- Email: [email protected]
270 FK-506 inhibits IgE receptor mediated exocytosis from rat basophilic leukemia cells without affecting pi hydrolysis or calcium uptake
VOLUME NUMBER
269
S AND
Abstracts
87 1. PART 2
IS AN INHIBITOROF
271
ENDD
J. wi B.S.. 1,. BQ&& M.D..S. J0hns.o~. B.S.. M.S. I&Q, M.D, a. Coil. B.S.-and L.J. Ross N.D, Denver, CO. Human serum was found to contain an inhibitor of constitutive interleukin 1 (IL-l) production by human umbilical vein endothelial cells (EC). Purification of the serum activity by anion exchange molecular sieve HPLC, and chromatography, hydroxyl apatite chromatography yielded material 82% pure with a MW of 17 kD by SDS-PAGE. Amino acid sequencing revealed inhibitor the purified to be transthyretin (TTR), a liver-derived protein. Up to a 75% reduction in the production of spontaneous IL1 activity in EC supernatants occurred after co-culture with 10 pg/ml TTR. TTR was subsequently found by ELISA to inhibit LPS-stimulated IL-l production by cells of the human monocytic line THP-1 by leukemia 40.8+30.3%. Inhibition of IL-l secretion correlated with decreased IL-1 mRNA synthesis. inhibition of In addition, peripheral blood monocyte IL-l production (20.8%) and mRNA synthesis was also appreciated. This novel inhibitory effect of TTR on the production of EC IL1 activity suggests a previously unrecognized endogenous anti-inflammatory mediator.
270
R mm EXGCYTGSIS FROM RAT BASOPHILIC
WM
IN-ED LEUKEMIA
OUT AFFBCI’ING PI HYDROLYSIS OR UPTAKE. T. Hultscb IU.D .. M. Kaliner. M.D.,
Y. Fuiiro, M.D.. R. Nussenblatt. M.D.. and R. J. Hohman, PhD. Bethesda, Maryland FK-506 is a new immunosuppressive drug isolated from the fungus Streptomyces tsukubaenis. Even though FK-506 bears no structural resemblance to cyclosporin A (CSA), these two drugs have nearly identical immunosuppressive properties. Moreover, FK-506 has an identical spectrum of activity to CSA with respect to specific inhibition of cytokine transcription from Tlymphocytes. However, FK-506 is - 100 more potent than CSA both in vitro and in viva. As was previously shown for CSA (Hultsch et. al, Journal of Immunology; 144:26592664, 1990), FK-506 inhibits IgE receptor mediated exocytosis from rat basophilic leukemia (RBL) cells. m-506 is 100 times more tent than CSA at inhibiting activation of RBL cells (I J for CSA and FK-506 is 200 nM and 2 nM respectively), and neither drug inhibits IgE receptor mediated phosphatidyl-inositol hydrolysis or calcium uptake. Since both CSA and FK-506 belong to the same (new) class of enzyme inhibitors, peptidyl-prolyl cistrans isomerase inhibitors, and both drugs have similar immunosuppressive properties, it is likely that i) peptidyl prolyl cis-trans isomerascs are critically involved in the activation of mast cells as well as T-lymphocytes; ii) mast cells may play a role in graft rejection reaction; iii) both drugs may be potentially useful in the treatment of some mast cell mediated diseases.
207
ALLERGEN-INDUCED LATE-PH&E REACTION (LPIQ SITES CONTAIN IL-3 AND GM-CSF ACTIVITY. W, Massev. M. .Dj;, Bi, Fri&an. M.D.. M. Kate. M.D.. L,M, Lichtenstem. Ph.D. M. Liu. M.D.. R. Schleimet
Ph.D. Baltimore. MD. ’ Antigen (Ag) challenge in skin blister chambers and segmentalairway challenge lung models was performed to determine whether interleukin-3 (IL-3)/granulocytemonocyte colony stimulating factor (GM-CSF) activity is found at sites of allergic inflammation in vim Activity was quantitated using proliferation of the IL-3/GM-CSF responsive M-07 cell line. Results are expressed as 8 of maximal proliferation. Fluid from the Ag andvehicle control (C) challenged dermal sites was collected hourly for 12 hr from 9 patientswith allergicrhinitis. IL-3/GM-CSF activity was elevated at hr 4 (median Ag=4% vs C=2%, p
272 IL-8 lNlUBlTS HISTAMINERELEASE INDUCED BY HISTAMINE RELEASING FACT0RS (BRF-s), CoNNEcIlvE TXWJE ACINATlNG PEPTlDE m (CTAP-ill) AND IL3 FROMHUMANBASOPHUS. m Stony Bmok, New York. Ihe objective of this study was to investigatethe.influence of IL-8 oo histaminedease horn basophitsinducedby HRF-s. FaCtorsh%IedwgeaudemOnnuClearCGllsupanetnnt.alliOM excbaqe chromatogrephy(QMA) void (unbound)nmmiai from HRF ulxk. (HRF-1). pified CrAP-Hl and IL-3. Paipkd biocd leukocyte.SwereiL?&ted~2Odorors.dorors.ireleasedfrein kophiiswasmeasumdbymdkqmakassay.Wedetemkd thatIL8itself,at concen@tionsof1O-‘tolO-~~M.docsno1 releaseb&amine, (s+%). HRP CNderekased biiine. in 19 of 2Odonorsintherangcof6.7%to1OO%.Wbenkuphilswae preineuhated with IL-8 (10-7 to l&11 M) for 5 min. followed by a 40 min. inabioo wttb HRF, b&amine lelease was inhibited in
12af19daaofs.llmrsage.ofiahiiticmwssbZO%ta8O% dqeadmg on the donor. l-be.eoncentmtirmrangeof IL-8 showing inhibition was 10-7to 10-10M with 109 being tlx mostcommon. Cmle HRP is a mixture of many different factcas.To specify which factor(s) may be inhibited by L-8, HTtF-1, purifii CTAe-IUandL3werestndkd.Hiinedease.by RR&land CMP-BI(5@nl)wasiahibitedbylL-8ia9eutaf lOdonors, and 5 out of 10 donols lx!qeetive.ly. IL-3 (soa U/ml) released histamineio 3 out of 8 donas.. all wexeinbibii by IL-K For IL8thec~enaationandntngeofinhibitionwenthcsameas shownwithHRFaude.Bowever,wefoundhistamine.releaseby anti-I@3and F-Met-Lcu-Phe was not inhibited by IL-S. IL8 appearsto be.a spcclfic inhibitor of qtekiae like moleculeshaving todowithbiinereteasebutdoesnotdiseriminatehetween them.IflL-8inhibitsthehismmineteleaseinducedbytluae factorsinviw,~ismPybcanimpormnt~meckanismin tbe rekasabiity of l&mine containing cells. An imbalanceof theaet%orsmayplayaroleintbe@qene&efdiseascsin which histamineis an important mediamr.
269
S AND
Abstracts
87 1. PART 2
IS AN INHIBITOROF
271
ENDD
J. wi B.S.. 1,. BQ&& M.D..S. J0hns.o~. B.S.. M.S. I&Q, M.D, a. Coil. B.S.-and L.J. Ross N.D, Denver, CO. Human serum was found to contain an inhibitor of constitutive interleukin 1 (IL-l) production by human umbilical vein endothelial cells (EC). Purification of the serum activity by anion exchange molecular sieve HPLC, and chromatography, hydroxyl apatite chromatography yielded material 82% pure with a MW of 17 kD by SDS-PAGE. Amino acid sequencing revealed inhibitor the purified to be transthyretin (TTR), a liver-derived protein. Up to a 75% reduction in the production of spontaneous IL1 activity in EC supernatants occurred after co-culture with 10 pg/ml TTR. TTR was subsequently found by ELISA to inhibit LPS-stimulated IL-l production by cells of the human monocytic line THP-1 by leukemia 40.8+30.3%. Inhibition of IL-l secretion correlated with decreased IL-1 mRNA synthesis. inhibition of In addition, peripheral blood monocyte IL-l production (20.8%) and mRNA synthesis was also appreciated. This novel inhibitory effect of TTR on the production of EC IL1 activity suggests a previously unrecognized endogenous anti-inflammatory mediator.
270
R mm EXGCYTGSIS FROM RAT BASOPHILIC
WM
IN-ED LEUKEMIA
OUT AFFBCI’ING PI HYDROLYSIS OR UPTAKE. T. Hultscb IU.D .. M. Kaliner. M.D.,
Y. Fuiiro, M.D.. R. Nussenblatt. M.D.. and R. J. Hohman, PhD. Bethesda, Maryland FK-506 is a new immunosuppressive drug isolated from the fungus Streptomyces tsukubaenis. Even though FK-506 bears no structural resemblance to cyclosporin A (CSA), these two drugs have nearly identical immunosuppressive properties. Moreover, FK-506 has an identical spectrum of activity to CSA with respect to specific inhibition of cytokine transcription from Tlymphocytes. However, FK-506 is - 100 more potent than CSA both in vitro and in viva. As was previously shown for CSA (Hultsch et. al, Journal of Immunology; 144:26592664, 1990), FK-506 inhibits IgE receptor mediated exocytosis from rat basophilic leukemia (RBL) cells. m-506 is 100 times more tent than CSA at inhibiting activation of RBL cells (I J for CSA and FK-506 is 200 nM and 2 nM respectively), and neither drug inhibits IgE receptor mediated phosphatidyl-inositol hydrolysis or calcium uptake. Since both CSA and FK-506 belong to the same (new) class of enzyme inhibitors, peptidyl-prolyl cistrans isomerase inhibitors, and both drugs have similar immunosuppressive properties, it is likely that i) peptidyl prolyl cis-trans isomerascs are critically involved in the activation of mast cells as well as T-lymphocytes; ii) mast cells may play a role in graft rejection reaction; iii) both drugs may be potentially useful in the treatment of some mast cell mediated diseases.
207
ALLERGEN-INDUCED LATE-PH&E REACTION (LPIQ SITES CONTAIN IL-3 AND GM-CSF ACTIVITY. W, Massev. M. .Dj;, Bi, Fri&an. M.D.. M. Kate. M.D.. L,M, Lichtenstem. Ph.D. M. Liu. M.D.. R. Schleimet
Ph.D. Baltimore. MD. ’ Antigen (Ag) challenge in skin blister chambers and segmentalairway challenge lung models was performed to determine whether interleukin-3 (IL-3)/granulocytemonocyte colony stimulating factor (GM-CSF) activity is found at sites of allergic inflammation in vim Activity was quantitated using proliferation of the IL-3/GM-CSF responsive M-07 cell line. Results are expressed as 8 of maximal proliferation. Fluid from the Ag andvehicle control (C) challenged dermal sites was collected hourly for 12 hr from 9 patientswith allergicrhinitis. IL-3/GM-CSF activity was elevated at hr 4 (median Ag=4% vs C=2%, p
272 IL-8 lNlUBlTS HISTAMINERELEASE INDUCED BY HISTAMINE RELEASING FACT0RS (BRF-s), CoNNEcIlvE TXWJE ACINATlNG PEPTlDE m (CTAP-ill) AND IL3 FROMHUMANBASOPHUS. m Stony Bmok, New York. Ihe objective of this study was to investigatethe.influence of IL-8 oo histaminedease horn basophitsinducedby HRF-s. FaCtorsh%IedwgeaudemOnnuClearCGllsupanetnnt.alliOM excbaqe chromatogrephy(QMA) void (unbound)nmmiai from HRF ulxk. (HRF-1). pified CrAP-Hl and IL-3. Paipkd biocd leukocyte.SwereiL?&ted~2Odorors.dorors.ireleasedfrein kophiiswasmeasumdbymdkqmakassay.Wedetemkd thatIL8itself,at concen@tionsof1O-‘tolO-~~M.docsno1 releaseb&amine, (s+%). HRP CNderekased biiine. in 19 of 2Odonorsintherangcof6.7%to1OO%.Wbenkuphilswae preineuhated with IL-8 (10-7 to l&11 M) for 5 min. followed by a 40 min. inabioo wttb HRF, b&amine lelease was inhibited in
12af19daaofs.llmrsage.ofiahiiticmwssbZO%ta8O% dqeadmg on the donor. l-be.eoncentmtirmrangeof IL-8 showing inhibition was 10-7to 10-10M with 109 being tlx mostcommon. Cmle HRP is a mixture of many different factcas.To specify which factor(s) may be inhibited by L-8, HTtF-1, purifii CTAe-IUandL3werestndkd.Hiinedease.by RR&land CMP-BI(5@nl)wasiahibitedbylL-8ia9eutaf lOdonors, and 5 out of 10 donols lx!qeetive.ly. IL-3 (soa U/ml) released histamineio 3 out of 8 donas.. all wexeinbibii by IL-K For IL8thec~enaationandntngeofinhibitionwenthcsameas shownwithHRFaude.Bowever,wefoundhistamine.releaseby anti-I@3and F-Met-Lcu-Phe was not inhibited by IL-S. IL8 appearsto be.a spcclfic inhibitor of qtekiae like moleculeshaving todowithbiinereteasebutdoesnotdiseriminatehetween them.IflL-8inhibitsthehismmineteleaseinducedbytluae factorsinviw,~ismPybcanimpormnt~meckanismin tbe rekasabiity of l&mine containing cells. An imbalanceof theaet%orsmayplayaroleintbe@qene&efdiseascsin which histamineis an important mediamr.