CRYOBIOLOGY
16, 112-117
Effect
( 1979 )
of Endocellular Neutrophil
Cryoprotectant upon Polymorphonuclear Function during Storage at Low
TATSUHISA “Laboratory School
YAMASHITA,”
of
Temperature
NORIKO
of Physiological Medicine, Juntendo
IMAIZUMI,’
Chemistry University,
Transfusion of large numbers of viable polymorphonuclear neutrophils ( PMNs) is therapeutically effective in neutropenic patients with gram-negative bacteremia, leukemia, and bone marrow aplasia induced by radiation and/or drugs. Increased use of leukocyte transfusions has led investigators to try to freeze preserve leukocytes (2-10). We studied the effect of commonly employed cryoprotective agents such as dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon ceil function to select a cryoprotectant of choice for freeze preservation of leukocytes. In the present communication, guinea pig PMN integrity after treatment with cryoprotective agents was assessed by trypan blue exclusion, in vitro directed migration (chemotaxis), and phagocytosis. MATERIALS
AND
METHODS
Leukocytes. Polymorphonuclear neutrophi1 exudates were elicited in fasted guinea pigs by intraperitoneal injection of 100 ml of 0.12% glycogen in 0.9%; saline. Thirteen to fourteen hours later, the animal was killed with ether and 100 ml of citratesaline (0.474 sodium citrate in 0.85% sodium chloride) was injected intraperito20,
Received 1978.
August
4, 1978;
accepted
December
112 OOlI-2240/79/020112-06$02.00/0 Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
and tBlood Bunkyo-ku,
AND
SHINJI
YUASAt
Transfusion Service, Tokyo, Japan 113
neally. The abdomen was kneaded for 1 to 2 min, perforated, ‘and the fluid ( 140 to 160 ml) was withdrawn. The exudates were filtered through three layers of gauze and centrifuged at 1OOg for 10 minutes. After removal of the supernatant, the pellets were suspended in 1.5 to 2.0 ml of phosphate-buffered saline without divalent cations [pH 7.4 PBS( - )] . The red blood cells underwent hypotonic lysis by exposure of the cell suspension to 10 to 20 volumes of 0.2%) saline for 60 set, followed by addition of an equal voIume of 1.61% saline. After centrifugation, the cells were washed twice with PBS( - ) and finally suspended mat ,a ‘desired concentration in phosphate-buffered saline containing 1 mM MgS04 and 0.07 mM CaClz (PBS). Four to five milliliters of PMN suspension containing 8 to 9 X 10’ cells/ml were usually obtained from a single animal. More than 95% of PMNs excluded the vit.al dye trypan blue after suspension in 0.13% trypan blue. Differential cell counts employing Wright’s stain showed that 97 to 98p of cells were PMNs. Centrifugation was carried out in a refrigerated 6entrifuge using swing-out cups. All glassware used in an isolation and modification of PMNs was siliconized. Chemoattractant. Escherichiu coli CH/2 were grown for 20 hr at 37°C with vigor-
EFFECT OF CRYOPROTECTANT ous shaking in 14 Sakaguchi flasks containing 200 ml of polypepton medium (polypepton, 10 g; NaCI, 3 g; MgS01*7H20, 0.25 g; CaCI,, 0.01 g; glucose, 2 g; KH2P04, 1.3 g; Tris (hydroxymethyl) aminomethane, 7.3 g/liter, pH 7.4). Organisms were concentrated into one tube by centrifugation at 95OOg for 20 min, washed twice with PBS ( .- ), and finally suspended in PBS. The bacterial suspension (ca. 120 ml) was four times sonicated in ice for 2 min using a Branson sonic generator (20 k cycles/set) with a 2 min pause between cycles. After centrifugation of the sonicated suspension at 9500g for 20 min, supernatant was diluted with one to two8 volumes of PBS and u.sed as a chemoattractant. Treatment of PMNs with cryoprotectants. One milliliter of PMN suspension ( lo7 PMN) was incubated between 0” and 4°C with 0.0,5 to 0.2 ml of cryoprotectant containing O.l5 to 0 ml of deionized distilled water. Final concentrations of the cryoprotectant were 4.2, 8.3, and 16.7$%. In one group at the end of 1 hr, and in the other at the end of 20 hr, the PMNs were washed twice with a large excess of PBS. After adjusting the PMN count to 5 x 106/ml, trypan blue exclusion, chemotaxis and phagocytosis of cryoprotectant-treated PMNs were assayed. PMNs, used as a control, were treated under the same conditions except that 0.2 ml water was used instead of the different cryoprotectants. Chemotaxis assay. Chemotaxis was assayed by employing a modified Boyden chamber (1) .with Sartorius Membranfilter (its pore size and diameter were 3 pm and 25 mm, respectively) to separate the chamber into compartments and by measuring the distance from the top of the filter to the furthest two cells at the same focal plane according to the method of Zigmond and Hirsch (11). Two milliliters of bacterial chemoattractant solution were placed in the lower compartment, and 1 ml of PMN suspension (5 X lo6 cells/ml) was introduced into the unner comnartment. After L
ON GRANULOCYTES
113
incubation at 37°C for 40 min, the suspension was removed, the filter was fixed with 4% formalin for more than 1 hr, and stained with hematoxylin for 5 to 10 min. After the filter was cleared with xylene, the distance from the starting surface to the cell front was measured with the micrometer on a fine focusing adjuster. The results were expressed as an average migration distance per 10 random fields in duplicate at a magnification of 600x. Phagocytosis. The ability of PMNs to phagocytize bacteria was determined by microscopic examination of Gram stained smears of PMNs incubated with heat-killed (20 min at 100°C) bacteria. To the incubation mixture containing 0.5 ml of PMN suspension ( lo7 cells/ml), 0.3 ml PBS and 0.1 ml fresh homologous serum in a siliconized Pyrex tube was added 0.1 ml of a saline suspension containing 5 X lo7 streptococci aureus per ml. The tubes were constantly shaken at 37°C. Aliquots of the mixture were removed at 5, 15, and 30 min, smeared on the glass slides, fixed with methanol for ca. 3 min and stained with Gram stain. Ingestive ability was represented as the ratio of PMNs with intracellular bacteria to total PMNs enumerated. RESULTS Effect of cryoprotectant on trypan blue dye exclusion. Greater than 90% of PMNs incubated with all concentrations of each of the three cryoprotectants excluded trypan blue dye. Effect of cryoprotectant on chemotaxis. The chemotaxis of PMNs in the presence of agents after exposure to different concentrations of cryoprotective agents at 0 to 4°C is indicated in Table 1. An inhibitory effect of cryoprotectant on the chemotaxis increased as cryoprotectant concentration increased with complete inhibition of chemotaxis at 16.7%. Among protectants used, glycerol showed the strongest inhibitory action as compared with the other two agents and almost completely inhibited
114
YAMASHITA,
IMAIZUMI,
AND
TABLE
YUASA
1
Chemotaxis
and Viability of P?vlNs after I-hr and 90-hr Storage at 0 to 4°C Presence of Different Concentrations of Cryoprotectants
Agents
Concentretion % M
Neutrophil migrations (70 of control) 1-hr t!XPOSUR
in the
Viability (%I
20-hr exposure
I-hr
exposure
20-hr
exposure
DMSO
4.2 (0.59) 8.3 (1.17) 16.7 (2.35)
78 f 41 f 4fl
5 8
52 zk 2 25 + 4 6&l
92 f 94 f 95 f
Glycerol
4.2 (0.57) 8.3 (1.14) 16.7 (2.28)
63 f 13f2 6fl
8
36 z!z 4 llf5 5&l
92 f 7 (94 f 92 i 6 (97 f 96 z!c 1 (83 f
4.2 (0.75) 8.3 (1.49) 16.7 (2.98)
80 + 8 55 f 5 6&l
50 f 8 35 + 3 8zt4
94 zt 2 (94 f 3) 97 f 2 (93 f 2) 91 f 5 (83 zk 4)
94 f 92 f 85 f
3 (85 f 6) 1 (86 * 2) 8 (70 + 7)
75 f
97 f
89 f
4 (85 f
Ethylene
Control Migration 0 Migration b Viability parentheses.
glycol
100 and viability values represent distance of controls is 71 f of PMNs incubated at 37°C
10
6 (95 f 4)b 3 (97 zk 2) 2 (83 f 5)
2 (91 f
4) 2) 2)
4)
95 f 89 f 89 f
1 (91 i 5 (89 f 5 (72 f
88 f 4 (85 i 91 f 2 (90 f 95 f 5 (78 f
the mean & standard deviation of three experiments. 12 pm in 40 min. for 1 hr in the presence of agents after the storage
the chemotaxis at 8.3% even after 1 hr incubation, As the chemotaxis assays were carried out at 37°C for 40 min, PMNs were further incubated at 37°C for 1 hr to check whether the temperature accelerated the impairment by the cryoprotectant ‘of PMNs, resulting in the chemotaxis inhibition. However, no remarkable changes were observed in viability although the viability of PMNs exposed to 16.7% agents decreased to some extent by incubation at 37°C. These results suggest the possibility that the inhibition of chemotaxis is not due to death of cells but due to the modification by cryoprotectant of mechanisms involved in the chemotaxis. Recovery of chemotaxis after the remoual of cryoprotectants with washing. To study whether the chemotaxis inhibition by cryoprotectants is reversibly eliminated or not, PMNs were washed with PBS( - ) to remove agents after an exposure to different concentrations of cryoprotectants during two time periods. As can be seen in Table 2, chemotaxis was suppressed by
4)b 6) 8) 7) 5) 3)
4)
is given
in
approximately 1070 with two washings even in freshly prepared control PMNs. When control PMNs were kept at 4°C for 20 hr, approximately 20% inhibition in chemotaxis was observed even without washing and about 40% inhibition with washing, although most PMNs excluded trypan blue dye. When exposed to 4.2% cryoprotectants, chemot’axis was restored with washing almost to the same level of migration as that of the washed control regardless of the exposure time and the kind of cryoprotectant except 20-hr exposure to glycerol, suggesting that PMNs are hardly affected by cryoprotectants at this concentration. With 8.3% cryoprotectant-treated PMNs, chemotactic inhibition was ‘dependent on the agents and exposure time. The DMSO-treated PMNs retained nearly the same chemotaxis as washed control PMNs even during 20-hr Ethylene glycol-treated PMNs exposure. were inhibited by about 30% with 1-hr exposure and considerably more with 2O-hr exposure. In glycerol, So% inhibition was
EFFECT
OF
CRYOPROTECTANT
ON
observed following 1 hr treatment with complete inhibition after 20-hr exposure. However, viability tests showed that at least 90% PMNs were viable in I-hr exposure regardless of the sort of cryoprotectants while greater than 60% PMNs were viable in 20-hr exposure. With 16.7y0 cryoprotectants, no recovery of chemotaxis was observed Iat all by the removal of agents even after I-hr exposure and moreover the death of a considerable number of PMNs was observed. Phagocytosis by PMNs after an exposure to cryoprotectants. The ingestive ability of PMNs washed after a treatment with cryoprotectants was studied. As seen in Table 3, the percentage of the cells containing bacteria increased with increasing incubation time. Heat-killed PMNs did not show such an increase in the percentage of ceils with bacteria, indicating a true ingestion of bacteria by cryoprotectant-treated PMNs. Ingestive ability of control PMNs was not as affected as chemotaxis by storage and washing. With 1 hr exposure to cryoprotectants, no essential differences
Agents
and Viability
of PMNs
after
Concentration (%I
115
were observed in the ingestive ability within 8.370: and even at 16.770 the ingestion of bacteria by PMNs was considerable, Phago,cytic ability, however, almost dis‘appeared in ethylene glycol. With 20-hr exposure, the ingestion was not affected at 4.2%) concentrations, but was considerably suppressed especially in glycerol at 8.3%. PMNs exposed to 8.3%1 DMSO, however, retained the ingestive ability well. DISCUSSION
Most of PMNs could exclude the trypan blue after 20 hour exposure to cryoprotectants even at a high concentration if the washing procedure was omitted ( Table 1) . On the other hand, when PMNs were washed after an exposure to cryoprotectant at a high concentration, 50 to 80%’ of PMNs could not exclude the dye. However, the ability to exclude dye was hardly affected by washing after short exposure to 8.3% concentrations of cryoprotectants (Table 2). This would indicate that cells become very fragile with cryoprotectants
TABLE Chemotaxis
GRANULOCYTES
2
the Removal
of Cryoprotectants
with
Neutrophil migrations (70 of control) 1-hr
exposure
20-hr
Washing Viability (%I
exposure
1.hr
exposure
20-hr exposure
DMSO
4.2 8.3 16.7
88 f 73 l 18 f
6 10 7
69 f 51 f 5&l
7 11
94 zt 1 89 k 7 55 z!z 5
90 f 75 f 17 f
Glycerol
4.2 8.3 16.7
89 f 45 f Qf5
13 9
41 f 5f2 3fl
8
93 f 2 91 f6 85 f 7
85 f 5 67 zt 9 47 f 12
4.2 8.3 16 7
81 rt 3 61 f 10 7*3
64 l 25 f 5+2
10 8
96 f 89 f 55 f
3 7 4
89 f 61 f 29 i
6 10 12
60 f 75 f
8 3
94 f 96 f
4 2
92 f 96 f
7 3
Ethylene
Control
glycol
Washed Unwashed
93 f 100
The mean f standard deviation of 4 samples. a Percentages of neutrophil migration are calculated PMNs after preparation, their migration value being
8
as compared to t.he chemotaxis 65 i 8 pm in 40 min.
of the unwashed
5 7 6
fresh
116
YAMASHITA,
IMAIZUMI,
AND
TABLE Ingestive Agents
Ability
of I’MNs
Washed
with
YUASA
3
PBS
after
Concentration (%)
an Exposure Ingestion
5
15 1-hr
30
time
to Cryoptrotcctant bin) 5
-_ PMNs
exposed
15 ZO-hr
exposed
30 PMNs
DMSO
4.2 8.3 16.7
5*3 6k3 2&l
21 f 7 17 f 5 11 f3
39 f 30 f 21 f
6 9 5
9f4 2f5 -
21 f 14 f -
9 6
30 i 22 f -
8 5
Glycerol
4.2 8.3 16.7
8f6 7fl 2f2
27 f 25 f 12 f
35 f 30 f 17 f
7 10 8
5*1 lf2 -
22 f 7fl -
7
29 f 8zt8 -
9
4.2 8.3 16.7
5f4 5f3 lf2
19 f 2 25 ic 5 4f3
34 f 9 33 f 8 3zk2
10 f 5 3z!z2 -
21 f 8f3 -
6
34 f 16 f -
10 5
17 f 23 i
38 f 34 f
7*7 10 f
23 f 26 f
6 5
36 f 38 f
9 3
Ethylene
Control
glycol
Washed Unwashed
7&5 11 f 8
10 10 6
8 7
8 9
5
The mean f standard deviation of three experiments. Values represent the percent of cells containing bacteria.
especially at a high concentration and are easily damaged by washing, i.e. by the osmotic effects of removing cryoprotective agents. Therefore, further studies of a better technique for adding cryoprotectant to a ‘desired concentration and then removing it without producing a significant impairment of’ PMN function need to be done. Chemotactic response decreased with washing and storage (control in Table 2) and was also sensitive to an exposure to cryoprotectant (Table 2). On the other hand, the ingestive ability was not so affected by washing, storage and cryoprotectants ‘as chemotaxis (Table 3). It seems to be of functional significance that the ‘ability to ingest bacteria is less damaged by washing and cryoprotectants than chemotactic response because phagocytosis is the function of primary concern in PMN transfusion. To put the above findings in another way, the inhibition of chemotactic response appears to be a more sensitive indicator of impaired leukocyte function.
SUMMARY
The effect of cryoprotective agents dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon the function of polymorphonuclear neutro,phils ( PMNs ) during storage between 0” and 4°C was investigated. Increasing concentration of each cryoprotectant caused an increasing inhibition of chemotaxis with complete inhibition at 16.7%. At this concentration most PMNs were still able to exclude trypan blue dye. Chemotaxis was not inhibited if PMNs were exposed to 4.2 or 8.3% concentrations of cryoprotectants for 1 hr, and washed subsequently. However, the recovery of chemotaxis was not observed at 16.7% after 1 hr exposure to cryoprotectants. Moreover, a considerable number of PMNs could not exclude the dye. This would indicate that cells become fragile with cryoprotectants at a high concentration and the PMNs are easily damaged by washing. With 20 hr exposure PMNs, the inhibitory effect on chemotaxis was removed by wash-
EFFECT
OF
CRYOPROTECTANT
ing when a 4.2% #agent was used, but using an 8.376 agent, chemotaxis was not restored but PMNs exposed to DMSO displayed almost the same chemotaxis as a control. On the other hand, the ability of PMNs to ingest bacteria ‘was not so markedly inhibited as the chemotaxis. With 1 hr exposure to cryoprotectants, the ingestive ability was hardly affected within 8.3%. As for 20 hr exposure, the same ingestive ability as tha.t ‘of a control was observed in all cases using a 4.2% concentration. However, using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability. Judging from the above findings, DMSO would be suitable as Ia crypotrotective ‘agent although the problem on toxicity remains to be resolved. ACKNOWLEDGMENT This work was supported (Nos. 771 and 7807) from
ON
4.
5.
6.
7.
8. in part Juntendo
by grants University.
REFERENCES 1. Boyden, S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. Exp. Med. 115, 453466 (1962). 2. Bouroncle, B. A., Aschenbrand, J. F., and Todd, R. F. Comparative study of the effectiveness of dimethyl sulfoxide and polyvinylpyrrolidone in the preservation of human leukemic blood cells at -80°C. Cryobiology 6, 409-415 ( 1970). 3. Cavins, J. A., Djerassi, I., Roy, A. J., and
9.
10.
11.
GRANULOCYTES
117
Klein, E. Preservation of viable human granulocytes at low temperature in dimethyl sulfoxide. Cryobiology 2, 129-133 ( 1955 ). Crowley, J. P., Skrabut, E. M., and Valeri, C. R. The recovery, structure, and function of human blood leukocytes after freezepreservation. Cyobiology 11, 395-409 (1974). Crowley, J. P., Skrabut, E. M., and Valeri, C. R. The determination of leukocyte phagocytic oxidase activity by measurement of the initial rate of stimulated oxygen consumption. J. Lab. Clin. Med. 86, 586594 ( 1975). French, J. E., Flor, W. J., Grissom, M. P., Parker, J. L., Sajko, G., and Ewald, W. G. Recovery, structure, and function of dog granulocytes after freeze-preservation with dimethylsulfoxide. Cryobiology 14, l-14 (1977). Lionetti, F. J., Hunt, S. M., Gore, J. M., and Curby, W. A. Cryopreservation of human granulocytes. Cryobiology 12, 181-191 (1975). Malinin, T. I. Injury of human polymorphonuclear granulocytes frozen in the presence of cryoprotective agents. CryobioZogy 9, 123-130 (1972). McCullough, J., Carter, S. J., and Quie, P. G. Effects of anticoagulants and storage on granulocyte function in bank blood. Blood 43, 207-217 (1974). Rowe, A. W., and Cohen, E. Phagocytic activity and antigenic integrity of leukocytes preserved with dimethylsulfoxide at a cryogenic temperature (-196°C). VOX Sang. 10, 382-384 (1965). Zigmond, S. H., and Hirsch, J. G. Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis. ExptZ. Cell Res. 73, 383-393 (1972).
16, 112-117
Effect
( 1979 )
of Endocellular Neutrophil
Cryoprotectant upon Polymorphonuclear Function during Storage at Low
TATSUHISA “Laboratory School
YAMASHITA,”
of
Temperature
NORIKO
of Physiological Medicine, Juntendo
IMAIZUMI,’
Chemistry University,
Transfusion of large numbers of viable polymorphonuclear neutrophils ( PMNs) is therapeutically effective in neutropenic patients with gram-negative bacteremia, leukemia, and bone marrow aplasia induced by radiation and/or drugs. Increased use of leukocyte transfusions has led investigators to try to freeze preserve leukocytes (2-10). We studied the effect of commonly employed cryoprotective agents such as dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon ceil function to select a cryoprotectant of choice for freeze preservation of leukocytes. In the present communication, guinea pig PMN integrity after treatment with cryoprotective agents was assessed by trypan blue exclusion, in vitro directed migration (chemotaxis), and phagocytosis. MATERIALS
AND
METHODS
Leukocytes. Polymorphonuclear neutrophi1 exudates were elicited in fasted guinea pigs by intraperitoneal injection of 100 ml of 0.12% glycogen in 0.9%; saline. Thirteen to fourteen hours later, the animal was killed with ether and 100 ml of citratesaline (0.474 sodium citrate in 0.85% sodium chloride) was injected intraperito20,
Received 1978.
August
4, 1978;
accepted
December
112 OOlI-2240/79/020112-06$02.00/0 Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
and tBlood Bunkyo-ku,
AND
SHINJI
YUASAt
Transfusion Service, Tokyo, Japan 113
neally. The abdomen was kneaded for 1 to 2 min, perforated, ‘and the fluid ( 140 to 160 ml) was withdrawn. The exudates were filtered through three layers of gauze and centrifuged at 1OOg for 10 minutes. After removal of the supernatant, the pellets were suspended in 1.5 to 2.0 ml of phosphate-buffered saline without divalent cations [pH 7.4 PBS( - )] . The red blood cells underwent hypotonic lysis by exposure of the cell suspension to 10 to 20 volumes of 0.2%) saline for 60 set, followed by addition of an equal voIume of 1.61% saline. After centrifugation, the cells were washed twice with PBS( - ) and finally suspended mat ,a ‘desired concentration in phosphate-buffered saline containing 1 mM MgS04 and 0.07 mM CaClz (PBS). Four to five milliliters of PMN suspension containing 8 to 9 X 10’ cells/ml were usually obtained from a single animal. More than 95% of PMNs excluded the vit.al dye trypan blue after suspension in 0.13% trypan blue. Differential cell counts employing Wright’s stain showed that 97 to 98p of cells were PMNs. Centrifugation was carried out in a refrigerated 6entrifuge using swing-out cups. All glassware used in an isolation and modification of PMNs was siliconized. Chemoattractant. Escherichiu coli CH/2 were grown for 20 hr at 37°C with vigor-
EFFECT OF CRYOPROTECTANT ous shaking in 14 Sakaguchi flasks containing 200 ml of polypepton medium (polypepton, 10 g; NaCI, 3 g; MgS01*7H20, 0.25 g; CaCI,, 0.01 g; glucose, 2 g; KH2P04, 1.3 g; Tris (hydroxymethyl) aminomethane, 7.3 g/liter, pH 7.4). Organisms were concentrated into one tube by centrifugation at 95OOg for 20 min, washed twice with PBS ( .- ), and finally suspended in PBS. The bacterial suspension (ca. 120 ml) was four times sonicated in ice for 2 min using a Branson sonic generator (20 k cycles/set) with a 2 min pause between cycles. After centrifugation of the sonicated suspension at 9500g for 20 min, supernatant was diluted with one to two8 volumes of PBS and u.sed as a chemoattractant. Treatment of PMNs with cryoprotectants. One milliliter of PMN suspension ( lo7 PMN) was incubated between 0” and 4°C with 0.0,5 to 0.2 ml of cryoprotectant containing O.l5 to 0 ml of deionized distilled water. Final concentrations of the cryoprotectant were 4.2, 8.3, and 16.7$%. In one group at the end of 1 hr, and in the other at the end of 20 hr, the PMNs were washed twice with a large excess of PBS. After adjusting the PMN count to 5 x 106/ml, trypan blue exclusion, chemotaxis and phagocytosis of cryoprotectant-treated PMNs were assayed. PMNs, used as a control, were treated under the same conditions except that 0.2 ml water was used instead of the different cryoprotectants. Chemotaxis assay. Chemotaxis was assayed by employing a modified Boyden chamber (1) .with Sartorius Membranfilter (its pore size and diameter were 3 pm and 25 mm, respectively) to separate the chamber into compartments and by measuring the distance from the top of the filter to the furthest two cells at the same focal plane according to the method of Zigmond and Hirsch (11). Two milliliters of bacterial chemoattractant solution were placed in the lower compartment, and 1 ml of PMN suspension (5 X lo6 cells/ml) was introduced into the unner comnartment. After L
ON GRANULOCYTES
113
incubation at 37°C for 40 min, the suspension was removed, the filter was fixed with 4% formalin for more than 1 hr, and stained with hematoxylin for 5 to 10 min. After the filter was cleared with xylene, the distance from the starting surface to the cell front was measured with the micrometer on a fine focusing adjuster. The results were expressed as an average migration distance per 10 random fields in duplicate at a magnification of 600x. Phagocytosis. The ability of PMNs to phagocytize bacteria was determined by microscopic examination of Gram stained smears of PMNs incubated with heat-killed (20 min at 100°C) bacteria. To the incubation mixture containing 0.5 ml of PMN suspension ( lo7 cells/ml), 0.3 ml PBS and 0.1 ml fresh homologous serum in a siliconized Pyrex tube was added 0.1 ml of a saline suspension containing 5 X lo7 streptococci aureus per ml. The tubes were constantly shaken at 37°C. Aliquots of the mixture were removed at 5, 15, and 30 min, smeared on the glass slides, fixed with methanol for ca. 3 min and stained with Gram stain. Ingestive ability was represented as the ratio of PMNs with intracellular bacteria to total PMNs enumerated. RESULTS Effect of cryoprotectant on trypan blue dye exclusion. Greater than 90% of PMNs incubated with all concentrations of each of the three cryoprotectants excluded trypan blue dye. Effect of cryoprotectant on chemotaxis. The chemotaxis of PMNs in the presence of agents after exposure to different concentrations of cryoprotective agents at 0 to 4°C is indicated in Table 1. An inhibitory effect of cryoprotectant on the chemotaxis increased as cryoprotectant concentration increased with complete inhibition of chemotaxis at 16.7%. Among protectants used, glycerol showed the strongest inhibitory action as compared with the other two agents and almost completely inhibited
114
YAMASHITA,
IMAIZUMI,
AND
TABLE
YUASA
1
Chemotaxis
and Viability of P?vlNs after I-hr and 90-hr Storage at 0 to 4°C Presence of Different Concentrations of Cryoprotectants
Agents
Concentretion % M
Neutrophil migrations (70 of control) 1-hr t!XPOSUR
in the
Viability (%I
20-hr exposure
I-hr
exposure
20-hr
exposure
DMSO
4.2 (0.59) 8.3 (1.17) 16.7 (2.35)
78 f 41 f 4fl
5 8
52 zk 2 25 + 4 6&l
92 f 94 f 95 f
Glycerol
4.2 (0.57) 8.3 (1.14) 16.7 (2.28)
63 f 13f2 6fl
8
36 z!z 4 llf5 5&l
92 f 7 (94 f 92 i 6 (97 f 96 z!c 1 (83 f
4.2 (0.75) 8.3 (1.49) 16.7 (2.98)
80 + 8 55 f 5 6&l
50 f 8 35 + 3 8zt4
94 zt 2 (94 f 3) 97 f 2 (93 f 2) 91 f 5 (83 zk 4)
94 f 92 f 85 f
3 (85 f 6) 1 (86 * 2) 8 (70 + 7)
75 f
97 f
89 f
4 (85 f
Ethylene
Control Migration 0 Migration b Viability parentheses.
glycol
100 and viability values represent distance of controls is 71 f of PMNs incubated at 37°C
10
6 (95 f 4)b 3 (97 zk 2) 2 (83 f 5)
2 (91 f
4) 2) 2)
4)
95 f 89 f 89 f
1 (91 i 5 (89 f 5 (72 f
88 f 4 (85 i 91 f 2 (90 f 95 f 5 (78 f
the mean & standard deviation of three experiments. 12 pm in 40 min. for 1 hr in the presence of agents after the storage
the chemotaxis at 8.3% even after 1 hr incubation, As the chemotaxis assays were carried out at 37°C for 40 min, PMNs were further incubated at 37°C for 1 hr to check whether the temperature accelerated the impairment by the cryoprotectant ‘of PMNs, resulting in the chemotaxis inhibition. However, no remarkable changes were observed in viability although the viability of PMNs exposed to 16.7% agents decreased to some extent by incubation at 37°C. These results suggest the possibility that the inhibition of chemotaxis is not due to death of cells but due to the modification by cryoprotectant of mechanisms involved in the chemotaxis. Recovery of chemotaxis after the remoual of cryoprotectants with washing. To study whether the chemotaxis inhibition by cryoprotectants is reversibly eliminated or not, PMNs were washed with PBS( - ) to remove agents after an exposure to different concentrations of cryoprotectants during two time periods. As can be seen in Table 2, chemotaxis was suppressed by
4)b 6) 8) 7) 5) 3)
4)
is given
in
approximately 1070 with two washings even in freshly prepared control PMNs. When control PMNs were kept at 4°C for 20 hr, approximately 20% inhibition in chemotaxis was observed even without washing and about 40% inhibition with washing, although most PMNs excluded trypan blue dye. When exposed to 4.2% cryoprotectants, chemot’axis was restored with washing almost to the same level of migration as that of the washed control regardless of the exposure time and the kind of cryoprotectant except 20-hr exposure to glycerol, suggesting that PMNs are hardly affected by cryoprotectants at this concentration. With 8.3% cryoprotectant-treated PMNs, chemotactic inhibition was ‘dependent on the agents and exposure time. The DMSO-treated PMNs retained nearly the same chemotaxis as washed control PMNs even during 20-hr Ethylene glycol-treated PMNs exposure. were inhibited by about 30% with 1-hr exposure and considerably more with 2O-hr exposure. In glycerol, So% inhibition was
EFFECT
OF
CRYOPROTECTANT
ON
observed following 1 hr treatment with complete inhibition after 20-hr exposure. However, viability tests showed that at least 90% PMNs were viable in I-hr exposure regardless of the sort of cryoprotectants while greater than 60% PMNs were viable in 20-hr exposure. With 16.7y0 cryoprotectants, no recovery of chemotaxis was observed Iat all by the removal of agents even after I-hr exposure and moreover the death of a considerable number of PMNs was observed. Phagocytosis by PMNs after an exposure to cryoprotectants. The ingestive ability of PMNs washed after a treatment with cryoprotectants was studied. As seen in Table 3, the percentage of the cells containing bacteria increased with increasing incubation time. Heat-killed PMNs did not show such an increase in the percentage of ceils with bacteria, indicating a true ingestion of bacteria by cryoprotectant-treated PMNs. Ingestive ability of control PMNs was not as affected as chemotaxis by storage and washing. With 1 hr exposure to cryoprotectants, no essential differences
Agents
and Viability
of PMNs
after
Concentration (%I
115
were observed in the ingestive ability within 8.370: and even at 16.770 the ingestion of bacteria by PMNs was considerable, Phago,cytic ability, however, almost dis‘appeared in ethylene glycol. With 20-hr exposure, the ingestion was not affected at 4.2%) concentrations, but was considerably suppressed especially in glycerol at 8.3%. PMNs exposed to 8.3%1 DMSO, however, retained the ingestive ability well. DISCUSSION
Most of PMNs could exclude the trypan blue after 20 hour exposure to cryoprotectants even at a high concentration if the washing procedure was omitted ( Table 1) . On the other hand, when PMNs were washed after an exposure to cryoprotectant at a high concentration, 50 to 80%’ of PMNs could not exclude the dye. However, the ability to exclude dye was hardly affected by washing after short exposure to 8.3% concentrations of cryoprotectants (Table 2). This would indicate that cells become very fragile with cryoprotectants
TABLE Chemotaxis
GRANULOCYTES
2
the Removal
of Cryoprotectants
with
Neutrophil migrations (70 of control) 1-hr
exposure
20-hr
Washing Viability (%I
exposure
1.hr
exposure
20-hr exposure
DMSO
4.2 8.3 16.7
88 f 73 l 18 f
6 10 7
69 f 51 f 5&l
7 11
94 zt 1 89 k 7 55 z!z 5
90 f 75 f 17 f
Glycerol
4.2 8.3 16.7
89 f 45 f Qf5
13 9
41 f 5f2 3fl
8
93 f 2 91 f6 85 f 7
85 f 5 67 zt 9 47 f 12
4.2 8.3 16 7
81 rt 3 61 f 10 7*3
64 l 25 f 5+2
10 8
96 f 89 f 55 f
3 7 4
89 f 61 f 29 i
6 10 12
60 f 75 f
8 3
94 f 96 f
4 2
92 f 96 f
7 3
Ethylene
Control
glycol
Washed Unwashed
93 f 100
The mean f standard deviation of 4 samples. a Percentages of neutrophil migration are calculated PMNs after preparation, their migration value being
8
as compared to t.he chemotaxis 65 i 8 pm in 40 min.
of the unwashed
5 7 6
fresh
116
YAMASHITA,
IMAIZUMI,
AND
TABLE Ingestive Agents
Ability
of I’MNs
Washed
with
YUASA
3
PBS
after
Concentration (%)
an Exposure Ingestion
5
15 1-hr
30
time
to Cryoptrotcctant bin) 5
-_ PMNs
exposed
15 ZO-hr
exposed
30 PMNs
DMSO
4.2 8.3 16.7
5*3 6k3 2&l
21 f 7 17 f 5 11 f3
39 f 30 f 21 f
6 9 5
9f4 2f5 -
21 f 14 f -
9 6
30 i 22 f -
8 5
Glycerol
4.2 8.3 16.7
8f6 7fl 2f2
27 f 25 f 12 f
35 f 30 f 17 f
7 10 8
5*1 lf2 -
22 f 7fl -
7
29 f 8zt8 -
9
4.2 8.3 16.7
5f4 5f3 lf2
19 f 2 25 ic 5 4f3
34 f 9 33 f 8 3zk2
10 f 5 3z!z2 -
21 f 8f3 -
6
34 f 16 f -
10 5
17 f 23 i
38 f 34 f
7*7 10 f
23 f 26 f
6 5
36 f 38 f
9 3
Ethylene
Control
glycol
Washed Unwashed
7&5 11 f 8
10 10 6
8 7
8 9
5
The mean f standard deviation of three experiments. Values represent the percent of cells containing bacteria.
especially at a high concentration and are easily damaged by washing, i.e. by the osmotic effects of removing cryoprotective agents. Therefore, further studies of a better technique for adding cryoprotectant to a ‘desired concentration and then removing it without producing a significant impairment of’ PMN function need to be done. Chemotactic response decreased with washing and storage (control in Table 2) and was also sensitive to an exposure to cryoprotectant (Table 2). On the other hand, the ingestive ability was not so affected by washing, storage and cryoprotectants ‘as chemotaxis (Table 3). It seems to be of functional significance that the ‘ability to ingest bacteria is less damaged by washing and cryoprotectants than chemotactic response because phagocytosis is the function of primary concern in PMN transfusion. To put the above findings in another way, the inhibition of chemotactic response appears to be a more sensitive indicator of impaired leukocyte function.
SUMMARY
The effect of cryoprotective agents dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon the function of polymorphonuclear neutro,phils ( PMNs ) during storage between 0” and 4°C was investigated. Increasing concentration of each cryoprotectant caused an increasing inhibition of chemotaxis with complete inhibition at 16.7%. At this concentration most PMNs were still able to exclude trypan blue dye. Chemotaxis was not inhibited if PMNs were exposed to 4.2 or 8.3% concentrations of cryoprotectants for 1 hr, and washed subsequently. However, the recovery of chemotaxis was not observed at 16.7% after 1 hr exposure to cryoprotectants. Moreover, a considerable number of PMNs could not exclude the dye. This would indicate that cells become fragile with cryoprotectants at a high concentration and the PMNs are easily damaged by washing. With 20 hr exposure PMNs, the inhibitory effect on chemotaxis was removed by wash-
EFFECT
OF
CRYOPROTECTANT
ing when a 4.2% #agent was used, but using an 8.376 agent, chemotaxis was not restored but PMNs exposed to DMSO displayed almost the same chemotaxis as a control. On the other hand, the ability of PMNs to ingest bacteria ‘was not so markedly inhibited as the chemotaxis. With 1 hr exposure to cryoprotectants, the ingestive ability was hardly affected within 8.3%. As for 20 hr exposure, the same ingestive ability as tha.t ‘of a control was observed in all cases using a 4.2% concentration. However, using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability. Judging from the above findings, DMSO would be suitable as Ia crypotrotective ‘agent although the problem on toxicity remains to be resolved. ACKNOWLEDGMENT This work was supported (Nos. 771 and 7807) from
ON
4.
5.
6.
7.
8. in part Juntendo
by grants University.
REFERENCES 1. Boyden, S. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J. Exp. Med. 115, 453466 (1962). 2. Bouroncle, B. A., Aschenbrand, J. F., and Todd, R. F. Comparative study of the effectiveness of dimethyl sulfoxide and polyvinylpyrrolidone in the preservation of human leukemic blood cells at -80°C. Cryobiology 6, 409-415 ( 1970). 3. Cavins, J. A., Djerassi, I., Roy, A. J., and
9.
10.
11.
GRANULOCYTES
117
Klein, E. Preservation of viable human granulocytes at low temperature in dimethyl sulfoxide. Cryobiology 2, 129-133 ( 1955 ). Crowley, J. P., Skrabut, E. M., and Valeri, C. R. The recovery, structure, and function of human blood leukocytes after freezepreservation. Cyobiology 11, 395-409 (1974). Crowley, J. P., Skrabut, E. M., and Valeri, C. R. The determination of leukocyte phagocytic oxidase activity by measurement of the initial rate of stimulated oxygen consumption. J. Lab. Clin. Med. 86, 586594 ( 1975). French, J. E., Flor, W. J., Grissom, M. P., Parker, J. L., Sajko, G., and Ewald, W. G. Recovery, structure, and function of dog granulocytes after freeze-preservation with dimethylsulfoxide. Cryobiology 14, l-14 (1977). Lionetti, F. J., Hunt, S. M., Gore, J. M., and Curby, W. A. Cryopreservation of human granulocytes. Cryobiology 12, 181-191 (1975). Malinin, T. I. Injury of human polymorphonuclear granulocytes frozen in the presence of cryoprotective agents. CryobioZogy 9, 123-130 (1972). McCullough, J., Carter, S. J., and Quie, P. G. Effects of anticoagulants and storage on granulocyte function in bank blood. Blood 43, 207-217 (1974). Rowe, A. W., and Cohen, E. Phagocytic activity and antigenic integrity of leukocytes preserved with dimethylsulfoxide at a cryogenic temperature (-196°C). VOX Sang. 10, 382-384 (1965). Zigmond, S. H., and Hirsch, J. G. Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis. ExptZ. Cell Res. 73, 383-393 (1972).
