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77 Evaluation of platelet activating factor (PAF) metabolism after nasal allergen challenge
J. ALLERGY
158 Abstracts
pen.7;~-l”sj scores.Xk?d la
“~sJIIuluuIIs” “y “‘w(5 v1 sy*p”lw e wasperkned before( pre-WC) apd 10 and 3ominutesafter I& , . -~tbelavageswercassayedforAHactivirv. ?&al fluids fron 8 ‘e~~Bwereas!iayedfor PAFandirsnon-a&e metaboliteIyso-P.r .T.4ME+&eraseactivity (TA!!yIE)andprorkns. were deterrmnedasindi~ararof vascularpennetity. In addition -laodinDz !PG&) wasaJJIyedasindicator of mastcm and ~ho~olipase & ( PL& ) acktion The baselinevalues ( preWC) for .&I. TXvfE , PGD2and tot& symptoms were( ti SE) 32.6-+2.5pmol~mf13Ormn.l6+0.3cpmx1~.97.6,+2.1 pgml. aadO.93fl.3rqectively. Graqolienchalieogeresulredin values( at 10‘) of 1% t 28 pmoi i mi I 30 mio ( p c 0.01) , 6.72 0.9cpmr10J~p~0.001).375.8+69.5pgrml(p~0.001) ,aad 7.42 3.2(0< O.OOl)fortbeseoeramsefs.A&ificamea~~Uel haease of @oteinWasobsewed: .I dilar in& of &-PAL: wasobsen~edarlOmin~44.3vs193.7np;~mf.r,: O.Ol).pAFwaa detectedin pre-WC lavagefromXl pa&& a& in post&C Iav e fmm 3/Spatients( mean0.8 vs 2.5 ngktl. XX) At 30 min alI 3 e valuesdecreasedbut remainedover the pre+iPC v&es. PM was not detectedY 30 min. PosGX?C.#I aad Ivso-PAF values were correIatedCr = 0.84. pc 0 001). .ti leveis’were Mated wirh T.LLIEandpr~reinl&ls indi&g apiasmaticinflux of AH in the nose.Wecoockderbattire innaaalfluidimkatetbat Theabsence of PAFmsgenaqid inaaivation of thismediatorand raisedtiequestionofksmodeofaction.
78 RR8OLUTiCtN OF THE B-ADRENOCEPTOR @AR) RNXJLATLNGAClWITYINAiWulwGCtiXS#L~CONURRIvEDpfoM~~Im DITlONED-fLCI@
.
.
M.WS.O. . ~ Stern and Kunos (J. Biol. Chem. 1988) by co-culturing A549 human lung cells with IM9 human lymphoid cells showed a 3- to S-fold increase in the density of BAR in A549 cells. We confirmed these findings (Szentivanyi et aL, Cytokine 1989) except that a) ZfpLC yielded 4 distinct fractions (activity peaks) instead of the reported 3; b) Peaks II and III showed BAR upregulating activity; and c) Peak I yielded a significant &AR downregulating effect. These differences may reflect the higher resolution capacity of ion exchange HPLC (Robicsek et aL 1989,199O)over the reported gel permeation HPLC The elution profile of the DAR regulating activity of LCM of IM9 cells by DEAE ion exchange HPLC was developed by incubating the LCM with A549 cells as adrenergic effector cells for 24 hrs. The activity peaks were distinguished by: a) retention time on ion exchange column; b) ability to significantly increase or decrease BAR density relative to that of untreated A549 cells; and c) immunoneutralization of the A549 BAR upregulating or downregulating effect of LCM peaks by antibodies to lymphokines. BAR densities were measured by pH]dihydroalprenolol. Activity Peaks II and III showed a 311 and 196% increase, whereas Peak I showed a 59% decrease in OAR concentrations of untreated A549 cells (100%).
CL!N IMMbNiiL .iANUARY 1997
&' t COPPER INHIBITS ANTI-IcrEAND HISTAMINE A23187-INDUEED IONOPHORE RELEASE.(H.R.) FROM HUMAN BASOPHILS. & Tedeschi, M.D., M. Lorini, M.Tech., A. A~-$&f+ _ Miadonna, M.D., Milano, Italy Aim of the study was to evaluate the from human copper on H.R. effect of with anti--igE stimulated basophils (l/5000) and Ca2+ ionophore A23187 (5 suspensions were prepared PM). Leukocyte sedimentation of peripheral by dextran and incubated wit:? venous blood (n=30), c&SO, and concentrations of different measured by a H.R. was CUCl, cu2A salts Both fluorimetric method. k r! a dosesianificantlv reduced H.R. related fashion. The concentration which inhibited 50% (IC50) of anti-fgE-induced H.R. was 0.5 uM for CuSO, and i.5 UM for of 6a2+ ionophoreThe ' IC30 CUCl,. was 4 uM for both CUSQ, induced H.R. effect on and CUCl, I The inhibitory anti--IgE-induced H.R. did not change appreciably when the cells were washed after incubation with CuZ+, whereas the H.R. effect on Cal+ ionophore-induced was no more observed after celi washing. Increased extracellular CaCl, concentraof CUCl, on the effect reduced tions but Schild plot anti-IgE-induced H.R., that Cu2+ 1.5 not a revealed analysis Cap+. inhibitor of competitive D,O, and enmicrotubules which aggregates antagonized Cuzi activity. hances H.R., Cu%+ i.5 that indicate results These hibits H.R. from human basophils
80 EFFECT OF Na+,K+ INHIBITION ON LEUKOCYTE BISTAHIKE RELEASE (LHR) .
P. Gs~&.&&e. B.S.. E. w, skoner M.D, Pittsburgh,
and D. Pennsvlvania
Pre&ous studies ha&shown that a Na”,K+ ATPase inhibitor is present in plasBna of allergic subjects, in whom enhanced LHR was reported. The purpose of th$s ftudy was to determine the effect of
Na ,K ATPase inhibition on LHB. Leukocytes obtained from 12 allergic rhinitis (ALL) and 5 non-allerqic /NON-ALL) subjects were incubated for j0 minut& with anti-IgE antibody (100 us/ml) or control following a 0,10,2ti,30,6~,126 and 180 minute preincubation with 3 mM ouabain or with control buffer. Cell supernatants were assayed for histamine by radioimmunoassay and expressed as a % of total LH. Mean (+ 1SEM) anti-IgE induced LHR, in the
presence
and absence
of ouabain,
respectively, for ALL subiects was 32.2 i 6 and 24.1 15 (10 min), 35.1 + 6 and 24.7 f 4 (20 min), 34.6 + 6 and 27.3 + 4 (30 min), 40.8 + 8 and 32.2 It: 7 (60 min), 57.1 + 13 and 30.6 + 6 (120 min, ~~0.05)~ and 42.7 + 6 and 36.5 + 5 (180 min). Spontaneous UiR in ALL subjects was also increased by a 60 minute preincubation with ouabain (2.2 + 0.5 vs 1.0 k 0.2 for control, pcO.05). Ouabain did Got significantly alter LHR in NON-ALL subjects. These data show that ouabain induced a significant increase in both spontaneous an? i?duced LHR in ALL subjects. In vivo Na ,K ATPase inhibition in ALL may have significant effects on LHR.
158 Abstracts
pen.7;~-l”sj scores.Xk?d la
“~sJIIuluuIIs” “y “‘w(5 v1 sy*p”lw e wasperkned before( pre-WC) apd 10 and 3ominutesafter I& , . -~tbelavageswercassayedforAHactivirv. ?&al fluids fron 8 ‘e~~Bwereas!iayedfor PAFandirsnon-a&e metaboliteIyso-P.r .T.4ME+&eraseactivity (TA!!yIE)andprorkns. were deterrmnedasindi~ararof vascularpennetity. In addition -laodinDz !PG&) wasaJJIyedasindicator of mastcm and ~ho~olipase & ( PL& ) acktion The baselinevalues ( preWC) for .&I. TXvfE , PGD2and tot& symptoms were( ti SE) 32.6-+2.5pmol~mf13Ormn.l6+0.3cpmx1~.97.6,+2.1 pgml. aadO.93fl.3rqectively. Graqolienchalieogeresulredin values( at 10‘) of 1% t 28 pmoi i mi I 30 mio ( p c 0.01) , 6.72 0.9cpmr10J~p~0.001).375.8+69.5pgrml(p~0.001) ,aad 7.42 3.2(0< O.OOl)fortbeseoeramsefs.A&ificamea~~Uel haease of @oteinWasobsewed: .I dilar in& of &-PAL: wasobsen~edarlOmin~44.3vs193.7np;~mf.r,: O.Ol).pAFwaa detectedin pre-WC lavagefromXl pa&& a& in post&C Iav e fmm 3/Spatients( mean0.8 vs 2.5 ngktl. XX) At 30 min alI 3 e valuesdecreasedbut remainedover the pre+iPC v&es. PM was not detectedY 30 min. PosGX?C.#I aad Ivso-PAF values were correIatedCr = 0.84. pc 0 001). .ti leveis’were Mated wirh T.LLIEandpr~reinl&ls indi&g apiasmaticinflux of AH in the nose.Wecoockderbattire innaaalfluidimkatetbat Theabsence of PAFmsgenaqid inaaivation of thismediatorand raisedtiequestionofksmodeofaction.
78 RR8OLUTiCtN OF THE B-ADRENOCEPTOR @AR) RNXJLATLNGAClWITYINAiWulwGCtiXS#L~CONURRIvEDpfoM~~Im DITlONED-fLCI@
.
.
M.WS.O. . ~ Stern and Kunos (J. Biol. Chem. 1988) by co-culturing A549 human lung cells with IM9 human lymphoid cells showed a 3- to S-fold increase in the density of BAR in A549 cells. We confirmed these findings (Szentivanyi et aL, Cytokine 1989) except that a) ZfpLC yielded 4 distinct fractions (activity peaks) instead of the reported 3; b) Peaks II and III showed BAR upregulating activity; and c) Peak I yielded a significant &AR downregulating effect. These differences may reflect the higher resolution capacity of ion exchange HPLC (Robicsek et aL 1989,199O)over the reported gel permeation HPLC The elution profile of the DAR regulating activity of LCM of IM9 cells by DEAE ion exchange HPLC was developed by incubating the LCM with A549 cells as adrenergic effector cells for 24 hrs. The activity peaks were distinguished by: a) retention time on ion exchange column; b) ability to significantly increase or decrease BAR density relative to that of untreated A549 cells; and c) immunoneutralization of the A549 BAR upregulating or downregulating effect of LCM peaks by antibodies to lymphokines. BAR densities were measured by pH]dihydroalprenolol. Activity Peaks II and III showed a 311 and 196% increase, whereas Peak I showed a 59% decrease in OAR concentrations of untreated A549 cells (100%).
CL!N IMMbNiiL .iANUARY 1997
&' t COPPER INHIBITS ANTI-IcrEAND HISTAMINE A23187-INDUEED IONOPHORE RELEASE.(H.R.) FROM HUMAN BASOPHILS. & Tedeschi, M.D., M. Lorini, M.Tech., A. A~-$&f+ _ Miadonna, M.D., Milano, Italy Aim of the study was to evaluate the from human copper on H.R. effect of with anti--igE stimulated basophils (l/5000) and Ca2+ ionophore A23187 (5 suspensions were prepared PM). Leukocyte sedimentation of peripheral by dextran and incubated wit:? venous blood (n=30), c&SO, and concentrations of different measured by a H.R. was CUCl, cu2A salts Both fluorimetric method. k r! a dosesianificantlv reduced H.R. related fashion. The concentration which inhibited 50% (IC50) of anti-fgE-induced H.R. was 0.5 uM for CuSO, and i.5 UM for of 6a2+ ionophoreThe ' IC30 CUCl,. was 4 uM for both CUSQ, induced H.R. effect on and CUCl, I The inhibitory anti--IgE-induced H.R. did not change appreciably when the cells were washed after incubation with CuZ+, whereas the H.R. effect on Cal+ ionophore-induced was no more observed after celi washing. Increased extracellular CaCl, concentraof CUCl, on the effect reduced tions but Schild plot anti-IgE-induced H.R., that Cu2+ 1.5 not a revealed analysis Cap+. inhibitor of competitive D,O, and enmicrotubules which aggregates antagonized Cuzi activity. hances H.R., Cu%+ i.5 that indicate results These hibits H.R. from human basophils
80 EFFECT OF Na+,K+ INHIBITION ON LEUKOCYTE BISTAHIKE RELEASE (LHR) .
P. Gs~&.&&e. B.S.. E. w, skoner M.D, Pittsburgh,
and D. Pennsvlvania
Pre&ous studies ha&shown that a Na”,K+ ATPase inhibitor is present in plasBna of allergic subjects, in whom enhanced LHR was reported. The purpose of th$s ftudy was to determine the effect of
Na ,K ATPase inhibition on LHB. Leukocytes obtained from 12 allergic rhinitis (ALL) and 5 non-allerqic /NON-ALL) subjects were incubated for j0 minut& with anti-IgE antibody (100 us/ml) or control following a 0,10,2ti,30,6~,126 and 180 minute preincubation with 3 mM ouabain or with control buffer. Cell supernatants were assayed for histamine by radioimmunoassay and expressed as a % of total LH. Mean (+ 1SEM) anti-IgE induced LHR, in the
presence
and absence
of ouabain,
respectively, for ALL subiects was 32.2 i 6 and 24.1 15 (10 min), 35.1 + 6 and 24.7 f 4 (20 min), 34.6 + 6 and 27.3 + 4 (30 min), 40.8 + 8 and 32.2 It: 7 (60 min), 57.1 + 13 and 30.6 + 6 (120 min, ~~0.05)~ and 42.7 + 6 and 36.5 + 5 (180 min). Spontaneous UiR in ALL subjects was also increased by a 60 minute preincubation with ouabain (2.2 + 0.5 vs 1.0 k 0.2 for control, pcO.05). Ouabain did Got significantly alter LHR in NON-ALL subjects. These data show that ouabain induced a significant increase in both spontaneous an? i?duced LHR in ALL subjects. In vivo Na ,K ATPase inhibition in ALL may have significant effects on LHR.