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Mast cells and tissue water
Experimental
437
Cell Research 11, 437-443 (1956)
MAST CELLS AND TISSUE STUDIES
ON
LIVING
CONNECTIVE CHEEK
0. WEGELIUS*
WATER1
TISSUE
IN
THE
HAMSTER
POUCH
and G. ASBOE-HANSEN
The Connective Tissue Research Laboratory, University Copenhagen, Denmark
Institute
of Medical
Anatomy,
Received April 16, 1956
INTRAPERITONEAL injection of water into rats is followed by disruption of the mast cells in the mesentery [6]. This phenomenon has also been observed in ovomucoid-induced oedemas in the rat [4] and in human urticaria factitia [ 11. Studying mast cells under the influence of water in the phase contrast microscope Zollinger [lo] observed swelling of the cell body as well as of the individual cytoplasmic granules. At the same time there was a decrease in the metachromasia normally resulting from toluidine blue staining. Prolonged treatment with physiological saline produced similar changes of the mast cells. On injection of physiological saline and Tyrode solution into the peritoneal cavity of rats Fawcett [S] noted that most mast cells remained unaffected, but occasional cells released their granules. Tissue sections treated with hyaluronidase do not show a subsidence of the metachromasia in the mast cells [9,1]. After having injected hyaluronidase into the subcutaneous tissue of the hamster Compton [5] studied fixed connective tissue specimens. In other experiments, he studied unfixed tissue treated with hyaluronidase. He did not observe any alteration of the metachromasia of the mast cells upon staining with safranin. By contrast, AsboeHansen [2] observed degranulation of the mast cells in biopsy specimens of human, normal and myxoedematous, skin injected intracutaneously with hyaluronidase. The phenomenon was explained as cellular activity in restoration of a normal ground substance. Since mast cells abound wherever new formation of connective tissue takes place [3], not least in oedemas during organization, and since they 1 Aided by grants Association against Odense Johann and 2 Home address:
from Eli Lilly and Co., Indianapolis, Indiana, Rheumatic Diseases, the Danish Anti-Cancer Hanne Weimann, n8e Seedorff’s Foundation. Maria Sjukhus, Helsingfors, Finland.
U.S.A., the National Danish League, and Merchant in
Experimental
Cell Research 11
0. Wegelius and G. Asboe-Hansen
438
TABLE
I
Influence of various fluids on living tissue mast cells. Changes in mast cells within 30 minutes
Fluid Sterile water
Degranulation Disruption Granule swelling
Physiological saline
Degranulation
Tyrode sol.
Degranulation
2 per cent saline sol.
Shrinkage
Hyaluronidase in physiological saline (100 and 1000 VFW/ml)*
Rapid degranulation
I-Iyaluronidase in Tyrode sol. (100 and 1000 VFW/ml)
Rapid degranulation
Hyaluronic acid 1 and 2 per cent in physiological saline
Insignificant
Traumatization
Degranulation Disruption Granule clumping
* Viscosity-reducing
(tissue oedema)
changes
-
units.
appear to play a role in the formation of the connectiveltissue ground substance [l&which is a most important fluid depot-it seemed relevant to study the response of living connective-tissue mast cells to altered fluid conditions in the tissues. Recently the authors showed that systemic application of histamine and chemical histamine-liberator substances caused widespread and marked degranulation of the mast cells in the connective tissue of the hamster cheek pouch [7]. MATERIAL
AND
METHODS
The test object was the hamster (Mesocricetus auratus) cheek pouch. The method, previously described 181, permits detailed microscopic study of the morphology of mast cells in living connective tissue with intact circulation-partly after staining with toluidine blue in physiological saline, 1: 100 to 1 :lOO,OOO, and partly in unstained connective tissue-in the bright field and phase contrast microscope, by means of water immersion objectives, and physiological saline as immersion fluid. Experimental
Cell Research 11
~139
Mast cells and tissue water
The present paper concerns the local effect of various fluids upon the connectivetissue mast cells. One of the two cheek pouches of the hamster was invariably used for control. Both cheek pouches may be drawn out simultaneously and each spread over a hole in a disc of cork which permits translumination. The fluids were injected between the two layers of the cheek pouch or dropped onto the lower membrane of the outspread, split pouch. The control fluid was allowed to act for an equal length of time on the contra-lateral cheek pouch of the same hamster. By dropping the fluid directly onto the denuded connective tissue, the traumatic effect of injecting is avoided to some extent. In order to approach physiological conditions as far as possible, the hamsters were placed on a warming stage mounted on the microscope. The depolymerizing effect of hyaluronidase upon hyaluronic acid in the connectivetissue ground substance was utilized partly by injecting the connective tissue with the enzyme and partly by dropping a concentrated solution of testicular hyaluronidasel onto the denuded connective tissue of the divided cheek pouch. One of the preparations used contained 100 viscosity-reducing units per mg and another, highly concentrated one 3000 V.R.U./mg or about 27,000 V.R.U./mg N. Hyaluronic acid, produced from bovine vitreous humour and from hutnan umbilical cord2 was injected-in 1 and 2 per cent solutions-into the connective tissue of the cheek pouch or dropped onto the connective-tissue aspect of the tnembrane. To obtain a local tissue oedema by a method as physiological as possible, the cheek pouch was traumatized by rubbing it a few times between the thumb ancl index finger. RESULTS Injection of the fluids into the connective tissue of the cheek pouches invariably caused disruption, i.e. irreversible damage, of some mast cells. Perceptibly fewer disintegrated cells were seen after the (hopping procedure. ,\fter application of wafer a considerably larger number of cells in various stages of degrannlafion were observed than in the directly stained control pouches. This phenomenon apparently reflects an active granule release by the living cell. In some of the cells the granules appeared swollen. Many granules \vere found lying free in the connective-tissue ground substance after cell disrupfion, showing intense metachromasia; they retained their colour unchanged for hours (Fig. 4). Varying numbers of macrophages containing in their cytoplasm metachromatic granules were observed, a phenomenon which was interpreted as evidence of phagocytosis of emitted mast-cell granules. These granules retained their metachromasia for some time within the cytoplasm of the phagocytizing cells (Fig. 5). 1 Kindly z Kindly
supplied supplied
by A/B Leo, Hllsingborg, Sweden. by Wyeth International Ltd., Philadelphia,
Pa., LT.S.A.
Experimental Cell Research 11
440
Experimental
0. Wegelius and G. Asboe-Hansen
Cell Research 11
Mast cells and tissue water
441
After treatment with physiological saline and Tyrode solution a considerable increase in the number of degranulated cells was observed as early as 20-30 minutes after application. These cells were fairly often surrounded by a zone of intercellular substance showing more intense metachromasia than more distant areas. Entirely degranulated cells showed a basic pattern like a honeycomb or sponge (cf. Fig. 3). Thirty minutes after application of a 2 per cent saline solution the mast cells revealed less degranulation than in the control pouches treated with physiological saline. The cells were smaller and their granular structure seemed to be denser. Hyaluronidase caused marked degranulation occurring even within 10 minutes, at which time the control specimens still had well-granulated cells. Even in these specimens a few cells were well stained and metachromatic after a fairly long time of treatment. Similarly, the granules remaining in partially degranulated cells showed normal metachromasia (Fig. 3). The size of the cells was not definitely altered. No extracellular metachromasia was noticed, nor any signs of phagocytosis. Hyaluronic acid, 1 and 2 per cent in physiological saline, did not induce any significant changes of the mast cells within 40 minutes. The cells stood out intensely metachromatic and densely granulated (Figs. 1, 2). After traumatization of a cheek pouch mast cells with conglomerated granules and more or less degranulated were observed in considerably larger numbers than in the control pouch; in addition, a large number of disintegrated cells, as well as macrophages with phagocytized granules. DISCUSSION
The disruption following osmosis. The extra-cellular
upon application of water is probably due to granules originating from disrupted mast cells
Fig. 1. Microphotograph of mast cells of living connective tissue stained with toluidine blue 1: 40,000 in physiological saline and examined in incident light. Magnification approx. x 80. Notice accumulation around vessels. Fig. 2. Normal, metachromatically granulated mast cell. Staining: toluidine blue 1:40,000 in physiological saline. Magnification x 2900. Fig. 3. Almost completely degranulated mast cell influenced by hyaluronidase. Notice basic pattern like a honeycomb or a sponge. A few metachromatic granules (black) are left within the cell. Staining: toluidine blue 1: 40,000 in physiological saline. Magnification x 2900. Fig. 4. A mast cell of living tissue during*disruption. Staining: toluidine blue 1: 40,000 in physiological saline. Magnification x 2900. Fig. 5. Macrophage with phagocytized mast cell granules in its cytoplasm. Staining: toluidine blue 1: 40,000 in physiological saline. Magnification x 2900. 30* - 563705
Ezperimental
Cell Research 11
442
0. Wegelius and G. Asboe-Hansen
resisted the action of water. There seems to be reason to believe that the granule substance may come into the ground substance partly by a disruption caused by some violent-physical or chemical-action during which the cell perishes, and partly by physiological degranulation probably involving some alteration of the granules, e.g. with regard to water solubility, before or during the passage from within to outside the cell. The few cells, which resisted prolonged influence of physiological saline, Tyrode fluid and hyaluronidase, may have been protected from this influence by adipose tissue, thicker, fibrous connective-tissue layers, etc. An increase in the water content of the connective-tissue ground substance appears to entail a movement of the granule substance from the intra- to the extracellular space. The diffusion of the fluids in the tissues as well as the rate of hydration are evidently factors influencing the conditions in the experimental as well as the control specimens. The rapid release of granules from the mast cells after application of hyaluronidase may be interpreted as a compensatory reaction to the topical situation established by a breakdown of the hyaluronic acid of the ground substance, the removal of the components by diffusion, and an increase in the diffusivity of water in the tissues. This view is supported by the fact that the cells are altered very slowly by the influence of a hyaluronic acid solution. The last-mentioned fluid is possibly the real control fluid, yielding a physiological environment for the mast cells. The fact that mechanical trauma of the cheek pouch entails tissue oedema as well as degranulation of the mast cells, supports the interpretation of the degranulation as a physiological response to increased tissue water. It may be comparable to the degranulating effect of histamine and histamine liberators, which also cause tissue oedema. The implications of degranulation are not fully understood. Previous experiments have brought much evidence that mast cells form mutinous ground-substance components of the hyaluronic acid type which represent a hydrated gel in the connective tissue. The present observations indicate that free water in the tissues is immediately converted into connective-tissue in the physicoground substance by a granule release. Any disturbance chemical balance between the mast cells and the intercellular matrix may immediately elicit a mast-cell response. The perivascular accumulation of mast cells is consistent with the view that they are to some extent functionally linked to vascular permeability, being ever ready to change a perivascular oedema into a mutinous ground substance. Experimental
Cell Research 11
443
Mast cells and tissue water SUMMARY
Applied topically, water entails granule swelling and active degranulation of living mast cells of the master cheek pouch, and, to some extent, irreversible disruption. Physiological saline and Tyrode solution cause degranulation alone, 2 per cent saline solution shrinkage. Hyaluronidase brings about rapid degranulation, hyaluronic acid insignificant or very slow alterations of the mast cells. Mechanical trauma is followed by degranulation, granule cluml~ing as well as disruption. Injection of the fluids provokes traumatization phenomena; dropping on the denuded connective tissue is more lenient and is preferred. The authors advance the view that the mast cells contain and are able to release mucopolysaccharide material to the intercellular substance in response to tissue oedema. Free water in the tissues may be bound and converted into mutinous substance. REFERENCES G., Ada dermafo-uenereol. 30, 338 (1950). J. Inuesf. Dermatol. 15, 25 (1950). Internatl. Rev. Cytology 3, 415 (1954).
1. ASBOE-HANSEN,
2. ~ 3. ~
4. BENDITT, 422 5. COMPTON, 6. FAWCETT, 7. WEGEL&, 8. WEGELIUS, 9. WISLOCKI, 10. ZOLLINGER,
E. P., BADER, S., ARASE, M., CORLEY, C., and LAM, K. B., Federation (1954). k. S.; Am. J. And. 91, 301 (1952). D. W., And. Rec. 121. 29 (1955). 0. and ASBOE-HAN&N, d., (I, press). 0. and HJELMMAN, G., Acta pathol. microbial. stand. 36, 304 (1955). G. B., BUNTING, H., and DEMPSEY, E. W., Am. J. And. 81, 1 (1947). H. U., Experientia 6, 384 (1950).
Experimental
Proc. 13,
Cell Research 11
437
Cell Research 11, 437-443 (1956)
MAST CELLS AND TISSUE STUDIES
ON
LIVING
CONNECTIVE CHEEK
0. WEGELIUS*
WATER1
TISSUE
IN
THE
HAMSTER
POUCH
and G. ASBOE-HANSEN
The Connective Tissue Research Laboratory, University Copenhagen, Denmark
Institute
of Medical
Anatomy,
Received April 16, 1956
INTRAPERITONEAL injection of water into rats is followed by disruption of the mast cells in the mesentery [6]. This phenomenon has also been observed in ovomucoid-induced oedemas in the rat [4] and in human urticaria factitia [ 11. Studying mast cells under the influence of water in the phase contrast microscope Zollinger [lo] observed swelling of the cell body as well as of the individual cytoplasmic granules. At the same time there was a decrease in the metachromasia normally resulting from toluidine blue staining. Prolonged treatment with physiological saline produced similar changes of the mast cells. On injection of physiological saline and Tyrode solution into the peritoneal cavity of rats Fawcett [S] noted that most mast cells remained unaffected, but occasional cells released their granules. Tissue sections treated with hyaluronidase do not show a subsidence of the metachromasia in the mast cells [9,1]. After having injected hyaluronidase into the subcutaneous tissue of the hamster Compton [5] studied fixed connective tissue specimens. In other experiments, he studied unfixed tissue treated with hyaluronidase. He did not observe any alteration of the metachromasia of the mast cells upon staining with safranin. By contrast, AsboeHansen [2] observed degranulation of the mast cells in biopsy specimens of human, normal and myxoedematous, skin injected intracutaneously with hyaluronidase. The phenomenon was explained as cellular activity in restoration of a normal ground substance. Since mast cells abound wherever new formation of connective tissue takes place [3], not least in oedemas during organization, and since they 1 Aided by grants Association against Odense Johann and 2 Home address:
from Eli Lilly and Co., Indianapolis, Indiana, Rheumatic Diseases, the Danish Anti-Cancer Hanne Weimann, n8e Seedorff’s Foundation. Maria Sjukhus, Helsingfors, Finland.
U.S.A., the National Danish League, and Merchant in
Experimental
Cell Research 11
0. Wegelius and G. Asboe-Hansen
438
TABLE
I
Influence of various fluids on living tissue mast cells. Changes in mast cells within 30 minutes
Fluid Sterile water
Degranulation Disruption Granule swelling
Physiological saline
Degranulation
Tyrode sol.
Degranulation
2 per cent saline sol.
Shrinkage
Hyaluronidase in physiological saline (100 and 1000 VFW/ml)*
Rapid degranulation
I-Iyaluronidase in Tyrode sol. (100 and 1000 VFW/ml)
Rapid degranulation
Hyaluronic acid 1 and 2 per cent in physiological saline
Insignificant
Traumatization
Degranulation Disruption Granule clumping
* Viscosity-reducing
(tissue oedema)
changes
-
units.
appear to play a role in the formation of the connectiveltissue ground substance [l&which is a most important fluid depot-it seemed relevant to study the response of living connective-tissue mast cells to altered fluid conditions in the tissues. Recently the authors showed that systemic application of histamine and chemical histamine-liberator substances caused widespread and marked degranulation of the mast cells in the connective tissue of the hamster cheek pouch [7]. MATERIAL
AND
METHODS
The test object was the hamster (Mesocricetus auratus) cheek pouch. The method, previously described 181, permits detailed microscopic study of the morphology of mast cells in living connective tissue with intact circulation-partly after staining with toluidine blue in physiological saline, 1: 100 to 1 :lOO,OOO, and partly in unstained connective tissue-in the bright field and phase contrast microscope, by means of water immersion objectives, and physiological saline as immersion fluid. Experimental
Cell Research 11
~139
Mast cells and tissue water
The present paper concerns the local effect of various fluids upon the connectivetissue mast cells. One of the two cheek pouches of the hamster was invariably used for control. Both cheek pouches may be drawn out simultaneously and each spread over a hole in a disc of cork which permits translumination. The fluids were injected between the two layers of the cheek pouch or dropped onto the lower membrane of the outspread, split pouch. The control fluid was allowed to act for an equal length of time on the contra-lateral cheek pouch of the same hamster. By dropping the fluid directly onto the denuded connective tissue, the traumatic effect of injecting is avoided to some extent. In order to approach physiological conditions as far as possible, the hamsters were placed on a warming stage mounted on the microscope. The depolymerizing effect of hyaluronidase upon hyaluronic acid in the connectivetissue ground substance was utilized partly by injecting the connective tissue with the enzyme and partly by dropping a concentrated solution of testicular hyaluronidasel onto the denuded connective tissue of the divided cheek pouch. One of the preparations used contained 100 viscosity-reducing units per mg and another, highly concentrated one 3000 V.R.U./mg or about 27,000 V.R.U./mg N. Hyaluronic acid, produced from bovine vitreous humour and from hutnan umbilical cord2 was injected-in 1 and 2 per cent solutions-into the connective tissue of the cheek pouch or dropped onto the connective-tissue aspect of the tnembrane. To obtain a local tissue oedema by a method as physiological as possible, the cheek pouch was traumatized by rubbing it a few times between the thumb ancl index finger. RESULTS Injection of the fluids into the connective tissue of the cheek pouches invariably caused disruption, i.e. irreversible damage, of some mast cells. Perceptibly fewer disintegrated cells were seen after the (hopping procedure. ,\fter application of wafer a considerably larger number of cells in various stages of degrannlafion were observed than in the directly stained control pouches. This phenomenon apparently reflects an active granule release by the living cell. In some of the cells the granules appeared swollen. Many granules \vere found lying free in the connective-tissue ground substance after cell disrupfion, showing intense metachromasia; they retained their colour unchanged for hours (Fig. 4). Varying numbers of macrophages containing in their cytoplasm metachromatic granules were observed, a phenomenon which was interpreted as evidence of phagocytosis of emitted mast-cell granules. These granules retained their metachromasia for some time within the cytoplasm of the phagocytizing cells (Fig. 5). 1 Kindly z Kindly
supplied supplied
by A/B Leo, Hllsingborg, Sweden. by Wyeth International Ltd., Philadelphia,
Pa., LT.S.A.
Experimental Cell Research 11
440
Experimental
0. Wegelius and G. Asboe-Hansen
Cell Research 11
Mast cells and tissue water
441
After treatment with physiological saline and Tyrode solution a considerable increase in the number of degranulated cells was observed as early as 20-30 minutes after application. These cells were fairly often surrounded by a zone of intercellular substance showing more intense metachromasia than more distant areas. Entirely degranulated cells showed a basic pattern like a honeycomb or sponge (cf. Fig. 3). Thirty minutes after application of a 2 per cent saline solution the mast cells revealed less degranulation than in the control pouches treated with physiological saline. The cells were smaller and their granular structure seemed to be denser. Hyaluronidase caused marked degranulation occurring even within 10 minutes, at which time the control specimens still had well-granulated cells. Even in these specimens a few cells were well stained and metachromatic after a fairly long time of treatment. Similarly, the granules remaining in partially degranulated cells showed normal metachromasia (Fig. 3). The size of the cells was not definitely altered. No extracellular metachromasia was noticed, nor any signs of phagocytosis. Hyaluronic acid, 1 and 2 per cent in physiological saline, did not induce any significant changes of the mast cells within 40 minutes. The cells stood out intensely metachromatic and densely granulated (Figs. 1, 2). After traumatization of a cheek pouch mast cells with conglomerated granules and more or less degranulated were observed in considerably larger numbers than in the control pouch; in addition, a large number of disintegrated cells, as well as macrophages with phagocytized granules. DISCUSSION
The disruption following osmosis. The extra-cellular
upon application of water is probably due to granules originating from disrupted mast cells
Fig. 1. Microphotograph of mast cells of living connective tissue stained with toluidine blue 1: 40,000 in physiological saline and examined in incident light. Magnification approx. x 80. Notice accumulation around vessels. Fig. 2. Normal, metachromatically granulated mast cell. Staining: toluidine blue 1:40,000 in physiological saline. Magnification x 2900. Fig. 3. Almost completely degranulated mast cell influenced by hyaluronidase. Notice basic pattern like a honeycomb or a sponge. A few metachromatic granules (black) are left within the cell. Staining: toluidine blue 1: 40,000 in physiological saline. Magnification x 2900. Fig. 4. A mast cell of living tissue during*disruption. Staining: toluidine blue 1: 40,000 in physiological saline. Magnification x 2900. Fig. 5. Macrophage with phagocytized mast cell granules in its cytoplasm. Staining: toluidine blue 1: 40,000 in physiological saline. Magnification x 2900. 30* - 563705
Ezperimental
Cell Research 11
442
0. Wegelius and G. Asboe-Hansen
resisted the action of water. There seems to be reason to believe that the granule substance may come into the ground substance partly by a disruption caused by some violent-physical or chemical-action during which the cell perishes, and partly by physiological degranulation probably involving some alteration of the granules, e.g. with regard to water solubility, before or during the passage from within to outside the cell. The few cells, which resisted prolonged influence of physiological saline, Tyrode fluid and hyaluronidase, may have been protected from this influence by adipose tissue, thicker, fibrous connective-tissue layers, etc. An increase in the water content of the connective-tissue ground substance appears to entail a movement of the granule substance from the intra- to the extracellular space. The diffusion of the fluids in the tissues as well as the rate of hydration are evidently factors influencing the conditions in the experimental as well as the control specimens. The rapid release of granules from the mast cells after application of hyaluronidase may be interpreted as a compensatory reaction to the topical situation established by a breakdown of the hyaluronic acid of the ground substance, the removal of the components by diffusion, and an increase in the diffusivity of water in the tissues. This view is supported by the fact that the cells are altered very slowly by the influence of a hyaluronic acid solution. The last-mentioned fluid is possibly the real control fluid, yielding a physiological environment for the mast cells. The fact that mechanical trauma of the cheek pouch entails tissue oedema as well as degranulation of the mast cells, supports the interpretation of the degranulation as a physiological response to increased tissue water. It may be comparable to the degranulating effect of histamine and histamine liberators, which also cause tissue oedema. The implications of degranulation are not fully understood. Previous experiments have brought much evidence that mast cells form mutinous ground-substance components of the hyaluronic acid type which represent a hydrated gel in the connective tissue. The present observations indicate that free water in the tissues is immediately converted into connective-tissue in the physicoground substance by a granule release. Any disturbance chemical balance between the mast cells and the intercellular matrix may immediately elicit a mast-cell response. The perivascular accumulation of mast cells is consistent with the view that they are to some extent functionally linked to vascular permeability, being ever ready to change a perivascular oedema into a mutinous ground substance. Experimental
Cell Research 11
443
Mast cells and tissue water SUMMARY
Applied topically, water entails granule swelling and active degranulation of living mast cells of the master cheek pouch, and, to some extent, irreversible disruption. Physiological saline and Tyrode solution cause degranulation alone, 2 per cent saline solution shrinkage. Hyaluronidase brings about rapid degranulation, hyaluronic acid insignificant or very slow alterations of the mast cells. Mechanical trauma is followed by degranulation, granule cluml~ing as well as disruption. Injection of the fluids provokes traumatization phenomena; dropping on the denuded connective tissue is more lenient and is preferred. The authors advance the view that the mast cells contain and are able to release mucopolysaccharide material to the intercellular substance in response to tissue oedema. Free water in the tissues may be bound and converted into mutinous substance. REFERENCES G., Ada dermafo-uenereol. 30, 338 (1950). J. Inuesf. Dermatol. 15, 25 (1950). Internatl. Rev. Cytology 3, 415 (1954).
1. ASBOE-HANSEN,
2. ~ 3. ~
4. BENDITT, 422 5. COMPTON, 6. FAWCETT, 7. WEGEL&, 8. WEGELIUS, 9. WISLOCKI, 10. ZOLLINGER,
E. P., BADER, S., ARASE, M., CORLEY, C., and LAM, K. B., Federation (1954). k. S.; Am. J. And. 91, 301 (1952). D. W., And. Rec. 121. 29 (1955). 0. and ASBOE-HAN&N, d., (I, press). 0. and HJELMMAN, G., Acta pathol. microbial. stand. 36, 304 (1955). G. B., BUNTING, H., and DEMPSEY, E. W., Am. J. And. 81, 1 (1947). H. U., Experientia 6, 384 (1950).
Experimental
Proc. 13,
Cell Research 11