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Respiration of rat peritoneal mast cells during histamine release induced by antigen-antibody reaction
Q
160
Experimental
RESPIRATION CELLS
DURING
1968
RELEASE
ANTIGEN-ANTIBODY
Academic Press Inc.
Cell Research 49, 160-168 (196S)
OF RAT PERITONEAL HISTAMINE
by
MAST INDUCED
BY
REACTION
N. CHAKRAVARTY Department
of Pharmacology, Received
University
of Umed, Umed, Sweden
March 24, 1967’
?IHE oxygen dependence of anaphylactic histamine release in guinea pig tissue, first demonstrated by Parrot [19], has since been confirmed in many laboratories [l, 14, 17, 221. Histamine release in rat tissue was found to be resistant to oxygen lack in Tyrode solution which contains glucose [ 11, but an inhibitory effect of anoxia could be demonstrated in the absence of glucose [S]. A likely explanation for the inhibition of histamine release by anoxia is that oxygen is required for maintaining an active energy metabolism which is involved in the release process. If histamine release requires the expenditure of extra energy this could be provided by oxidative metabolism, and a stimulation of oxygen uptake in close time relation to the histamine release would be expected. The respiration of guinea pig lung during anaphylactic histamine release has been studied in the past with variable results. The author found no appreciable change [2] while others have reported some stimulation [ll, 161. The mast cells, which store and release histamine [la, 201, constitute only a minute fraction of the respiring tissue in guinea pig lung. ,4 measure of lung respiration cannot therefore reflect changes in the oxygen uptake by the mast cells. The possibility that the respiration of a pure population of mast cells might be influenced during anaphylactic histamine release thus remained to be explored. The experiments reported here using pure rat peritoneal mast cells show that, under suitable experimental conditions, a transient stimulation of respiration occurs immediately after contact with the antigen which triggers anaphylactic histamine release.
MATERIAL
Male W&tar rats weighing tions of 25 mg egg albumin 1 Revised Experimental
version
received
Cell Research 49
AND
METHODS
200-250 g were sensitized by two subcutaneous injecon consecutive days along with H. pertussis vaccine
May 22, 1967.
Mast cell respiration
during
histamine
release
161
(2 x 10’0 bacilEPalso subcutaneouslyPon the first day. After an interval of IO-20 days peritoneal cell suspensions were collected pooling cells from two rats for each experiment. Mast cells were isolated from the mixed peritoneal cell suspension in concentrated human albumin solution as described previously [3, 71. The Cartesian diver technique [9, 12, 231 was used for the respiration studies. Silicone coated ampulla divers were especially adapted to permit mixing of mast cells with antigen within the diver. Details of this method have already been described 141. After a brief record of the initial respiration rate at 37°C the cells were brought in contact with the antigen solution placed as a drop on the wall of the diver, and the respiration measurement continued. The time between killing of the rats and the beginning of the respiration measurement was about 4 h. The cells were kept cold (0-4°C) throughout this interval except for the brief period of filling the diver, The cells in the diver were viewed under a dissection microscope after each experiment and after some they were stained with toluidine blue for closer inspection by introducing a fixing staining solution [7] into the diver. Histamine release following incubation with antigen was determined separateI\ in a sample of the mast cell suspension using the same method as for mixed peritoneal cell suspension [5]. The cells were incubated for 5 min initially for warming up to 37°C and for another 5 min with antigen. Histamine was tested by the biological method on atropinized guinea pig ileum.
RESULTS With the experimental set up used here it was possible to measure accurately changes in the respiration rate of mast cells caused by the addition of antigen. No attempt was made to measure the absolute respiration rate. However, in order to ensure that the method gives a reliable measure of mast cell respiration the absolute values were determined by this method in 3 experiments using Sprague-Dawley rats. The size of the sample was determined by counting the number of mast cells in a test tube before and after transferring a portion of the cells to the diver. The difference (.?O,OOO200,000 cells) was taken to be the number Lvithin the diver. The mean respiration rate by this method in presence of 5 mM glucose was found to be 0.41 X 1OP PI/cell/h. This agrees quite \vell with the respiration rate (0.47 x 1OP $/cell/h) of Sprague-Dawley rat mast cells found previousl> [7 I. .A larger error by the present method is inevitable because of the pipetting lechniquc and the indirect method of counting, and the absolute respiration rate obtained by the previous method [7] in xvhich the size of the sample was determined by counting the cells lvithin the diver must be considered more accurate. The size of the sample, however, has less relevance to the present set up because oxygen uptake by the same cell sample is determined before and after mixing with antigen. Experimental
Cell
Research
49
162
N. Chakravarty
The effect of antigen on the sensitized mast cells was remarkably different depending on whether glucose was present in the suspending medium or not. Antigen in low concentrations (0.08-0.16 mg/ml) caused very characteristic changes in the oxygen uptake of the mast cells in all 4 experiments in which no glucose was added to the medium (Figs l-4). An increase of oxygen uptake amounting to 26-37 per cent stimulation was observed as soon as reading could be resumed after mixing with antigen. The interval between the addition of antigen and the first reading after that was about 5 min. The increased oxygen uptake was maintained for 15-20 min after mixing. The respiration rate then returned to the original level or very close to it. The change in presence of glucose was, in contrast, less noticeable and less characteristic. As illustrated in Fig. 5 the antigen caused only a slight stimulation (8-9 per cent) of the oxygen uptake and the two phases, so characteristic in the absence of glucose, could not be distinguished. The control experiments, in which the antigen (egg albumin: 0.14-0.18 mg/ml) was added to unsensitized mast cells, showed either no effect or a slight depression of respiration (Fig. 6). Glucose was added to the medium in one of the controls and omitted from the other. Further details of the experiments are presented in Table 1. The concentration of the antigen in the side-drop was 3.5 to 8 times higher than the concentration after complete mixing. With this range of values, as pointed out before [4], the concentration of antigen with which the cells come in contact immediately on mixing cannot be greatly different from the final concentration after complete diffusion. In some experiments however, the concentration at the time of contact of the cells with the antigen could be about double the final concentration, It may be seen that the mixing pressures were relatively low, thus keeping any disturbance of gas exchange after mixing to a minimum [4]. The histamine release from sensitized cells with antigen was
Figs l-4.-Oxygen uptake of sensitized mast cells in a glucose-free medium before and after contact with antigen. Figs l-4 represent Expts 1-4 respectively in Table 1. The respiration was stimulated (26-37 per cent) for 15-20 min after contact with antigen, and then returned to the original level. The dotted line is a projection of the initial slope. In Expt 3 (Fig. 3) the manometer was briefly opened to air between the third and the fourth readings after mixing. Fig. 5.-The effect of antigen on the oxygen uptake of sensitized mast cells in presence Antigen was added at the arrow. This figure represents Expt 5 in Table 1.
of glucose.
Fig. 6.-The at the arrow.
was added
Abscissae: Experimental
effect of antigen (egg albumin) on unsensitized This figure represents Expt 7 in Table 1.
minutes; Cell
ordinates: Research
oxygen 49
uptake
x 10-l
~1.
mast
cells.
Egg
albumin
Masf cell respiration
during
histamine
release
163
1 .I
0.6 -
o.0.: /
‘~ANTIGEN
. /
I:
02 -
: / 1’: : :
: 1’ 1’ 1’ : 1’ : 1’ t’
/ 4
/ <
30
Fig.
1.
Fig. 2
/I
J
/
/
.f
Fig.
60
5.
j-
0.7:
O.!
j08
/ i\ J
./ j-
0.2:
ANTIGEN
/
06
ANTIGEN
/
./’
.I / 2/
/
/60
Fig.
120
3.
c
L
I-
60
Fig.
120
4.
0
30
Fig. Experimentnl
60
90
6.
Cell Research
49
N. Chakravarty
Fig. ‘I.-Photomicrograph of unsensitized (Expt 8 in Table 1). Stained with toluidine normal.
mast cells in the diver after blue. Microscopic enlargement:
incubation with antigen x 160. The cells appear
I;ig. 8.-Photomicrograph of sensitized mast cells in the diver after incubation with (Expt 1 in Table 1). Stained with toluidine blue. Microscopic enlargement: x 160. The slightly swollen with irregular margin and protruding granules.
antigen cells are
21-U per cent, the spontaneous release varying from an undetectable amount to 6 per cent. The purity of the cells is shown in the last column. The contamination was low: 98-100 per cent of the cells were mast cells. In some experiments a fixing staining solution was introduced into the diver at the end of respiration measurements and the cells were photographed. Fig. 7 shows unsensitized mast cells and Fig. 8 sensitized mast experiment cells, both after exposure to antigen. The cells in the control (Fig. 7) look normal with sharp outline and densely packed granules. The sensitized cells (Fig. 8) also appear to be quite well preserved. The only differences from the control cells are an irregular margin with protruding granules and some amount of swelling of the mast cells. The effect of egg albumin on histamine release from the unsensitized cells have not been Experimental
Cell
Research
49
Mast cell respiration during histnmine release
165
observed in the two control experiments reported here (Expts 7 and 8, Table 1) but this question has been investigated separately. 0.1 mgjml, 0.5 mg/ml and 1.0 mg/ml egg albumin gave 2.0, 2.1 and 4.5 per cent histamine release respectively from Wistar rat mast cells, the spontaneous release 1.
TABLE
96Increase (+) or Glucose Gas in volume medium of for diver cells ,Zxpt (pl) (mM)
Final Cont. of cone. of Initial Mixing Initial antigen antigen pressure: pressure: oxygen cm uptake: in side- after cm drop mixing Brodie’s Brodie’s x lo-* SOl. (mg/ml) (mglml) sol. /4/h
Oxygen decrease uptake (-) in after Histamine Purity oxygen adding uptake release’ 01 antigen: after p mast x10-” adding Sponta- With cell PI/h antigen neous antigen (96)
Sessitized masl cells 1 2 3 4 5 6
13.4 5.8 12.2 9.7 13.5 2l.G
0 0 0 0 5 5
0.7 0.7 0.7 0.8 0.7 0.7
0.0s 0.09 0.16 0.10 0.15 0.20
-56 t17 -38 -13 -15 -12
t33 tfi5 t 27 t 28 t 68 t56
7.50 3.56 5.03 4.06 14.62 9.15
9.67 4.48 6.38 5.56 16.00 9.86
+29 t26 -27 t37 t 9.4 f 7.8
1.0 1.1
0.18 0.14
-69 -35
$32 t 32
4.47 2.44
4.02 2.34
-10 - 4.1
2.3 5.4 i 3.0 5.11 6.2 3.8
32 21 15 24 44 41
99.5 99 98 100 99 99
Unsensifizrd mast cells , m 4
13.3 9.4
0 5
99.5 100
a Histamine release was tested with a sample of the isolated cells using the same antigen cont. as the final coat. in the diver.
being undetectable ( < 1.2 per cent). The histamine release which the concentrations of egg albumin, used in the control experiments, could give is thus negligible. DISCUSSION
Anaphylactic histamine release from mast cells was accompanied by 30 per cent stimulation of their respiration in the absence of glucose. The stimulation was transient lasting 15 to 20 min after which normal respiration was restored. The change was very characteristic and was uniformly seen in all the 4 experiments. The mean increase in oxygen uptake was 1.49 x 1O-2 l/h which is far above the slight variations in the slope that may occur in
166
N. Chakravarty
control divers without cells. The mean gas exchange after mixing in contro1 divers was 0.066 X 10e2 ,ul/h [4]. This value would be interpreted as increased gas uptake but is of another order of magnitude differing from increased respiratory uptake by the mast cells by a factor of 22. Whenrespiring cells are used the slope changes in simulated mixing experiments were even less or none at all as shown in a previous paper [4]. Mast cell respiration was only slightly stimulated (8-9 per cent) in presence of glucose. The two phases, clearly seen in the absence of glucose, were not observed now, and it would hardly be possible to distinguish them at this low level of stimulation. Exposure of unsensitized mast cells to antigen did not change their oxygen uptake appreciably or caused a slight depression (10 per cent). Mongar and Perera [ 131, using a micro capillary respirometer, were unable to show any increase in mast cell respiration during antigen induced histamine release. Their absolute value for mast cell respiration in presence of glucose and with air as the gas phase was 1.2 x 1O-5 ,ul/cell/h. This is about 25 times higher than the value found by us [7] for mast cells from SpragueDawley rats. This factor would be even higher if the values in glucose-free medium were compared. A part of the difference may be due to the strain difference, because mast cells from Wistar rats used by them [ 131 may have a higher respiration rate. The respiration rate of Wistar rat mast cells has indeed been found in a recent study by Kahl and Netter [lo] to be 0.89 X 10~~ pi/cell/h which is about double as high as the respiration of mast cells from Sprague-Dawley rats. Even after taking this higher respiration rate into account the values of Mongar and Perera differ by a factor around 10. The basal respiration of rat mast cells found by us agrees in the order of magnitude with that of Kieler (unpublished observation, see [7]), Kahl and Netter [lo] and in general with the respiration of leucocytes [18]. The respiration rate reported by Mongar and Perera [13] thus seems to be around 10 times too high and at this level any change caused by antigen would hardly be detectable. The antigen used by them was also different (2 per cent horse serum). Inspection of the mast cells at the end of the experiment showed that the antigen caused in general mild or moderate changes in the cells (Figs 7 it and 8). Judging from studies using light [ 5 ] and electron [6] microscopy appears that the antigen causes some swelling of the cell with redistribution of granules many of which protrude through the cell membrane and are in contact with the extracellular fluid. The cell membrane usually shows localized disruption releasing a few granules. More severe changes are less frequent, especially with low concentrations of antigen. The typical mast cell Experimental
Cell Research
49
Mast cell respiration
during
histamine
release
167
in anaphylaxis thus shows some morphological and functional disturbances but its metabolic activity-judging from the respiration rate utilizing endogenous substrate-remains unimpaired after a brief period of initial stimulation. In attempting to interpret the brief respiratory stimulation one is faced with the question: what is the physiological significance of the stimulation and why does it become more apparent in the absence of glucose? Histamine is released within a few seconds of contact with the releaser [21]. If the triggering mechanism itself requires extra energy the respiratory stimulation, which persists for 15-20 min, might restore the energy balance again. This view would be compatible with the hypothesis that oxidative energ? metabolism is directly concerned with the histamine release process. The unmasking of the stimulant effect on respiration in the absence of glucose seems to be consistent with the metabolic pattern of the mast cell. Glucose causes 60 per cent stimulation of mast cell respiration [7]. It is quite conceivable that at this enhanced rate of oxygen uptake the stimulatory mechanism set in motion by antigen-antibody reaction may be less pronounced and largely masked by the more effective stimulation produced by glucose itself. Glucose might also influence the antigen effect on respiration in another way. The mast cell has an active glycolytic pathway-both aerobic and anaerobic [3]. By stimulating aerobic glycolysis glucose could provide an alternate source of energy and thus indirectly suppress the stimulant ef‘fect on respiration. The mast cell seems to be rich in endogenous substrate [3, 71, which could very well provide the fuel for the short-lived stimulation of respiration in a glucose-free medium.
SUMMARY
The respiration of isolated peritoneal mast cells from sensitized rats was stimulated 26-37 per cent above the basal level immediately after contact with antigen which released 21-32 per cent of their histamine content. The stimulation was short-lived lasting 15 to 20 minutes. This phenomenon was observed characteristically in the absence of glucose in the medium. In presence of glucose the stimulation was much less pronounced. The finding is consistent with the view that oxidative energy metabolism is directly involved in the process of anaphylactic histamine release. This work
was supported
by the Swedish
Medical
Research
Council
(Project
No.
13X-532-02). Experimental
Cell
Research
49
168
N. Chakravariy REFERENCES N., Acta Physiol. Stand. Am. .I. Physiol. 203, 1193 (1962). J. Cell Biol. 25, 123 (1965). Exptl Cell Res. 47, 278 (1967). To be published (1967).
1. CHAKRAVARTY,
2. 3. 4. 5. 6.
-~ -~ --~
-~ CHAKRAVARTY,
N.,
GLJSTAFSOX,
G. and
48, 146 (1960).
PIHL,
E.,
Acta
Pathol.
Microbial.
Stand.
In press
(1967). 7. CHAKRAVARTY, N. and ZEUTHEN, E., J. Cell Biol. 25, 113 (1965). 8. DIAMANT, B., Acta Physiol. Stand. 55, 11 (1962). 9. HOLTER, H., Compt. Rend. Lab. Carlsberg, Ser. Chim. 24, 399 (1943). 10. KAHL, G.-F. and NETTER, K. J., Naun. Arch. Pharm. Exptl Path. 256, 55 (1967). 11. KAISER, E., RAAB, W. and FARKOUH, E., K/in. Wochschr. 43, 1118 (1965). 12. LINDERSTRBM-LANG, K., Compt. Rend. Lab. Carlsberg, Ser. Chim. 24, 333 (1943). 13. MONGAR, J. L. and PERERA, B. A. V., Immunol. 8, 511 (1965). 14. MOSGAR, J. L. and SCHILD, H. O., J. Physiol. (London) 135, 301 (1957). 15. MOTA, I. and VUGMAN, I., Nature 177, 427 (1956). 16. MOUSSATCHS, H. and DANON, A. P., Anais Acad. Brasil. Cien. 28, 23 (1956).
17. -Experientia 16, 118 (1960). 18. OREN, R., FARNHAM, K., MILOFSKY, E. and KARNOVSKY, M. L., J. Cell Biol. 17, 487 (1963). 19. PARROT, J. L., Compt. Rend. Sot. Biol. (Paris) 136, 361 (1942). 20. RILEY, J. F. and WEST, G. B., J. Physiol. (London) 120, 528 (1953). 21. U~NKS: B., Ann. N.Y. Acad. Sci. 116, 880 (1964). 22. YAMASAKI, H., MURAOKA, S. and ENDO, K., Japan. .I. Pharmacol. 10, 21 (1960). 23. ZEUTHEN, I?., J. Embryol. Expfl Morphol. 1, 239 (1953).
Experimenlal
Cell
Research
49
160
Experimental
RESPIRATION CELLS
DURING
1968
RELEASE
ANTIGEN-ANTIBODY
Academic Press Inc.
Cell Research 49, 160-168 (196S)
OF RAT PERITONEAL HISTAMINE
by
MAST INDUCED
BY
REACTION
N. CHAKRAVARTY Department
of Pharmacology, Received
University
of Umed, Umed, Sweden
March 24, 1967’
?IHE oxygen dependence of anaphylactic histamine release in guinea pig tissue, first demonstrated by Parrot [19], has since been confirmed in many laboratories [l, 14, 17, 221. Histamine release in rat tissue was found to be resistant to oxygen lack in Tyrode solution which contains glucose [ 11, but an inhibitory effect of anoxia could be demonstrated in the absence of glucose [S]. A likely explanation for the inhibition of histamine release by anoxia is that oxygen is required for maintaining an active energy metabolism which is involved in the release process. If histamine release requires the expenditure of extra energy this could be provided by oxidative metabolism, and a stimulation of oxygen uptake in close time relation to the histamine release would be expected. The respiration of guinea pig lung during anaphylactic histamine release has been studied in the past with variable results. The author found no appreciable change [2] while others have reported some stimulation [ll, 161. The mast cells, which store and release histamine [la, 201, constitute only a minute fraction of the respiring tissue in guinea pig lung. ,4 measure of lung respiration cannot therefore reflect changes in the oxygen uptake by the mast cells. The possibility that the respiration of a pure population of mast cells might be influenced during anaphylactic histamine release thus remained to be explored. The experiments reported here using pure rat peritoneal mast cells show that, under suitable experimental conditions, a transient stimulation of respiration occurs immediately after contact with the antigen which triggers anaphylactic histamine release.
MATERIAL
Male W&tar rats weighing tions of 25 mg egg albumin 1 Revised Experimental
version
received
Cell Research 49
AND
METHODS
200-250 g were sensitized by two subcutaneous injecon consecutive days along with H. pertussis vaccine
May 22, 1967.
Mast cell respiration
during
histamine
release
161
(2 x 10’0 bacilEPalso subcutaneouslyPon the first day. After an interval of IO-20 days peritoneal cell suspensions were collected pooling cells from two rats for each experiment. Mast cells were isolated from the mixed peritoneal cell suspension in concentrated human albumin solution as described previously [3, 71. The Cartesian diver technique [9, 12, 231 was used for the respiration studies. Silicone coated ampulla divers were especially adapted to permit mixing of mast cells with antigen within the diver. Details of this method have already been described 141. After a brief record of the initial respiration rate at 37°C the cells were brought in contact with the antigen solution placed as a drop on the wall of the diver, and the respiration measurement continued. The time between killing of the rats and the beginning of the respiration measurement was about 4 h. The cells were kept cold (0-4°C) throughout this interval except for the brief period of filling the diver, The cells in the diver were viewed under a dissection microscope after each experiment and after some they were stained with toluidine blue for closer inspection by introducing a fixing staining solution [7] into the diver. Histamine release following incubation with antigen was determined separateI\ in a sample of the mast cell suspension using the same method as for mixed peritoneal cell suspension [5]. The cells were incubated for 5 min initially for warming up to 37°C and for another 5 min with antigen. Histamine was tested by the biological method on atropinized guinea pig ileum.
RESULTS With the experimental set up used here it was possible to measure accurately changes in the respiration rate of mast cells caused by the addition of antigen. No attempt was made to measure the absolute respiration rate. However, in order to ensure that the method gives a reliable measure of mast cell respiration the absolute values were determined by this method in 3 experiments using Sprague-Dawley rats. The size of the sample was determined by counting the number of mast cells in a test tube before and after transferring a portion of the cells to the diver. The difference (.?O,OOO200,000 cells) was taken to be the number Lvithin the diver. The mean respiration rate by this method in presence of 5 mM glucose was found to be 0.41 X 1OP PI/cell/h. This agrees quite \vell with the respiration rate (0.47 x 1OP $/cell/h) of Sprague-Dawley rat mast cells found previousl> [7 I. .A larger error by the present method is inevitable because of the pipetting lechniquc and the indirect method of counting, and the absolute respiration rate obtained by the previous method [7] in xvhich the size of the sample was determined by counting the cells lvithin the diver must be considered more accurate. The size of the sample, however, has less relevance to the present set up because oxygen uptake by the same cell sample is determined before and after mixing with antigen. Experimental
Cell
Research
49
162
N. Chakravarty
The effect of antigen on the sensitized mast cells was remarkably different depending on whether glucose was present in the suspending medium or not. Antigen in low concentrations (0.08-0.16 mg/ml) caused very characteristic changes in the oxygen uptake of the mast cells in all 4 experiments in which no glucose was added to the medium (Figs l-4). An increase of oxygen uptake amounting to 26-37 per cent stimulation was observed as soon as reading could be resumed after mixing with antigen. The interval between the addition of antigen and the first reading after that was about 5 min. The increased oxygen uptake was maintained for 15-20 min after mixing. The respiration rate then returned to the original level or very close to it. The change in presence of glucose was, in contrast, less noticeable and less characteristic. As illustrated in Fig. 5 the antigen caused only a slight stimulation (8-9 per cent) of the oxygen uptake and the two phases, so characteristic in the absence of glucose, could not be distinguished. The control experiments, in which the antigen (egg albumin: 0.14-0.18 mg/ml) was added to unsensitized mast cells, showed either no effect or a slight depression of respiration (Fig. 6). Glucose was added to the medium in one of the controls and omitted from the other. Further details of the experiments are presented in Table 1. The concentration of the antigen in the side-drop was 3.5 to 8 times higher than the concentration after complete mixing. With this range of values, as pointed out before [4], the concentration of antigen with which the cells come in contact immediately on mixing cannot be greatly different from the final concentration after complete diffusion. In some experiments however, the concentration at the time of contact of the cells with the antigen could be about double the final concentration, It may be seen that the mixing pressures were relatively low, thus keeping any disturbance of gas exchange after mixing to a minimum [4]. The histamine release from sensitized cells with antigen was
Figs l-4.-Oxygen uptake of sensitized mast cells in a glucose-free medium before and after contact with antigen. Figs l-4 represent Expts 1-4 respectively in Table 1. The respiration was stimulated (26-37 per cent) for 15-20 min after contact with antigen, and then returned to the original level. The dotted line is a projection of the initial slope. In Expt 3 (Fig. 3) the manometer was briefly opened to air between the third and the fourth readings after mixing. Fig. 5.-The effect of antigen on the oxygen uptake of sensitized mast cells in presence Antigen was added at the arrow. This figure represents Expt 5 in Table 1.
of glucose.
Fig. 6.-The at the arrow.
was added
Abscissae: Experimental
effect of antigen (egg albumin) on unsensitized This figure represents Expt 7 in Table 1.
minutes; Cell
ordinates: Research
oxygen 49
uptake
x 10-l
~1.
mast
cells.
Egg
albumin
Masf cell respiration
during
histamine
release
163
1 .I
0.6 -
o.0.: /
‘~ANTIGEN
. /
I:
02 -
: / 1’: : :
: 1’ 1’ 1’ : 1’ : 1’ t’
/ 4
/ <
30
Fig.
1.
Fig. 2
/I
J
/
/
.f
Fig.
60
5.
j-
0.7:
O.!
j08
/ i\ J
./ j-
0.2:
ANTIGEN
/
06
ANTIGEN
/
./’
.I / 2/
/
/60
Fig.
120
3.
c
L
I-
60
Fig.
120
4.
0
30
Fig. Experimentnl
60
90
6.
Cell Research
49
N. Chakravarty
Fig. ‘I.-Photomicrograph of unsensitized (Expt 8 in Table 1). Stained with toluidine normal.
mast cells in the diver after blue. Microscopic enlargement:
incubation with antigen x 160. The cells appear
I;ig. 8.-Photomicrograph of sensitized mast cells in the diver after incubation with (Expt 1 in Table 1). Stained with toluidine blue. Microscopic enlargement: x 160. The slightly swollen with irregular margin and protruding granules.
antigen cells are
21-U per cent, the spontaneous release varying from an undetectable amount to 6 per cent. The purity of the cells is shown in the last column. The contamination was low: 98-100 per cent of the cells were mast cells. In some experiments a fixing staining solution was introduced into the diver at the end of respiration measurements and the cells were photographed. Fig. 7 shows unsensitized mast cells and Fig. 8 sensitized mast experiment cells, both after exposure to antigen. The cells in the control (Fig. 7) look normal with sharp outline and densely packed granules. The sensitized cells (Fig. 8) also appear to be quite well preserved. The only differences from the control cells are an irregular margin with protruding granules and some amount of swelling of the mast cells. The effect of egg albumin on histamine release from the unsensitized cells have not been Experimental
Cell
Research
49
Mast cell respiration during histnmine release
165
observed in the two control experiments reported here (Expts 7 and 8, Table 1) but this question has been investigated separately. 0.1 mgjml, 0.5 mg/ml and 1.0 mg/ml egg albumin gave 2.0, 2.1 and 4.5 per cent histamine release respectively from Wistar rat mast cells, the spontaneous release 1.
TABLE
96Increase (+) or Glucose Gas in volume medium of for diver cells ,Zxpt (pl) (mM)
Final Cont. of cone. of Initial Mixing Initial antigen antigen pressure: pressure: oxygen cm uptake: in side- after cm drop mixing Brodie’s Brodie’s x lo-* SOl. (mg/ml) (mglml) sol. /4/h
Oxygen decrease uptake (-) in after Histamine Purity oxygen adding uptake release’ 01 antigen: after p mast x10-” adding Sponta- With cell PI/h antigen neous antigen (96)
Sessitized masl cells 1 2 3 4 5 6
13.4 5.8 12.2 9.7 13.5 2l.G
0 0 0 0 5 5
0.7 0.7 0.7 0.8 0.7 0.7
0.0s 0.09 0.16 0.10 0.15 0.20
-56 t17 -38 -13 -15 -12
t33 tfi5 t 27 t 28 t 68 t56
7.50 3.56 5.03 4.06 14.62 9.15
9.67 4.48 6.38 5.56 16.00 9.86
+29 t26 -27 t37 t 9.4 f 7.8
1.0 1.1
0.18 0.14
-69 -35
$32 t 32
4.47 2.44
4.02 2.34
-10 - 4.1
2.3 5.4 i 3.0 5.11 6.2 3.8
32 21 15 24 44 41
99.5 99 98 100 99 99
Unsensifizrd mast cells , m 4
13.3 9.4
0 5
99.5 100
a Histamine release was tested with a sample of the isolated cells using the same antigen cont. as the final coat. in the diver.
being undetectable ( < 1.2 per cent). The histamine release which the concentrations of egg albumin, used in the control experiments, could give is thus negligible. DISCUSSION
Anaphylactic histamine release from mast cells was accompanied by 30 per cent stimulation of their respiration in the absence of glucose. The stimulation was transient lasting 15 to 20 min after which normal respiration was restored. The change was very characteristic and was uniformly seen in all the 4 experiments. The mean increase in oxygen uptake was 1.49 x 1O-2 l/h which is far above the slight variations in the slope that may occur in
166
N. Chakravarty
control divers without cells. The mean gas exchange after mixing in contro1 divers was 0.066 X 10e2 ,ul/h [4]. This value would be interpreted as increased gas uptake but is of another order of magnitude differing from increased respiratory uptake by the mast cells by a factor of 22. Whenrespiring cells are used the slope changes in simulated mixing experiments were even less or none at all as shown in a previous paper [4]. Mast cell respiration was only slightly stimulated (8-9 per cent) in presence of glucose. The two phases, clearly seen in the absence of glucose, were not observed now, and it would hardly be possible to distinguish them at this low level of stimulation. Exposure of unsensitized mast cells to antigen did not change their oxygen uptake appreciably or caused a slight depression (10 per cent). Mongar and Perera [ 131, using a micro capillary respirometer, were unable to show any increase in mast cell respiration during antigen induced histamine release. Their absolute value for mast cell respiration in presence of glucose and with air as the gas phase was 1.2 x 1O-5 ,ul/cell/h. This is about 25 times higher than the value found by us [7] for mast cells from SpragueDawley rats. This factor would be even higher if the values in glucose-free medium were compared. A part of the difference may be due to the strain difference, because mast cells from Wistar rats used by them [ 131 may have a higher respiration rate. The respiration rate of Wistar rat mast cells has indeed been found in a recent study by Kahl and Netter [lo] to be 0.89 X 10~~ pi/cell/h which is about double as high as the respiration of mast cells from Sprague-Dawley rats. Even after taking this higher respiration rate into account the values of Mongar and Perera differ by a factor around 10. The basal respiration of rat mast cells found by us agrees in the order of magnitude with that of Kieler (unpublished observation, see [7]), Kahl and Netter [lo] and in general with the respiration of leucocytes [18]. The respiration rate reported by Mongar and Perera [13] thus seems to be around 10 times too high and at this level any change caused by antigen would hardly be detectable. The antigen used by them was also different (2 per cent horse serum). Inspection of the mast cells at the end of the experiment showed that the antigen caused in general mild or moderate changes in the cells (Figs 7 it and 8). Judging from studies using light [ 5 ] and electron [6] microscopy appears that the antigen causes some swelling of the cell with redistribution of granules many of which protrude through the cell membrane and are in contact with the extracellular fluid. The cell membrane usually shows localized disruption releasing a few granules. More severe changes are less frequent, especially with low concentrations of antigen. The typical mast cell Experimental
Cell Research
49
Mast cell respiration
during
histamine
release
167
in anaphylaxis thus shows some morphological and functional disturbances but its metabolic activity-judging from the respiration rate utilizing endogenous substrate-remains unimpaired after a brief period of initial stimulation. In attempting to interpret the brief respiratory stimulation one is faced with the question: what is the physiological significance of the stimulation and why does it become more apparent in the absence of glucose? Histamine is released within a few seconds of contact with the releaser [21]. If the triggering mechanism itself requires extra energy the respiratory stimulation, which persists for 15-20 min, might restore the energy balance again. This view would be compatible with the hypothesis that oxidative energ? metabolism is directly concerned with the histamine release process. The unmasking of the stimulant effect on respiration in the absence of glucose seems to be consistent with the metabolic pattern of the mast cell. Glucose causes 60 per cent stimulation of mast cell respiration [7]. It is quite conceivable that at this enhanced rate of oxygen uptake the stimulatory mechanism set in motion by antigen-antibody reaction may be less pronounced and largely masked by the more effective stimulation produced by glucose itself. Glucose might also influence the antigen effect on respiration in another way. The mast cell has an active glycolytic pathway-both aerobic and anaerobic [3]. By stimulating aerobic glycolysis glucose could provide an alternate source of energy and thus indirectly suppress the stimulant ef‘fect on respiration. The mast cell seems to be rich in endogenous substrate [3, 71, which could very well provide the fuel for the short-lived stimulation of respiration in a glucose-free medium.
SUMMARY
The respiration of isolated peritoneal mast cells from sensitized rats was stimulated 26-37 per cent above the basal level immediately after contact with antigen which released 21-32 per cent of their histamine content. The stimulation was short-lived lasting 15 to 20 minutes. This phenomenon was observed characteristically in the absence of glucose in the medium. In presence of glucose the stimulation was much less pronounced. The finding is consistent with the view that oxidative energy metabolism is directly involved in the process of anaphylactic histamine release. This work
was supported
by the Swedish
Medical
Research
Council
(Project
No.
13X-532-02). Experimental
Cell
Research
49
168
N. Chakravariy REFERENCES N., Acta Physiol. Stand. Am. .I. Physiol. 203, 1193 (1962). J. Cell Biol. 25, 123 (1965). Exptl Cell Res. 47, 278 (1967). To be published (1967).
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Cell
Research
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