428
Preliminary
notes
Rat mast cells have been found to generate the superoxide anion and to release superoxide dismutase concomitantly with deWe thank A. Celis for supplying the HGPRT mutant. granulation in response to immunologic and We also acknowledge the support of the Landsfornon-immunologic stimulation [ 11. In order eningen til Kraeftens Bekiempelse, the Danish Natural Science Research Council and Aarhus Universitets to study the granular source of superoxide Forskningsfond. generation, it became necessary to obtain mast cell granules with their perigranular References membranes intact. Several techniques for 1. Furusawa, M, Nishimura, J, Yamaizumi, M & this preparation have been described [2-4], Okada, Y, Nature 249 (1974) 449. 2. Loyter, A, Zakai, N & Kulka, R G, J cell biol 66 but the resultant preparations are generally (1975) 292. contaminated with significant quantities of 3. Schlegel, R A & Rechsteiner, M C, Cell 5 (1975) membrane-free granules. Therefore a tech371. 4. Wassermann, M, Zakai, N, Loyter, A & Kulka, R nique capable of reproducibly providing G, Cell 7 (1976) 551. relatively pure preparations of intact gran5. Wille, W & Willecke, K, FEBS lett 65 (1976) 59. 6. Nishimura, T, Furusawa, M, Yamaizumi, M & ules has been developed. Okada, Y, Cell struct and func 1 (1976) 197. jetted material may not suffice for biochemical work [ 111.
7. Wassermann, M, Loyter, A & Kulka, R G, J cell sci. In press (1977). 8. Kaltoft, K, Zeuthen, J, Engbaek, F, Piper, P W & Celis, J E, Proc natl acad sci US 73 (1976) 2793. 9. Fenwick, R G & Caskey, C T, Cell 5 (1975) I IS. 10. Pontecorvo, G, Riddle, P N & Hales, A, Nature 265 (1977) 257. 11. Celis, J E, Brookhaven symp bio129 (1977) 178. Received December 9, 1977 Revised version received April 3, 1978 Accepted April 14, 1978
Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l 152.0428$02.00/0
Isolation of membrane-bound rat mast cell granules G. D. RAPHAEL, W. R. HENDERSON and M. KALINER, Laboratory of Clinical Investigation, National National USA
Institute of Allergy and infectious Diseases, Institutes of Health, Bethesda, MD 20014,
A technique for obtaining membranebound rat peritoneal mast cell granules in high yield is described. Mast cells purified by centrifugation into 38 % BSA gradients were sonicated in Caz+, Mg2+-free Tyrode’s-gelatin buffer, incubated in EDTA for I5 min at 37 “C, and differentially centrifuged through a 0.34 M sucrose cushion to yield a granular preparation with >80% of the granules bound by perigranular membranes. The perigranular membranes were demonstrated morphologically by light and electron microscopy and functionally by histamine distribution. Summary.
EIP Cell Res 115 (I 978)
Materials
and Methods
Mast cells were obtained as described f51 from 200 gram male Sprague-Dawley rats using Tyidde’s buffer prepared without Ca*+ or MeZ+ and containine 0. I % gelatin (Ca2+, Mg*+-free TyLgel). Purified mast cell preparations were prepared from mixed peritoneal cell preparations by centrifugation into 38 % BSA cushions [6]. The resultant mast cell populations were >85% homogeneous as determined by toluidine blue staining 171.Secretorv granules from the nurified mast ceil preparations were-obtained free of perigranular membranes by osmotic lysis [ 1, 81. Secretory granules with intact perigranular membranes were isolated bv the followine techniaue: ourified mast cells were suspended in-2.0 ml *of da’+, ME*+-free Tvr-ael at 4 “C in 15X75 mm nolvoronvlene tubes and soni~ated for I2 set using a mifiotip sonicator with a power setting of 2.5 (Model WI85 F, Heat Systems-Ultrasonics, Inc., Plainview, NY). EDTA (0.22 ml, 25 mM, Ca2+, Mg*+-free Tyr-gel) was added to each tube and the suspensions were incubated at 37°C for 15 min. After vortexing for I min, the suspension were layered over 2.0 ml of 0.34 M sucrose and centrifuged at 300 g for 10 min at 4°C. The pellet resulting from this initial separation has been designated the cellular debris pellet. The remaining granule-rich supernatants were centrifuged at 2 000 g for 20 min at 4 “C and the resultant oellet desienated the granular pellet. The supernatants remaining after the 2 000 g centrifugation were relatively granule-free. The pellets obtained from the second centrifugation usually contained >80% membrane-bound granules. Histamine was determined employing an atropinized guinea pig ileum as described [9]. Samples were prepared for electron microscopy by fixation in 3% glutaraldehyde, Maraglas embedding, and staining with 0~0~. A Philips EM 300 (Philips Electronics Instruments, Mt. Vernon, NY) was used.
Preliminary
notes
429
Fig. I. (a) A low magnification electron micrograph of the purified granule suspension containing more than 80% intact membrane-bound rat mast cell granules.
(6) Electron micrograph of mast cell granules isolated with intact perigranular membranes (lef panel) and free of membranes (right panel).
Results and Discussion
solutions as described [2]. Analysis of the preparations obtained revealed mixed populations of granules by toluidine blue staining. A portion (50 %) of the granules stained
Initial attempts at preparing suspensions of mast cell granules with intact perigranular membranes employed sonication in sucrose
Exp Cell Res II5 (1978)
430
Preliminary
notes
Table 1 Histamine content % total Granular pellet Supernatant
364 83
81 19
Two individual mast cell preparations (41X IO6 cells, >90% homogenous; and 15~10~ cells, >8.5% homogenous) were subjected to the procedures as outlined in the Materials section. During the isolation procedure, an additional 75 kg of histamine fractionated with the cellular debris pellet indicating that the final yield of histamine-contaking granules is only 67 % of the starting pool. As the granule pellet is suspended in cation-containing buffer, histamine remains associated onlv with granules contained within functionally intact p&granular membranes. The observation that 81% of the histamine remains in the granule fraction confirms the observations made by electron microscopy. The data represents the results of two individual experiments. The histamine was released from the granule preparation by repeated freeze-thawing.
intensely blue and were more compact than the remaining lightly stained granules. This was consistent with data reported elsewhere [2, lo] from which the conclusion was drawn that the darkly staining granules are membrane-bound while the lightly staining granules are membrane-free. Since a more highly purified preparation of intact granules was desired, modifications of this technique were explored. Sonication in Tyrode’s-gelatin (containing Ca2+, MgZ+) rather than in sucrose eliminated two or three centrifugation steps along with their obligatory loss of granular integrity. The optimal time of sonication was determined by comparing 6, 12, 18, 24 and 30 set sonication periods at a power setting of 2.5 (resulting in a maximum energy output of 50-60). The granular pellet from the preparation sonicated for 12 set contained a nearly homogeneous population of compact granules which stained intensely with toluidine blue. In comparison, Exp
Cell
Res
II.5 (1978)
longer periods of sonication resulted in mixed populations of granules. Analysis of the cellular debris pellet after 12 set of sonication revealed numerous mast cells with partially disrupted cell membranes, but apparently containing a full complement of granules. In an attempt to recover these granules, several additional modifications were studied. We have consistently observed that granules released from mast cells adhere both to mast cell membranes and to each other, and we reasoned that divalent cations may be necessary for this phenomenon. Therefore, we substituted Ca2+, Mg*+-free Tyrode’s-gel in the initial sonication phase, added EDTA to the sonicated preparations (final concentration, 2.5 mM) and incubated the samples for 15 min at 37 “C. These procedures were designed to permit dissociation of the granules and resulted in an increased yield of darkly staining granules in the granular pellet. However, examination of the cellular debris pellet still revealed the presence of numerous partially disrupted mast cells. Therefore, in an attempt to further disperse these cells, the sonicated preparations were centrifuged into 0.34 M sucrose cushions. These modifications resulted in a granular pellet greatly enriched in quantity and containing >80% compact, darkly staining granules. In contrast to our earlier observations, the cellular debris pellet contained few partially disrupted mast cells. An electron microscopic examination of a representative granular preparation is shown in fig. 1, and is contrasted with granules obtained free of their perigranular membranes by osmotic lysis [ 1, 81. The mast cell granule consists of a basic protein-heparin matrix to which are bound low molecular weight moieties such as histamine [8]. Histamine is released from the
Preliminary notes granular matrix upon the addition of cations by a simple ion exchange mechanism [8]. In addition to morphological examination, the capacity of salt to elute histamine from the granules was employed to test the integrity of the perigranular membranes. The distribution of histamine throughout the granular isolation procedure is shown in table 1. The granular pellet was shown to contain 81% of the total histamine. As the granular isolation is carried out in cationcontaining buffer, the persistence of 81% of the histamine in the granule pellet indicates that the perigranular membranes were functionally intact in that portion of the granules. Therefore, the following lines of evidence suggest that the granular preparations were obtained with intact perigranular membranes: 1. >80% of the granules have a compact, darkly staining appearance after toluidine blue staining. 2. >80% of the granules contained perigranular membranes by electron microscopy . 3. Histamine remained associated with the granular preparation in the presence of cations, indicating functionally intact perigranular membranes [4].
43 1
7. Valentine, M D, Bach, M, Stechschulte, D J & Austen, K F, Methods in immunology and immunochemistry (ed C A Williams & M W Chase) p. 108. Academic Press, New York (1976). 8. Uvnls, B, Aborg, C H & Bergendorff, A, Acta physiol Stand 78, suppl. 336 (1970) 3. 9. Stechschulte, D J, Austen, K F & Block, K J, J exp med 125 (1%7) 127. 10. Anderson, P & Uvntis, B, Acta physiol Stand 94 (1975) 63. Received December 30, 1977 Revised version received March 20, 1978 Accepted March 20, 1978
Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l 152-0431$02.00/0
Aggregation of homozygous Brachyury (T) cells in the culture supernatant of wild-type or mutant embryos K. 0. YANAGISAWA
and H. FUJIMOTO, Labo-
ratory of Cell Biology, Mitsubishi-Kasei Life Sciences, Machida-shi, Minamiooya, Japan
Institute of Tokyo 194,
Dissociated cells from wild-type or homozygous mutant (T/T) embryos of mice were cultured in the culture supematant of +I+ or T/T embryos on a gyratory shaker. The aggregation was promoted by the culture supematant in the following combinations; +/+ cells in -+I+ culture supematant, +I+ cells in T/T supematant and T/T cells in +I+ supematant. When T/T cells were cultured in T/T supematant, however, only a slight promotion of the aggregation was observed.
Summary.
The authors eratefullv acknowledae the hem of Dr Alan Rosenthil Mr Thomas Blake,-Ms Kerstin Cehrs, Dr Timothv T&he and Dr Millicent Donlon in nrenaration and interpretation of the electron micrographs and MS Patricia Silva for her excellent preparation of the manuscript.
Mutations at the T/t locus of the mouse cause abnormalities in the organizational and morphogenetic processes in early embryogenesis [ 11. Histological and immunological studies suggest that alleles at the References T/t locus may affect cell-cell interaction [2, 1. Henderson, W R & Kaliner, M, J clin invest 61 31. Among these homozygous mutants, the (1978) 187. T/T embryos are distinguishable by their 2. Anderson, P, Rohlich, P, Slorach, S A & Uvnls, B, Acta physiol Stand 91 (1974) 145. abnormal morphology at 8 days of gesta3. Saeki, K, Endo, K & Yamasaki, H, Japan j tion. We have been studying the effect of pharmacol22 (1972) 27. 4. Grossman, N & Diamant, B, Agents and actions 6 the T mutation at the cellular level. (1976) 394. It was previously shown that dissociated 5. Austen, K F. Bloch, K J, Baker, A R & Amason, B G, Proc sot exp biol med 120 (1965) 542. T/T cells have a different adhesiveness as 6. Sullivan, T J, Parker, K L, Stenson, W & Parker, compared to +I+ cells [4]. Since aggregaC W, J immunol 114 (1975) 1473. Exp Cell Res I I5 (1978)
Preliminary
notes
Rat mast cells have been found to generate the superoxide anion and to release superoxide dismutase concomitantly with deWe thank A. Celis for supplying the HGPRT mutant. granulation in response to immunologic and We also acknowledge the support of the Landsfornon-immunologic stimulation [ 11. In order eningen til Kraeftens Bekiempelse, the Danish Natural Science Research Council and Aarhus Universitets to study the granular source of superoxide Forskningsfond. generation, it became necessary to obtain mast cell granules with their perigranular References membranes intact. Several techniques for 1. Furusawa, M, Nishimura, J, Yamaizumi, M & this preparation have been described [2-4], Okada, Y, Nature 249 (1974) 449. 2. Loyter, A, Zakai, N & Kulka, R G, J cell biol 66 but the resultant preparations are generally (1975) 292. contaminated with significant quantities of 3. Schlegel, R A & Rechsteiner, M C, Cell 5 (1975) membrane-free granules. Therefore a tech371. 4. Wassermann, M, Zakai, N, Loyter, A & Kulka, R nique capable of reproducibly providing G, Cell 7 (1976) 551. relatively pure preparations of intact gran5. Wille, W & Willecke, K, FEBS lett 65 (1976) 59. 6. Nishimura, T, Furusawa, M, Yamaizumi, M & ules has been developed. Okada, Y, Cell struct and func 1 (1976) 197. jetted material may not suffice for biochemical work [ 111.
7. Wassermann, M, Loyter, A & Kulka, R G, J cell sci. In press (1977). 8. Kaltoft, K, Zeuthen, J, Engbaek, F, Piper, P W & Celis, J E, Proc natl acad sci US 73 (1976) 2793. 9. Fenwick, R G & Caskey, C T, Cell 5 (1975) I IS. 10. Pontecorvo, G, Riddle, P N & Hales, A, Nature 265 (1977) 257. 11. Celis, J E, Brookhaven symp bio129 (1977) 178. Received December 9, 1977 Revised version received April 3, 1978 Accepted April 14, 1978
Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l 152.0428$02.00/0
Isolation of membrane-bound rat mast cell granules G. D. RAPHAEL, W. R. HENDERSON and M. KALINER, Laboratory of Clinical Investigation, National National USA
Institute of Allergy and infectious Diseases, Institutes of Health, Bethesda, MD 20014,
A technique for obtaining membranebound rat peritoneal mast cell granules in high yield is described. Mast cells purified by centrifugation into 38 % BSA gradients were sonicated in Caz+, Mg2+-free Tyrode’s-gelatin buffer, incubated in EDTA for I5 min at 37 “C, and differentially centrifuged through a 0.34 M sucrose cushion to yield a granular preparation with >80% of the granules bound by perigranular membranes. The perigranular membranes were demonstrated morphologically by light and electron microscopy and functionally by histamine distribution. Summary.
EIP Cell Res 115 (I 978)
Materials
and Methods
Mast cells were obtained as described f51 from 200 gram male Sprague-Dawley rats using Tyidde’s buffer prepared without Ca*+ or MeZ+ and containine 0. I % gelatin (Ca2+, Mg*+-free TyLgel). Purified mast cell preparations were prepared from mixed peritoneal cell preparations by centrifugation into 38 % BSA cushions [6]. The resultant mast cell populations were >85% homogeneous as determined by toluidine blue staining 171.Secretorv granules from the nurified mast ceil preparations were-obtained free of perigranular membranes by osmotic lysis [ 1, 81. Secretory granules with intact perigranular membranes were isolated bv the followine techniaue: ourified mast cells were suspended in-2.0 ml *of da’+, ME*+-free Tvr-ael at 4 “C in 15X75 mm nolvoronvlene tubes and soni~ated for I2 set using a mifiotip sonicator with a power setting of 2.5 (Model WI85 F, Heat Systems-Ultrasonics, Inc., Plainview, NY). EDTA (0.22 ml, 25 mM, Ca2+, Mg*+-free Tyr-gel) was added to each tube and the suspensions were incubated at 37°C for 15 min. After vortexing for I min, the suspension were layered over 2.0 ml of 0.34 M sucrose and centrifuged at 300 g for 10 min at 4°C. The pellet resulting from this initial separation has been designated the cellular debris pellet. The remaining granule-rich supernatants were centrifuged at 2 000 g for 20 min at 4 “C and the resultant oellet desienated the granular pellet. The supernatants remaining after the 2 000 g centrifugation were relatively granule-free. The pellets obtained from the second centrifugation usually contained >80% membrane-bound granules. Histamine was determined employing an atropinized guinea pig ileum as described [9]. Samples were prepared for electron microscopy by fixation in 3% glutaraldehyde, Maraglas embedding, and staining with 0~0~. A Philips EM 300 (Philips Electronics Instruments, Mt. Vernon, NY) was used.
Preliminary
notes
429
Fig. I. (a) A low magnification electron micrograph of the purified granule suspension containing more than 80% intact membrane-bound rat mast cell granules.
(6) Electron micrograph of mast cell granules isolated with intact perigranular membranes (lef panel) and free of membranes (right panel).
Results and Discussion
solutions as described [2]. Analysis of the preparations obtained revealed mixed populations of granules by toluidine blue staining. A portion (50 %) of the granules stained
Initial attempts at preparing suspensions of mast cell granules with intact perigranular membranes employed sonication in sucrose
Exp Cell Res II5 (1978)
430
Preliminary
notes
Table 1 Histamine content % total Granular pellet Supernatant
364 83
81 19
Two individual mast cell preparations (41X IO6 cells, >90% homogenous; and 15~10~ cells, >8.5% homogenous) were subjected to the procedures as outlined in the Materials section. During the isolation procedure, an additional 75 kg of histamine fractionated with the cellular debris pellet indicating that the final yield of histamine-contaking granules is only 67 % of the starting pool. As the granule pellet is suspended in cation-containing buffer, histamine remains associated onlv with granules contained within functionally intact p&granular membranes. The observation that 81% of the histamine remains in the granule fraction confirms the observations made by electron microscopy. The data represents the results of two individual experiments. The histamine was released from the granule preparation by repeated freeze-thawing.
intensely blue and were more compact than the remaining lightly stained granules. This was consistent with data reported elsewhere [2, lo] from which the conclusion was drawn that the darkly staining granules are membrane-bound while the lightly staining granules are membrane-free. Since a more highly purified preparation of intact granules was desired, modifications of this technique were explored. Sonication in Tyrode’s-gelatin (containing Ca2+, MgZ+) rather than in sucrose eliminated two or three centrifugation steps along with their obligatory loss of granular integrity. The optimal time of sonication was determined by comparing 6, 12, 18, 24 and 30 set sonication periods at a power setting of 2.5 (resulting in a maximum energy output of 50-60). The granular pellet from the preparation sonicated for 12 set contained a nearly homogeneous population of compact granules which stained intensely with toluidine blue. In comparison, Exp
Cell
Res
II.5 (1978)
longer periods of sonication resulted in mixed populations of granules. Analysis of the cellular debris pellet after 12 set of sonication revealed numerous mast cells with partially disrupted cell membranes, but apparently containing a full complement of granules. In an attempt to recover these granules, several additional modifications were studied. We have consistently observed that granules released from mast cells adhere both to mast cell membranes and to each other, and we reasoned that divalent cations may be necessary for this phenomenon. Therefore, we substituted Ca2+, Mg*+-free Tyrode’s-gel in the initial sonication phase, added EDTA to the sonicated preparations (final concentration, 2.5 mM) and incubated the samples for 15 min at 37 “C. These procedures were designed to permit dissociation of the granules and resulted in an increased yield of darkly staining granules in the granular pellet. However, examination of the cellular debris pellet still revealed the presence of numerous partially disrupted mast cells. Therefore, in an attempt to further disperse these cells, the sonicated preparations were centrifuged into 0.34 M sucrose cushions. These modifications resulted in a granular pellet greatly enriched in quantity and containing >80% compact, darkly staining granules. In contrast to our earlier observations, the cellular debris pellet contained few partially disrupted mast cells. An electron microscopic examination of a representative granular preparation is shown in fig. 1, and is contrasted with granules obtained free of their perigranular membranes by osmotic lysis [ 1, 81. The mast cell granule consists of a basic protein-heparin matrix to which are bound low molecular weight moieties such as histamine [8]. Histamine is released from the
Preliminary notes granular matrix upon the addition of cations by a simple ion exchange mechanism [8]. In addition to morphological examination, the capacity of salt to elute histamine from the granules was employed to test the integrity of the perigranular membranes. The distribution of histamine throughout the granular isolation procedure is shown in table 1. The granular pellet was shown to contain 81% of the total histamine. As the granular isolation is carried out in cationcontaining buffer, the persistence of 81% of the histamine in the granule pellet indicates that the perigranular membranes were functionally intact in that portion of the granules. Therefore, the following lines of evidence suggest that the granular preparations were obtained with intact perigranular membranes: 1. >80% of the granules have a compact, darkly staining appearance after toluidine blue staining. 2. >80% of the granules contained perigranular membranes by electron microscopy . 3. Histamine remained associated with the granular preparation in the presence of cations, indicating functionally intact perigranular membranes [4].
43 1
7. Valentine, M D, Bach, M, Stechschulte, D J & Austen, K F, Methods in immunology and immunochemistry (ed C A Williams & M W Chase) p. 108. Academic Press, New York (1976). 8. Uvnls, B, Aborg, C H & Bergendorff, A, Acta physiol Stand 78, suppl. 336 (1970) 3. 9. Stechschulte, D J, Austen, K F & Block, K J, J exp med 125 (1%7) 127. 10. Anderson, P & Uvntis, B, Acta physiol Stand 94 (1975) 63. Received December 30, 1977 Revised version received March 20, 1978 Accepted March 20, 1978
Printed in Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/l 152-0431$02.00/0
Aggregation of homozygous Brachyury (T) cells in the culture supernatant of wild-type or mutant embryos K. 0. YANAGISAWA
and H. FUJIMOTO, Labo-
ratory of Cell Biology, Mitsubishi-Kasei Life Sciences, Machida-shi, Minamiooya, Japan
Institute of Tokyo 194,
Dissociated cells from wild-type or homozygous mutant (T/T) embryos of mice were cultured in the culture supematant of +I+ or T/T embryos on a gyratory shaker. The aggregation was promoted by the culture supematant in the following combinations; +/+ cells in -+I+ culture supematant, +I+ cells in T/T supematant and T/T cells in +I+ supematant. When T/T cells were cultured in T/T supematant, however, only a slight promotion of the aggregation was observed.
Summary.
The authors eratefullv acknowledae the hem of Dr Alan Rosenthil Mr Thomas Blake,-Ms Kerstin Cehrs, Dr Timothv T&he and Dr Millicent Donlon in nrenaration and interpretation of the electron micrographs and MS Patricia Silva for her excellent preparation of the manuscript.
Mutations at the T/t locus of the mouse cause abnormalities in the organizational and morphogenetic processes in early embryogenesis [ 11. Histological and immunological studies suggest that alleles at the References T/t locus may affect cell-cell interaction [2, 1. Henderson, W R & Kaliner, M, J clin invest 61 31. Among these homozygous mutants, the (1978) 187. T/T embryos are distinguishable by their 2. Anderson, P, Rohlich, P, Slorach, S A & Uvnls, B, Acta physiol Stand 91 (1974) 145. abnormal morphology at 8 days of gesta3. Saeki, K, Endo, K & Yamasaki, H, Japan j tion. We have been studying the effect of pharmacol22 (1972) 27. 4. Grossman, N & Diamant, B, Agents and actions 6 the T mutation at the cellular level. (1976) 394. It was previously shown that dissociated 5. Austen, K F. Bloch, K J, Baker, A R & Amason, B G, Proc sot exp biol med 120 (1965) 542. T/T cells have a different adhesiveness as 6. Sullivan, T J, Parker, K L, Stenson, W & Parker, compared to +I+ cells [4]. Since aggregaC W, J immunol 114 (1975) 1473. Exp Cell Res I I5 (1978)
