Control of Granulopoiesis in Man 111. INHIBITION OF COLONY FORMATION BY DENSE LEUKOCYTES
'
F. L. BAKER, H. E. BROXMEYER AND P. R. GALBRAITH Department of Medicine, Queen's University, Kingston, Ontario, Canada
ABSTRACT
Cellular feeder layers, prepared from normal blood leukocytes, usually stimulate human marrow to form colonies. A significant increase in the stimulating activity of unseparated leukocyte feeder layers is brought about following the removal of dense leukocytes in a manner which avoids enrichment of any remaining cell type. Restoration of dense leukocytes to a dense leukocyte depleted leukocyte feeder layer results in the reduction of stimulating activity to that of an unseparated leukocyte feeder; however, addition of dense leukocytes to unseparated leukocyte feeder layers has no effect on the stimulatory activity, over the range o f concentrations used in this study.
A characteristic, common to virtually all physiological systems, is one of regulation and control. There are numerous examples in which the target organ is regulated by a combination of negative and positive feedback loops, and the regulation of granulopoiesis in vivo likely involves both inhibitory and stimulatory feedback loops (Moore et al., '73). Certain leukocytes possess stimulatory activity and the cell responsible for the production of colony stimulating factor (CSF) has been tentatively identified as the monocyte (Moore et al., '73). However, the role that the monocyte plays in the regulation of granulopoiesis in vivo is not fully understood. The regulation of granulopoiesis, by granulocytes, via a negative feedback loop appears to be a reasonable a priori assumption as evidence has been accumulated which shows that granulocytes inhibit granulopoiesis in vitro (Haskill et a]., '72, Paran et al., '69; Rytomaa et al., '68). As with the monocyte the role that granulocytes might play in the physiology of granulopoiesis is not understood. This paper describes a series of experiments which support our previous work (Haskill et al., '72) and provides additional evidence of granulocyte induced inhibition of colony formation, J. CELL.PHYSIOL., 86: 337-342.
METHODS
Cells Blood, drawn from normal donors, was collected in heparinized vacutainers and allowed to settle for one to one and a half hours. The leukocyte rich plasma was drawn off using a pasteur pipette. Cells were centrifuged down (250 X g for 10 minutes) and washed three times in single strength CMRL-1066 media plus 10% fetal calf serum. A cell count was performed on the second last wash and a stock leukocyte suspension containing 20 x 106cells per ml was made u p after the last wash. Marrow, aspirated from the sternum of informed normal individuals, was centrifuged (500 X g for 10 minutes) and the buffy coat drawn off using a pasteur pipette. Marrow cells were washed and counted in the same way as the blood leukocytes, except that the marrow cell stock suspension contained 2 X 10' cells per ml. Aliquots, taken from the stock cell suspensions were used to prepare the plating suspension, which contained either 2 X log unseparated blood leukocytes per ml or 2 X lo5 unseparated marrow cells per ml. Received June 21, '74. Accepted Feb. 6, 75. 'Supported by Grant 263, The Ontario Cancer Treatment and Research Foundation.
337
338
F. L. BAKER, H. E. BROXMEYER AND P. R. GALBRAITH
Cell separations Cells were separated into two fractions (one containing cells lighter than, and the other containing cells more dense than 1.070 gm/cc), using a single density cut procedure (Shortman et al., '72, Moore et al., '71). The cells to be separated were pelletted by centrifugation at 500 X g for 10 minutes and resuspended in 7 ml of a balanced salt solution containing bovine serum albumin (BSA) of the desired density. Two ml of a lighter density BSA solution (density 1.047 gm,/cc) was laid over the BSA cell suspension to prevent a cell-air interface from forming during centrifugation (4000 X g for 10 minutes). Following centrifugation, the buoyant cells were recovered and reconstituted to their starting volume: e.g. if 5 ml of the stock leukocyte suspension was separated, then the buoyant fraction would be reconstituted to 5 ml. This procedure was followed so that enrichment of the buoyant fraction with any cell type did not occur. The dense cells were reconstituted in a similar manner. The osmolarity of the BSA solutions was measured with a Hewlett Packard model 301A vapour pressure osmometer and brought to 270 mosm. This instrument was used instead of a freezing point depression osometer because Williams et al. ('72) demonstrated that the latter instrument is in error when measuring concentrated solutions of BSA.
(100 X I , 2 m l MEM amino acids (50 X ) , (without glutamine), and 20 ml antibioticantimycotic mixture (100 X ). Feeder layers prepared from unseparated leukocytes contained 2 X lo6 cells per ml per plate, but feeders made from buoyant leukocytes contained fewer cells: the actual number depended upon the numbers of dense cells removed during the separation procedure. The upper or culture layer ( 1 ml) containing the colony-forming cells differed from the feeder layer only in that it contained 0.3% agar and was seeded with 2 X lo5 unseparated marrow cells per ml per plate. Thirty-five mm Falcon #lo08 petri dishes were used throughout.
CSA assay A sigmoid dose response relationship exists between colony formation by normal murine marrow and feeder CSA (Metcalf and Stanley, '69). Consequently feeder CSA may be assayed using an overlayer containing a constant number of marrow cells. As unseparated human marrow was used as the source of target cells in this study, caution had to be exercised in its use because of its content of adherent cells which elaborate CSF. With the marrow concentrations used in this study ( 2 X lo5 cells per ml), aggregates of 20 to 30 cells grow spontaneously, but the spontaneous formation of colonies (aggregates of 50 or more cells) seldom occurred. In the absence of spontaneous colony formation, an exogenous source of stimulator is required to induce colony formation and the numIn uitro culture system ber of colonies so formed is related to the The two layer semi-solid agar culture number of leukocytes plated in the feeder system for mouse marrow cells, modified layer (Galbraith and Broxmeyer '74). by Pike and Robinson ('70) to support the Spontaneous colony formation was not growth of human marrow cells, was used present in any of the cultures reported in this study. The lower, or feeder, layer in this paper. Marrow cells were plated at 2 X lo5cells (1 ml) consisted of 0.5% agar (Difco) in modified CMRL-1066 medium (Gibco) per ml per plate and a single marrow was with 10% heat-inactivated fetal calf serum used to assay both the control and test (Gibco) and blood leukocytes. The modi- feeder layers in each experiment. Colony fications made to the medium involved counts (> 50 cells) were scored on three adding the following ingredients per litre to five plates usually after the seventh day of double strength CMRL-1066 medium : of incubation and occasionally on the fourteenth day. An Olympus model CK inverted 40 ml nonessential amino acids (100 X ) , 20 ml sodium pyruvate (100 X ) , 56 ml microscope, fitted with a mechanical stage, sodium bicarbonate (7.5% ), 4 ml vitamin was used at 40 X to count colonies. Small B12 (1000 pg/ml), 20 ml 1-Glutamine colonies were examined at 100 X to ensure (200 mM), 1 ml MEM vitamin Solution that they contained at least 50 cells.
339
INHIBITION OF COLONY FORMATION BY P M N
Inhibitory effects of blood granulocytes in vitro The inhibitory property of dense leukocytes was demonstrated by removing them and subsequently comparing the CSA elaborated by the remaining cells to that by unseparated leukocytes : a technique which is, in principle, the same as the method employed to demonstrate the presence of serum inhibitors. Blood drawn from healthy normal donors was used to prepare sets of leukocyte feeder layers. Each set consisted of two parts: a control part, and a test part. The control part consisted of feeder layers containing 2 X 10' cells per ml per plate of unseparated blood leukocytes. Test feeder layers were made from buoyant leukocytes (density < 1.070 gm/cc) such that they contained the same numbers of buoyant cells per ml per plate as did the control part. CSA of the feeder layers was assayed using unseparated human marrow cells at 2 X lo5cells per ml per plate. Removal of dense leukocytes, in a manner which avoids concentrating the remaining buoyant leukocytes resulted in a significant increase in feeder CSA: the increase being between 2- and 15-fold RESULTS (table 1 ) . This procedure has been perDensity cut separations formed more than 30 times with identical Between 30% and 50% of the cells sub- results. jected to the density cut were recovered in Dose response characteristics the buoyant fraction and of these less than of t h e inhibition 5% were granulocytes, 50 to 80% lymphoTo study the dose response charactercytes, 10 to 40% monocytes and about 2% basophils. The rest of the cells were istics of dense cell induced inhibition of recovered in the dense fraction, which con- granulopoiesis in vitro feeder layers contained greater than 90% granulocytes, 1 taining a fixed number of buoyant cells to 3% eosinophils, less than 2% mono- with additions of dense cells were prepared. The feeder layers contained buoyant cytes and up to 10% lymphocytes.
Incubation conditions The cell cultures were incubated i n a modified National dual chamber incubator at 37°C. in a humidified atmosphere containing 5 % CO,. The pH of the culture media was checked by comparing the color of the media against a set of La Motte indicator standards and was maintained at 7.2 I 0.1 pH units. Bottled gas, containing 5% CO, in air was used to gas the incubator. A flow rate of 40 ml per minute, per chamber was chosen; however, this low flow rate is insufficient to establish a significant pressure gradient between the two chambers, and as there are no check valves between the two chambers of the incubator to prevent backward flow, the 2-minute 100% COZ flush (used to quickly raise the CO, content following the opening of a chamber) purged the unopened chamber as well. After our incubator was modified to prevent accidental overgassing of the unopened chamber with 100% CO,, we achieved better control over the culture conditions and increased colony formation resulted.
TABLE 1
C S A (Colonies per plate
?
S E M ) o f feeder layers m a d e f r o m :
Unseparated leukocytes
Buoyant leukocytes density < 1.070 gm/cc
45.5 ? 2.5 34.8 ? 4.6 62.8 % 8.4 25.8 C 1.8 7.6 C 1.2 38.3% 0.9
113.6? 10.7 61.2k 6.2 140.3% 8.2 71.2% 2.8 115 % 2.9 175.6e17.8
Probability of difference being a chance occurrence determined by students' test
0.01 0.01
< 0.001 < 0.001 < 0.001 < 0.001
340
F. L. BAKER, H . E. BROXMEYER AND P. R. GALBRAITH
cells (density < 1.070 gm/cc) plus additions of 0 to 160% of the dense cells (density > 1.070 gm/cc) originally present. (Hence, a n addition of 100% of the dense cells originally present in a n unseparated leukocyte feeder, to a feeder layer prepared from buoyant cells would result in a feeder layer whose composition should be indistinguishable from that of a n unseparated layer. ) The dose response relationship obtained indicates that CSA is significantly reduced following the addition of small numbers of dense leukocytes (fig. 1 ) . When 100% of the dense cells were restored, CSA of the feeder layer was the same a s that prepared from unseparted leukocytes. Further increases in the numbers of added dense cells did not reduce CSA further. Qualitatively similar findings have been obtained on at least eight separate occasions. 401
L
U m
’“1 0
20
40
60
80
100
120
140
160
Feeder Layer Composition Co/o dense leukocytes)
Fig. 1 Dose response relationship between leukocyte feeder layer composition and stimulating activity. Abscissa, feeder layer composed of buoyant cells plus 0 to 160% of the dense cells present in a feeder layer prepared from unseparated cells. Ordinate, number of colonies ( > 50 cells) formed per plate & SEM. Shaded area, colony formation 2 SEM stimulated by unseparated feeder layers.
TABLE 2
Colony f o r m a t i o n (Colonies per plate ( .t. S E M ) ) b y m a r r o w cultures prepared f r o m : Unseparated marrow
Separated marrow buoyant fraction
45.5 -t. 2.5 38.3 -t. 0.9
80%3 77.5 % 3.5
P
'
F. L. BAKER, H. E. BROXMEYER AND P. R. GALBRAITH Department of Medicine, Queen's University, Kingston, Ontario, Canada
ABSTRACT
Cellular feeder layers, prepared from normal blood leukocytes, usually stimulate human marrow to form colonies. A significant increase in the stimulating activity of unseparated leukocyte feeder layers is brought about following the removal of dense leukocytes in a manner which avoids enrichment of any remaining cell type. Restoration of dense leukocytes to a dense leukocyte depleted leukocyte feeder layer results in the reduction of stimulating activity to that of an unseparated leukocyte feeder; however, addition of dense leukocytes to unseparated leukocyte feeder layers has no effect on the stimulatory activity, over the range o f concentrations used in this study.
A characteristic, common to virtually all physiological systems, is one of regulation and control. There are numerous examples in which the target organ is regulated by a combination of negative and positive feedback loops, and the regulation of granulopoiesis in vivo likely involves both inhibitory and stimulatory feedback loops (Moore et al., '73). Certain leukocytes possess stimulatory activity and the cell responsible for the production of colony stimulating factor (CSF) has been tentatively identified as the monocyte (Moore et al., '73). However, the role that the monocyte plays in the regulation of granulopoiesis in vivo is not fully understood. The regulation of granulopoiesis, by granulocytes, via a negative feedback loop appears to be a reasonable a priori assumption as evidence has been accumulated which shows that granulocytes inhibit granulopoiesis in vitro (Haskill et a]., '72, Paran et al., '69; Rytomaa et al., '68). As with the monocyte the role that granulocytes might play in the physiology of granulopoiesis is not understood. This paper describes a series of experiments which support our previous work (Haskill et al., '72) and provides additional evidence of granulocyte induced inhibition of colony formation, J. CELL.PHYSIOL., 86: 337-342.
METHODS
Cells Blood, drawn from normal donors, was collected in heparinized vacutainers and allowed to settle for one to one and a half hours. The leukocyte rich plasma was drawn off using a pasteur pipette. Cells were centrifuged down (250 X g for 10 minutes) and washed three times in single strength CMRL-1066 media plus 10% fetal calf serum. A cell count was performed on the second last wash and a stock leukocyte suspension containing 20 x 106cells per ml was made u p after the last wash. Marrow, aspirated from the sternum of informed normal individuals, was centrifuged (500 X g for 10 minutes) and the buffy coat drawn off using a pasteur pipette. Marrow cells were washed and counted in the same way as the blood leukocytes, except that the marrow cell stock suspension contained 2 X 10' cells per ml. Aliquots, taken from the stock cell suspensions were used to prepare the plating suspension, which contained either 2 X log unseparated blood leukocytes per ml or 2 X lo5 unseparated marrow cells per ml. Received June 21, '74. Accepted Feb. 6, 75. 'Supported by Grant 263, The Ontario Cancer Treatment and Research Foundation.
337
338
F. L. BAKER, H. E. BROXMEYER AND P. R. GALBRAITH
Cell separations Cells were separated into two fractions (one containing cells lighter than, and the other containing cells more dense than 1.070 gm/cc), using a single density cut procedure (Shortman et al., '72, Moore et al., '71). The cells to be separated were pelletted by centrifugation at 500 X g for 10 minutes and resuspended in 7 ml of a balanced salt solution containing bovine serum albumin (BSA) of the desired density. Two ml of a lighter density BSA solution (density 1.047 gm,/cc) was laid over the BSA cell suspension to prevent a cell-air interface from forming during centrifugation (4000 X g for 10 minutes). Following centrifugation, the buoyant cells were recovered and reconstituted to their starting volume: e.g. if 5 ml of the stock leukocyte suspension was separated, then the buoyant fraction would be reconstituted to 5 ml. This procedure was followed so that enrichment of the buoyant fraction with any cell type did not occur. The dense cells were reconstituted in a similar manner. The osmolarity of the BSA solutions was measured with a Hewlett Packard model 301A vapour pressure osmometer and brought to 270 mosm. This instrument was used instead of a freezing point depression osometer because Williams et al. ('72) demonstrated that the latter instrument is in error when measuring concentrated solutions of BSA.
(100 X I , 2 m l MEM amino acids (50 X ) , (without glutamine), and 20 ml antibioticantimycotic mixture (100 X ). Feeder layers prepared from unseparated leukocytes contained 2 X lo6 cells per ml per plate, but feeders made from buoyant leukocytes contained fewer cells: the actual number depended upon the numbers of dense cells removed during the separation procedure. The upper or culture layer ( 1 ml) containing the colony-forming cells differed from the feeder layer only in that it contained 0.3% agar and was seeded with 2 X lo5 unseparated marrow cells per ml per plate. Thirty-five mm Falcon #lo08 petri dishes were used throughout.
CSA assay A sigmoid dose response relationship exists between colony formation by normal murine marrow and feeder CSA (Metcalf and Stanley, '69). Consequently feeder CSA may be assayed using an overlayer containing a constant number of marrow cells. As unseparated human marrow was used as the source of target cells in this study, caution had to be exercised in its use because of its content of adherent cells which elaborate CSF. With the marrow concentrations used in this study ( 2 X lo5 cells per ml), aggregates of 20 to 30 cells grow spontaneously, but the spontaneous formation of colonies (aggregates of 50 or more cells) seldom occurred. In the absence of spontaneous colony formation, an exogenous source of stimulator is required to induce colony formation and the numIn uitro culture system ber of colonies so formed is related to the The two layer semi-solid agar culture number of leukocytes plated in the feeder system for mouse marrow cells, modified layer (Galbraith and Broxmeyer '74). by Pike and Robinson ('70) to support the Spontaneous colony formation was not growth of human marrow cells, was used present in any of the cultures reported in this study. The lower, or feeder, layer in this paper. Marrow cells were plated at 2 X lo5cells (1 ml) consisted of 0.5% agar (Difco) in modified CMRL-1066 medium (Gibco) per ml per plate and a single marrow was with 10% heat-inactivated fetal calf serum used to assay both the control and test (Gibco) and blood leukocytes. The modi- feeder layers in each experiment. Colony fications made to the medium involved counts (> 50 cells) were scored on three adding the following ingredients per litre to five plates usually after the seventh day of double strength CMRL-1066 medium : of incubation and occasionally on the fourteenth day. An Olympus model CK inverted 40 ml nonessential amino acids (100 X ) , 20 ml sodium pyruvate (100 X ) , 56 ml microscope, fitted with a mechanical stage, sodium bicarbonate (7.5% ), 4 ml vitamin was used at 40 X to count colonies. Small B12 (1000 pg/ml), 20 ml 1-Glutamine colonies were examined at 100 X to ensure (200 mM), 1 ml MEM vitamin Solution that they contained at least 50 cells.
339
INHIBITION OF COLONY FORMATION BY P M N
Inhibitory effects of blood granulocytes in vitro The inhibitory property of dense leukocytes was demonstrated by removing them and subsequently comparing the CSA elaborated by the remaining cells to that by unseparated leukocytes : a technique which is, in principle, the same as the method employed to demonstrate the presence of serum inhibitors. Blood drawn from healthy normal donors was used to prepare sets of leukocyte feeder layers. Each set consisted of two parts: a control part, and a test part. The control part consisted of feeder layers containing 2 X 10' cells per ml per plate of unseparated blood leukocytes. Test feeder layers were made from buoyant leukocytes (density < 1.070 gm/cc) such that they contained the same numbers of buoyant cells per ml per plate as did the control part. CSA of the feeder layers was assayed using unseparated human marrow cells at 2 X lo5cells per ml per plate. Removal of dense leukocytes, in a manner which avoids concentrating the remaining buoyant leukocytes resulted in a significant increase in feeder CSA: the increase being between 2- and 15-fold RESULTS (table 1 ) . This procedure has been perDensity cut separations formed more than 30 times with identical Between 30% and 50% of the cells sub- results. jected to the density cut were recovered in Dose response characteristics the buoyant fraction and of these less than of t h e inhibition 5% were granulocytes, 50 to 80% lymphoTo study the dose response charactercytes, 10 to 40% monocytes and about 2% basophils. The rest of the cells were istics of dense cell induced inhibition of recovered in the dense fraction, which con- granulopoiesis in vitro feeder layers contained greater than 90% granulocytes, 1 taining a fixed number of buoyant cells to 3% eosinophils, less than 2% mono- with additions of dense cells were prepared. The feeder layers contained buoyant cytes and up to 10% lymphocytes.
Incubation conditions The cell cultures were incubated i n a modified National dual chamber incubator at 37°C. in a humidified atmosphere containing 5 % CO,. The pH of the culture media was checked by comparing the color of the media against a set of La Motte indicator standards and was maintained at 7.2 I 0.1 pH units. Bottled gas, containing 5% CO, in air was used to gas the incubator. A flow rate of 40 ml per minute, per chamber was chosen; however, this low flow rate is insufficient to establish a significant pressure gradient between the two chambers, and as there are no check valves between the two chambers of the incubator to prevent backward flow, the 2-minute 100% COZ flush (used to quickly raise the CO, content following the opening of a chamber) purged the unopened chamber as well. After our incubator was modified to prevent accidental overgassing of the unopened chamber with 100% CO,, we achieved better control over the culture conditions and increased colony formation resulted.
TABLE 1
C S A (Colonies per plate
?
S E M ) o f feeder layers m a d e f r o m :
Unseparated leukocytes
Buoyant leukocytes density < 1.070 gm/cc
45.5 ? 2.5 34.8 ? 4.6 62.8 % 8.4 25.8 C 1.8 7.6 C 1.2 38.3% 0.9
113.6? 10.7 61.2k 6.2 140.3% 8.2 71.2% 2.8 115 % 2.9 175.6e17.8
Probability of difference being a chance occurrence determined by students' test
0.01 0.01
< 0.001 < 0.001 < 0.001 < 0.001
340
F. L. BAKER, H . E. BROXMEYER AND P. R. GALBRAITH
cells (density < 1.070 gm/cc) plus additions of 0 to 160% of the dense cells (density > 1.070 gm/cc) originally present. (Hence, a n addition of 100% of the dense cells originally present in a n unseparated leukocyte feeder, to a feeder layer prepared from buoyant cells would result in a feeder layer whose composition should be indistinguishable from that of a n unseparated layer. ) The dose response relationship obtained indicates that CSA is significantly reduced following the addition of small numbers of dense leukocytes (fig. 1 ) . When 100% of the dense cells were restored, CSA of the feeder layer was the same a s that prepared from unseparted leukocytes. Further increases in the numbers of added dense cells did not reduce CSA further. Qualitatively similar findings have been obtained on at least eight separate occasions. 401
L
U m
’“1 0
20
40
60
80
100
120
140
160
Feeder Layer Composition Co/o dense leukocytes)
Fig. 1 Dose response relationship between leukocyte feeder layer composition and stimulating activity. Abscissa, feeder layer composed of buoyant cells plus 0 to 160% of the dense cells present in a feeder layer prepared from unseparated cells. Ordinate, number of colonies ( > 50 cells) formed per plate & SEM. Shaded area, colony formation 2 SEM stimulated by unseparated feeder layers.
TABLE 2
Colony f o r m a t i o n (Colonies per plate ( .t. S E M ) ) b y m a r r o w cultures prepared f r o m : Unseparated marrow
Separated marrow buoyant fraction
45.5 -t. 2.5 38.3 -t. 0.9
80%3 77.5 % 3.5
P
