Cti+]i. Thus, NK cells represent a new imponanr cellular target for the action of C-C chemokines.
PULMONARY SURFACTANT SUPPRESSES PRODUCTION OF INFLAMMATORY MEDIATORS BY HUMAN ALVEOLAR MACROPHAGES (AMs) AND LUNG PARENCHYMAL CELLS. MJ Thomassen, JM Antal, LT Divis, DP Meeker and HP Wiedemann. Cleveland Cliic Foundation, Cleveland, OH 44195 In sepsis-related Adult Respiratory Distress Syndrome, pulmonary homeostasis is disrupted by numerous peptide and lipid inflammatory mediators. ‘Under such conditions surfactant abnormalities occur. We have reported that both synthetic surfactant (ExosurQ and the modified bovine surfactant (Survanta) inhibit endotoxin-stimulated cytokine (TNF,IL-l,IL-6) secretion from human AMs (Am J Respir Cell Mol Biol 10:399, 1994). In this study, we investigated whether lipid inflammatory mediators were also senstive to regulation by surfactant. The effect of snrfactant on PGEz secreted by endotoxin (LPS) stimulated AMs and IL-1 stimulated lung fibroblasts (CCD 18Lu) was evaluated and yielded the following results: LFs4m”lated AMS IL1 stimulated fibroblasts (ainhibition of PG& secretion) 62*11 46flO EXoSUti sulvanta 45f6 22+2 Both surfactant preparations suppress PGEz secretion from AMs and lung fibroblasts.
A68
A71
CYTOKINE,
P3 October 2: InJammation
Vol. 6, No. 5 (September
1994: 539-582)
and Cytokines
A66 SELECTIVE INDUCTION OF VASCULAR CELL ADHESION MOLECULE-l AND AMPLIFICATION OF IL-6 PRODUCTION BY IL-13 IN ENDOTHELIAL (EC) AND MESOTHELIAL (MC) CELLS. M. Sironi, F.L. Sciacca, C. Matteucci, M. Conni , A. Vecchi, A. Minty, D. Caput, P. Ferrara, F. Colotta, A. Mantovani. 1st. Ric. Farmacol. “Mario Negri”, Milano, Italy. In vitro exposure to IL-13 of vascular EC induced surface expression of VCAM-1. At optimal concentrations (lo-50 rig/ml) and exposure times (24h), IL-13 was a 2 to 3-fold less effective inducer of VCAM-1 than IL-l, used as reference EC activator. When IL-13 was combined with IL-l, almost additive induction of VCAM was observed. Induction of VCAM-1 by IL-1 3 was selective in that E-selectin and ICAMwere unaffected. IL-13 caused a modest reduction of IL-l induction of E-selectin and ICAM-I. Surface expression of VCAM-1 on IL-13-treated cells was associated with mRNA induction, with predominance of transcripts encoding the 7 lg domain form of this molecule. IL-13 inhibited cytokine production in monocytes but was a weak inducer and amplifier (in concert with IL-l) of IL-6 expression in EC. MC were stimulated to express VCAM-1 and IL-6 by IL-13. Thus, IL-13 elicits a spectrum of responses in EC remarkably similar to that of IL-4.
LYMPHOTOXIN-a REQUIRES HIGH EXPRESSION OF THE ~55.TNF RECEPTOR FOR EFFICIENT CELL SIGNALING A. SUNDAN, A: MEDVEDEV AND T. ESPEVIK. Institute for Cancer Research, MTS, Univ. of Trondheim, N-7005 Trondheim, Norway
AfiS . ---
Tumor Necrosis Factor-u (TNF) and Lymphotoxin-a (LT) are pleiotropiccytokines which both mediatetheir effects by binding to the two known TNF receptors, -the ~55 and the ~75 TNFR. These receptors are almost ubiquitously expressed on cells, and TNF and LT bind the receptors with comparable affinities. However, the expression of the two receptor types are independentlyregulated,and the ratio of the two receptors expressed on cells varies. Here we present experiments demonstrating that whereas some human ceils,i.e. the FS4 and WHEI ~1.13,are equally sensitive to TNF and LT, other human cells such as the KYM-1 and Sii480 cells were much more resistant towards LT than TNF. Bindingexperiments with iodinatedLT and TNF indicated that both cytokines were bound to the these cell lines in a similar manner with comparable association constants and number of binding sites. However, binding experiments in the presence of antibodies which block the binding to one or the other TNFR indicated that the Fs4 and WEHI cells expressed a high proportion of p55 compared to ~75 receptors, whereas the KYM-1 and SW480 cells expressed high numbers of ~75 compared to p55 TNFR. Cells expressmg a high proportion of ~75 TNFR therfore appears to be resistant to LT. The relative expression of the two TNFR may therfore be a way for the cells to discriminate between suceptibility towards TNF and LT. We also present data indicatingthat whereas TNF have a relatively high on-off rate in the Interactionwith the ~75 TNFR, the rate of dissociation of LT is much lower. The resistance towards LT of some cell linesexpressing a high proportion of the p75 TNFR may therfore be due fo a lack of ability to pass LT on from the ~75 to the ~55 TNFR, wheras such a mechanism may operate in the case 01TNF.
A67
A70 SOLUBLE TNF RECEPTOR MEDIATED REDUCTION OF MIP-la EXPRESSION IN BLEOMYCIN-INDUCED LUNG INJURY. R. E. Smith, S.H. Phan, R.M. Stricter, and S.L. Kunkel(Sponsor). Depts. of Pathology and Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, 48109-0602. Bleomycin-induced lung injury, a model of interstitial lung disease, is a T-cell dependent pulmonary inflammatory response characterized by an increase in leukocyte infiltration, fibroblast proliferation and collagen synthesis. Cytokines such as TNF are believed to have a central role in the emanation of the inflammatory response to bleomycin. Previously, we have demonstrated that intratracheal challenge of CBA/J mice with bleomycin resulted in a significant time-dependent increase in MIP-la protein levels both in whole lung homogenates and broncho-alveolar lavage fluid (BALF) with detectable protein peaking at days 2 and 12 post-bleomycin instillation. In addition, anti-MIP-la antibodies significantly reduced inflammatory cell content and fibrosis. We now demonstrate that treatment of bleomycin challenged mice with soluble TNF receptor (sTNFr) reduces MIP-la production (measured by ELISA) in the mouse lung. TNF levels (measured by bioassay) in BALF fluid were significantly elevated, compared to controls, 2. 8, and 16 days postbleomycin injection. CBA/J mice challenged with 0.025 units of bleomycin were injected intraperitoneally with 100 ug of IgG or sTNPr either at time of bleomycin instillation or at 6 days post-instillation. Lung homogenates from these mice demonstrated a significant reduction in MI&-laprotein in lung homogenates fmm 611 f 166 pg/animal (mean f SEM) (IgG) to 302 f 139 pg/animal (sTNPr) at 2 days and a reduction from 956 f 150 pg/animaJ to 706 f 147 pg/animal at 10 days post-bleomycin instillation. These results suggest MP-la production is mediated by TNFa.
HUMAN C3a ENHANCES TUMOR NECROSIS FACTOR, INTERLEUKIN1, AND INTERLEUKIN-6 PRODUCTION IN ENDOTOXIN-STIMULATED HUMAN PBMC. T. Takabayashi, C. Dinarello, N. Margolis, E. Vannier, B. Clark, J. Burke and J. Gelfand New England Medical Center, Boston, MA 02111 and Massachusetts General Hospital, Boston, MA 02114 Anaphylatoxins and cytokines have been implicated in the pathophysiology of sepsis. We therefore investigated the effect of purified human C3a on protein synthesis and gene expression of TNF, IL-l, and IL-6 in human PBMC. C3a by itself did not induce protein synthesis and gene expression for these cytokines. C3a at 1 to 10 yg/ml, however, significantly increased LPS-induced TNF production by 285-361%, IL-1R by 457.576%, and IL-6 by 285542%) compared with stimulation with LPS alone. Moreover, C3a at 5 pglml enhanced and sustained TNF, IL-113, and IL-5 MRNA levels in LPS-stimulated PBMC. These results suggest that C3a may enhance the sustain inflammatory effects on host responses to Gram-negative infections by up-regulating TNF, IL-lB, and IL-6 production in PBMC.
RECEPTORS AND SIGNALING MECHANISMS OF MONOCYTE CHEMOTACTIC PROTEIN(MCP)-1, MCP-2 AND MCP-3 IN HUMAN MONOCYTES AND NK CELLS. S. Sozzani, D. Zhou, G. Bianchi, M. Rieppi, W. Luini, G. Iamorte, P. Allavena and A. Mantovani Istituto di Ricerche Fannacologiche ‘Mario Negri’, Milan, Italy MCP-1, MCP-2 and MCP-3 are closely related proteins (60.70% homology) part of the C-C branch of the chemokine family. At chemotactic concentrations (50-100 @ml), MCP-1 and MCP-3 induced a rapid accumulation of [3H]arachidonic acid which was dependent on extracellular [Ca2+]i and important for chemotaxis. Amchidonate release was increased by pretreatment with PAF and was associated with the activation and translocation of cPLAz to the membrane fraction. On the contrary, MCP-2 did not induce appreciable change in [Ca2+]i nor activated PLAz. Pertussis toxin (PTox) blocked calcium fluxes, actin polymerization, PLA? activation and chemotaxis by MCP-1 and MCP-3 but it was ineffective in inhibiting MCP2 action. These results together with receptor binding studies suggest that MCP-1 and MCP-3 share a common receptor on monocytes while MCP-2 uses a specific receptor. MCP-1, MCP-2 and MCP-3 also were active in inducing NK cell (CD3., CD14. CD16+, CD56+) migration. The migration was higher in IL-Zactivated cells, required a positive chemoattractant gradient and was blocked by PTox. Similarly to monocytes, MCP-1 induced a concentration-dependent rise in [Cti+]i. Thus, NK cells represent a new imponanr cellular target for the action of C-C chemokines.
PULMONARY SURFACTANT SUPPRESSES PRODUCTION OF INFLAMMATORY MEDIATORS BY HUMAN ALVEOLAR MACROPHAGES (AMs) AND LUNG PARENCHYMAL CELLS. MJ Thomassen, JM Antal, LT Divis, DP Meeker and HP Wiedemann. Cleveland Cliic Foundation, Cleveland, OH 44195 In sepsis-related Adult Respiratory Distress Syndrome, pulmonary homeostasis is disrupted by numerous peptide and lipid inflammatory mediators. ‘Under such conditions surfactant abnormalities occur. We have reported that both synthetic surfactant (ExosurQ and the modified bovine surfactant (Survanta) inhibit endotoxin-stimulated cytokine (TNF,IL-l,IL-6) secretion from human AMs (Am J Respir Cell Mol Biol 10:399, 1994). In this study, we investigated whether lipid inflammatory mediators were also senstive to regulation by surfactant. The effect of snrfactant on PGEz secreted by endotoxin (LPS) stimulated AMs and IL-1 stimulated lung fibroblasts (CCD 18Lu) was evaluated and yielded the following results: LFs4m”lated AMS IL1 stimulated fibroblasts (ainhibition of PG& secretion) 62*11 46flO EXoSUti sulvanta 45f6 22+2 Both surfactant preparations suppress PGEz secretion from AMs and lung fibroblasts.
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A71