Clinical and Experimental Allergy, 1992, Volume 22. pages 401-409
Inhibition profiles of sodium cromoglycate and nedocromil sodium on mediator release from mast cells of human skin, lung, tonsil, adenoid and intestine Y OKAYAMA, R. C. BENYON, P. H. REES, M. A. LOWMAN, K. HILLIER Lttid M. K. CHURCH Immunopharmacology Group. Clinical Pharmacology. Centre Block, Southampton General Hospital, Southampton. U.K. Summary We have studied an aspect of the functional heterogeneity ofhuman mast cells, namely responsiveness to the inhibitory effects of sodium cromoglycate and nedocromil sodium. The effects of these drugs were examined on the release of histamine and PGD: frotn mast cells of human skin, lung, tonsils, adenoids and intestine. A high concentration. 1000 /iM, of sodium cromoglycate was required to significantly inhibit histamine release from lung and tonsillar mast celts. Nedocromil sodium, 1000 /iM, was more effective than sodium cromoglycate against histamine release from lung, tonsillar and adenoidal cells. Both compounds showed tachyphylaxis in lung and tonsiltar mast cells but not in adenoidal and intestinal mast cells. In contrast, in intestinal mast cells, tbe effect of nedocromil sodium was weaker and more variable than sodium cromoglycate. Skin mast cells differed from mast cells of the other anatomical sites in being unresponsive to sodium cromoglycate and nedocromil sodium. Our results confirm that high concentrations of sodium cromoglycate and nedocromil sodium are required to achieve even modest inhibition of mediator release from human mast cells under in vitro conditions. Notwithstanding this, the results also indicate that differences exist among skin, lung, lonsillar. adenoidal and intestinal mast cells with respect to their sensitivity to sodium cromoglycate and nedocromil sodium, thus extending our knowledge of functional heterogeneity within the human masl cell populations. Clinical and E-xperimcntal Allergy, \o\. 22, pp. 401^09. Submitted 13 February 1991; revised 17 October 1991; accepted 25 October 1991.
Introduction Cross-linkage of membrane bound IgE by allergen triggers masl cells to release a wide spectrum of preformed and newly-generated mediators which are considered to play an important role in the development of the itnmediatc hypersensitivity response [1-3]. An understanding of the biochemistry of this mediator release is required for rational development of future drug therapies for human hypersensitivity reactions. Most prospective drugs for the treatment ofhuman allergic disease are assessed initially in animal models of anaphylaxis. Correspondence: Dr Yoshimichi Okayama. Immunopharmacology Group. Clinical Pharmacology. Centre Block. Southampton General Hospital. Southampton SO9 4XY. U.K.
predominantly in vitro tests using rat peritoneal mast cells. However, it is now generally accepted that mast cells from different species, and even from different tissues within a given species, are functionally heterogeneous [4,5]. In particular, they may vary in their responsiveness to particular anti-allergic agents such as sodium cromoglycate (SCG) and nedocromil sodium (Nedo). Taken together, the results of previous studies give a hint to the complexity ofhuman mast cell responsiveness to SCG. In mast cells dispersed from human lung tissue, SCG is a weak inhibitor of bistamine and prostaglandin (PG)D2 release [6-9] and shows tachyphylaxis [9,10]. Conversely, in skin mast cells SCG has no inhibitory effect on IgE-dependenl histamine release [4,11.12]. In human intestinal mast cells. Fox et al. [13] reported that
402
y, Okavama et al.
SCG has no effect on histamine release, while Befus et al. [14] reported it to be an effective inhibitor only at high concentration (1 niM). Nedocromil sodium, a pyranoquinoline dicarboxylic acid derivative which differs structurally and physicochemically from SCG. shows a similar spectrum of activity in human lung mast cells. Nedo is significantly more active in preventing mediator release from bronchoalveolar lavage mast cells than mast cells dispersed from lung parenchyma and is an order of magnitude more potent than SCG in both cell types [10]. In common with SCG, tachyphylaxis to the inhibitory effects of Ncdo is seen only in lung parenchymal mast cells [10]. Nedo has been shown to be a weak inhibitor of IgE-dependent histamine release from human intestinal mucosal mast cells only when pre-incubated with the cells before challenge [14]. Liu et al. have reported tachyphylaxis of SCG and Nedo with isolated mast cells from human coionic muscle but not with those of human colonic mucosa [15]. In this study, we have undertaken detailed investigation of the characteristics of inhibition by high concentrations of SCG and Nedo of mediator release from mast cells dispersed from a variety of human tissues using similar enzymatic techniques. We show heterogeneity in responsiveness of different mast cell subpopulations to these drugs which may have important implications for fiiture development of allergic therapies.
Materials and methods Material Sodium cromoglycate (SCG) and nedocromil sodium (Nedo) were donated by Fisons Pharmaceuticals. U.K. Goat anti-human IgE was prepared as described previously [I]. Mouse monoclonal anti-human IgE GE-1 was purchased from ICN Biomedical Ltd, U.K. Chymopapain (papaya latex), pronase (Streptomyces gri.scus), collagenase([ype 1). hyaluronidase (type I), bovine serum albumin (BSA) (fraction V) and A'-2-hydroxyethyl-piperazine-A''-2-ethanesulphonic acid (HEPES) were purchased from Sigma. U.K. Foetal calf serum (FCS). RPMI 1640 medium, Eaglesminimumessential medium (MEM) containing 25 mM HEPES and L-glutamine was obtained from GIBCO. Scotland. ['H]PGD. ( - 37 kBq) was from Amersham International. U.K. Ail other chemicals were purchased from BDH, Poole. U.K. The composition of HEPES buffered salts solution (HBSS) was (in mM): NaCl. 137: D-glucose, 5 0; NaH2PO4. 0 4; KCl. 2 7; MgCl:, 0 5; CaCU, 1-8 and HEPES 10. The pH was adjusted to 7 2 with 2 M sodium hydroxide and I 0% FCS added.
Methods Dispersal of human skin, lung, adenoidal and tonsillar intestinal cells Foreskin: Skin mast cells were dispersed from foreskin by an enzymatic procedure described previously [3]. Samples of foreskin from children (1-9 years) were placed immediately in RPMI 1640 medium, stored at 4 C. and used within 24 hr of circumcision. Fragments of 0 5-2-0 m m \ obtained by chopping with scissors, were washed twice with MEM containing 2-0% foetal calf serum (MEM/FCS) before incubation (75 min, 37 C) in the same buffer (I g tissue/10 ml buffer) containing collagenase. 15 mg/ml, hyaluronidase. 0 75 mg/ ml, and BSA. 35 mg/ml. Cells dispersed by this procedure were separated from undissociated tissue by filtration through nylon gauze (150 (ivn) and washed twice with MEM/FCS by cenlrifugation at 500 j? for 5 min at 20 C. Mast celt numbers were assessed by counting in a Neubauer haemocytometer after metachromatic staining with Kimura stain [16]. Lung: Macroscopically normal human lung tissue, obtained within 1 hr of resection, was dissected free from major bronchi and blood vessels. The dispersal method subsequently used was similar to that used for foreskin except that two 40 min incubations with pronase. 2 mg/ ml. and chymopapain, 0 5 mg/ml were used [lj. Tonsils and adenoids: Surgical specimens removed by tonsillectomy or adenoidectomy of children (4-12 years) were used. The dispersal method subsequently used was the same as for foreskin except that a single I hr digestion was used [17]. Colon: Colectomy specimens obtained from patients (55-92 years of age) undergoing resection for colorectal carcinoma were dissected to separate histologically normal mucosa. muscularis mucosa, and submucosa from the underlying muscle layers. Cell dispersion was performed as described for foreskin, except that I mmol/I of dithiothreitol was included in all digestion and wash stages in order to remove mucus, and FCS, 20%, was substituted for BSA. 35 mg/ml, in the digestion mixture. Purification of skin ami hmg mast cells Skin mast cells were purified by density sedimentation through Percoll. Dispersed cells were resuspended in MEM/FCS and layered onto a discontinuous gradient of 60-80% isotonic Percol! (density 1 076 I I g/ml) which was centrifuged at 500 j? and 4 C for 20 min. The mast cells pooled from the bottom of the gradient and 70/80% Percoll interface constituted >80% of nucleated cells [3]. Density sedimentation techniques were not applicable to lung mast cells due to the nature of the contaminating cells. Lung mast cells were purified by affinity magnetic selection. Dispersed cells (in a purity of approximately 5%) were incubated for 30 min at 4 C with mouse
Effect of SCG and nedocromil on human mast eells
403
monoclonal anti-IgE antibody {1/500 dilution) which was i release and assay Duplicate 0 4 ml cell replieates not involved in cell activation. Following washing cells containing 4 8x lO'* mast cells were warmed to 37 C were resuspended in 500 ^i\ ofa suspension of magnetic before addition of 0 05 ml each of drug or HBSS and beads coated with goat anti-mouse IgG {Dynabeads anti-IgE. Release reactions were allowed to proceed for M-450., Dynal, Norway) for 60 min at 4 C. Positively 15 min. Because of potential degradation of P G D T by stained cells were removed by two 15 min exposures to a protein, ECS was omitled from the buffer in these magnetic field. The masl cells obtained were more than experiments. PGD^ was measured in duplieate 0-1 ml V>O"/u pure (T. C. Hunt and M. K. Church, manuscript in aiiquots of cell supernatant or diluted eell supernatant to preparation). which 01 ml of ['HJPGD. (10000 d.p.m.) was added followed by 01 ml of a l/lOO dilution of anti-PGD: Histamine release studies Immunologieal histamine serum. After incubation overnight at 4 C, 0 2 ml of release was assessed in duplicate tubes containing 2 dextran coated charcoal was added and the tubes were 4x 10^ mast cells ineubated for 15 min with anti-tgE. centrifuged at 1500j< for 15 min at 4 C. The supernatant Reactions were temiinated by addition of 0 5 ml ice-cold was removed and 'H eounted by liquid scintillation calcium-free HBSS and eentrifugation at 250 j? for 10 min spectrometry. The limit of detection of P G D T in this assay at 4 C. The supernatant was acidified to 5"4P trichloroacewas 10 pg per tube. tic acid (TCA) and histamine measured speetrofluorimeThe effects of SCG and Nedo on purified .sk in and lung mast trieally. Spontaneous release was estimated in duplicate cell.s A volume of 0 02 ml of polyclonal anti-IgE was lubes to whieh no anti-IgE had been added. Total added to reaction tubes and warmed to 37 C. At zerohistamine was measured in parallel duplicate tubes by time, pre-warmed purified mast cell suspensions, eontaindisintegrating the cells in 5% TCA. Net histamine release ing2 X IO''purified mastceilsof human skin or lung, were by anti-TgE was expressed as a pereentage of total added to each drug solution {SCG 1000/IM. Nedo 100/IM) histamine and corrected for spontaneous release. or buffer. After pre-ineubation for 15 min or 0 min, 0-48 In time-course studies, a volume of 0 02 ml of anti-TgE min cell suspension was removed into reaetion tubes to was added to reaetion tubes and warmed lo 37 C. Al zero initiate histamine release. Release reaetions were stopped Lime, pre-warmed cell suspensions, containing 2 4 x 10** 15 min later. mast eells of human skin or lung or eolon or tonsils or adenoids, were added to eaeh drug solution (SCG 1000 /(M, Nedo 10 /JM) or butler. After 45, 30, 15, 5, 0 min, Statistical analy.ses 0 48 ml cell suspension was removed into the reaction Results are expressed as the mean + s.e.m. Differences tubes to initiate histamine release. Release reactions were between means were tested for significance with Student's stopped 15 min later. /-test for paired data. Differences were eonsidered signifiIn concentration-response studies., duplieate 0 4 ml cell eant when the probability {P) was < 0 05. suspensions containing 2-4 x lO'* mast cells from skin. lung and lonsils were pre-ineubated for 15 min at 37 C Results with 0 05 ml of HBSS (eontroi) or 5 min with 0-05 ml of Nedo (0-1. I (). 100, 1000 /JM) before challenge while Time-course studies 005 ml of SCG (10, 100, 1000 /(M) were added simultaThe effects of SCG (1000 /XM) and Nedo (10 /^M) in neously with 0 05 ml of anti-IgE to 0 4 ml cell suspenlime-course studies in human mast cells arc shown in sions. The mast cells from eolon and adenoids were preFigs 1 and 2. incubated for 15 min with HBSS (control) or Nedo {01., In human lung mast cells, both eompounds were 10, 100, 1000 //M) or SCG (10. 100, 1000 yM) before maximally active when added simultaneously with antichallenge. Release reaetions were allowed lo proceed IgE challenge. A similar ellect was seen with SCG in before termination at 15 min. tonsillar mast cells while the inhibitory effects of Nedo Dilutions of 10-1000 /^M SCG and 01-1000 ^M Nedo increased with pre-incubation periods of up to 5 min. Tn alone did not atfeet spontaneous release from human bolh of these cell types. SCG and Nedo showed marked mast cells examined. tachyphylaxis with prolonged incubation. In eross-tachyphylaxis studies, a volume of 0 20 ml of In intestinal and adenoidal mast cells, the effects of eell suspensions containing 2 4x10"* mast eells was SCG increased with pre-incubation to a maximum inhibiincubated for 30 min at 37 C with 0 20 ml SCG (final tion of 49 3 ± 7 4% and 24-4 + 3-7% at 15 min. Eurther concentration 1 mM) or buffer. After this time, O-I ml of prolongation of the incubation time in adenoidal mast anti-IgE plus drug or buffer was added and the release cells led to a decay of activity. While the effects of Nedo reaction allowed to proceed for 15 min.
404
Y. Okavamaei
al.
50
50 r
40
40
30
30
20
B
[0
20 10
5
15
30
45
Pre-incubotion time with SCG, I mM (mm)
Fig. 1. Time-course for inhibition of IgE-dcpcndent histamine release from human dispersed skin ( A ) , lung (O), tonsillar (O)adenoidal (D) and intestinal (•) mast cells by SCG. 1-0 mM. Each result is the mean + s.c.m. of six (skin), four (lung), 10 11 (tonsils), six (adenoids) and five (intestine) experiments. Mean nel control histamine releases from skin. lung, tonsillar. adenoidal and intestinal mast cells were 19-6+1-7%. I7-5±1-I%. 22-2+ 0-4%,20'7 + 0-6%, and 13-4+2-2'!^, respectively. Spontaneous releases from skin, lung, lonsillar, adenoidal and iniestinal mast cells were 4-8 + 0-7%, 3-9 + 0-2%., 5-6 + 0-7%, 4-3+0-7%! and 1O'1± I'9%i respectively. Asterisks refer to significant difference of histamine release in presence or absence of drugs {*P
Inhibition profiles of sodium cromoglycate and nedocromil sodium on mediator release from mast cells of human skin, lung, tonsil, adenoid and intestine Y OKAYAMA, R. C. BENYON, P. H. REES, M. A. LOWMAN, K. HILLIER Lttid M. K. CHURCH Immunopharmacology Group. Clinical Pharmacology. Centre Block, Southampton General Hospital, Southampton. U.K. Summary We have studied an aspect of the functional heterogeneity ofhuman mast cells, namely responsiveness to the inhibitory effects of sodium cromoglycate and nedocromil sodium. The effects of these drugs were examined on the release of histamine and PGD: frotn mast cells of human skin, lung, tonsils, adenoids and intestine. A high concentration. 1000 /iM, of sodium cromoglycate was required to significantly inhibit histamine release from lung and tonsillar mast celts. Nedocromil sodium, 1000 /iM, was more effective than sodium cromoglycate against histamine release from lung, tonsillar and adenoidal cells. Both compounds showed tachyphylaxis in lung and tonsiltar mast cells but not in adenoidal and intestinal mast cells. In contrast, in intestinal mast cells, tbe effect of nedocromil sodium was weaker and more variable than sodium cromoglycate. Skin mast cells differed from mast cells of the other anatomical sites in being unresponsive to sodium cromoglycate and nedocromil sodium. Our results confirm that high concentrations of sodium cromoglycate and nedocromil sodium are required to achieve even modest inhibition of mediator release from human mast cells under in vitro conditions. Notwithstanding this, the results also indicate that differences exist among skin, lung, lonsillar. adenoidal and intestinal mast cells with respect to their sensitivity to sodium cromoglycate and nedocromil sodium, thus extending our knowledge of functional heterogeneity within the human masl cell populations. Clinical and E-xperimcntal Allergy, \o\. 22, pp. 401^09. Submitted 13 February 1991; revised 17 October 1991; accepted 25 October 1991.
Introduction Cross-linkage of membrane bound IgE by allergen triggers masl cells to release a wide spectrum of preformed and newly-generated mediators which are considered to play an important role in the development of the itnmediatc hypersensitivity response [1-3]. An understanding of the biochemistry of this mediator release is required for rational development of future drug therapies for human hypersensitivity reactions. Most prospective drugs for the treatment ofhuman allergic disease are assessed initially in animal models of anaphylaxis. Correspondence: Dr Yoshimichi Okayama. Immunopharmacology Group. Clinical Pharmacology. Centre Block. Southampton General Hospital. Southampton SO9 4XY. U.K.
predominantly in vitro tests using rat peritoneal mast cells. However, it is now generally accepted that mast cells from different species, and even from different tissues within a given species, are functionally heterogeneous [4,5]. In particular, they may vary in their responsiveness to particular anti-allergic agents such as sodium cromoglycate (SCG) and nedocromil sodium (Nedo). Taken together, the results of previous studies give a hint to the complexity ofhuman mast cell responsiveness to SCG. In mast cells dispersed from human lung tissue, SCG is a weak inhibitor of bistamine and prostaglandin (PG)D2 release [6-9] and shows tachyphylaxis [9,10]. Conversely, in skin mast cells SCG has no inhibitory effect on IgE-dependenl histamine release [4,11.12]. In human intestinal mast cells. Fox et al. [13] reported that
402
y, Okavama et al.
SCG has no effect on histamine release, while Befus et al. [14] reported it to be an effective inhibitor only at high concentration (1 niM). Nedocromil sodium, a pyranoquinoline dicarboxylic acid derivative which differs structurally and physicochemically from SCG. shows a similar spectrum of activity in human lung mast cells. Nedo is significantly more active in preventing mediator release from bronchoalveolar lavage mast cells than mast cells dispersed from lung parenchyma and is an order of magnitude more potent than SCG in both cell types [10]. In common with SCG, tachyphylaxis to the inhibitory effects of Ncdo is seen only in lung parenchymal mast cells [10]. Nedo has been shown to be a weak inhibitor of IgE-dependent histamine release from human intestinal mucosal mast cells only when pre-incubated with the cells before challenge [14]. Liu et al. have reported tachyphylaxis of SCG and Nedo with isolated mast cells from human coionic muscle but not with those of human colonic mucosa [15]. In this study, we have undertaken detailed investigation of the characteristics of inhibition by high concentrations of SCG and Nedo of mediator release from mast cells dispersed from a variety of human tissues using similar enzymatic techniques. We show heterogeneity in responsiveness of different mast cell subpopulations to these drugs which may have important implications for fiiture development of allergic therapies.
Materials and methods Material Sodium cromoglycate (SCG) and nedocromil sodium (Nedo) were donated by Fisons Pharmaceuticals. U.K. Goat anti-human IgE was prepared as described previously [I]. Mouse monoclonal anti-human IgE GE-1 was purchased from ICN Biomedical Ltd, U.K. Chymopapain (papaya latex), pronase (Streptomyces gri.scus), collagenase([ype 1). hyaluronidase (type I), bovine serum albumin (BSA) (fraction V) and A'-2-hydroxyethyl-piperazine-A''-2-ethanesulphonic acid (HEPES) were purchased from Sigma. U.K. Foetal calf serum (FCS). RPMI 1640 medium, Eaglesminimumessential medium (MEM) containing 25 mM HEPES and L-glutamine was obtained from GIBCO. Scotland. ['H]PGD. ( - 37 kBq) was from Amersham International. U.K. Ail other chemicals were purchased from BDH, Poole. U.K. The composition of HEPES buffered salts solution (HBSS) was (in mM): NaCl. 137: D-glucose, 5 0; NaH2PO4. 0 4; KCl. 2 7; MgCl:, 0 5; CaCU, 1-8 and HEPES 10. The pH was adjusted to 7 2 with 2 M sodium hydroxide and I 0% FCS added.
Methods Dispersal of human skin, lung, adenoidal and tonsillar intestinal cells Foreskin: Skin mast cells were dispersed from foreskin by an enzymatic procedure described previously [3]. Samples of foreskin from children (1-9 years) were placed immediately in RPMI 1640 medium, stored at 4 C. and used within 24 hr of circumcision. Fragments of 0 5-2-0 m m \ obtained by chopping with scissors, were washed twice with MEM containing 2-0% foetal calf serum (MEM/FCS) before incubation (75 min, 37 C) in the same buffer (I g tissue/10 ml buffer) containing collagenase. 15 mg/ml, hyaluronidase. 0 75 mg/ ml, and BSA. 35 mg/ml. Cells dispersed by this procedure were separated from undissociated tissue by filtration through nylon gauze (150 (ivn) and washed twice with MEM/FCS by cenlrifugation at 500 j? for 5 min at 20 C. Mast celt numbers were assessed by counting in a Neubauer haemocytometer after metachromatic staining with Kimura stain [16]. Lung: Macroscopically normal human lung tissue, obtained within 1 hr of resection, was dissected free from major bronchi and blood vessels. The dispersal method subsequently used was similar to that used for foreskin except that two 40 min incubations with pronase. 2 mg/ ml. and chymopapain, 0 5 mg/ml were used [lj. Tonsils and adenoids: Surgical specimens removed by tonsillectomy or adenoidectomy of children (4-12 years) were used. The dispersal method subsequently used was the same as for foreskin except that a single I hr digestion was used [17]. Colon: Colectomy specimens obtained from patients (55-92 years of age) undergoing resection for colorectal carcinoma were dissected to separate histologically normal mucosa. muscularis mucosa, and submucosa from the underlying muscle layers. Cell dispersion was performed as described for foreskin, except that I mmol/I of dithiothreitol was included in all digestion and wash stages in order to remove mucus, and FCS, 20%, was substituted for BSA. 35 mg/ml, in the digestion mixture. Purification of skin ami hmg mast cells Skin mast cells were purified by density sedimentation through Percoll. Dispersed cells were resuspended in MEM/FCS and layered onto a discontinuous gradient of 60-80% isotonic Percol! (density 1 076 I I g/ml) which was centrifuged at 500 j? and 4 C for 20 min. The mast cells pooled from the bottom of the gradient and 70/80% Percoll interface constituted >80% of nucleated cells [3]. Density sedimentation techniques were not applicable to lung mast cells due to the nature of the contaminating cells. Lung mast cells were purified by affinity magnetic selection. Dispersed cells (in a purity of approximately 5%) were incubated for 30 min at 4 C with mouse
Effect of SCG and nedocromil on human mast eells
403
monoclonal anti-IgE antibody {1/500 dilution) which was i release and assay Duplicate 0 4 ml cell replieates not involved in cell activation. Following washing cells containing 4 8x lO'* mast cells were warmed to 37 C were resuspended in 500 ^i\ ofa suspension of magnetic before addition of 0 05 ml each of drug or HBSS and beads coated with goat anti-mouse IgG {Dynabeads anti-IgE. Release reactions were allowed to proceed for M-450., Dynal, Norway) for 60 min at 4 C. Positively 15 min. Because of potential degradation of P G D T by stained cells were removed by two 15 min exposures to a protein, ECS was omitled from the buffer in these magnetic field. The masl cells obtained were more than experiments. PGD^ was measured in duplieate 0-1 ml V>O"/u pure (T. C. Hunt and M. K. Church, manuscript in aiiquots of cell supernatant or diluted eell supernatant to preparation). which 01 ml of ['HJPGD. (10000 d.p.m.) was added followed by 01 ml of a l/lOO dilution of anti-PGD: Histamine release studies Immunologieal histamine serum. After incubation overnight at 4 C, 0 2 ml of release was assessed in duplicate tubes containing 2 dextran coated charcoal was added and the tubes were 4x 10^ mast cells ineubated for 15 min with anti-tgE. centrifuged at 1500j< for 15 min at 4 C. The supernatant Reactions were temiinated by addition of 0 5 ml ice-cold was removed and 'H eounted by liquid scintillation calcium-free HBSS and eentrifugation at 250 j? for 10 min spectrometry. The limit of detection of P G D T in this assay at 4 C. The supernatant was acidified to 5"4P trichloroacewas 10 pg per tube. tic acid (TCA) and histamine measured speetrofluorimeThe effects of SCG and Nedo on purified .sk in and lung mast trieally. Spontaneous release was estimated in duplicate cell.s A volume of 0 02 ml of polyclonal anti-IgE was lubes to whieh no anti-IgE had been added. Total added to reaction tubes and warmed to 37 C. At zerohistamine was measured in parallel duplicate tubes by time, pre-warmed purified mast cell suspensions, eontaindisintegrating the cells in 5% TCA. Net histamine release ing2 X IO''purified mastceilsof human skin or lung, were by anti-TgE was expressed as a pereentage of total added to each drug solution {SCG 1000/IM. Nedo 100/IM) histamine and corrected for spontaneous release. or buffer. After pre-ineubation for 15 min or 0 min, 0-48 In time-course studies, a volume of 0 02 ml of anti-TgE min cell suspension was removed into reaetion tubes to was added to reaetion tubes and warmed lo 37 C. Al zero initiate histamine release. Release reaetions were stopped Lime, pre-warmed cell suspensions, containing 2 4 x 10** 15 min later. mast eells of human skin or lung or eolon or tonsils or adenoids, were added to eaeh drug solution (SCG 1000 /(M, Nedo 10 /JM) or butler. After 45, 30, 15, 5, 0 min, Statistical analy.ses 0 48 ml cell suspension was removed into the reaction Results are expressed as the mean + s.e.m. Differences tubes to initiate histamine release. Release reactions were between means were tested for significance with Student's stopped 15 min later. /-test for paired data. Differences were eonsidered signifiIn concentration-response studies., duplieate 0 4 ml cell eant when the probability {P) was < 0 05. suspensions containing 2-4 x lO'* mast cells from skin. lung and lonsils were pre-ineubated for 15 min at 37 C Results with 0 05 ml of HBSS (eontroi) or 5 min with 0-05 ml of Nedo (0-1. I (). 100, 1000 /JM) before challenge while Time-course studies 005 ml of SCG (10, 100, 1000 /(M) were added simultaThe effects of SCG (1000 /XM) and Nedo (10 /^M) in neously with 0 05 ml of anti-IgE to 0 4 ml cell suspenlime-course studies in human mast cells arc shown in sions. The mast cells from eolon and adenoids were preFigs 1 and 2. incubated for 15 min with HBSS (control) or Nedo {01., In human lung mast cells, both eompounds were 10, 100, 1000 //M) or SCG (10. 100, 1000 yM) before maximally active when added simultaneously with antichallenge. Release reaetions were allowed lo proceed IgE challenge. A similar ellect was seen with SCG in before termination at 15 min. tonsillar mast cells while the inhibitory effects of Nedo Dilutions of 10-1000 /^M SCG and 01-1000 ^M Nedo increased with pre-incubation periods of up to 5 min. Tn alone did not atfeet spontaneous release from human bolh of these cell types. SCG and Nedo showed marked mast cells examined. tachyphylaxis with prolonged incubation. In eross-tachyphylaxis studies, a volume of 0 20 ml of In intestinal and adenoidal mast cells, the effects of eell suspensions containing 2 4x10"* mast eells was SCG increased with pre-incubation to a maximum inhibiincubated for 30 min at 37 C with 0 20 ml SCG (final tion of 49 3 ± 7 4% and 24-4 + 3-7% at 15 min. Eurther concentration 1 mM) or buffer. After this time, O-I ml of prolongation of the incubation time in adenoidal mast anti-IgE plus drug or buffer was added and the release cells led to a decay of activity. While the effects of Nedo reaction allowed to proceed for 15 min.
404
Y. Okavamaei
al.
50
50 r
40
40
30
30
20
B
[0
20 10
5
15
30
45
Pre-incubotion time with SCG, I mM (mm)
Fig. 1. Time-course for inhibition of IgE-dcpcndent histamine release from human dispersed skin ( A ) , lung (O), tonsillar (O)adenoidal (D) and intestinal (•) mast cells by SCG. 1-0 mM. Each result is the mean + s.c.m. of six (skin), four (lung), 10 11 (tonsils), six (adenoids) and five (intestine) experiments. Mean nel control histamine releases from skin. lung, tonsillar. adenoidal and intestinal mast cells were 19-6+1-7%. I7-5±1-I%. 22-2+ 0-4%,20'7 + 0-6%, and 13-4+2-2'!^, respectively. Spontaneous releases from skin, lung, lonsillar, adenoidal and iniestinal mast cells were 4-8 + 0-7%, 3-9 + 0-2%., 5-6 + 0-7%, 4-3+0-7%! and 1O'1± I'9%i respectively. Asterisks refer to significant difference of histamine release in presence or absence of drugs {*P
