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Effect of cyclooxygenase inhibition on tumor necrosis factor induced lung injury in awake sheep
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RELEASE OF ACTIVE TGF,Tl FROM KUPFFER CELLS ISOLATED FROM RATS WITH ALCOHOLIC LIVER FIBROSIS: A POSSIBLE PARACRINE MECHANISM OF LIVER FIBROGENESIS. g. ~-Tsukamoto and M. Matsuoka. Hepatooancreatic Res Lab. VA Med Ctr. Martinez. CA and Deut of internal Med, UC Davis School of Med, CA 94553 TGFfll stimulates collagen production of hepatic lipocytes, perisinusoidal cells believed to be involved in production of extracellular matrices in the liver. Kupffer cells, resident macrophages in the liver are known to release inflammatory mediators. The present study examined release of TGFfll by Kupffer cells isolated from a rat model of alcoholic liver fibrosis (ALF) in order to explore a possible paracrine effect The Kupffer cell-conditioned medium on hepatic lipocytes. (KCM) from rats with ALF but not that from pair-fed controls contained the active TGF@l (1 pg/ml) as assessed by stimulation of hepatic lipocyte collagen production and neutralization of this effect with specific anti-TGFBl (from Dr. Jim The KCM from the rats with ALF Dasch, Collagen Corporation). also contained the large amount of the latent TGFfll (lo-100 Northern blotting anq2hybridization of Kupffer cell pg/ml). mRNA from these animals with P-labeled TGFBl clearly demonstrated the presence of the expected 2.5kb mRNA for this cytokine These results demonstrate expression and release of TGF,91 by Kupffer cells and suggest the possibility that the active TGF,91 from Kupffer cells may provide the paracrine stimulatory effect on hepatic lipocytes during the active liver fibrogenesis.
368 TRANSFECTANTS RESF’ONSIVENBSS.
DIFFERENTIAL
HOST
RESPONSES
TO REPEATED
LPS ADMINISTRA-
TIONCORRELATES TO PLASMA TNF AND IL-6 LEVELS H Wei. Y Fona, MA Marano. JE Gershenwald. LL Moldawer &SF Lowry Lab. of Surg. Metab., Cornell U. Med. Coil., NY, NY 10021. Administration of liootmlvsaccaride (LPS) results in the induction of an acute phase response, mediated principally thiough the release of cytokines.
However,
repeated exposure to LPS attenuates many of these host responses. The
oresent studv examined whether various comoonents of this acute-ohase resnonse ire uniformly observed, concurrently with &ered TNF or IL-6 ievels. Fkmale Wistar rats (150-200 g) received daily ip injections of E. co/i 0127:88 LPS according to the following schedule: 0.1 mg/kg (day]), 0.5 mg/kg (day2). I mg/kg (day3). 5 mg/kg (days4-14). Food intake and body weight were measured daily. Several hematologic parameters and cytokines were measured on days 1, 5 and 14. Sham-treated rats received saline injections. Peak TNF and IL-6 levels were measured 90 mins and 4 hrs after LPS,.respectively. Albumin Fibrinogen WBC IL-6 TNF Dav fa/ ) Wf ) Hct (x103) 3.5,?8 0.53& 45146 Ill8 0.49f3.07’ 10/19* 71/241 315 I4 l 12130’ 86/132 0.69/0.76 I/I Sham-treated/LPS-rreated * pqO.05 vs sham Food intake and body weight gain declined after the first LPS injection, but returned to baseline after nine days despite continued LPS administration. PostLPS plasma IL-6 and TNF responses declined markedly after the first LPS injection. Plasma fibrinogen levels also returned to normal. However, plasma albumin, HCT and WBC remained abnormal despite restoration of pre-LPS BW gain and food intake, and attenuated cytokine responses. Despite declining TNF and IL-6 levels following repeated LPS administration, the presence of persistent hypoalbuminemia and hematologic changes (anemia and leukocytosis) suggests that these responses may not be entirely TNF- or IL-6-mediated.
371 SECRETING mck.
LATENT
TGF+l
SUPPRESS IMMUNE L. Wa&&Ld +, M. Swrn+. A.
I. Fm. d M. PaGenentech, Inc., So. San Francisco, CA 94080 and + National Cancer Institute, Bethesda, MD 20892. Although several studies have provided insight into the immunoregulatory properties of active TGF-PI, similar studies which examine the primary product of TGF-pt synthesizing cells, that is, latent TGF-PI, have not been performed. In order to characterize this TGF-PI precursor, we have prepared and analyzed transfectant CHO cells which synthesize latent TGF-PI and demonstrate the immunosuppressive properties of TGF-p1 under physiological conditions. We report that transfected CHO cells secreting latent TGF-PI (CHOTTGF+l) inhibit cytotoxic T lymphocyte (CTL) generation against parental CHO targets in vifro, presumably via conversion of latent TGF-Pl to its active form. CTLs generated against parental CHO cells showed only slightly depressed ability to lyse CHO/TGF-Pl targets compared to parental CHO targets suggesting that Class I surface expression was maintained on the CHO cells after transfection. Moreover, nude mice injected with the CHO/TGF!31 cells but not untreated conuol animals show significantly depressed NK activity against YAC-1 target cells. Animals which received the CHO/TGF-fil cells showed elevated serum levels of TGF-P1 above those of normal mice and which when activated by acidification inhibit in ‘IGFp bioassays. In general, the expression and synthesis of TGF-Pt in vivo rendered the transfectant CHO cells tumorigenic; an observation which requires further inquiry. Currently we are testing the effects of exogenously added purified recombinant latent TGF-P1 on CTL generation in vitro.
EFFECT FACTOR
OF CYCLOOXYGENASE INHIBITION ON TUMOR NECROSIS INDUCED LUNG INJURY IN AWAKE SHEEP. A.P. Wheeler,
W.D. Hardie. K.L. Brigham. G.R. Bernard. Center for Lung Research, Vanderbilt Univ. School of Medicine, Nashville, TN 37232. In awake, chronically instrumented sheep, human recombinant tumor necrosis factor alpha (TNFa) causes pulmonary hypertension, hypoxemia, leukopenia, decreased dynamic compliance (Cdyn), increased resistance to airflow across the lung (RL), exudation of protein-rich lung lymph and the release of prostanoid constrictors thromboxane and PGEa. To assess the effect of cyclooxygenase inhibition on TNFa-induced lung injury, 8 sheep received ibuprofen (IBU) (14 mg/kg) 1 hour before TNFa (IOpg/kg) or TNFo: alone in random order, paired experiments. Pulmonary artery pressure (Pp.), lung mechanics, lymph flow (QL) and arachidonic acid metabolites in lymph were measured. Alone, TNFa caused a 35% reduction in Cdyn and a 400% increase in RL within I5 minutes of initiating the infusion. IBU delayed the fall in Cdyn and completely prevented increases in RL. Within 30 minutes of starting the TNFu infusion, AaPOa increased by 50% in TNFa controls, but was unchanged in the IBU group. In TNFa controls, Ppa doubled and QL tripled within I hour of initiating the TNFa! and remained elevated for 4 hours. IBU prevented early increases in QL and Pp., but not increases in QL and Ppa beyond I hour after initiating TNFa. The peripheral leukocyte count declined by 65% in both groups, reaching a nadir 30 minutes after starting the TNFor infusion. Increases in Iuna lvmoh thromboxane. nrostacvclin and PGEz were prevented by pre-treatmeit with IBU. We &cludethat the increases in RL and AaPOa, initial elevations in Ppa and QL, (but not leukooenial. followine TNFa are mediated. at least in Dart. bv cyclo&yge&se products, and can be preventeh with IBU pre-ireatmeni. Supported by: Am Heart Assoc Student Res., HL07123, HLI9153, HL27274, and Cetus and Upjohn Corporations.
369
312
HISTOPATHOLOGICAL OBSERVATIONS OF A RAT GLIOMA INJECTION OF HUMAN FOLLOWING AN INTRALESIONAL RECOMBINANT INTERLEUKIN-2 (rIL2). R.G. Watts and R.E. Merchant. Medical College of Virginia, Richmond, VA 23298. Our previous studies indicated that a single intracerebral (IC) injection of 6 x 104U rIL2 (Cetus) into normal rat brain increases vascular permeability at the injection site that was sustained over 8 days. The present study examined vessels in and around a primary brain tumor following an intralesional injection of rIL2. Fischer rats received an IC injection of 104 RT-2 glioma cells (mean survival 17 days) into the right parietal cortex. At days 7 and 10, tumor-bearing animals had blood-brain barrier (BBB) disruption around the tumor; evidenced by extravasation of endogenous IgG. There was necrosis, neovascularization, hemorrhage and leukocytic infiltration within the tumor. Glioma-bearing rats were injected intralesionally with 6 x 1dU rIL-2 7 days after tumor inoculation and sacrificed 3, 4, or 6 days later. Within the tumor there was no apparent histologic differences from untreated glioma, however, animals injected with the rIL2 showed an increased IgG extravasation that extended beyond the tumor site and into the contralateral cortex and white matter. Because malignant glioma is a pervasive disease whereby tumor cells invade the entire brain, an IC injection of rIL2 could prove to be clinically useful for increasing cerebrovascular permeability and delivery of chemotherapeutic agents into areas of the brain which have a normally functioning BBB.
MULTIPLE INTRAPERITONEAL INJECTIONS OF INTERLEUKIN-16 (IL-l) INDUCE INHIBITION a INSULIN RELEASE FROn THE PERFUSED RAT PANCREAS. L. Wogensen, J. Nerup, S. Helqvist, F. Pociot, J. Johannesen and T. Mandrup-roulsen. Steno Memorial Hospital, Gentofte, Denmark. Interleukin-18 stimulates insulin and glucagon release from the perfused pancreas and induces B cell specific ultramorphological changes. The aim of this study was to test whether pretreatment in vivo with daily injections of IL-l (4ug/kg) (N=17) or NaCl (N= 13) for 5 days affected insulin and glucagon release from the intact pancreas. On the 5th day, pancreata were isolated and perfused with 11 (O-72 min) and 20 mM D-glucose (72-84 min). Saline or IL-l (15ng/ml) was added fror,~ 12 to 72 min. Glands isolated from rats pretreated with IL-l showed a significant decrease in insulin release from O-72 min (p
/ SECOND
INTERNATIONAL
WORKSHOP
ON
CYTOKINES
367
370
RELEASE OF ACTIVE TGF,Tl FROM KUPFFER CELLS ISOLATED FROM RATS WITH ALCOHOLIC LIVER FIBROSIS: A POSSIBLE PARACRINE MECHANISM OF LIVER FIBROGENESIS. g. ~-Tsukamoto and M. Matsuoka. Hepatooancreatic Res Lab. VA Med Ctr. Martinez. CA and Deut of internal Med, UC Davis School of Med, CA 94553 TGFfll stimulates collagen production of hepatic lipocytes, perisinusoidal cells believed to be involved in production of extracellular matrices in the liver. Kupffer cells, resident macrophages in the liver are known to release inflammatory mediators. The present study examined release of TGFfll by Kupffer cells isolated from a rat model of alcoholic liver fibrosis (ALF) in order to explore a possible paracrine effect The Kupffer cell-conditioned medium on hepatic lipocytes. (KCM) from rats with ALF but not that from pair-fed controls contained the active TGF@l (1 pg/ml) as assessed by stimulation of hepatic lipocyte collagen production and neutralization of this effect with specific anti-TGFBl (from Dr. Jim The KCM from the rats with ALF Dasch, Collagen Corporation). also contained the large amount of the latent TGFfll (lo-100 Northern blotting anq2hybridization of Kupffer cell pg/ml). mRNA from these animals with P-labeled TGFBl clearly demonstrated the presence of the expected 2.5kb mRNA for this cytokine These results demonstrate expression and release of TGF,91 by Kupffer cells and suggest the possibility that the active TGF,91 from Kupffer cells may provide the paracrine stimulatory effect on hepatic lipocytes during the active liver fibrogenesis.
368 TRANSFECTANTS RESF’ONSIVENBSS.
DIFFERENTIAL
HOST
RESPONSES
TO REPEATED
LPS ADMINISTRA-
TIONCORRELATES TO PLASMA TNF AND IL-6 LEVELS H Wei. Y Fona, MA Marano. JE Gershenwald. LL Moldawer &SF Lowry Lab. of Surg. Metab., Cornell U. Med. Coil., NY, NY 10021. Administration of liootmlvsaccaride (LPS) results in the induction of an acute phase response, mediated principally thiough the release of cytokines.
However,
repeated exposure to LPS attenuates many of these host responses. The
oresent studv examined whether various comoonents of this acute-ohase resnonse ire uniformly observed, concurrently with &ered TNF or IL-6 ievels. Fkmale Wistar rats (150-200 g) received daily ip injections of E. co/i 0127:88 LPS according to the following schedule: 0.1 mg/kg (day]), 0.5 mg/kg (day2). I mg/kg (day3). 5 mg/kg (days4-14). Food intake and body weight were measured daily. Several hematologic parameters and cytokines were measured on days 1, 5 and 14. Sham-treated rats received saline injections. Peak TNF and IL-6 levels were measured 90 mins and 4 hrs after LPS,.respectively. Albumin Fibrinogen WBC IL-6 TNF Dav fa/ ) Wf ) Hct (x103) 3.5,?8 0.53& 45146 Ill8 0.49f3.07’ 10/19* 71/241 315 I4 l 12130’ 86/132 0.69/0.76 I/I Sham-treated/LPS-rreated * pqO.05 vs sham Food intake and body weight gain declined after the first LPS injection, but returned to baseline after nine days despite continued LPS administration. PostLPS plasma IL-6 and TNF responses declined markedly after the first LPS injection. Plasma fibrinogen levels also returned to normal. However, plasma albumin, HCT and WBC remained abnormal despite restoration of pre-LPS BW gain and food intake, and attenuated cytokine responses. Despite declining TNF and IL-6 levels following repeated LPS administration, the presence of persistent hypoalbuminemia and hematologic changes (anemia and leukocytosis) suggests that these responses may not be entirely TNF- or IL-6-mediated.
371 SECRETING mck.
LATENT
TGF+l
SUPPRESS IMMUNE L. Wa&&Ld +, M. Swrn+. A.
I. Fm. d M. PaGenentech, Inc., So. San Francisco, CA 94080 and + National Cancer Institute, Bethesda, MD 20892. Although several studies have provided insight into the immunoregulatory properties of active TGF-PI, similar studies which examine the primary product of TGF-pt synthesizing cells, that is, latent TGF-PI, have not been performed. In order to characterize this TGF-PI precursor, we have prepared and analyzed transfectant CHO cells which synthesize latent TGF-PI and demonstrate the immunosuppressive properties of TGF-p1 under physiological conditions. We report that transfected CHO cells secreting latent TGF-PI (CHOTTGF+l) inhibit cytotoxic T lymphocyte (CTL) generation against parental CHO targets in vifro, presumably via conversion of latent TGF-Pl to its active form. CTLs generated against parental CHO cells showed only slightly depressed ability to lyse CHO/TGF-Pl targets compared to parental CHO targets suggesting that Class I surface expression was maintained on the CHO cells after transfection. Moreover, nude mice injected with the CHO/TGF!31 cells but not untreated conuol animals show significantly depressed NK activity against YAC-1 target cells. Animals which received the CHO/TGF-fil cells showed elevated serum levels of TGF-P1 above those of normal mice and which when activated by acidification inhibit in ‘IGFp bioassays. In general, the expression and synthesis of TGF-Pt in vivo rendered the transfectant CHO cells tumorigenic; an observation which requires further inquiry. Currently we are testing the effects of exogenously added purified recombinant latent TGF-P1 on CTL generation in vitro.
EFFECT FACTOR
OF CYCLOOXYGENASE INHIBITION ON TUMOR NECROSIS INDUCED LUNG INJURY IN AWAKE SHEEP. A.P. Wheeler,
W.D. Hardie. K.L. Brigham. G.R. Bernard. Center for Lung Research, Vanderbilt Univ. School of Medicine, Nashville, TN 37232. In awake, chronically instrumented sheep, human recombinant tumor necrosis factor alpha (TNFa) causes pulmonary hypertension, hypoxemia, leukopenia, decreased dynamic compliance (Cdyn), increased resistance to airflow across the lung (RL), exudation of protein-rich lung lymph and the release of prostanoid constrictors thromboxane and PGEa. To assess the effect of cyclooxygenase inhibition on TNFa-induced lung injury, 8 sheep received ibuprofen (IBU) (14 mg/kg) 1 hour before TNFa (IOpg/kg) or TNFo: alone in random order, paired experiments. Pulmonary artery pressure (Pp.), lung mechanics, lymph flow (QL) and arachidonic acid metabolites in lymph were measured. Alone, TNFa caused a 35% reduction in Cdyn and a 400% increase in RL within I5 minutes of initiating the infusion. IBU delayed the fall in Cdyn and completely prevented increases in RL. Within 30 minutes of starting the TNFu infusion, AaPOa increased by 50% in TNFa controls, but was unchanged in the IBU group. In TNFa controls, Ppa doubled and QL tripled within I hour of initiating the TNFa! and remained elevated for 4 hours. IBU prevented early increases in QL and Pp., but not increases in QL and Ppa beyond I hour after initiating TNFa. The peripheral leukocyte count declined by 65% in both groups, reaching a nadir 30 minutes after starting the TNFor infusion. Increases in Iuna lvmoh thromboxane. nrostacvclin and PGEz were prevented by pre-treatmeit with IBU. We &cludethat the increases in RL and AaPOa, initial elevations in Ppa and QL, (but not leukooenial. followine TNFa are mediated. at least in Dart. bv cyclo&yge&se products, and can be preventeh with IBU pre-ireatmeni. Supported by: Am Heart Assoc Student Res., HL07123, HLI9153, HL27274, and Cetus and Upjohn Corporations.
369
312
HISTOPATHOLOGICAL OBSERVATIONS OF A RAT GLIOMA INJECTION OF HUMAN FOLLOWING AN INTRALESIONAL RECOMBINANT INTERLEUKIN-2 (rIL2). R.G. Watts and R.E. Merchant. Medical College of Virginia, Richmond, VA 23298. Our previous studies indicated that a single intracerebral (IC) injection of 6 x 104U rIL2 (Cetus) into normal rat brain increases vascular permeability at the injection site that was sustained over 8 days. The present study examined vessels in and around a primary brain tumor following an intralesional injection of rIL2. Fischer rats received an IC injection of 104 RT-2 glioma cells (mean survival 17 days) into the right parietal cortex. At days 7 and 10, tumor-bearing animals had blood-brain barrier (BBB) disruption around the tumor; evidenced by extravasation of endogenous IgG. There was necrosis, neovascularization, hemorrhage and leukocytic infiltration within the tumor. Glioma-bearing rats were injected intralesionally with 6 x 1dU rIL-2 7 days after tumor inoculation and sacrificed 3, 4, or 6 days later. Within the tumor there was no apparent histologic differences from untreated glioma, however, animals injected with the rIL2 showed an increased IgG extravasation that extended beyond the tumor site and into the contralateral cortex and white matter. Because malignant glioma is a pervasive disease whereby tumor cells invade the entire brain, an IC injection of rIL2 could prove to be clinically useful for increasing cerebrovascular permeability and delivery of chemotherapeutic agents into areas of the brain which have a normally functioning BBB.
MULTIPLE INTRAPERITONEAL INJECTIONS OF INTERLEUKIN-16 (IL-l) INDUCE INHIBITION a INSULIN RELEASE FROn THE PERFUSED RAT PANCREAS. L. Wogensen, J. Nerup, S. Helqvist, F. Pociot, J. Johannesen and T. Mandrup-roulsen. Steno Memorial Hospital, Gentofte, Denmark. Interleukin-18 stimulates insulin and glucagon release from the perfused pancreas and induces B cell specific ultramorphological changes. The aim of this study was to test whether pretreatment in vivo with daily injections of IL-l (4ug/kg) (N=17) or NaCl (N= 13) for 5 days affected insulin and glucagon release from the intact pancreas. On the 5th day, pancreata were isolated and perfused with 11 (O-72 min) and 20 mM D-glucose (72-84 min). Saline or IL-l (15ng/ml) was added fror,~ 12 to 72 min. Glands isolated from rats pretreated with IL-l showed a significant decrease in insulin release from O-72 min (p