Acta physiol. scand. 1976. 97. 222-232 From the Department of Pharmacology, Karolinska Institutet, Stockholm, Sweden
Potentiation of Anaphylactic Histamine Release from Isolated Rat Pleural Mast Cells by Rat Serum Phospholipids BY and B. UVNAS A. SYDBOM Received 18 November 1975
Abstract
SYDBOM,A. and B. UVNAS.Potentiation of anaphylactic histamine release from isolated rat pleural mast cells by rat serum phospholipids. Acta physiol. scand. 1976. 97. 222-232. Isolation of sensitized rat mast cells by density gradient centrifugation in Ficoll decreases the histamine release obtained when they are subsequently exposed to antigen. The histamine release from such isolated cells is potentiated by the addition of 2 7 , boiled rat serum. This potentiation is dose-dependent and has a temperature optimum of about 25°C. The potentiating activity was localized to the serum phospholipid fraction. Of the pure phospholipids studied (LPC, PC, PE, PI, PS and S M ) only phosphatidylserine and lysophosphatidylcholine were found to potentiate the histamine release. The mechanism behind this potentiation is discussed and it is suggested that the potentiation by phosphatidylserine and lysophosphatidylcholine is due to a requirement of these phospholipids for the ion exchange (Na+, K+ and Ca++) o r the adenylcyclase activity essential for the histamine release process.
During early attempts to isolate rat peritoneal mast cells in our laboratory it was observed that such isolated cells had lost their reactivity to degranulating agents like compound 48/80 and antigens (Uvnas and Thon 1959). Such cells also showed a high level of spontaneous histamine release. The isolation procedure involved density gradient centrifugation in Ficoll and several subsequent washings in isotonic saline solution. The presence of 2% rat serum throughout the isolation procedure was found to prevent the loss of cell reactivity. Later, we noticed that albumin (1 mg/ml) could replace rat serum in maintaining the mast cell reactivity to compound 48/80 (Uvnas and Thon 1961), but did not suffice to maintain the response of sensitized mast cells to purified antigens. Evidently, another serum factorpossibly in the lipid fraction-was essential for the development of an antigen-induced mast cell reaction (Uvnas 1968). Other investigators have also reported that the antigen-induced histamine release from isolated rat mast cells is lower than the release from mast cells in mixed peritoneal cell suspensions (Johnson and Moran 1966, Norn 1968). It has also been known for a long time that isolated peritoneal mast cells do not respond to dextran (Lagunoff and Benditt 1960, Goth and Knoohuizen 1962), despite the fact that dextran induces histamine release in 222
POTENTIATION OF HISTAMINE RELEASE
223
in vioo systems (Goth 1967). Goth and Knoohuizen (1962) reported that histamine was released when a lipid extract from rabbit brain was added together with dextran to peritoneal mast cells, even though the lipid extract by itself did not cause any histamine release. Lichtenstein and Osler (1966) reported an enhancing effect of a heat-stable factor in human serum on histamine release from leucocytes induced by ragweed pollen antigen. During isolation of leucocytes the reactivity was lost and could be restored by adding extracts from normal leucocytes (Lichtenstein 1968). The potentiating factor from rabbit brain tissue has been identified as phosphatidylserine (Goth et at. 1971). Later several authors have reported a potentiating effect of phosphatidylserine, phosphatidylinositol or phosphatidylethanolamine on histamine release induced by antigen or dextran (Mongar and Svec 1972a, 1972 b, Foreman and Mongar 1972, Garland and Mongar 1974, Chakravarty et at. 1973, Stechschulte and Austen 1973, 1974). The present paper describes the potentiating effect of bovine phosphatidylserine and lysophosphatidylcholine on the antigen-induced histamine release from isolated rat pleural mast cells. The potentiating effect of rat serum is tentatively ascribed to the presence of the same phospholipids.
Materials and methods Egg albumin was purchased from Difco Laboratories, Detroit, Michigan, USA, Ficoll from AB Pharmacia,
Uppsala, Sweden, and human serum albumin (free from preservatives) from AB Kabi, Stockholm, Sweden. Pertussis vaccine (nr. 299) (2 x IO'O bact./ml) was obtained from SBL, Stockholm, Sweden. Unisil (200-325 mesh) was purchased from Clarkson Chem. Inc., Williarnsport, USA, and Eastman chromatogram sheet 6061 without fluorescence indicator from Eastman Kodak Co., Rochester, N.Y., USA. Phospholipid references were: Phosphatidylserine (type 111 from ox brain) from Sigma Chem. Co., St. Louis, USA (chr. pure ex. bovine brain) Koch Light Labs., Colnbrook, Bucks, England and (chr. pure) Grand Island Biological Comp., Grand Island, N.Y., USA. Phosphatidylethanolaminefrom Koch Light Labs. and Grand Island Biological Co. Phosphatidylcholine (L-a-lecithin from egg, type 111 E) from Sigma Chem. Co. Phosphatidylinositol (chr. pure) from Grand Island Biological Co., Supelco Inc., Bellefonte, Penn., USA and Analabs Inc., North Haven, Connecticut, USA. Lysophosphatidylcholine and sphingomyelin from Sigma Chem. Co. All other chemicals were obtained from the usual commercial sources. Abbreviations used: LPC PC PE PI PS SM
lysophosphatidylcholine phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine sphingomyelin
Preparation of rat serum Male Sprague-Dawley rats were anaesthetized with ether, the carotids were cut and the blood was collected in a beaker and allowed to stand for 1 h at 20°C and then overnight at 4°C. The serum was centrifuged (400 x g) and stored in 5 ml portions at - 20°C. If the serum was to be boiled it was first diluted with saline t o 20% (v/v) and then heated in a boiling water-bath for 10 min. After centrifugation, the supernatant was stored in 2 ml portions at - 20°C. Purification of the serum factor Extracrion of lipids. The total lipids were extracted from rat serum essentially according to Nelson and Freeman (1959). Chloroform-methanol ( I : I ) (332 ml) was added to 20 ml of rat serum under a stream of nitrogen and
224
A. SYDBOM AND B. UVNLS
protected from light. After agitation, the mixture was incubated for IS min at 60°C. The mixture was cooled to 0°C and diluted with chloroform to a final volume of 500 ml. The precipitated proteins were separated from the supernatant by filtration. The volume of the filtrate was adjusted to 500 ml by adding chloroform and it was then transferred to a separating funnel and 100 ml of distilled water was added. After agitation and phase separation for 3 h at 4”C, the lower phase was separated and evaporated to dryness and the upper phase was stored frozen under nitrogen. Separation of phospholipids. Phospholipids were separated from neutral lipids by chromatography on silicic acid according to Colacicco and Rapport (1967). Eight grams of Unisil silicic acid (200-325 mesh) was dispersed in 20 ml of chloroform-methanol-ammonia, 80: 20: 2 (v/v), poured into a glass column (diameter 1 cm), and washed with 100 ml of the solvent mixture. The residue of the lower phase from the extraction was dissolved in 2 ml of chloroform-methanol-ammonia 80:20:2 (v/v) and put on to the column. After elution with 150 ml of chloroform-methanol-ammonia 80: 20 :2 (v/v), the phospholipids were eluted with 100 ml of ethanol and collected in 10 fractions of 10 ml each. The fractions were evaporated to dryness. Thin layer chromatography (t.1.c.). Eastman precoated “chromatoplates”, with a 0.1 mm silicic acid layer, were used. The plates were activated for 60 min at 110°C. Samples were dissolved in small volumes of chloroform or chloroform-methanol, 1 : 1 (v/v), and applied to the plates. The solvent system used was chloroform-methanol-acetic acid-water, 5 0 :25: 7: 3 (v/v) (Skipsky et al. 1963) or, in some experiments, modified to 50:25:9:1 to improve the separation of PS from PI. The chromatoplate was developed for about 2 h and the spots were visualized with iodine vapour. T o distinguish PS from PI, which had very close RF-values (PS-0.90. PI-O.85), the plates were sprayed with 0.2 g of ninhydrin dissolved in 95 ml of n-butanol and 5 ml of acetic acid (10% in water (v/v)) and developed for 20 min at 105°C. PS and PE and the corresponding lysocompounds appeared as pink spots. Dragendorffs reagent was used to detect choline-containing phospholipids, i.e. PC, SM and LPC. Samples were taken from each fraction during the extraction procedure and examined for the presence of phospholipids using t.1.c. Sensitization of rats
Male Sprague-Dawley rats (180-200 g) were injected S.C. in the abdomen with 2 x 0.5 ml of egg albumin (100 mg/ml) and S.C. in the neck with 2 x 0.5 ml of pertussis vaccine as an adjuvant. Mast cells were taken from the rats 2 to 4 weeks after the injections. Isolation of mast cells
Mast cells from the pleural cavities of sensitized rats (about 300-350 g) were isolated essentially as described by Thon and Uvnas (1967). After separation by gradient centrifugation on Ficoll (350 x g) the cells were washed three times at 20°C with an isotonic saline solution containing 10 % (v/v) Sorensen’s phosphate buffer (Na,HPO,+ KH,PO,, 67 mM), pH 6.3, and human serum albumin (I mglml). Pleural mast cells were used since they were regularly observed to be considerably better sensitized than the peritoneal mast cells. Incubation procedure The isolated, sensitized mast cells were counted in a Biirker chamber and suspended in the incubation medium (about 10 000 cells/ml). Unless otherwise stated, the mast cells were incubated with antigen (5-10 ,ug/ml) for 10 min at 25°C in 2.0 ml of medium containing boiled rat serum to a final concentration of 2% (v/v). The standard incubation medium was an isotonic saline solution (NaCI 137 mM, KCI 2.7 mM, CaCI, 1.8 mM, MgCI, 1 mM) containing 10% (v/v) Sorensen’s phosphate buffer (67 mM), pH 6.7 and human serum albumin (0.5 mglml). The incubation was terminated by placing the tubes in ice-water and then centrifuging them (350 x g) for 10 min at 4°C. The supernatants were decanted and 0.2 ml of 0.1 M HCI was added to the cell residues. The supernatants and the cell residues were heated in a boiling water bath for 5 min. By adding 1.8 ml of incubation medium t o the cell residues the volume was readjusted to 2 ml. Histamine was determined in the supernatants and the residues by the method of Shore et al. (1959), omitting the extraction procedure (Bergendorff and Uvnas 1972). Histamine release was expressed as a percentage of the total histamine originally present in the cells. The spontaneous release has been deducted from the values presented. The spontaneous histamine release, calculated from 34 experiments, was 5.4+ 0.2% (meanfSEM) and 5.7f0.4% respectively in the absence and in the presence of 2 % rat serum in the medium.
225
POTENTIATION OF HISTAMINE RELEASE
a
60W
ul
a W _I
40W
I
5
5ul
E
20-
c W z r Y W a -
0-
0
5
10 ANTIGEN
15
20
( pg /ml)
$ I
F Fig. 1. a) Effect of antigen concentration on histamine release from mixed thoracic cells. b) Effect of antigen concentration on histamine release from isolated mast cells. 0-0, Antigen alone; 0-0, antigen in the presence of 2 % rat serum. M+_S.E.M. (n=4).
v)
f 20c W u C W
a
_ _ - --- - 5 - - - - - -
- _ _ _ _ *_ _
00
5
10 ANTIGEN
15
20
( g g /ml )
Potentiation of histamine release All fractions obtained from the lipid extractions and the column chromatography were tested for their potentiating effect on antigen-induced histamine release from rat mast cells. The protein precipitate wss washed with distilled water, then suspended in 10 ml of distilled water and freeze-dried. The residues from the lipid fractions were usually dissolved in a small volume of distilled water and then freeze-dried. The potentiation was calculated using the formula given by Mongar and Svec (1972 b):
Pot
~
~.
(release with antigen +extract) - (release with extract alone) (release with antigen alone) - (spontaneous release)
Results The potentiating effect of rat serum on histamine release induced by antigen Mixed pleural cells challenged with antigen (egg albumin, 10 ,ug/ml) released 30% of their histamine; the histamine release on exposure to antigen in the presence of 2% rat serum was 54% (Fig. 1 a). Isolated mast cells were much less sensitive to the antigen and released only 8 % of their histamine. However, the addition of 2 % rat serum to the medium also promoted the release in this case-to about 27% (Fig. 1 b). Fig. 2 shows the dose-response curve for rat serum. Maximal release was obtained in the presence of 2 % rat serum. The cells isolated from the pleural cavity were found to be more reactive to the antigen in the presence of rat serum than were the peritoneal mast cells (Table I) and therefore pleural mast cells were used throughout the study. The temperature optimum for the release was between 20 and 30°C (Fig. 3). The time I5 - 765876
226
A. SYDBOM AND B. UVNKS
Fig. 2. Effect of rat serum concentration on histamine release from isolated mast cells incubated with antigen (10 ,ug/ml). 0 , Antigen alone; 0 , antigen in the presence of rat serum. M+S.E.M. Figures within brackets show number of experiments. 2
I
0
3
4
SERUM (PER CENT )
30W v)
9 -I @
20-
z W
Ei!
1
10-
,i--n----[j
I-
2 W
0
U
!?
Fig. 3. Effect of temperature o n histamine release from isolated mast cells incubated with antigen (10 ,ug/ml). 0-0, Antigen alone; 0-0, antigen in the presence of 2 % rat serum. MkS.E.M. (n=3).
4’ 6-0-
15 20 25 30 35 40 O C
course for the histamine release at 25°C is shown in Fig. 4. Maximum release is reached after 3 minutes incubation. The serum factor was found to be heat-stable, i.e. the activity was not reduced after boiling the serum or after keeping it at 20°C for 70 h. Human serum, boiled and treated in the same way as the rat serum, also showed a potentiating activity, but was less effective (Fig. 5). Extraction of the serum factor from rat serum
Lipids were extracted from rat serum using chloroform-methanol. The protein precipitate had no detectable potentiating activity. After a Folch extraction of the supernatant both the lower and the upper (more polar) phases showed potentiating activity. When the residue
Fig. 4. Time course of the histamine release from isolated mast cells incubated with antigen (10 ,ug/ml). A-A, Spontaneous release; 0-0, release with antigen alone; 0-0, release with antigen in the presence of 2 % rat serum. M S.E.M. (n = 4).
L
Y
go.. 0
- A ,
,
,
I
2
3
,
,
4 - 5
10 MIN
221
POTENTIATION OF HISTAMINE RELEASE
TABLE 1. The release of histamine from rat mast cells, isolated from the thoracic cavity or the peritoneal cavity, after incubation with antigen (lOpg/ml) in the presence of rat serum (2%). Histamine release percent, + Thoracic mast cells Peritoneal mast cells +
**
23.1 k 3.0** 12.1k2.3
Means and standard errors (n = 8) are given. 0.001 < p
Potentiation of Anaphylactic Histamine Release from Isolated Rat Pleural Mast Cells by Rat Serum Phospholipids BY and B. UVNAS A. SYDBOM Received 18 November 1975
Abstract
SYDBOM,A. and B. UVNAS.Potentiation of anaphylactic histamine release from isolated rat pleural mast cells by rat serum phospholipids. Acta physiol. scand. 1976. 97. 222-232. Isolation of sensitized rat mast cells by density gradient centrifugation in Ficoll decreases the histamine release obtained when they are subsequently exposed to antigen. The histamine release from such isolated cells is potentiated by the addition of 2 7 , boiled rat serum. This potentiation is dose-dependent and has a temperature optimum of about 25°C. The potentiating activity was localized to the serum phospholipid fraction. Of the pure phospholipids studied (LPC, PC, PE, PI, PS and S M ) only phosphatidylserine and lysophosphatidylcholine were found to potentiate the histamine release. The mechanism behind this potentiation is discussed and it is suggested that the potentiation by phosphatidylserine and lysophosphatidylcholine is due to a requirement of these phospholipids for the ion exchange (Na+, K+ and Ca++) o r the adenylcyclase activity essential for the histamine release process.
During early attempts to isolate rat peritoneal mast cells in our laboratory it was observed that such isolated cells had lost their reactivity to degranulating agents like compound 48/80 and antigens (Uvnas and Thon 1959). Such cells also showed a high level of spontaneous histamine release. The isolation procedure involved density gradient centrifugation in Ficoll and several subsequent washings in isotonic saline solution. The presence of 2% rat serum throughout the isolation procedure was found to prevent the loss of cell reactivity. Later, we noticed that albumin (1 mg/ml) could replace rat serum in maintaining the mast cell reactivity to compound 48/80 (Uvnas and Thon 1961), but did not suffice to maintain the response of sensitized mast cells to purified antigens. Evidently, another serum factorpossibly in the lipid fraction-was essential for the development of an antigen-induced mast cell reaction (Uvnas 1968). Other investigators have also reported that the antigen-induced histamine release from isolated rat mast cells is lower than the release from mast cells in mixed peritoneal cell suspensions (Johnson and Moran 1966, Norn 1968). It has also been known for a long time that isolated peritoneal mast cells do not respond to dextran (Lagunoff and Benditt 1960, Goth and Knoohuizen 1962), despite the fact that dextran induces histamine release in 222
POTENTIATION OF HISTAMINE RELEASE
223
in vioo systems (Goth 1967). Goth and Knoohuizen (1962) reported that histamine was released when a lipid extract from rabbit brain was added together with dextran to peritoneal mast cells, even though the lipid extract by itself did not cause any histamine release. Lichtenstein and Osler (1966) reported an enhancing effect of a heat-stable factor in human serum on histamine release from leucocytes induced by ragweed pollen antigen. During isolation of leucocytes the reactivity was lost and could be restored by adding extracts from normal leucocytes (Lichtenstein 1968). The potentiating factor from rabbit brain tissue has been identified as phosphatidylserine (Goth et at. 1971). Later several authors have reported a potentiating effect of phosphatidylserine, phosphatidylinositol or phosphatidylethanolamine on histamine release induced by antigen or dextran (Mongar and Svec 1972a, 1972 b, Foreman and Mongar 1972, Garland and Mongar 1974, Chakravarty et at. 1973, Stechschulte and Austen 1973, 1974). The present paper describes the potentiating effect of bovine phosphatidylserine and lysophosphatidylcholine on the antigen-induced histamine release from isolated rat pleural mast cells. The potentiating effect of rat serum is tentatively ascribed to the presence of the same phospholipids.
Materials and methods Egg albumin was purchased from Difco Laboratories, Detroit, Michigan, USA, Ficoll from AB Pharmacia,
Uppsala, Sweden, and human serum albumin (free from preservatives) from AB Kabi, Stockholm, Sweden. Pertussis vaccine (nr. 299) (2 x IO'O bact./ml) was obtained from SBL, Stockholm, Sweden. Unisil (200-325 mesh) was purchased from Clarkson Chem. Inc., Williarnsport, USA, and Eastman chromatogram sheet 6061 without fluorescence indicator from Eastman Kodak Co., Rochester, N.Y., USA. Phospholipid references were: Phosphatidylserine (type 111 from ox brain) from Sigma Chem. Co., St. Louis, USA (chr. pure ex. bovine brain) Koch Light Labs., Colnbrook, Bucks, England and (chr. pure) Grand Island Biological Comp., Grand Island, N.Y., USA. Phosphatidylethanolaminefrom Koch Light Labs. and Grand Island Biological Co. Phosphatidylcholine (L-a-lecithin from egg, type 111 E) from Sigma Chem. Co. Phosphatidylinositol (chr. pure) from Grand Island Biological Co., Supelco Inc., Bellefonte, Penn., USA and Analabs Inc., North Haven, Connecticut, USA. Lysophosphatidylcholine and sphingomyelin from Sigma Chem. Co. All other chemicals were obtained from the usual commercial sources. Abbreviations used: LPC PC PE PI PS SM
lysophosphatidylcholine phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine sphingomyelin
Preparation of rat serum Male Sprague-Dawley rats were anaesthetized with ether, the carotids were cut and the blood was collected in a beaker and allowed to stand for 1 h at 20°C and then overnight at 4°C. The serum was centrifuged (400 x g) and stored in 5 ml portions at - 20°C. If the serum was to be boiled it was first diluted with saline t o 20% (v/v) and then heated in a boiling water-bath for 10 min. After centrifugation, the supernatant was stored in 2 ml portions at - 20°C. Purification of the serum factor Extracrion of lipids. The total lipids were extracted from rat serum essentially according to Nelson and Freeman (1959). Chloroform-methanol ( I : I ) (332 ml) was added to 20 ml of rat serum under a stream of nitrogen and
224
A. SYDBOM AND B. UVNLS
protected from light. After agitation, the mixture was incubated for IS min at 60°C. The mixture was cooled to 0°C and diluted with chloroform to a final volume of 500 ml. The precipitated proteins were separated from the supernatant by filtration. The volume of the filtrate was adjusted to 500 ml by adding chloroform and it was then transferred to a separating funnel and 100 ml of distilled water was added. After agitation and phase separation for 3 h at 4”C, the lower phase was separated and evaporated to dryness and the upper phase was stored frozen under nitrogen. Separation of phospholipids. Phospholipids were separated from neutral lipids by chromatography on silicic acid according to Colacicco and Rapport (1967). Eight grams of Unisil silicic acid (200-325 mesh) was dispersed in 20 ml of chloroform-methanol-ammonia, 80: 20: 2 (v/v), poured into a glass column (diameter 1 cm), and washed with 100 ml of the solvent mixture. The residue of the lower phase from the extraction was dissolved in 2 ml of chloroform-methanol-ammonia 80:20:2 (v/v) and put on to the column. After elution with 150 ml of chloroform-methanol-ammonia 80: 20 :2 (v/v), the phospholipids were eluted with 100 ml of ethanol and collected in 10 fractions of 10 ml each. The fractions were evaporated to dryness. Thin layer chromatography (t.1.c.). Eastman precoated “chromatoplates”, with a 0.1 mm silicic acid layer, were used. The plates were activated for 60 min at 110°C. Samples were dissolved in small volumes of chloroform or chloroform-methanol, 1 : 1 (v/v), and applied to the plates. The solvent system used was chloroform-methanol-acetic acid-water, 5 0 :25: 7: 3 (v/v) (Skipsky et al. 1963) or, in some experiments, modified to 50:25:9:1 to improve the separation of PS from PI. The chromatoplate was developed for about 2 h and the spots were visualized with iodine vapour. T o distinguish PS from PI, which had very close RF-values (PS-0.90. PI-O.85), the plates were sprayed with 0.2 g of ninhydrin dissolved in 95 ml of n-butanol and 5 ml of acetic acid (10% in water (v/v)) and developed for 20 min at 105°C. PS and PE and the corresponding lysocompounds appeared as pink spots. Dragendorffs reagent was used to detect choline-containing phospholipids, i.e. PC, SM and LPC. Samples were taken from each fraction during the extraction procedure and examined for the presence of phospholipids using t.1.c. Sensitization of rats
Male Sprague-Dawley rats (180-200 g) were injected S.C. in the abdomen with 2 x 0.5 ml of egg albumin (100 mg/ml) and S.C. in the neck with 2 x 0.5 ml of pertussis vaccine as an adjuvant. Mast cells were taken from the rats 2 to 4 weeks after the injections. Isolation of mast cells
Mast cells from the pleural cavities of sensitized rats (about 300-350 g) were isolated essentially as described by Thon and Uvnas (1967). After separation by gradient centrifugation on Ficoll (350 x g) the cells were washed three times at 20°C with an isotonic saline solution containing 10 % (v/v) Sorensen’s phosphate buffer (Na,HPO,+ KH,PO,, 67 mM), pH 6.3, and human serum albumin (I mglml). Pleural mast cells were used since they were regularly observed to be considerably better sensitized than the peritoneal mast cells. Incubation procedure The isolated, sensitized mast cells were counted in a Biirker chamber and suspended in the incubation medium (about 10 000 cells/ml). Unless otherwise stated, the mast cells were incubated with antigen (5-10 ,ug/ml) for 10 min at 25°C in 2.0 ml of medium containing boiled rat serum to a final concentration of 2% (v/v). The standard incubation medium was an isotonic saline solution (NaCI 137 mM, KCI 2.7 mM, CaCI, 1.8 mM, MgCI, 1 mM) containing 10% (v/v) Sorensen’s phosphate buffer (67 mM), pH 6.7 and human serum albumin (0.5 mglml). The incubation was terminated by placing the tubes in ice-water and then centrifuging them (350 x g) for 10 min at 4°C. The supernatants were decanted and 0.2 ml of 0.1 M HCI was added to the cell residues. The supernatants and the cell residues were heated in a boiling water bath for 5 min. By adding 1.8 ml of incubation medium t o the cell residues the volume was readjusted to 2 ml. Histamine was determined in the supernatants and the residues by the method of Shore et al. (1959), omitting the extraction procedure (Bergendorff and Uvnas 1972). Histamine release was expressed as a percentage of the total histamine originally present in the cells. The spontaneous release has been deducted from the values presented. The spontaneous histamine release, calculated from 34 experiments, was 5.4+ 0.2% (meanfSEM) and 5.7f0.4% respectively in the absence and in the presence of 2 % rat serum in the medium.
225
POTENTIATION OF HISTAMINE RELEASE
a
60W
ul
a W _I
40W
I
5
5ul
E
20-
c W z r Y W a -
0-
0
5
10 ANTIGEN
15
20
( pg /ml)
$ I
F Fig. 1. a) Effect of antigen concentration on histamine release from mixed thoracic cells. b) Effect of antigen concentration on histamine release from isolated mast cells. 0-0, Antigen alone; 0-0, antigen in the presence of 2 % rat serum. M+_S.E.M. (n=4).
v)
f 20c W u C W
a
_ _ - --- - 5 - - - - - -
- _ _ _ _ *_ _
00
5
10 ANTIGEN
15
20
( g g /ml )
Potentiation of histamine release All fractions obtained from the lipid extractions and the column chromatography were tested for their potentiating effect on antigen-induced histamine release from rat mast cells. The protein precipitate wss washed with distilled water, then suspended in 10 ml of distilled water and freeze-dried. The residues from the lipid fractions were usually dissolved in a small volume of distilled water and then freeze-dried. The potentiation was calculated using the formula given by Mongar and Svec (1972 b):
Pot
~
~.
(release with antigen +extract) - (release with extract alone) (release with antigen alone) - (spontaneous release)
Results The potentiating effect of rat serum on histamine release induced by antigen Mixed pleural cells challenged with antigen (egg albumin, 10 ,ug/ml) released 30% of their histamine; the histamine release on exposure to antigen in the presence of 2% rat serum was 54% (Fig. 1 a). Isolated mast cells were much less sensitive to the antigen and released only 8 % of their histamine. However, the addition of 2 % rat serum to the medium also promoted the release in this case-to about 27% (Fig. 1 b). Fig. 2 shows the dose-response curve for rat serum. Maximal release was obtained in the presence of 2 % rat serum. The cells isolated from the pleural cavity were found to be more reactive to the antigen in the presence of rat serum than were the peritoneal mast cells (Table I) and therefore pleural mast cells were used throughout the study. The temperature optimum for the release was between 20 and 30°C (Fig. 3). The time I5 - 765876
226
A. SYDBOM AND B. UVNKS
Fig. 2. Effect of rat serum concentration on histamine release from isolated mast cells incubated with antigen (10 ,ug/ml). 0 , Antigen alone; 0 , antigen in the presence of rat serum. M+S.E.M. Figures within brackets show number of experiments. 2
I
0
3
4
SERUM (PER CENT )
30W v)
9 -I @
20-
z W
Ei!
1
10-
,i--n----[j
I-
2 W
0
U
!?
Fig. 3. Effect of temperature o n histamine release from isolated mast cells incubated with antigen (10 ,ug/ml). 0-0, Antigen alone; 0-0, antigen in the presence of 2 % rat serum. MkS.E.M. (n=3).
4’ 6-0-
15 20 25 30 35 40 O C
course for the histamine release at 25°C is shown in Fig. 4. Maximum release is reached after 3 minutes incubation. The serum factor was found to be heat-stable, i.e. the activity was not reduced after boiling the serum or after keeping it at 20°C for 70 h. Human serum, boiled and treated in the same way as the rat serum, also showed a potentiating activity, but was less effective (Fig. 5). Extraction of the serum factor from rat serum
Lipids were extracted from rat serum using chloroform-methanol. The protein precipitate had no detectable potentiating activity. After a Folch extraction of the supernatant both the lower and the upper (more polar) phases showed potentiating activity. When the residue
Fig. 4. Time course of the histamine release from isolated mast cells incubated with antigen (10 ,ug/ml). A-A, Spontaneous release; 0-0, release with antigen alone; 0-0, release with antigen in the presence of 2 % rat serum. M S.E.M. (n = 4).
L
Y
go.. 0
- A ,
,
,
I
2
3
,
,
4 - 5
10 MIN
221
POTENTIATION OF HISTAMINE RELEASE
TABLE 1. The release of histamine from rat mast cells, isolated from the thoracic cavity or the peritoneal cavity, after incubation with antigen (lOpg/ml) in the presence of rat serum (2%). Histamine release percent, + Thoracic mast cells Peritoneal mast cells +
**
23.1 k 3.0** 12.1k2.3
Means and standard errors (n = 8) are given. 0.001 < p
