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670 Human fibroblasts regulate the density and function of human neutrophils in culture
VOLUME NUMBER
87 1, PART 2
669 CHARACTERlZATlONOFTHEA549B-AD~N~~R(BAR) REGULATING PROTEIN COMPONENTS OF LYMPHCONDI~ONED MEDIUM (LCM) 0F iM9 CELLS: S. Robi-
Abstracts
671
COMPARISON OF BETA-ADRENERGICANTAGONISTSON GUlNEAPIGTRACEALSMODTH~: EFFECTSOF LIPID SOLUBILITY, BETA-l-SELECTIVITY. INTRINSIC SYMPATHOMfMETlC ACTlVlTY, AND ALPHAADRENERGIC ANTAGONISM. MA OConnell. MD: AR Henrv. MD: and TR &uahan. MD, Aurora, CO With tfie widespread clinical use of beta-adrenergic antagonists (BAA), potential pharmacologic differences between agents and their effects on bronchial hyperreactivity (BHR) are important. BAA are now available that possess differences in lipid solubility; beta-l-receptor selectivity (BRS); intrinsic sympathomimetic activity (ISA); and alpha-adrenergic antagonism. Utilizing an in-vitro model with isolated guinea pig tracheal ring sections, we studied the effects of propranolol (PRO), metoprolol (MET), nadolol (NAD), atenolol (ATE), acebutol (ACE), pindolol (PIN), and labetalol (LAB) at concentrations therapeutic in humans. Two-way analysis of variance with multiple comparisons analysis revealed highly significant differences between the individual BAA and their effects on both intrinsic bronchoconstrictor activity (IBA) on tracheal smooth muscle (p c.001) and histamineinduced bronchoconstriction (HIB) (p<.oOl). NAD and ATE demonstrated the greatest IBA (p-=.05). LAB, ATE, and NAD tended to potentiate HIB the most. PROBIT multiple regression analysis showed that hydrophilic agents potentiated IBA and HIB the most (x2=26.96, df=2, f~O01) and agents with BRS the least (x2=0.58, df=2, p=NS). This data suggests that pharmacologic differences between BAA have a significant impact on tracheal smooth muscle. Based on our data, a lipophilic BAA possessing BRS would be least likely to induce bronchoconstriction.
872
CQHPARISOW OFEFFICACY AHI SAFETY OFALBJJTEROL AMINISTERED BY POWER-DRIVEII KREULIZRR @II) VERSUHETERRD DOSE INHLW (RDI) WITRAEROCMMR ANDIWK IN YOUNG CEILDREW WITNASTRHA. N.LEE,D; G.RACRELEFSKY,HD: R.E.KOBAYASliI,tlD; b.L.KC+AYASRI,!lD; S.C.SIEGRL,HD; R.N.KATZ,HD; S.L.SPECTOR,KD ARDA.S.ROER,!UI. OHARA, m ANDLos ANGELES, CA. Thirty three children (38 + 24 wontbsof age) with acute asthmaticsynptos were ranhwized in a double blind placebo controlled fashion into either group il or 12. GroupI1 received albuterol ID1 at 0.03 q/kg/dose (weandose of 0.45 wg, saxiwuwof 0.54 wg) followed by placebo PlMtreatmentfor 10 win. Group12 received placebo WI puffs followed by albwterol via PDNat 0.1 wq/kg/dose(weandose of 1.45 nq, waxiwuwof 2.5 wg). Eachset of treatwents was given every 30 win or until an iwprovewentwas noted(maxim of 4 sets). Beartrate (HR),respiratoryrate (RR), pulse oxiseter (FOX)were weaswredat baseline (BSL), 0 win, 30 rin, 6 60 tin post treatment(pf). Thephysicianscore(PX) was obtained at BSLand 30 min PT.
M.D.. J.F, csek. Pb.D..r. &&nev. Ph.D.. J. 78ritva.D.. W. Lockev. M.D. and A. Szentivanvi. M.D.> Tampa, Florida In studying the protein components of the activity peaks of LCM of IM9 cells responsible for their regulatory effects on BARS of A549 human lung cells, two approaches were employed: a) .immunoneutralization using polyclonal or monoclonal antibodies to cytokines synthesized and released by lymphocytes and b) measurement of A549 SAR concentrations to recombinant cytokines for determining whether they have the capacity to upregulate or downregulate OARS. Of the two upregulating activity peaks, Peak II was neutralized by anti-human ILla and ILlI3 antibodies, but not Peak III. Antibodies against IL-2, IL3, IL-4, IL-S, IL6, IFN-a, IFN-y, TNFa, TNF-l3, GM-CSF, a-, B- and y-endorphins, [MET] and [Leuj-enkephalins, luteinizing hormone, prolactin, chorionic gonadotropin (B-chain), ACTH, growth hormone, TSH, epidermal-, fibroblast- and platelet-derived growth factors, were ineffective against upregulating Peaks II and III. Nor were they effective against downregulating Peak I. Thus, Peak II appears to be IL1 whereas immunoneutralization did not provide information on Peak I and Peak III. This is also supported by the effects of recombinant cytokines on AS49 BAR since only r-ILa, r-IL-a, and rIFN-y increased RAR densities. In comparison with immunoneutralization, the regulating effect of IFN-y remains at this time unexplained.
670
HUMANFIBROBLASTS REGULATE THE DENSITY AND FDNCTION OF HUMANNEUTROPHILS IN CULTURE. Carolyn J. Ling, M.D., William F. Owen Jr., M.D., K. Frank Austen, M.D., Boston, Massachusetts. Previously, we have demonstrated that human neutrophils (PMN) cocultured for up to --in vitro 72h with the human fibroblast cell line HF-15 have markedly enhanced survival as compared to replicate samples freshly isolated from the peripheral blood. The fibroblast-conditioned medium from HF-15 cultures are equally capable of maintaining PMN survival in a dose-dependent manner that is distinct from GM-CSF. To further characterize some of the phenotypic changes that occur with fibroblast coculture, freshly isolated and replicate cultured PMN were simultaneously assessed for changes in metrizamide gradient density profile and PI&P-stimulated superoxide production. PMN were assessed at Oh and after 48h. After 48h in culture PMN exhibited a 200X increase in PML.P-stimulated superoxide production (n=3; P~0.01) as compared to freshly isolated replicate PMN. In addition, there was a concomitant shift in the density gradient profile of cultured PMN such that 50+8% of PMN cultured for 48h acquired a hypodense phenotype as compared to 4.6+2.5% for freshly isolated replicate samples (n=6). In summary, coculture of human PMN with human fibroblasts is associated with acquisition of a hypodense phenotype and priming for enhanced ligand-specific response with respect to PMLPstimulated superoxide production. Furthermore, acquisition of the hypodense phenotype may be a means of characterizing subpopulations of PMN with different biochemical properties.
307
GROUP PSBkINE 0 n1NoTts 30 IIIBUTRS (N) RR RR POXPSCBR RR FOXRR RR FOXPSC2 ‘r:X( i#1(16) 138 44 92%6.9 146 40 94%143 38 96%4.4 130,36 97% i ! ]#2(17)112540 93%6.3 128 37 94%127 33 97%3.7 126132 961
* beats/nin,t breaths/sin A decreasein RRand PSCwas noted in both groups post treatlent. Concurrently, RRand POXincreased in both groups. Noneof the abovechangeswere statistically significant. In conclusion, albuterol treatment in dosagesusedwith Aerocbawberplus wask is as effective and safe as albuterol by PM for acute astbedtic attack in youngchildren.
87 1, PART 2
669 CHARACTERlZATlONOFTHEA549B-AD~N~~R(BAR) REGULATING PROTEIN COMPONENTS OF LYMPHCONDI~ONED MEDIUM (LCM) 0F iM9 CELLS: S. Robi-
Abstracts
671
COMPARISON OF BETA-ADRENERGICANTAGONISTSON GUlNEAPIGTRACEALSMODTH~: EFFECTSOF LIPID SOLUBILITY, BETA-l-SELECTIVITY. INTRINSIC SYMPATHOMfMETlC ACTlVlTY, AND ALPHAADRENERGIC ANTAGONISM. MA OConnell. MD: AR Henrv. MD: and TR &uahan. MD, Aurora, CO With tfie widespread clinical use of beta-adrenergic antagonists (BAA), potential pharmacologic differences between agents and their effects on bronchial hyperreactivity (BHR) are important. BAA are now available that possess differences in lipid solubility; beta-l-receptor selectivity (BRS); intrinsic sympathomimetic activity (ISA); and alpha-adrenergic antagonism. Utilizing an in-vitro model with isolated guinea pig tracheal ring sections, we studied the effects of propranolol (PRO), metoprolol (MET), nadolol (NAD), atenolol (ATE), acebutol (ACE), pindolol (PIN), and labetalol (LAB) at concentrations therapeutic in humans. Two-way analysis of variance with multiple comparisons analysis revealed highly significant differences between the individual BAA and their effects on both intrinsic bronchoconstrictor activity (IBA) on tracheal smooth muscle (p c.001) and histamineinduced bronchoconstriction (HIB) (p<.oOl). NAD and ATE demonstrated the greatest IBA (p-=.05). LAB, ATE, and NAD tended to potentiate HIB the most. PROBIT multiple regression analysis showed that hydrophilic agents potentiated IBA and HIB the most (x2=26.96, df=2, f~O01) and agents with BRS the least (x2=0.58, df=2, p=NS). This data suggests that pharmacologic differences between BAA have a significant impact on tracheal smooth muscle. Based on our data, a lipophilic BAA possessing BRS would be least likely to induce bronchoconstriction.
872
CQHPARISOW OFEFFICACY AHI SAFETY OFALBJJTEROL AMINISTERED BY POWER-DRIVEII KREULIZRR @II) VERSUHETERRD DOSE INHLW (RDI) WITRAEROCMMR ANDIWK IN YOUNG CEILDREW WITNASTRHA. N.LEE,D; G.RACRELEFSKY,HD: R.E.KOBAYASliI,tlD; b.L.KC+AYASRI,!lD; S.C.SIEGRL,HD; R.N.KATZ,HD; S.L.SPECTOR,KD ARDA.S.ROER,!UI. OHARA, m ANDLos ANGELES, CA. Thirty three children (38 + 24 wontbsof age) with acute asthmaticsynptos were ranhwized in a double blind placebo controlled fashion into either group il or 12. GroupI1 received albuterol ID1 at 0.03 q/kg/dose (weandose of 0.45 wg, saxiwuwof 0.54 wg) followed by placebo PlMtreatmentfor 10 win. Group12 received placebo WI puffs followed by albwterol via PDNat 0.1 wq/kg/dose(weandose of 1.45 nq, waxiwuwof 2.5 wg). Eachset of treatwents was given every 30 win or until an iwprovewentwas noted(maxim of 4 sets). Beartrate (HR),respiratoryrate (RR), pulse oxiseter (FOX)were weaswredat baseline (BSL), 0 win, 30 rin, 6 60 tin post treatment(pf). Thephysicianscore(PX) was obtained at BSLand 30 min PT.
M.D.. J.F, csek. Pb.D..r. &&nev. Ph.D.. J. 78ritva.D.. W. Lockev. M.D. and A. Szentivanvi. M.D.> Tampa, Florida In studying the protein components of the activity peaks of LCM of IM9 cells responsible for their regulatory effects on BARS of A549 human lung cells, two approaches were employed: a) .immunoneutralization using polyclonal or monoclonal antibodies to cytokines synthesized and released by lymphocytes and b) measurement of A549 SAR concentrations to recombinant cytokines for determining whether they have the capacity to upregulate or downregulate OARS. Of the two upregulating activity peaks, Peak II was neutralized by anti-human ILla and ILlI3 antibodies, but not Peak III. Antibodies against IL-2, IL3, IL-4, IL-S, IL6, IFN-a, IFN-y, TNFa, TNF-l3, GM-CSF, a-, B- and y-endorphins, [MET] and [Leuj-enkephalins, luteinizing hormone, prolactin, chorionic gonadotropin (B-chain), ACTH, growth hormone, TSH, epidermal-, fibroblast- and platelet-derived growth factors, were ineffective against upregulating Peaks II and III. Nor were they effective against downregulating Peak I. Thus, Peak II appears to be IL1 whereas immunoneutralization did not provide information on Peak I and Peak III. This is also supported by the effects of recombinant cytokines on AS49 BAR since only r-ILa, r-IL-a, and rIFN-y increased RAR densities. In comparison with immunoneutralization, the regulating effect of IFN-y remains at this time unexplained.
670
HUMANFIBROBLASTS REGULATE THE DENSITY AND FDNCTION OF HUMANNEUTROPHILS IN CULTURE. Carolyn J. Ling, M.D., William F. Owen Jr., M.D., K. Frank Austen, M.D., Boston, Massachusetts. Previously, we have demonstrated that human neutrophils (PMN) cocultured for up to --in vitro 72h with the human fibroblast cell line HF-15 have markedly enhanced survival as compared to replicate samples freshly isolated from the peripheral blood. The fibroblast-conditioned medium from HF-15 cultures are equally capable of maintaining PMN survival in a dose-dependent manner that is distinct from GM-CSF. To further characterize some of the phenotypic changes that occur with fibroblast coculture, freshly isolated and replicate cultured PMN were simultaneously assessed for changes in metrizamide gradient density profile and PI&P-stimulated superoxide production. PMN were assessed at Oh and after 48h. After 48h in culture PMN exhibited a 200X increase in PML.P-stimulated superoxide production (n=3; P~0.01) as compared to freshly isolated replicate PMN. In addition, there was a concomitant shift in the density gradient profile of cultured PMN such that 50+8% of PMN cultured for 48h acquired a hypodense phenotype as compared to 4.6+2.5% for freshly isolated replicate samples (n=6). In summary, coculture of human PMN with human fibroblasts is associated with acquisition of a hypodense phenotype and priming for enhanced ligand-specific response with respect to PMLPstimulated superoxide production. Furthermore, acquisition of the hypodense phenotype may be a means of characterizing subpopulations of PMN with different biochemical properties.
307
GROUP PSBkINE 0 n1NoTts 30 IIIBUTRS (N) RR RR POXPSCBR RR FOXRR RR FOXPSC2 ‘r:X( i#1(16) 138 44 92%6.9 146 40 94%143 38 96%4.4 130,36 97% i ! ]#2(17)112540 93%6.3 128 37 94%127 33 97%3.7 126132 961
* beats/nin,t breaths/sin A decreasein RRand PSCwas noted in both groups post treatlent. Concurrently, RRand POXincreased in both groups. Noneof the abovechangeswere statistically significant. In conclusion, albuterol treatment in dosagesusedwith Aerocbawberplus wask is as effective and safe as albuterol by PM for acute astbedtic attack in youngchildren.