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Non-degranulating structural changes of rat mast cells induced by antigen and toluidine blue
Experimental Cell Research 63 (1970) 101-109
NON-DEGRANULATING CELLS INDUCED
STRUCTURAL BY ANTIGEN
P. G. KRUGER,
B. DIAMANT
CHANGES
OF RAT MAST
AND TOLUIDINE
BLUE
and L. SCHOLANDER
Department of Pharmacology, Karolinska Institute& Stockholm 60, Sweden
SUMMARY The effect of antigen and toluidine blue on the morphology of sensitized and non-sensitized rat mast cells resp. were studied by light microscopy. The morphological changes on mast cells after incubation with these agents always consisted of degranulation of the cells. After local application of the agents, however, various intracellular morphological changes gradually occurred which rarely led to degranulation. The local effect of antigen and toluidine blue on the mast cell were in certain respects similar to those earlier described for ATP. It is proposed that degranulation induced by antigen and toluidine blue represent a cellular response which occurs when too much of the cell membrane suddenly is stimulated, whereas intracellular morphological changes without degranulation is consistent with a more discrete stimulation of the mast cell.
The importance of mast cells in allergic reactions became apparent through the work of Riley &West [25, 261,who showed that most tissue histamine is located in these cells. The mechanism by which these cells release their histamine is still a matter for discussion. Several observations are contradictory in the literature, possibly due to variations in experimental procedures. According to most authors the antigen-antibody reaction induces degranulation of the mast cell [l, 7, 21, 28, 371.Similarly, degranulation of rat mast cells has been reported after incubation of isolated cells or various mast cell rich tissues with histamine releasing agents like compound 48/80 [12, 13, 14, 18, 281,bee venom [16, 171 and Ascaris extract [9, 341. Experimental results concerning the action of compound 48/80 suggestedthat the extrusion of granules is an energy-requiring process [13, 201 and that the actual release is a passive exchange of histamine of the extruded granules for
cations in the surrounding medium [32, 361. This mechanism of degranulation and release has been assumed to be in principle identical for other energy-dependent degranulating agents [32]. Electron microscopic studies on rat mast cells exposed to 48/80, antigen, bee venom, decylamine and reserpin [4, 5, 6, 8, 191have revealed the appearance of swollen and less electron-dense granules within the cells in addition to degranulation. Morphological changes of living mast cells without degranulation have been described, however. Light microscopic studies on rat mast cells exposed to toluidine blue [30, 311, ATP [l l] and antigen (passive sensitization) [24] have shown that intracellular morphological changesmight occur without degranulation. Among these investigations, histamine release without noticeable degranulation was reported for mast cells incubated with ATP [lo, 111. The intracellular morphological Exptl Cell Res 63
102 P. G. Kriiger et al.
Fig. 1. A sensitized mast cell after incubation with a specific antigen. Note degranulation.
changes of mast cells after exposure to ATP consisted of changes of the refractory properties of perinuclear granules in addition to reversible alteration of the cell contour. Whether or not the mast cells in the body have a physiological function is still an unsolved problem. Earlier investigators assumed the mast cell to be unicellular glands (for ref. see [12]). Based on this hypothesis and on the morphological finding that the perivascular mast cells (in the brain of the hedgehog) [15] are located in close contact with the capillary endothelium with part of their cell membrane we have assumed that the mast cell surface might be stimulated only at a restricted area in situ. In the present investigation we have studied the effect of antigen and toluidine blue on the morphology of individual rat mast cells after local application of these agents to the cell membrane. MATERIALS
AND METHODS
Sensitized mast cells were obtained from male Sprague-Dawley rats (150-200 g) which were sensitized to egg albumin activated by pertussis vaccine according to Uvnls [33]. Non-sensitized mast cells ExptI Cell Res 63
were obtained from the peritoneal cavity of male Surague-Dawlev rats (300-400 .a) 1351.In the uresent stud; mast celis were not isolated from the-mixed cell susnension obtained from the rats. Before use. the mixed cell suspensions were washed three times with a buffered balanced salt solution (oH 7.0) [lo] containing human serum albumin (l”mg/ml). The cells were finally suspended in the same solution. In case of sensitized cells the solution used for the final suspension of the cells contained 0.1 mg/ml of human serum albumin and in addition rat serum (1 % final concentration) was added according to UvnBs [33]. The local annlication of toluidine blue and antigen to the mast ill membrane of individual cells was performed as described earlier [ll, 121 by the use of micropipettes. The concentration of antigen in the pipettes was 50-100 pg/ml of buffered salt solution. The concentration of toluidine blue amounted to 0.45 mg/ml. It was dissolved in the same balanced salt solution [lo] as that used for incubation of the cells. In certain experiments mixed cells were incubated with antigen or toluidine blue for 10 min at 37°C prior to the microscopic observations. Toluidine blue was obtained from Merck AG, Darmstadt, BRD (C.I. Nr. 52040, S. Nr. 1041); and egg albumin from Difco Laboratories, Detroit, Mich. Human serum albumin was kindly supplied bv AB Kebo. Sweden. - A Leitz orthoplan microscope was used equipped with Leitz planapochromat imm. oil lens 100/1.32 and a achr. 0.60/L 11 condenser. Kodak panatomic-X/ 135 fine grain film was used-without any filtersfor all microphotographs, developed in Kodak Microdol-X.
RESULTS Effect of antigen on morphology of sensitized rat mast cells Incubation of sensitized rat mast cells with a specific antigen in various concentrations (0.75lO,ug/ml) was found to result in degranulation of most of the cells (at least 80 % irrespectively of the concentration used) (fig. 1). Application of antigen (50-100 pug/ml) to a restricted area of the surface of a sensitized mast cell usually induced intracellular structural changes only. These consisted of sudden changes of the refractory properties of individual granules. Granules were involved one by one, by which they became less visible and the reaction proceeded towards the periphery of the cell starting with granules situated at
Non-degranulating response of rat mast cells
103
Fig. 2. The effect of local application of antigen (100 pg/ml) to the surface of a sensitized mast cell causing changes of the refractive properties of intracellular granules starting at the perinuclear area. No degranulation is observed. (a) Prior to application; (b) 30 set after application of antigen; (c) 2 min after application of antigen. The nucleus is visible and the cell volume increased. Granule-free ectoplasmic blebs are notable (arrows); (d) 5 min after application of antigen. The granule mass decreased towards its original volume. Granule-free ectoplasmic blebs are prominent (arrows); (e) 20 min after application of antigen. No further changes of the granule mass. One of the blebs has increased in size and the remaining two are not discernible. Exptl Cell Res 63
104 P. G. Krtiger et al. 48/80 [12] and included most of the cell surface. Thus, intracellular morphological changes without degranulation was found to be the usual response of sensitized mast cells to antigen applied locally to the cell surface. Effect of toluidine blue on morphology of non-sensitized mast cells
Fig. 3. After local application of a specific antigen (100 ,ug/ml) to the surface of a sensitized mast cell intracellular refractory changes were in a few instances followed by a degranulation of the cell (3 min after application of a comparatively large amount of antigen).
the perinuclear area. Varying from 15 set to 3 min and as a result of the refractive changes of the granules the nucleus became visible (fig. 2a-c). The cell volume increased, the cell surface became less smooth than originally and in addition granule-free ectoplasmic blebs sometimes started to appear (fig. 2~). No cytoplasmic movements were observed. After prolonged incubation (fig. 2 d; 5 min after application of antigen) the granule mass decreased towards its original volume and the granule-free blebs, when observed, became more prominent. As evident from fig. 2e (20 min after application of antigen) no further changes occurred except that one of the blebs increased in size and the remaining two were no longer discernible. In two instances when coarse pipettes were used and comparatively large amounts of antigen were administered the intracellular changes were followed by a degranulation process (fig. 3). This degranulating response occurred however much later as compared to the effect of local application of compound Exptl Cd Res 63
Mast cells incubated with toluidine blue seemedto respond with a degranulation process.This was evident especially after incubation of the cells with low concentrations of the dye. With 0.03 mg/ml of toluidine blue only a few granules were seen outside the cell sticking to the membrane. These granules were metachromatically stained while the rest of the cells were not affected by the dye (fig. 4a, b). With 0.3 mg/ml of the dye in the incubation medium (fig. 4c) a dense metachromatic staining of the whole mast cell occurred, which made recognition of intracellular details impossible. The uneven contour of the cell indicated that granules were sticking to the membrane. Local application of toluidine blue to the surface of a mast cell induced several intracellular morphological phenomena. The velocity by which these changes occurred and the duration of individual steps were dependent on the strength and the amount of the dye applied. The first effect observed was that the central part of the cell became light blue (fig. 5b; 1 min after application of toluidine blue). Then the refractory properties of the granules disappeared one by one. In contrast to the effect of antigen, toluidine blue started to affect granules at various parts of the cell at the same time (fig. 5~; 1 min 45 set after application). As the nucleus became more easily visible it became apparent that the initially observed blue part in the center of the cell came from the nucleus. The mast cell increased in volume as all granules
Non-degranulating
response of rat mast cells
105
Fig. 4. Mast cells incubated with toluidine blue causing degranulation of the cells. (a) Two mast cells incubated with 0.03 mg/ml toluidine blue, centrally focuset;.(b) the same cells focused at the cell periphery, upper surface; (c) one mast cell incubated with 0.3 mg/ml tohudme blue. Exptl Cell Res 63
106 P. G. Kriiger et al.
ules. The morphological changes noted for toluidine blue agree in general with the observations of Smith [31], who studied the effect of this dye on mast cells in the mesentery of anestethized rats. In contrast to ATP, toluidine blue as well as antigen always induced degranulation when the cells were incubated in a medium containing these agents. Degranulation of mast cells after local application of antigen and toluidine blue was infrequently noted, and only when comparatively large amounts were administered. Degranulation was then always preceded by intracellular structural changes. It is interesting to consider the suggestions of Robertson [27] that microtubules of aminesecreting cells harbour amine containing granules and that these tubules transverse the cell and connect with the outer milieu at the plasma membrane. This microtubular theory may well be applicable to the present light microscopic observations and would explain how refractive changes could begin at the perinuclear area of the cell (antigen) as well as why an initial effect was observed in the nucleus (toluidine blue). In fact, Padawer has demonstrated, by electronmicroscopy that rat mast cells contain microtubular systems [23]. DISCUSSION In caseof ATP [l l] it is known from studies The present results have shown that the local on incubated mast cells that intracellular morapplication of toluidine blue to non-sensitized phological changes of the same type as derat mast cells and antigen to sensitized cells scribed above occur together with histamine induced intracellular morphological changes. release. The question whether the observed Both agents resemblein this respect the action intracellular morphological changes which of ATP [l 11.Refractive changes of individual occurred in the present investigation without granules occurred, which in the case of tolui- degranulation reflect histamine release from dine blue were preceded by staining of the the mast cell must be left open until methods nucleus. With both agents a notable reversible become available which allow the registration swelling phenomenon of the granule mass of of histamine releasefrom a single cell directly the cells was frequently observed. In contrast through the microscope. to the effect of antigen or ATP [l 11,the refracFrom a morphological point of view it is tive changesinduced by toluidine blue gradu- thus possible to distinguish two different ally proceeded to affect all intracellular gran- types of response initiated in mast cells by of the cell gradually seemed to be involved (fig. 5d; 2 min 30 set and fig. 5e; 3 min after application). At this stage no metachromatic changes were observed and the cell could maintain the morphology shown in fig. 5e for at least 60 min. If, however, additional amount of dye was applied to such a cell the next stage consisted of the immediate metachromatic staining of granules at the cell periphery, starting at the point of administration (fig. 5f; 10 set after additional application of toluidine blue to the cell). Gradually, the granules took up the dye and became metachromatically stained (fig. 5g; 45 set) and the cell contracted (fig. 5h; 2 min 15 set after second application). Details of the cell interior were no longer possible to distinguish. The cell surface remained smooth. When sufficient amounts of toluidine blue were applied to the mast cell membrane initially, all the described morphological changes progressed without intermission and at a faster rate. Jn addition, when too much of the dye was administered by the use of coarse pipettes the cell responded with immediate partialdegranulation and resembled in appearance the cells shown in fig. 4a, b.
Exptl Cd Res 63
Fig. 5. The effect of 0.45 mg/ml toluidine blue applied locally to the mast cell. (a) Prior to application; (b) 1 min after application. The central part of the cell appeared light blue (arrow); (c) 1 min 45 set after application. Changes in the refractory properties of the granules are noted at various parts of the cell; (d) 2 min 30 set after application. The blue central part noted in fig. 56 appeared to be the nucleus. Note increase in cell volume; (e) 3 min after application. All granules are involved and the cell is maximally swollen. Five min after the first application of toluidine blue an additional amount was applied from the same pipette; (f) 10 set after the second application. Note metachromatic staining of the peripherally positioned granules; (g) 45 set after the second application; (h) 2 min 15 set after the second application. Note the dense staining and the decrease in cell volume. Exptl Cell Res 63
108 P. G. Kriiger et al. the local application of histamine releasing agents. The first type of response,represented by the action of compound 48180consists of an immediate extrusion of granules from the cell. As judged from light microscopic studies no other structural changes seem to occur [12]. The second type of response,represented by the action of local application of small amounts of antigen, ATP and toluidine blue, consists of comparatively slow changes of the morphology of the cell without degranulation. These changesare in certain respectscommon to all three agents and consist of initial staining of the nucleus (toluidine blue), disappearance of the refractive properties of the granules, increased visibility of the nucleus, increased roughness of the cell contour (ATP and antigen) and reversible increase of the cell volume (antigen and toluidine blue). Areas of granule free ectoplasm observed after local application of antigen to sensitized mast cells was not a consistent finding. Therefore we cannot discuss their significance. No blebs have so far been observed after local application of ATP [l l] or compound 48/80 [ 121to the cell surface. Similar blebs w-ereearlier reported for mast cells incubated with ATP or ADP [l 11. Further work is needed to elucidate if these blebs are reversible and if they are indications of cellular death [2]. The degranulating effect of antigen and toluidine blue on mast cells observed in the present study under certain defined experimental conditions seems to occur when too much of the cell membrane is stimulated by these agents. This effect contrasts with those intracellular morphological changes observed without degranulation after more discreet stimulation of the cell. Whether these latter changes represent the normal response of mast cells towards physiological stimulation in the body and whether these changes are consistent with histamine release from the cells remains to be elucidated. Exptl CeN Res 63
It should be pointed out that compound 48/80 does not seem to be in all respects a suitable model substance for the study of antigen-antibody reaction since the only observable response of the mast cell towards 48/80 is a visible degranulation irrespectively of the mode of stimulation [12]. This study was supported by the Swedish Medical Research Council (B69-14X-1004-04), the NIH, Bethesda 14 Md, (5-R05-TWOO306-03), Karolirska Institute& Stockholm, Sweden and the Scientific Research Council of Norway (E. 11:30-l).
REFERENCES 1. Archer, G T, Austr j exptl biol med sci 38 (1960) 147. 2. Bessis, M, Ciba Foundation symposium on cellular injury. Churchill, London (1964). 3. Bloom, G D, Diamant, B, Haegermark, 6 & Ritzen, M., Expt cell res 62 (1970) 61. 4. Bloom, G D, Fredholm, B & Haegermark, 6, Acta physiol Stand 71 (1967) 270. 5. Bloom, G D & Haegermark, 6, Exptl cell res 40 (1965) 637. 6. - Acta physiol Stand 71 (1967) 257. 7. Brocklehurst, W E, Humphrey, J H & Perry, W L M, J physiol 129 (1955) 205. 8. Chakravarty, N, Gustafson, G T & Phil, E, Acta path01 microbial Stand 71 (1967) 233. 9. Diamant, B, Acta physiol Stand 52 (1961) 8. 10. Diamant, B & Kruger, P G, Acta physiol Stand 71 (1967) 291. 11. - J histochem cytochem 16 No. 11 (1968) 707. 12. Diamant, B, Kruger, P G & UvnLs, B, Acta physiol Stand 79 (1970) 1. 13. Diamant, B & Uvnis, B, Acta physiol Stand 53 (1961) 315. Fawcett, D W, J exptl med 100 (1954) 217. :t Flood, P R & Kruger, P G, Acta anat. In press. 16: Fredholm, B, Biochem pharmacol 15, No. 2 (1966) 2037. 17. Fredholm, B & Haegermark, 6, Acta physiol Stand 69 (1966) 304. 18. Hiigberg, B & Uvniis, B, Acta physiol Stand 41 (1957) 345. 19. Jansson, S-E & Penttila, A, Stand clin invest 21 (1968) 93. 20. Junqueira, L C U & Beiguelman, B, Texas repts biol med13 (1955) 69. 21. Mota, I, Brit j pharmacol 12 (1957) 453. 22. - J physiol 147 (1959) 425. 23. Padawer, J, J cell biol 35 (1967) 180. 24. Perelmutter, L & Khera, K, Int arch allergy. In press. 25. Riley, J F, J pathol bacterial 65 (1953) 471. 26. Riley, J F & West, G B, J physiol 120 (1953) 528.
Non-degranulating 27. Robertson, J D, Proc int Wenner-Gren symp Stockholm 1965, vol. 5, pp. 3-27. Pergamon Press, Oxford (1966). 28. Saeki, K, Jap j pharm 14 (3) (1965) 375. 29. Schuster, J & Kunze, J, Arzn Forsch 19 (1969) 589. 30. Smith, D E, Am j physiol 193 (1958) 573. 31. - Science 128 (1958) 207. 32. Thon, I L JK Uvnh, B, Acta physiol Stand 71 (1967) 303. 33. Uvnls, B. To be published (1970).
response of rat mast cells
109
34. Uvnis, B, Diamant, B, Hbgberg, B & Thon, I L, Am j physiol 199, No. 3 (1960) 575. 35. Uvnls, B & Thon, I L, Exptl cell res 18 (1959) 512. 36. - Proc int Wenner-Gren symp Stockholm 1965 vol. 5, pp. 361-371. Pergamon, Oxford (1966). 37. Wegelius, 0 t Hjehnman, G, Acta path01 microbiol Stand 36 (1955) 309. Received February 9, 1970
Expil Cell Res 63
NON-DEGRANULATING CELLS INDUCED
STRUCTURAL BY ANTIGEN
P. G. KRUGER,
B. DIAMANT
CHANGES
OF RAT MAST
AND TOLUIDINE
BLUE
and L. SCHOLANDER
Department of Pharmacology, Karolinska Institute& Stockholm 60, Sweden
SUMMARY The effect of antigen and toluidine blue on the morphology of sensitized and non-sensitized rat mast cells resp. were studied by light microscopy. The morphological changes on mast cells after incubation with these agents always consisted of degranulation of the cells. After local application of the agents, however, various intracellular morphological changes gradually occurred which rarely led to degranulation. The local effect of antigen and toluidine blue on the mast cell were in certain respects similar to those earlier described for ATP. It is proposed that degranulation induced by antigen and toluidine blue represent a cellular response which occurs when too much of the cell membrane suddenly is stimulated, whereas intracellular morphological changes without degranulation is consistent with a more discrete stimulation of the mast cell.
The importance of mast cells in allergic reactions became apparent through the work of Riley &West [25, 261,who showed that most tissue histamine is located in these cells. The mechanism by which these cells release their histamine is still a matter for discussion. Several observations are contradictory in the literature, possibly due to variations in experimental procedures. According to most authors the antigen-antibody reaction induces degranulation of the mast cell [l, 7, 21, 28, 371.Similarly, degranulation of rat mast cells has been reported after incubation of isolated cells or various mast cell rich tissues with histamine releasing agents like compound 48/80 [12, 13, 14, 18, 281,bee venom [16, 171 and Ascaris extract [9, 341. Experimental results concerning the action of compound 48/80 suggestedthat the extrusion of granules is an energy-requiring process [13, 201 and that the actual release is a passive exchange of histamine of the extruded granules for
cations in the surrounding medium [32, 361. This mechanism of degranulation and release has been assumed to be in principle identical for other energy-dependent degranulating agents [32]. Electron microscopic studies on rat mast cells exposed to 48/80, antigen, bee venom, decylamine and reserpin [4, 5, 6, 8, 191have revealed the appearance of swollen and less electron-dense granules within the cells in addition to degranulation. Morphological changes of living mast cells without degranulation have been described, however. Light microscopic studies on rat mast cells exposed to toluidine blue [30, 311, ATP [l l] and antigen (passive sensitization) [24] have shown that intracellular morphological changesmight occur without degranulation. Among these investigations, histamine release without noticeable degranulation was reported for mast cells incubated with ATP [lo, 111. The intracellular morphological Exptl Cell Res 63
102 P. G. Kriiger et al.
Fig. 1. A sensitized mast cell after incubation with a specific antigen. Note degranulation.
changes of mast cells after exposure to ATP consisted of changes of the refractory properties of perinuclear granules in addition to reversible alteration of the cell contour. Whether or not the mast cells in the body have a physiological function is still an unsolved problem. Earlier investigators assumed the mast cell to be unicellular glands (for ref. see [12]). Based on this hypothesis and on the morphological finding that the perivascular mast cells (in the brain of the hedgehog) [15] are located in close contact with the capillary endothelium with part of their cell membrane we have assumed that the mast cell surface might be stimulated only at a restricted area in situ. In the present investigation we have studied the effect of antigen and toluidine blue on the morphology of individual rat mast cells after local application of these agents to the cell membrane. MATERIALS
AND METHODS
Sensitized mast cells were obtained from male Sprague-Dawley rats (150-200 g) which were sensitized to egg albumin activated by pertussis vaccine according to Uvnls [33]. Non-sensitized mast cells ExptI Cell Res 63
were obtained from the peritoneal cavity of male Surague-Dawlev rats (300-400 .a) 1351.In the uresent stud; mast celis were not isolated from the-mixed cell susnension obtained from the rats. Before use. the mixed cell suspensions were washed three times with a buffered balanced salt solution (oH 7.0) [lo] containing human serum albumin (l”mg/ml). The cells were finally suspended in the same solution. In case of sensitized cells the solution used for the final suspension of the cells contained 0.1 mg/ml of human serum albumin and in addition rat serum (1 % final concentration) was added according to UvnBs [33]. The local annlication of toluidine blue and antigen to the mast ill membrane of individual cells was performed as described earlier [ll, 121 by the use of micropipettes. The concentration of antigen in the pipettes was 50-100 pg/ml of buffered salt solution. The concentration of toluidine blue amounted to 0.45 mg/ml. It was dissolved in the same balanced salt solution [lo] as that used for incubation of the cells. In certain experiments mixed cells were incubated with antigen or toluidine blue for 10 min at 37°C prior to the microscopic observations. Toluidine blue was obtained from Merck AG, Darmstadt, BRD (C.I. Nr. 52040, S. Nr. 1041); and egg albumin from Difco Laboratories, Detroit, Mich. Human serum albumin was kindly supplied bv AB Kebo. Sweden. - A Leitz orthoplan microscope was used equipped with Leitz planapochromat imm. oil lens 100/1.32 and a achr. 0.60/L 11 condenser. Kodak panatomic-X/ 135 fine grain film was used-without any filtersfor all microphotographs, developed in Kodak Microdol-X.
RESULTS Effect of antigen on morphology of sensitized rat mast cells Incubation of sensitized rat mast cells with a specific antigen in various concentrations (0.75lO,ug/ml) was found to result in degranulation of most of the cells (at least 80 % irrespectively of the concentration used) (fig. 1). Application of antigen (50-100 pug/ml) to a restricted area of the surface of a sensitized mast cell usually induced intracellular structural changes only. These consisted of sudden changes of the refractory properties of individual granules. Granules were involved one by one, by which they became less visible and the reaction proceeded towards the periphery of the cell starting with granules situated at
Non-degranulating response of rat mast cells
103
Fig. 2. The effect of local application of antigen (100 pg/ml) to the surface of a sensitized mast cell causing changes of the refractive properties of intracellular granules starting at the perinuclear area. No degranulation is observed. (a) Prior to application; (b) 30 set after application of antigen; (c) 2 min after application of antigen. The nucleus is visible and the cell volume increased. Granule-free ectoplasmic blebs are notable (arrows); (d) 5 min after application of antigen. The granule mass decreased towards its original volume. Granule-free ectoplasmic blebs are prominent (arrows); (e) 20 min after application of antigen. No further changes of the granule mass. One of the blebs has increased in size and the remaining two are not discernible. Exptl Cell Res 63
104 P. G. Krtiger et al. 48/80 [12] and included most of the cell surface. Thus, intracellular morphological changes without degranulation was found to be the usual response of sensitized mast cells to antigen applied locally to the cell surface. Effect of toluidine blue on morphology of non-sensitized mast cells
Fig. 3. After local application of a specific antigen (100 ,ug/ml) to the surface of a sensitized mast cell intracellular refractory changes were in a few instances followed by a degranulation of the cell (3 min after application of a comparatively large amount of antigen).
the perinuclear area. Varying from 15 set to 3 min and as a result of the refractive changes of the granules the nucleus became visible (fig. 2a-c). The cell volume increased, the cell surface became less smooth than originally and in addition granule-free ectoplasmic blebs sometimes started to appear (fig. 2~). No cytoplasmic movements were observed. After prolonged incubation (fig. 2 d; 5 min after application of antigen) the granule mass decreased towards its original volume and the granule-free blebs, when observed, became more prominent. As evident from fig. 2e (20 min after application of antigen) no further changes occurred except that one of the blebs increased in size and the remaining two were no longer discernible. In two instances when coarse pipettes were used and comparatively large amounts of antigen were administered the intracellular changes were followed by a degranulation process (fig. 3). This degranulating response occurred however much later as compared to the effect of local application of compound Exptl Cd Res 63
Mast cells incubated with toluidine blue seemedto respond with a degranulation process.This was evident especially after incubation of the cells with low concentrations of the dye. With 0.03 mg/ml of toluidine blue only a few granules were seen outside the cell sticking to the membrane. These granules were metachromatically stained while the rest of the cells were not affected by the dye (fig. 4a, b). With 0.3 mg/ml of the dye in the incubation medium (fig. 4c) a dense metachromatic staining of the whole mast cell occurred, which made recognition of intracellular details impossible. The uneven contour of the cell indicated that granules were sticking to the membrane. Local application of toluidine blue to the surface of a mast cell induced several intracellular morphological phenomena. The velocity by which these changes occurred and the duration of individual steps were dependent on the strength and the amount of the dye applied. The first effect observed was that the central part of the cell became light blue (fig. 5b; 1 min after application of toluidine blue). Then the refractory properties of the granules disappeared one by one. In contrast to the effect of antigen, toluidine blue started to affect granules at various parts of the cell at the same time (fig. 5~; 1 min 45 set after application). As the nucleus became more easily visible it became apparent that the initially observed blue part in the center of the cell came from the nucleus. The mast cell increased in volume as all granules
Non-degranulating
response of rat mast cells
105
Fig. 4. Mast cells incubated with toluidine blue causing degranulation of the cells. (a) Two mast cells incubated with 0.03 mg/ml toluidine blue, centrally focuset;.(b) the same cells focused at the cell periphery, upper surface; (c) one mast cell incubated with 0.3 mg/ml tohudme blue. Exptl Cell Res 63
106 P. G. Kriiger et al.
ules. The morphological changes noted for toluidine blue agree in general with the observations of Smith [31], who studied the effect of this dye on mast cells in the mesentery of anestethized rats. In contrast to ATP, toluidine blue as well as antigen always induced degranulation when the cells were incubated in a medium containing these agents. Degranulation of mast cells after local application of antigen and toluidine blue was infrequently noted, and only when comparatively large amounts were administered. Degranulation was then always preceded by intracellular structural changes. It is interesting to consider the suggestions of Robertson [27] that microtubules of aminesecreting cells harbour amine containing granules and that these tubules transverse the cell and connect with the outer milieu at the plasma membrane. This microtubular theory may well be applicable to the present light microscopic observations and would explain how refractive changes could begin at the perinuclear area of the cell (antigen) as well as why an initial effect was observed in the nucleus (toluidine blue). In fact, Padawer has demonstrated, by electronmicroscopy that rat mast cells contain microtubular systems [23]. DISCUSSION In caseof ATP [l l] it is known from studies The present results have shown that the local on incubated mast cells that intracellular morapplication of toluidine blue to non-sensitized phological changes of the same type as derat mast cells and antigen to sensitized cells scribed above occur together with histamine induced intracellular morphological changes. release. The question whether the observed Both agents resemblein this respect the action intracellular morphological changes which of ATP [l 11.Refractive changes of individual occurred in the present investigation without granules occurred, which in the case of tolui- degranulation reflect histamine release from dine blue were preceded by staining of the the mast cell must be left open until methods nucleus. With both agents a notable reversible become available which allow the registration swelling phenomenon of the granule mass of of histamine releasefrom a single cell directly the cells was frequently observed. In contrast through the microscope. to the effect of antigen or ATP [l 11,the refracFrom a morphological point of view it is tive changesinduced by toluidine blue gradu- thus possible to distinguish two different ally proceeded to affect all intracellular gran- types of response initiated in mast cells by of the cell gradually seemed to be involved (fig. 5d; 2 min 30 set and fig. 5e; 3 min after application). At this stage no metachromatic changes were observed and the cell could maintain the morphology shown in fig. 5e for at least 60 min. If, however, additional amount of dye was applied to such a cell the next stage consisted of the immediate metachromatic staining of granules at the cell periphery, starting at the point of administration (fig. 5f; 10 set after additional application of toluidine blue to the cell). Gradually, the granules took up the dye and became metachromatically stained (fig. 5g; 45 set) and the cell contracted (fig. 5h; 2 min 15 set after second application). Details of the cell interior were no longer possible to distinguish. The cell surface remained smooth. When sufficient amounts of toluidine blue were applied to the mast cell membrane initially, all the described morphological changes progressed without intermission and at a faster rate. Jn addition, when too much of the dye was administered by the use of coarse pipettes the cell responded with immediate partialdegranulation and resembled in appearance the cells shown in fig. 4a, b.
Exptl Cd Res 63
Fig. 5. The effect of 0.45 mg/ml toluidine blue applied locally to the mast cell. (a) Prior to application; (b) 1 min after application. The central part of the cell appeared light blue (arrow); (c) 1 min 45 set after application. Changes in the refractory properties of the granules are noted at various parts of the cell; (d) 2 min 30 set after application. The blue central part noted in fig. 56 appeared to be the nucleus. Note increase in cell volume; (e) 3 min after application. All granules are involved and the cell is maximally swollen. Five min after the first application of toluidine blue an additional amount was applied from the same pipette; (f) 10 set after the second application. Note metachromatic staining of the peripherally positioned granules; (g) 45 set after the second application; (h) 2 min 15 set after the second application. Note the dense staining and the decrease in cell volume. Exptl Cell Res 63
108 P. G. Kriiger et al. the local application of histamine releasing agents. The first type of response,represented by the action of compound 48180consists of an immediate extrusion of granules from the cell. As judged from light microscopic studies no other structural changes seem to occur [12]. The second type of response,represented by the action of local application of small amounts of antigen, ATP and toluidine blue, consists of comparatively slow changes of the morphology of the cell without degranulation. These changesare in certain respectscommon to all three agents and consist of initial staining of the nucleus (toluidine blue), disappearance of the refractive properties of the granules, increased visibility of the nucleus, increased roughness of the cell contour (ATP and antigen) and reversible increase of the cell volume (antigen and toluidine blue). Areas of granule free ectoplasm observed after local application of antigen to sensitized mast cells was not a consistent finding. Therefore we cannot discuss their significance. No blebs have so far been observed after local application of ATP [l l] or compound 48/80 [ 121to the cell surface. Similar blebs w-ereearlier reported for mast cells incubated with ATP or ADP [l 11. Further work is needed to elucidate if these blebs are reversible and if they are indications of cellular death [2]. The degranulating effect of antigen and toluidine blue on mast cells observed in the present study under certain defined experimental conditions seems to occur when too much of the cell membrane is stimulated by these agents. This effect contrasts with those intracellular morphological changes observed without degranulation after more discreet stimulation of the cell. Whether these latter changes represent the normal response of mast cells towards physiological stimulation in the body and whether these changes are consistent with histamine release from the cells remains to be elucidated. Exptl CeN Res 63
It should be pointed out that compound 48/80 does not seem to be in all respects a suitable model substance for the study of antigen-antibody reaction since the only observable response of the mast cell towards 48/80 is a visible degranulation irrespectively of the mode of stimulation [12]. This study was supported by the Swedish Medical Research Council (B69-14X-1004-04), the NIH, Bethesda 14 Md, (5-R05-TWOO306-03), Karolirska Institute& Stockholm, Sweden and the Scientific Research Council of Norway (E. 11:30-l).
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