CRYOBIOLOGY
27, 137-142 (1990)
Cryopreservation
Enhances Interleukin-I Production Mononuclear Cells’
M. VENKATARAMAN2’**t
AND
in Human
M. P. WESTERMAN*
*Division of HemntologyJOncology, Department of Medicine, and tMicrobiology and Immunology, Mount Sinai Hospital Medical Center/University of Health Sciences/Chicago Medical School and Rush University Culiege of Health Sciences, Chicago, Illinois 60608
The effects of cryopreservation on bacterial lipopolysaccharide (LPS)-induced interleukin-1 (IL-l) production by unfractionated mononuclear cells (MN&), adherent cells (ACs), and nonadherent cells (NACs) were studied. Culture supematants from cryopreserved cells contained significantly larger concentrations of IL-l [MNCs, 211 2 50; ACs, 640 ? 41; NACs, 116 2 19 U/ml (mean * SEM)] as compared with supernatants from fresh cells (69 2 22, 427 i 69, and 72 i. 33 U/ml, respectively). In addition, supematants obtained from cocultures of autologous fresh and frozen cells contained much less than the expected quantities of IL-1 (78 ? 8%), indicating that suppressor cells in the fresh population are responsible for the decreased IL-1 content. The studies suggest that functional inactivation of cryosensitive suppressor monocytes is associated with an increase in IL-1 production by the other subset. The results provide further evidence that lack of active suppressor monocytes and increased IL-1 production may be responsible for the previously reported enhanced plaque-forming cell responses of cryopreserved cells from normal controls and from patients with lung cancer. 01990 Academic
I’ma,
Inc.
Monocytes play a major role in the regulation of humoral and cellular immune responses by providing either help or suppression (3, 16, 19). The helper and suppressor functions have been shown to be mediated through the production of interleukin-1 (IL-l) and prostaglandin E-2 (PGE-2), respectively, by distinct monocyte subsets (4, 5, 8, 11). Functional imbalance between these subsets, particularly in patients with malignant diseases, has been shown to result in increased PGE-2 production and decreased IL-I secretion (7, 10, 21).
We have recently observed that coculReceived December 16, 1988; accepted June 8, 1989. ’ These investigations were supported by funds from the Mount Sinai Hospital Medical Center Service Club, Chicago. ’ To whom correspondence and reprint requests should be addressed at Department of Medicine, Mount Sinai Hospital Medical Center, California Avenue and 15th Street, Chicago, IL 60608.
ture of fresh monocyte and B-cell-enriched fractions with autologous fresh T cells results in the generation of significantly smaller numbers of plaque-forming cells (PFCs) as compared with cocultures containing frozen monocytes and B cells plus fresh T cells (25, 26). This was observed both in normal controls and in patients with lung cancer. The presence of an increased number of monocytes in the fresh population and their subsequent inactivation during freezing are considered to be the primary cause for the difference. This concept is based on the observation that B- and Tcell function requires IL-l, and that monocytes exert their suppressor activity through the production of PGE-2 which in turn inhibits the secretion of IL-1 (10, 16, 28). If freezing does inactivate suppressor monocytes, as has been considered, it should then result in enhanced IL-1 production. Since there is no information regarding IL-1 production by cryopreserved cells, which could explain our previous findings
137 001l-2240190$3.00 Copytiht 0 199U by Academic Press, Inc. All rights of reproduction in any form reserved.
138
VENKATARAMAN
both in normal controls and in patients with lung cancer (25), we undertook the present study. MATERIALS
AND
METHODS
Isolation of Mononuclear Cells, Adherent Cells, and Nonadherent Cells
Fifty milliliters of venous blood from nine healthy control subjects (four males, ages 2439, and five females, ages 22-35) was collected and mononuclear cells (MNCs) were isolated by the FicollHypaque density gradient centrifugation method of Boyum (1). The cells were washed three times with RPM1 1640 medium supplemented with 5% heatinactivated fetal bovine serum (FBS; GIBCO, Grand Island, NY). Adherent cell (AC) and nonadherent cell (NAC) populations were fractionated from an aliquot of MNCs by using FBS-coated plastic Petri dishes as described by Kumagai et al. (9). The AC- and NAC-enriched populations contained more than 80% and less than 2% monocytes, respectively, as identified by nonspecific esterase staining. Freezing, Storage, and Thawing Procedures
Freezing, storage, and thawing of cells were done as described previously (25). In brief, washed cells were counted and diluted in the cold freezing medium consisted of 10% FBS, 10% dimethyl sulfoxide (Me,SO), and 80% RPM1 1640 medium. The cells were distributed in l-ml aliquots in precooled plastic vials. One vial of each individual’s cells was kept on ice to assay the responses of fresh cells. The remaining vials were placed in a Cryo-Med liquid nitrogen programmable freezer (Cryo-Med, Mt. Clemens, MI), and frozen at a rate of l”C/min to -3O”C, and then at 5”Clmin to -70°C. The frozen vials were thawed rapidly in a 45°C water bath, and both the frozen and fresh cells that were kept on ice were washed once and cultured simulta-
AND
WESTERMAN
neously without further counting of cells. The frozen and thawed samples contained greater that 98% recovered cells and also contained more than 98% viable cells as determined by the trypan blue exclusion technique . Culture Conditions for IL-1 Production
For IL-1 production, 2 X lo6 cells in I ml were cultured in 12 x 75-mm Falcon tubes in RPMI-1640 with 25 mM Hepes buffer and 2 n&f glutamine supplemented with 50 pg/ml gentamicin (Schering Corp., Kenilworth, NJ) and 10% FBS in the presence of 10 pg/ml lipopolysaccharide (Escherichia coli 055:B5, Difco Laboratories, Detroit, MI). The cultures were incubated on a rocker platform (Bellco Glass Co., Vineland, NJ) at 6 cycleslmin at 37°C in a humidified atmosphere of 5% CO2 in air. The culture supernatants were harvested at the end of 24 hr and stored at 4°C. IL-1 Assay
The IL-1 contents of the supernatants were determined by the procedure of Mizel et al. (13). In brief, thymocytes from 5- to g-week-old C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME) were cultured in 96well flat-bottomed culture plates at a concentration of 1.5 x lo6 cells/well in the above-described culture medium, which was further supplemented with 1 m&f sodium pyruvate, 5 x 10m5n/l 2-mercaptoethanol in the presence or absence of 1 &ml purified phytohemagglutinin (PHA; Burroughs Wellcome, Research Triangle Park, NC), and double dilutions of the supernatams. The cultures were incubated in a rocker platform at 6 cycleslmin at 37°C in a humidified atmosphere of 5% CO2 in air for 72 hr and the proliferative responses were measured after a 6-hr 13H]deoxythymidine pulse. The IL-1 concentrations (in U/ml) of experimental samples were calculated by linear regression from a standard curve obtained on the day of assay with human rIL-1 (generously provided by Dr. Lomedico,
CRYOPRESERVATION
ENHANCES
Hoffmann-La Roche, Nutley, NJ). No IL-2 could be detected in the supernatants from either fresh or cryopreserved cells as determined by the standard bioassay using murine IL-2-dependent cytotoxic T-cell lines. Statistical
INTERLEUKIN-I
I39
SECRETION
only between the supernatants from fresh and frozen ACs. IL-l Concentrations in Culture Supernatants from Cocultures and Cryopreserved MNC
of Fresh
Analyses
The increase in IL-I concentrations in supernatants from cryopreserved cells prompted us to postulate that a subset of cryosensitive suppressor cells present in RESULTS the fresh population may inhibit the other IL-1 Concentrations in Culture cells from producing IL- 1 to their maximum Supernatants from Fresh and capacity. If our assumption was correct, Cryopreserved Cells the presence of active suppressor cells in The concentrations of IL-I present in the the fresh population should then inhibit ILculture supernatants from fresh and cryo- 1 production by the relatively suppressorpreserved, unseparated MNCs, ACs, and free cryopreserved population. To verify NACs are shown in Table 1. The superna- this hypothesis, in addition to separate cultants from cryopreserved MNCs of all five tures of fresh and frozen cells (2 x IO6 subjects contained significantly (P < 0.02) cells/ml), a third culture containing a mixhigher concentrations of IL-l’ (211 5 50 ture of both fresh and frozen cells (1 x lo6 U/ml, mean + SEM) than those from their cells/ml each) was set up with lipopolysacrespective fresh MNCs (69 * 22 U/ml), charide. The supernatants were collected at Similarly, the supernatants from cryopre- the end of 24 hr and their IL-l contents served ACs (640 2 41 U/ml) and NACs (116 determined. In cocultures of fresh and frof 19 U/ml) contained much higher concen- zen cells, the expected values were calcutrations of IL-l than those from their cor- lated as described by others (2) assuming responding fresh cells (427 * 69 and 72 k 33 the proportional participation of both popU/ml, respectively). However, the ob- ulations (% expected value = quantity in served difference was significant (P < 0.02) coculture/half the sum of the quantity in The results were compared by the use of the Student’s t test for paired samples.
IL-1 Concentrations
TABLE 1 in Supematants from Fresh and Cryopreserved MNCs, ACs, and NAG” Supernalant ACs
MNCs
NACs
Subject
Fresh
Frozen
Fresh
Frozen
Fresh
Frozen
I 2 3 4 5
29b 44 151 76 47
89 160 338 320 148
588 480 356 195 518
711 755 533 588 612
36 44 204 40 37
71 142 168 124 75
Mean k SEM P value
69 + 22
211 + 50
427 + 69
640 + 41
72 + 33
co.02
co.02
116 + 19 >0.05
a IL-1 containing supematants from fresh and cryopreserved unfractionated MNCs, ACs, and NACs were obtained by culturing 2 x 106cells/ml in 12 X 7%mm tubes for 24 hr in the presence of 10 &ml lipopolysaccharide b U/ml.
140
VENKATARAMAN
each separate culture). The results of such experiments are shown in Table 2. As observed in previous experiments, the culture supernatants from frozen MNCs of all subjects contained significantly (P < 0.02) higher concentrations of IL-1 as compared with those from fresh cells. In three of four subjects, the supematants from cocultures of fresh and frozen cells contained 31-27% less than the expected quantities of IL-l, while the supernatant from the fourth subject contained 100% of the expected value. When compared as a group, the supernatants from cocultures of fresh plus frozen cells contained a significantly (P < 0.02) smaller amount of IL-1 than those from frozen ceils. DISCUSSION
The present studies were initiated to determine the mechanisms responsible for enhancement of the PFC responses both of cryopreserved unfractionated MNCs and of frozen non-T plus fresh T cells from normal controls and from patients with lung cancer (25, 26). The observed increase in PFC responses of frozen cells was considered to be caused by inactivation of a cryosensitive PGE-2-secreting subset of monocytes with-
AND
WESTERMAN
out any significant loss in function of the IL-l-producing subset (18, 28). Because monocytes mediate their suppressor activity primarily through the production of PGE-2 (5, lo), which is turn modulates the production of both IL-l and IL-2 required for Ig production (3, 11, 16, 20), cells devoid of such active PGE-Z-secreting cells should produce relatively larger quantities of IL-1 than cells containing active suppressors. As expected, the culture supernatants obtained from both frozen unseparated MNCs and frozen ACs (Table 1) contained significantly larger quantities of IL-1 than supernatants from fresh cells. The suppressive effect of a cryosensitive monocyte subset on IL- 1 production is also corroborated by the smaller-than-expected amounts of IL-1 present in supernatants from cocultures of fresh and frozen MNCs (Table 2). The suppressive effect on IL-1 production was 27-30% in three individuals and was not present in one subject. This could be due in part to donor-to-donor variations between the activities of ILl-producing and PGE-2-secreting subsets of monocytes in the mixed population of fresh and frozen cells. This supports the general concept that IL- 1, like other biolog-
TABLE 2 IL-1 Concentrations in Supernatants from Fresh, Cryopreserved, and a Combination of Fresh and Cryopreserved MNCs” Supernatant from MNCs Subject 1
2 3 4 Mean + SEM P value
Frozen
Fresh plus frozen
eib
105
80 168 143
139 253 236
59 (69) 76 (69) 210 (loo) 139 (73)
114 k 25 0.05’
121 f 34 (78 f 8)
27, 137-142 (1990)
Cryopreservation
Enhances Interleukin-I Production Mononuclear Cells’
M. VENKATARAMAN2’**t
AND
in Human
M. P. WESTERMAN*
*Division of HemntologyJOncology, Department of Medicine, and tMicrobiology and Immunology, Mount Sinai Hospital Medical Center/University of Health Sciences/Chicago Medical School and Rush University Culiege of Health Sciences, Chicago, Illinois 60608
The effects of cryopreservation on bacterial lipopolysaccharide (LPS)-induced interleukin-1 (IL-l) production by unfractionated mononuclear cells (MN&), adherent cells (ACs), and nonadherent cells (NACs) were studied. Culture supematants from cryopreserved cells contained significantly larger concentrations of IL-l [MNCs, 211 2 50; ACs, 640 ? 41; NACs, 116 2 19 U/ml (mean * SEM)] as compared with supernatants from fresh cells (69 2 22, 427 i 69, and 72 i. 33 U/ml, respectively). In addition, supematants obtained from cocultures of autologous fresh and frozen cells contained much less than the expected quantities of IL-1 (78 ? 8%), indicating that suppressor cells in the fresh population are responsible for the decreased IL-1 content. The studies suggest that functional inactivation of cryosensitive suppressor monocytes is associated with an increase in IL-1 production by the other subset. The results provide further evidence that lack of active suppressor monocytes and increased IL-1 production may be responsible for the previously reported enhanced plaque-forming cell responses of cryopreserved cells from normal controls and from patients with lung cancer. 01990 Academic
I’ma,
Inc.
Monocytes play a major role in the regulation of humoral and cellular immune responses by providing either help or suppression (3, 16, 19). The helper and suppressor functions have been shown to be mediated through the production of interleukin-1 (IL-l) and prostaglandin E-2 (PGE-2), respectively, by distinct monocyte subsets (4, 5, 8, 11). Functional imbalance between these subsets, particularly in patients with malignant diseases, has been shown to result in increased PGE-2 production and decreased IL-I secretion (7, 10, 21).
We have recently observed that coculReceived December 16, 1988; accepted June 8, 1989. ’ These investigations were supported by funds from the Mount Sinai Hospital Medical Center Service Club, Chicago. ’ To whom correspondence and reprint requests should be addressed at Department of Medicine, Mount Sinai Hospital Medical Center, California Avenue and 15th Street, Chicago, IL 60608.
ture of fresh monocyte and B-cell-enriched fractions with autologous fresh T cells results in the generation of significantly smaller numbers of plaque-forming cells (PFCs) as compared with cocultures containing frozen monocytes and B cells plus fresh T cells (25, 26). This was observed both in normal controls and in patients with lung cancer. The presence of an increased number of monocytes in the fresh population and their subsequent inactivation during freezing are considered to be the primary cause for the difference. This concept is based on the observation that B- and Tcell function requires IL-l, and that monocytes exert their suppressor activity through the production of PGE-2 which in turn inhibits the secretion of IL-1 (10, 16, 28). If freezing does inactivate suppressor monocytes, as has been considered, it should then result in enhanced IL-1 production. Since there is no information regarding IL-1 production by cryopreserved cells, which could explain our previous findings
137 001l-2240190$3.00 Copytiht 0 199U by Academic Press, Inc. All rights of reproduction in any form reserved.
138
VENKATARAMAN
both in normal controls and in patients with lung cancer (25), we undertook the present study. MATERIALS
AND
METHODS
Isolation of Mononuclear Cells, Adherent Cells, and Nonadherent Cells
Fifty milliliters of venous blood from nine healthy control subjects (four males, ages 2439, and five females, ages 22-35) was collected and mononuclear cells (MNCs) were isolated by the FicollHypaque density gradient centrifugation method of Boyum (1). The cells were washed three times with RPM1 1640 medium supplemented with 5% heatinactivated fetal bovine serum (FBS; GIBCO, Grand Island, NY). Adherent cell (AC) and nonadherent cell (NAC) populations were fractionated from an aliquot of MNCs by using FBS-coated plastic Petri dishes as described by Kumagai et al. (9). The AC- and NAC-enriched populations contained more than 80% and less than 2% monocytes, respectively, as identified by nonspecific esterase staining. Freezing, Storage, and Thawing Procedures
Freezing, storage, and thawing of cells were done as described previously (25). In brief, washed cells were counted and diluted in the cold freezing medium consisted of 10% FBS, 10% dimethyl sulfoxide (Me,SO), and 80% RPM1 1640 medium. The cells were distributed in l-ml aliquots in precooled plastic vials. One vial of each individual’s cells was kept on ice to assay the responses of fresh cells. The remaining vials were placed in a Cryo-Med liquid nitrogen programmable freezer (Cryo-Med, Mt. Clemens, MI), and frozen at a rate of l”C/min to -3O”C, and then at 5”Clmin to -70°C. The frozen vials were thawed rapidly in a 45°C water bath, and both the frozen and fresh cells that were kept on ice were washed once and cultured simulta-
AND
WESTERMAN
neously without further counting of cells. The frozen and thawed samples contained greater that 98% recovered cells and also contained more than 98% viable cells as determined by the trypan blue exclusion technique . Culture Conditions for IL-1 Production
For IL-1 production, 2 X lo6 cells in I ml were cultured in 12 x 75-mm Falcon tubes in RPMI-1640 with 25 mM Hepes buffer and 2 n&f glutamine supplemented with 50 pg/ml gentamicin (Schering Corp., Kenilworth, NJ) and 10% FBS in the presence of 10 pg/ml lipopolysaccharide (Escherichia coli 055:B5, Difco Laboratories, Detroit, MI). The cultures were incubated on a rocker platform (Bellco Glass Co., Vineland, NJ) at 6 cycleslmin at 37°C in a humidified atmosphere of 5% CO2 in air. The culture supernatants were harvested at the end of 24 hr and stored at 4°C. IL-1 Assay
The IL-1 contents of the supernatants were determined by the procedure of Mizel et al. (13). In brief, thymocytes from 5- to g-week-old C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME) were cultured in 96well flat-bottomed culture plates at a concentration of 1.5 x lo6 cells/well in the above-described culture medium, which was further supplemented with 1 m&f sodium pyruvate, 5 x 10m5n/l 2-mercaptoethanol in the presence or absence of 1 &ml purified phytohemagglutinin (PHA; Burroughs Wellcome, Research Triangle Park, NC), and double dilutions of the supernatams. The cultures were incubated in a rocker platform at 6 cycleslmin at 37°C in a humidified atmosphere of 5% CO2 in air for 72 hr and the proliferative responses were measured after a 6-hr 13H]deoxythymidine pulse. The IL-1 concentrations (in U/ml) of experimental samples were calculated by linear regression from a standard curve obtained on the day of assay with human rIL-1 (generously provided by Dr. Lomedico,
CRYOPRESERVATION
ENHANCES
Hoffmann-La Roche, Nutley, NJ). No IL-2 could be detected in the supernatants from either fresh or cryopreserved cells as determined by the standard bioassay using murine IL-2-dependent cytotoxic T-cell lines. Statistical
INTERLEUKIN-I
I39
SECRETION
only between the supernatants from fresh and frozen ACs. IL-l Concentrations in Culture Supernatants from Cocultures and Cryopreserved MNC
of Fresh
Analyses
The increase in IL-I concentrations in supernatants from cryopreserved cells prompted us to postulate that a subset of cryosensitive suppressor cells present in RESULTS the fresh population may inhibit the other IL-1 Concentrations in Culture cells from producing IL- 1 to their maximum Supernatants from Fresh and capacity. If our assumption was correct, Cryopreserved Cells the presence of active suppressor cells in The concentrations of IL-I present in the the fresh population should then inhibit ILculture supernatants from fresh and cryo- 1 production by the relatively suppressorpreserved, unseparated MNCs, ACs, and free cryopreserved population. To verify NACs are shown in Table 1. The superna- this hypothesis, in addition to separate cultants from cryopreserved MNCs of all five tures of fresh and frozen cells (2 x IO6 subjects contained significantly (P < 0.02) cells/ml), a third culture containing a mixhigher concentrations of IL-l’ (211 5 50 ture of both fresh and frozen cells (1 x lo6 U/ml, mean + SEM) than those from their cells/ml each) was set up with lipopolysacrespective fresh MNCs (69 * 22 U/ml), charide. The supernatants were collected at Similarly, the supernatants from cryopre- the end of 24 hr and their IL-l contents served ACs (640 2 41 U/ml) and NACs (116 determined. In cocultures of fresh and frof 19 U/ml) contained much higher concen- zen cells, the expected values were calcutrations of IL-l than those from their cor- lated as described by others (2) assuming responding fresh cells (427 * 69 and 72 k 33 the proportional participation of both popU/ml, respectively). However, the ob- ulations (% expected value = quantity in served difference was significant (P < 0.02) coculture/half the sum of the quantity in The results were compared by the use of the Student’s t test for paired samples.
IL-1 Concentrations
TABLE 1 in Supematants from Fresh and Cryopreserved MNCs, ACs, and NAG” Supernalant ACs
MNCs
NACs
Subject
Fresh
Frozen
Fresh
Frozen
Fresh
Frozen
I 2 3 4 5
29b 44 151 76 47
89 160 338 320 148
588 480 356 195 518
711 755 533 588 612
36 44 204 40 37
71 142 168 124 75
Mean k SEM P value
69 + 22
211 + 50
427 + 69
640 + 41
72 + 33
co.02
co.02
116 + 19 >0.05
a IL-1 containing supematants from fresh and cryopreserved unfractionated MNCs, ACs, and NACs were obtained by culturing 2 x 106cells/ml in 12 X 7%mm tubes for 24 hr in the presence of 10 &ml lipopolysaccharide b U/ml.
140
VENKATARAMAN
each separate culture). The results of such experiments are shown in Table 2. As observed in previous experiments, the culture supernatants from frozen MNCs of all subjects contained significantly (P < 0.02) higher concentrations of IL-1 as compared with those from fresh cells. In three of four subjects, the supematants from cocultures of fresh and frozen cells contained 31-27% less than the expected quantities of IL-l, while the supernatant from the fourth subject contained 100% of the expected value. When compared as a group, the supernatants from cocultures of fresh plus frozen cells contained a significantly (P < 0.02) smaller amount of IL-1 than those from frozen ceils. DISCUSSION
The present studies were initiated to determine the mechanisms responsible for enhancement of the PFC responses both of cryopreserved unfractionated MNCs and of frozen non-T plus fresh T cells from normal controls and from patients with lung cancer (25, 26). The observed increase in PFC responses of frozen cells was considered to be caused by inactivation of a cryosensitive PGE-2-secreting subset of monocytes with-
AND
WESTERMAN
out any significant loss in function of the IL-l-producing subset (18, 28). Because monocytes mediate their suppressor activity primarily through the production of PGE-2 (5, lo), which is turn modulates the production of both IL-l and IL-2 required for Ig production (3, 11, 16, 20), cells devoid of such active PGE-Z-secreting cells should produce relatively larger quantities of IL-1 than cells containing active suppressors. As expected, the culture supernatants obtained from both frozen unseparated MNCs and frozen ACs (Table 1) contained significantly larger quantities of IL-1 than supernatants from fresh cells. The suppressive effect of a cryosensitive monocyte subset on IL- 1 production is also corroborated by the smaller-than-expected amounts of IL-1 present in supernatants from cocultures of fresh and frozen MNCs (Table 2). The suppressive effect on IL-1 production was 27-30% in three individuals and was not present in one subject. This could be due in part to donor-to-donor variations between the activities of ILl-producing and PGE-2-secreting subsets of monocytes in the mixed population of fresh and frozen cells. This supports the general concept that IL- 1, like other biolog-
TABLE 2 IL-1 Concentrations in Supernatants from Fresh, Cryopreserved, and a Combination of Fresh and Cryopreserved MNCs” Supernatant from MNCs Subject 1
2 3 4 Mean + SEM P value
Frozen
Fresh plus frozen
eib
105
80 168 143
139 253 236
59 (69) 76 (69) 210 (loo) 139 (73)
114 k 25 0.05’
121 f 34 (78 f 8)
