American Journal of Hematology 6: 1-10 (1979)
Control Factors of Granulopoiesis in Human Serum Rex W. Bolin and William A. Robinson Department of Medicine, Section of Oncology, University of Colorado Medical Center, Denver
The role of serum factors in the modulation of production of colony-stimulating activity (CSA) has been investigated. A factor has been described, and partially characterized, in human serum that has the capacity to stimulate increased synthesis and release of CSA by human mononuclear cells (MNC). MNC RNA and protein synthesis are required to demonstrate this effect of serum, but DNA synthesis and mitotic division are not required. The factor in serum resulting in this effect is a heat-labile protein with a molecular weight slightly greater than that of CSA. Key words: CSA, serum, regulation, granulopoiesis
INTRODUCTION
The maturation of granulocytes in vitro requires the presence of granulopoietic factors, termed colony-stimulating factors (CSF), that are sometimes referred to collectively as colony-stimulating activity (CSA) [ 11 . The major source of CSA in humans has been identified as the monocyte-macrophage system [2-51 , although recently lung and placental tissues have been shown t o be potent sources as well [6,7] . Lymphocytes have been shown to release CSA in response to mitogens or prior sensitization with parasites [8-101 . Factors involved in the modulation of CSA levels are not well defined. It has been demonstrated that bacterial components of gram-negative and gram-positive organisms increase levels of CSA [ll-151 , and the granulocytes may act t o decrease levels by release of an inhibitory factor [16-181 or removal of bacterial stimuli [14-151. Lymphocytes may modulate release of CSA from monocytes as well [13] . This paper reports the modulation of CSA levels by components of homologous serum. Factors are described that appear to increase the rate of synthesis and subsequent release of CSA. MATERIALS AND METHODS Preparation of Mononuclear Cells From the Peripheral Blood
Blood from normal humans was collected in tubes containing preservative-free heparin using sterile techniques, after informed written consent was secured, as approved by the Hu-
Received for publication May 23, 1978; accepted December 30, 1978. Address reprint requests to William A . Robinson, MD, PhD, 4200 East Ninth Avenue, Denver , CO 80262.
0361-8609/79/0601-0001$02.00 0 1979 Alan R. Liss, Inc.
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man Research Committee, UCMC. Erythrocytes were allowed t o settle by gravity at room temperature for 1-2 hours and the leukocyte-rich plasma was aspirated. Leukocytes were removed from the plasma by centrifugation at 350g for 10 minutes at room temperature and resuspended in McCoy’s 5A medium with 15% fetal calf serum. The suspension was then layered over a cushion of Ficoll-Hypaque of density 1.077 gm/ml (Lymphoprep, Nyegaard, and Co., Oslo) and erythrocytes and granulocytes were removed by centrifugation at 400g for 35 minutes at room temperature. The mononuclear cell (MNC) population was then removed from the cushion interface and washed several times in McCoy’s 5A medium t o insure removal of plasma and platelets. The resultant cell population was generally 70% lymphocytes and 30% monocytes as judged by adherence properties. Preparation of Bone Marrow Cells
Precursors of human granulocytes (CFU-C) were obtained from bone marrow of normal paid volunteers (as approved by the Human Research Committee, UCMC, after informed written consent). Bone marrow samples (30 ml total) were aspirated through the posterior iliac crest under local anesthesia and placed in tubes containing a total of 600 units of preservative-free heparin. Samples were then layered over a cushion of FicoI1-Hypaque and prepared as described above for mononuclear cells. Fnllowing centrifugation, the cells at the interface were washed as above and rendered free of CSA-releasing monocytes by a process of adhering them t o a glass surface. Time of adherence was approximately 90 minutes. Liquid Culture Techniques
McCoy’s 5A medium with 15% fetal calf serum was used throughout these experiments. MNC were prepared as described above and incubated in 2-ml liquid cultures in 35-mm plastic petri dishes with the described additions. Cultures contained 2.5 X lo5 MNC/ml unless noted. Cultures were incubated for 48-1 10 hours as noted, at 37°C in 7.5% COz and 100% humidity. Following incubation, cells and debris were removed by centrifugation and the resultant conditioned medium was assayed for CSA as described below. CSA Assay
CSA was assayed by the method of Pike and Robinson [19], CFU-C, which respond to CSA, were derived from bone marrow as described above. Material t o be assayed was derived from the liquid culture experiments unless otherwise specified and 0.1 ml was plated in triplicate 35-mm petri dishes. To each dish was added 1 ml of McCoy’s 5A medium with 15% fetal calf serum containing 0.3% agar and 50,000 nucleated human bone marrow cells prepared as described above. Plates were then incubated at 37°C in 7.5% COz and 100% humidity for 12-14 days and counted with the aid of dissecting microscope. Levels of CSA are expressed as the mean number of colonies SE in the triplicate plates.
*
Characterization of Serum Factor
Aliquots (1 ml) were treated with 50 units RNase, DNase, and insoluble pronase and trypsin (Sigma). Treatment was for 12 hours at 37°C. Pronase and trypsin were then removed by centrifugation. No attempt was made to remove RNase or DNase. Samples (2 ml) of serum maintained at various pH were dialyzed against 100 volumes of buffer of the corresponding pH for 24 hours. The buffer was changed three times during this period. Different buffers were used for each pH range so as t o retain buffering capacity. Sodium acetate (10 mM) was used at pH 2-5, tris (10 mM) was used at pH 6-9, and tribasic sodium phosphate (10 mM) was used at pH 10-12. The samples were then neutralized
Serum Modulation of Granulopoiesis
3
by dialysis against three changes (100 volumes each) of 50 mM Tris at pH 7.2 for 24 hours and finally against distilled water for 48 hours. The distilled water was changed every 8-10 hours. All steps in the procedure were done at 4°C. Gel fractionation of serum was done using Sephadex G150 in a 100- X 2.5-cm column with a pressure head of about 10 cm. Dextran 2000 was used to determine the exclusion volume and thereafter 50 fractions of 2.5 ml each were collected and assayed as described. Isotope Labeling
Cultures of 1 X lo6 MNC/ml were prepared and incubated with only 15% fetal calf serum or with an addition of 15% human serum for the described periods of time. Cultures were then washed t o removed serum, free amino acids, and nucleotides and were resuspended in Hanks’ balanced salt solution. The cells were suspended in Hanks’ balanced salt solution t o control the specific activity of the radioactive compounds and reduce the background. Tritium-labeled thymidine (2pCi/ml, 10.3 Ci/mmole, Schwartz/Mann, Orangeburg, New York) or amino acid hydrolysate ( 5 pCi/ml, Schwartz/Mann, Orangeburg, New York) was added for 4 hours and then trichloracetic acid was added to a final concentration of 10%.After at least 30 minutes at 0°C the precipitate was removed by filtration and following repeated washing with cold 5% trichloracetic acid incorporated isotope counted in a liquid scintillation counter. Inhibitor Studies
The effect of actinomycin D, cycloheximide, puromycin, colchicine, and N-ethylmaleimide on the serum-mediated release of CSA by MNC was examined. Inhibitors were added as described and were removed prior to assay of CSA by dialysis against 5,000 volumes of distilled water for 72 hours with changes of distilled water every 8-10 hours. Dialysis was done at 4°C. Dialysis using the conditions described effectively removed the inhibitors.
80
TIME (IN HOURS)
Fig. 1. Effect of human serum on the production of colony-stimulating activity (CSA) by mononuclear cells (MNC). Cultures containing 2.5 X lo5 MNC per milliliter were prepared and 15% human serum was added to half of them. Periodically for 96 hours, samples were taken and assayed for CSA. CSA was assayed on human bone marrow and expressed as the mean colony count ? SE of three plates.
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RESULTS Effect of Human Serum on Release of CSA by MNC
Figure 1 shows the effect of 15% autologous human serum on the release of CSA by MNC over a 96-hour culture period. Human serum resulted in an acceleration of CSA release compared t o a culture containing only 15% fetal calf serum. Cultures not receiving human serum usually achieved levels of CSA that were comparable to levels of cultures receiving human serum after a period of 80-120 hours of incubation. Further experiments (Table I) were done to determine whether the effect of the serum was species-specific or whether autologous material was required. Mouse serum was not able to increase release of CSA by human cells although a dramatic increase was noted with mouse spleen cells. It is of interest that unpurified mouse peritoneal cavity cells did not increase release of CSA in response t o mouse or human serum (not shown). Also shown in Table I is that sera from a number of individual humans resulted in various degrees of CSA release from MNC of a single donor. Instances were commonly encountered where serum from a donor would not evoke additional release of CSA. To date more than 100 sera have been examined and about 40%had little effect on the stimulation of CSA by MNC. In addition, care had to be taken to exclude the effect of serum potentiation of preexisting CSA [20,21] , a phenomenon previously noted by others. This was routinely controlled by adding human serum t o MNC-conditioned medium after the cells were removed and comparing the apparent amount of CSA t o that of a parallel culture which received human serum at the start of culture. Any increase due t o human serum that was not cell-dependent (found when serum was added after MNC were removed) was subtracted from recorded CSA values. This correction has been made in all data presented here. When serum from a typical donor was added t o MNC-conditioned medium at a final concentration of 15% and then assayed for CSA, potentiation would usually result in an increase of 15-25 colonies. On the other hand, addition of 15% human serum to a culture containing MNC would typically result in sufficient CSA to pro-
TABLE I. Species Specificity of Serum-Mediated Release of CSA Conditioned mediuma Cell source Mouse Human A Mouse Human A Mouse Human A Human A Human A Human A
Serum source
__
__
Human A Mouse Mouse Human A Human B Human C Human D
CSA levels in conditioned medium 11 * 2 24 1 4 12 +- 2 1811 115 + - 4 45 * 3 68 ? 6 30 * 2 41 + _ 3
a
Conditioned medium was obtained by incubation of described cells and serum for 7 2 hours at 37°C. Mouse cells were derived from the spleen of CS7/bl mice and mouse sera were from a pool of several mice. Human cells were MNC derived as described in Materials and Methods. Sera were added a t 15%final concentration. Cells were used at 1 X lo6 per milliliter for mice and 3 X lo5 per milliliter for humans. CSA from human MNC was assayed using human bone marrow and mouse spleen cells using mouse bone marrow. Sera alone did not produce colonies. The data presented here are from one experiment.
S e r u m Modulation of Granulopoiesis
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TABLE 11. Effect of Enzyme Pretreatment of Serum on Capacity to Stimulate Release of CSA CSA levels in conditioned medium
Enzyme treatmenta
40 * 3 35 c 3 77 i 5 75 + 4 62 f 2
Trypsin Pronase RNase DNase None
aSamples of serum (1 ml) were treated with 50 units of insolubilized trypsin or pronase for 12 hours at 37"C, after which time the enzyme as removed. RNase and DNase (50 units each) were added in similar fashion and no attempt was made to inactivate the enzymes. The treated serum was then added t o cultures of MNC ( 3 X l o 5 cells per milliliter) to 15% and incubated 72 hours at 37°C. CSA was then assayed on human bone marrow.
5a
30
201 10
4b
5b
6b
7b
8b
I
90 TEMPERATURE "C (MAINTAINED 30 MINUTES)
Fig. 2. Effect of temperature on the serum component. Aliquots of serum were treated at each temperature for 30 minutes and then added t o liquid cultures containing 2.5 X lo5 MNC per milliliter. After 7 2 hours of incubation, the conditioned media were assayed for CSA levels on human bone marrow and CSA was expressed as a percentage of activity in a control liquid culture containing untreated serum.
duce 35-65 colonies. Sera were often encountered that would result in neither greater potentiation nor production of CSA. The reasons for this variability are not understood. Partial Characterization of Serum Component
Aliquots of human serum were pretreated with various enzymes t o determine the nature of the component enhancing the release of CSA from MNC (Table 11). Nucleases had no effect on the activity, whereas trypsin and pronase both reduced the activity. That the serum component is heat-labile can be seen in Figure 2. Aliquots of serum were maintained at the indicated temperature for 30 minutes and then incubated with MNC for 72 hours and CSA
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Bolin and Robinson
I
T
30(I)
wz
2 0 0
20(2:
W
m
5z
10-
I
4
I
I
6 8 I b l b pH (MAINTAINED 24 HOURS)
2
Fig. 3. Effect of pH treatment on the serum factor. Samples of serum were maintained at the indicated pH for 24 hours prior to incubation for 72 hours with 2.5 X lo5 MNC per milliliter in liquid culture. CSA was assayed on human bone marrow and expressed as the mean colony count SE .
*
I
0
rc
'$
MNC-DEPENDENT COMPONENT
40-
I
FRACTION NUMBER
Fig. 4.Fractionation of serum on Sephadex G150 to yield CSA MNC-dependent components. A portion (0.3 ml) of each 2.5-ml fraction was assayed for CSA by plating directly on human bone marrow. A second portion (0.4 ml) of each fraction was assayed for MNC-dependent components by incubation with 2.5 X lo5 MNC per milliliter for 48 hours prior to assay of 0.1 ml of the resultant conditioned medium on human bone marrow. The CSA in fractions 8-18 would have little effect on CSA levels in the MNC-dependent assay due to this dilution. Each point is the mean colony count of duplicate plates.
Serum Modulation of Granulopoiesis
x
1
7
DNA SYNTHESIS
P(ROTE NI
SYNTHESIS
24
48
7’2
HOUHS OF CULTURE
Fig. 5. Effect of human serum on the synthesis of DNA and protein by MNC. Cultures containing 1 X lo6 MNC per milliliter were treated with 15% human serum for periods up t o 72 hours and the amount of protein and DNA synthesis that occurred in a four-hour period was periodically determined as described.
was assayed. Temperatures of 50°C had little effect but higher temperatures caused a rapid loss of activity. Figure 3 shows the effect of pH treatment on the serum factor. Samples were maintained at the indicated pH for 24 hours, neutralized, and then incubated with MNC; CSA was assayed as before. The factor was clearly stable from pH 5-1 1 but activity was rapidly lost outside this range. Optimal stability resulted at pH 7-8. The molecular weight of the serum component in comparison to CSA was then determined (Fig. 4). Serum was applied to a column and fractions collected. A portion of each fraction was assayed for CSA directly, and a second portion was incubated with MNC, the resultant conditioned medium being assayed for CSA. As seen in Figure 4 the serum factor enhancing CSA release from MNC appeared t o elute prior t o elution of endogenous serum CSA, indicating an apparently higher molecular weight. Macromolecular Synthesis Requirements for Stimulation
Cultures containing 1 X lo6 MNC/ml, with and without human serum, were cultured for periods up t o 72 hours and the amount of protein and DNA synthesis were periodically determined (Fig. 5). Addition of human serum had no effect on the amount of DNA synthesis. Protein synthesis was variably affected. Stimulation of protein synthesis was usually noted, but instances of no stimulation or little stimulation were also encountered. The reasons for this variability were not determined. In further studies, various inhibitors of macromolecular synthesis were added t o MNC cultures containing human serum t o determine the requirements for the serum-mediated stimulation of CSA production. DNA-RNA synthesis was inhibited with actinomycin D, protein synthesis with puromycin and cycloheximide, and mitosis inhibited with colchicine. The
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TABLE 111. Effect of Various Compounds on Serum-Mediated Release of CSAa Inhibitor added to MNC cultures
Levels of CSA in conditioned medium
Colchicine (0.5 pg/ml) Actinomycin D (1pg/ml) Cycloheximide (50 pg/ml) Puromycin (25 pg/ml) N-Ethylmaleimide (50 pg/ml) None
56+6 1 0 0 0 52 + - 4
"Inhibitors were added to MNC cultures containing 15% human serum and incubated for 96 hours at 37"C, after which time the conditioned medium was dialyzed and subsequently assayed for CSA. Samples treated with inhibitors and then dialyzed did not inhibit growth of bone marrow when stimulated with a known source of CSA.
"1 80
Fig. 6. Effect of puromycin on the serum-stimulated production of CSA and MNC. Puromycin (25 pg/ml) was added to series of MNC cultures containing 15% human serum and 2.5 X lo5 cells per milliliter at times from 0 t o 48 hours after start of incubation. The experiment was terminated 60 hours after start of incubation. Following dialysis, CSA in the resultant conditioned medium was assayed on human bone marrow and expressed as the mean colony count i. SE of triplicate plates. In cultures not treated with serum, puromycin would likewise inhibit CSA production. Refer to Figure 1 for the relative production of CSA with and without addition of human serum.
effect of N-ethylmaleimide, which affects export of proteins among other actions, was also examined. Table 111 shows that all inhibitors except colchicine inhibited the release of CSA. Parallel cultures that did not receive human serum were also treated with inhibitors and the same basic effect on production of CSA was noted (not shown). An experiment was done t o determine if there was a point after which no additional protein synthesis was required, which would be an indication that an event had occurred
Serum Modulation of Granulopoiesis
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that had resulted in the activation of preformed CSA in the cells or serum. Human serum was added t o cultures at the start of incubation and puromycin was added at increasing intervals throughout the incubation period. Puromycin added prior to 48 hours after start of incubation resulted in decreased CSA release (Figure 6). DISCUSSI ON
These studies have demonstrated that human serum has the potential t o enhance CSA production by mononuclear cells in culture. This does not appear to be the result of potentiation of existing CSA as has been described previously [20,21] , since this effect is dependent on viable cells. The serum factor responsible for this effect does not appear t o be CSA itself, as evidenced by its apparent higher molecular weight. Active cellular processes are required for this serum-mediated stimulation of CSA release by MNC. CSA released in the absence of human serum seems to be subject t o the same controls. RNA and protein synthesis are clearly required for this effect, but mitotic processes do not appear necessary. Colchicine, which inhibits DNA synthesis as well as mitosis, has no effect on this phenomenon, while N-ethylmaleimide, cycloheximide, and puromycin all inhibited the noted effect of serum. Likewise, actinomycin D was effective in stopping the process. This was probably the result of inhibition of RNA and not DNA synthesis, since colchicine had no effect. These data might be interpreted as indicating that the serum factor resulted in increasing CSA production by stimulation at the transcriptional level. The exact mechanism, however, remains t o be determined and appears complex. It should be noted that in most instances, incubation of MNC cultures not receiving human serum eventually resulted in CSA levels reaching a final plateau equivalent t o those receiving human serum. This may be the result of a finite capacity of cells t o produce CSA that is simply reached more rapidly in the presence of human serum, but otherwise can be achieved at a slower rate through the utilization of components present in fetal calf serum. It is also possible that these results represent limitation of the in vitro conditions under which the cells were cultured and the CSA assayed. The importance and physiologic role of the serum factor described here has not been determined. Since MNC are constantly bathed in plasma in vivo, it may simply represent superior nutritive conditions. Another possibility is that the factor represents a humoral signal for CSA production and release by MNC. This view is supported by our findings that certain patients with neutropenia respond t o plasma infusions to produce normal levels of neutrophils [22] . Another possibility is that serum contains a precursor t o CSA that is subsequently activated by MNC. Data presented here would demand that such an activation require continued protein synthesis by MNC. The capacity of serum to potentiate existing sources of CSA, such as MNC-conditioned media, would be compatible with activation of a serum component by an enzyme produced by MNC [20,21]. Previous data from this laboratory have suggested a cooperative interaction between lymphocytes and monocytes t o produce increased amounts of CSA [13] . Serum may play a role in this interaction as well. ACKNOWLEDGMENTS
This study was supported by grants from the National Institutes of Health, National Cancer Institute (lROlCA11305-09 and CA05058-lo), American Cancer Society No. CH8G, National Institutes of Health Basic Oncology (CA13419), and by grant No. RR-51 from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health.
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REFERENCES 1. Metcalf D: Regulation of granulocyte and monocyte-macrophage proliferation by CSF: A review. Exp Hematol 1:185-201, 1973. 2. Moore MAS, Williams NJ, Metcalf D: Purification and characterization of the in vitro colony-forming cell in monkey hemopoietic tissue. J Cell Physiol 79:283-292, 1972. 3. Chervenick PA, LoBuglio AF: Human blood nionocytes: Stimulators of granulocyte and mononuclear colony formation in vitro. Science 178:164-166, 1972. 4. Golde DW, Cline MJ: Identification of the colony-stimulating cell in human peripheral blood. J Clin Invest 51:2981-2983, 1972. 5 . Golde DW, Findley TN, Cline MJ: Production of colony stimulating factor in human macrophages. Lancet 2:1397-1399, 1972. 6. Fojo SS, Wu MC, Gross MA, Yunis AA: The isolation and characterization of a colony stimulating factor from human lung. Clin Res 24(3):307A, 1976. 7. Burgess AW, Wilson EMA, Metcalf D: Stimulation by human placental-conditioned medium of hemopoietic colony formation by human marrow cells. Blood 49:573-583, 1977. 8. Cline MJ, Golde DW: Production of colony-stimulating activity by human lymphocytes. Nature 248: 703-704, 1974. 9. Ruscetti FW, Chervenick PA: Release of colony-stimulating activity from thymus-derived lymphocytes. J Clin Invest 55520-527,1975. 10. Ruscetti FW, Cypress RH, Chervenick PA: Specific release of neutrophilic- and eosinophilic-stimulating factors from sensitized lymphocytes. Blood 47:757-765, 1976. 11. Quesenberry P, Morley A, Stohlman F Jr, Richard K, Howard D, Smith MR: Effect of endotoxin on granulopoiesis and colony stimulating factor. New Engl J Med 286:227-232, 1974. 12. Golde DW, Cline MJ: Endotoxin-induced release of colony-stimulating activity in man. Proc SOCExp Biol Med 149:845-848, 1975. 13. Bolin RW, Robinson WA: Bacterial, serum and cellular modulation of granulopoietic activity. J Cell Physiol92:145-153, 1977. 14. Robinson WA, Entringer M , Bolin RW, Stonington OG: Bacterial stimulation and granulocyte inhibition of granulopoietic factor production. N Engl J Med 297: 1129-1134, 1977. 15. Mahmood T, Robinson WA: Granulocyte modulation of endotoxin-stimulated colony-stimulating activity (CSA) production. Blood 51 :879-887, 1978. 16. Rytomaa T, Kivimeni K: Control of granulocyte production. I. Chalone and antichalone, two specific humoral regulators. Cell Tissue Kinet 1:329-340, 1968. 17. Haskill JS, McKnight RD, Galbraith PR: Cell-cell interaction in vitro: Studied by density separation of colony-forming, stimulating and inhibiting cells from human bone marrow. Blood 40:394-399, 1972 18. Broxmeyer HE, Moore MAS, Ralph P: Cell-free granulocyte colony-inhibiting activity derived from human polymorphonuclear neutrophils. Exp Hematol 5:87-102, 1977. 19. Pike BL, Robinson WA: Human bone marrow colony growth in agai-gel. J Cell Physiol76:77-84, 1970. 20. Mabry J, Carbone PP, Bull JM: Amplification of colony-stimulating activity in human serum by interaction with CSA from other sources. Exp Hematol 3:354-361, 1975. 21. Metcalf D, MacDonald HR, Chester HM: Serum potentiation of granulocyte and macrophage colony formation in vitro. Exp Hematol 3:261-273, 1975. 22. Bolin RW, Robinson WA, Hays T: Studies in childhood neutropenia: Stimulation of granulopoiesis in human serum (Submitted for publication).
Control Factors of Granulopoiesis in Human Serum Rex W. Bolin and William A. Robinson Department of Medicine, Section of Oncology, University of Colorado Medical Center, Denver
The role of serum factors in the modulation of production of colony-stimulating activity (CSA) has been investigated. A factor has been described, and partially characterized, in human serum that has the capacity to stimulate increased synthesis and release of CSA by human mononuclear cells (MNC). MNC RNA and protein synthesis are required to demonstrate this effect of serum, but DNA synthesis and mitotic division are not required. The factor in serum resulting in this effect is a heat-labile protein with a molecular weight slightly greater than that of CSA. Key words: CSA, serum, regulation, granulopoiesis
INTRODUCTION
The maturation of granulocytes in vitro requires the presence of granulopoietic factors, termed colony-stimulating factors (CSF), that are sometimes referred to collectively as colony-stimulating activity (CSA) [ 11 . The major source of CSA in humans has been identified as the monocyte-macrophage system [2-51 , although recently lung and placental tissues have been shown t o be potent sources as well [6,7] . Lymphocytes have been shown to release CSA in response to mitogens or prior sensitization with parasites [8-101 . Factors involved in the modulation of CSA levels are not well defined. It has been demonstrated that bacterial components of gram-negative and gram-positive organisms increase levels of CSA [ll-151 , and the granulocytes may act t o decrease levels by release of an inhibitory factor [16-181 or removal of bacterial stimuli [14-151. Lymphocytes may modulate release of CSA from monocytes as well [13] . This paper reports the modulation of CSA levels by components of homologous serum. Factors are described that appear to increase the rate of synthesis and subsequent release of CSA. MATERIALS AND METHODS Preparation of Mononuclear Cells From the Peripheral Blood
Blood from normal humans was collected in tubes containing preservative-free heparin using sterile techniques, after informed written consent was secured, as approved by the Hu-
Received for publication May 23, 1978; accepted December 30, 1978. Address reprint requests to William A . Robinson, MD, PhD, 4200 East Ninth Avenue, Denver , CO 80262.
0361-8609/79/0601-0001$02.00 0 1979 Alan R. Liss, Inc.
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Bolin and Robinson
man Research Committee, UCMC. Erythrocytes were allowed t o settle by gravity at room temperature for 1-2 hours and the leukocyte-rich plasma was aspirated. Leukocytes were removed from the plasma by centrifugation at 350g for 10 minutes at room temperature and resuspended in McCoy’s 5A medium with 15% fetal calf serum. The suspension was then layered over a cushion of Ficoll-Hypaque of density 1.077 gm/ml (Lymphoprep, Nyegaard, and Co., Oslo) and erythrocytes and granulocytes were removed by centrifugation at 400g for 35 minutes at room temperature. The mononuclear cell (MNC) population was then removed from the cushion interface and washed several times in McCoy’s 5A medium t o insure removal of plasma and platelets. The resultant cell population was generally 70% lymphocytes and 30% monocytes as judged by adherence properties. Preparation of Bone Marrow Cells
Precursors of human granulocytes (CFU-C) were obtained from bone marrow of normal paid volunteers (as approved by the Human Research Committee, UCMC, after informed written consent). Bone marrow samples (30 ml total) were aspirated through the posterior iliac crest under local anesthesia and placed in tubes containing a total of 600 units of preservative-free heparin. Samples were then layered over a cushion of FicoI1-Hypaque and prepared as described above for mononuclear cells. Fnllowing centrifugation, the cells at the interface were washed as above and rendered free of CSA-releasing monocytes by a process of adhering them t o a glass surface. Time of adherence was approximately 90 minutes. Liquid Culture Techniques
McCoy’s 5A medium with 15% fetal calf serum was used throughout these experiments. MNC were prepared as described above and incubated in 2-ml liquid cultures in 35-mm plastic petri dishes with the described additions. Cultures contained 2.5 X lo5 MNC/ml unless noted. Cultures were incubated for 48-1 10 hours as noted, at 37°C in 7.5% COz and 100% humidity. Following incubation, cells and debris were removed by centrifugation and the resultant conditioned medium was assayed for CSA as described below. CSA Assay
CSA was assayed by the method of Pike and Robinson [19], CFU-C, which respond to CSA, were derived from bone marrow as described above. Material t o be assayed was derived from the liquid culture experiments unless otherwise specified and 0.1 ml was plated in triplicate 35-mm petri dishes. To each dish was added 1 ml of McCoy’s 5A medium with 15% fetal calf serum containing 0.3% agar and 50,000 nucleated human bone marrow cells prepared as described above. Plates were then incubated at 37°C in 7.5% COz and 100% humidity for 12-14 days and counted with the aid of dissecting microscope. Levels of CSA are expressed as the mean number of colonies SE in the triplicate plates.
*
Characterization of Serum Factor
Aliquots (1 ml) were treated with 50 units RNase, DNase, and insoluble pronase and trypsin (Sigma). Treatment was for 12 hours at 37°C. Pronase and trypsin were then removed by centrifugation. No attempt was made to remove RNase or DNase. Samples (2 ml) of serum maintained at various pH were dialyzed against 100 volumes of buffer of the corresponding pH for 24 hours. The buffer was changed three times during this period. Different buffers were used for each pH range so as t o retain buffering capacity. Sodium acetate (10 mM) was used at pH 2-5, tris (10 mM) was used at pH 6-9, and tribasic sodium phosphate (10 mM) was used at pH 10-12. The samples were then neutralized
Serum Modulation of Granulopoiesis
3
by dialysis against three changes (100 volumes each) of 50 mM Tris at pH 7.2 for 24 hours and finally against distilled water for 48 hours. The distilled water was changed every 8-10 hours. All steps in the procedure were done at 4°C. Gel fractionation of serum was done using Sephadex G150 in a 100- X 2.5-cm column with a pressure head of about 10 cm. Dextran 2000 was used to determine the exclusion volume and thereafter 50 fractions of 2.5 ml each were collected and assayed as described. Isotope Labeling
Cultures of 1 X lo6 MNC/ml were prepared and incubated with only 15% fetal calf serum or with an addition of 15% human serum for the described periods of time. Cultures were then washed t o removed serum, free amino acids, and nucleotides and were resuspended in Hanks’ balanced salt solution. The cells were suspended in Hanks’ balanced salt solution t o control the specific activity of the radioactive compounds and reduce the background. Tritium-labeled thymidine (2pCi/ml, 10.3 Ci/mmole, Schwartz/Mann, Orangeburg, New York) or amino acid hydrolysate ( 5 pCi/ml, Schwartz/Mann, Orangeburg, New York) was added for 4 hours and then trichloracetic acid was added to a final concentration of 10%.After at least 30 minutes at 0°C the precipitate was removed by filtration and following repeated washing with cold 5% trichloracetic acid incorporated isotope counted in a liquid scintillation counter. Inhibitor Studies
The effect of actinomycin D, cycloheximide, puromycin, colchicine, and N-ethylmaleimide on the serum-mediated release of CSA by MNC was examined. Inhibitors were added as described and were removed prior to assay of CSA by dialysis against 5,000 volumes of distilled water for 72 hours with changes of distilled water every 8-10 hours. Dialysis was done at 4°C. Dialysis using the conditions described effectively removed the inhibitors.
80
TIME (IN HOURS)
Fig. 1. Effect of human serum on the production of colony-stimulating activity (CSA) by mononuclear cells (MNC). Cultures containing 2.5 X lo5 MNC per milliliter were prepared and 15% human serum was added to half of them. Periodically for 96 hours, samples were taken and assayed for CSA. CSA was assayed on human bone marrow and expressed as the mean colony count ? SE of three plates.
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RESULTS Effect of Human Serum on Release of CSA by MNC
Figure 1 shows the effect of 15% autologous human serum on the release of CSA by MNC over a 96-hour culture period. Human serum resulted in an acceleration of CSA release compared t o a culture containing only 15% fetal calf serum. Cultures not receiving human serum usually achieved levels of CSA that were comparable to levels of cultures receiving human serum after a period of 80-120 hours of incubation. Further experiments (Table I) were done to determine whether the effect of the serum was species-specific or whether autologous material was required. Mouse serum was not able to increase release of CSA by human cells although a dramatic increase was noted with mouse spleen cells. It is of interest that unpurified mouse peritoneal cavity cells did not increase release of CSA in response t o mouse or human serum (not shown). Also shown in Table I is that sera from a number of individual humans resulted in various degrees of CSA release from MNC of a single donor. Instances were commonly encountered where serum from a donor would not evoke additional release of CSA. To date more than 100 sera have been examined and about 40%had little effect on the stimulation of CSA by MNC. In addition, care had to be taken to exclude the effect of serum potentiation of preexisting CSA [20,21] , a phenomenon previously noted by others. This was routinely controlled by adding human serum t o MNC-conditioned medium after the cells were removed and comparing the apparent amount of CSA t o that of a parallel culture which received human serum at the start of culture. Any increase due t o human serum that was not cell-dependent (found when serum was added after MNC were removed) was subtracted from recorded CSA values. This correction has been made in all data presented here. When serum from a typical donor was added t o MNC-conditioned medium at a final concentration of 15% and then assayed for CSA, potentiation would usually result in an increase of 15-25 colonies. On the other hand, addition of 15% human serum to a culture containing MNC would typically result in sufficient CSA to pro-
TABLE I. Species Specificity of Serum-Mediated Release of CSA Conditioned mediuma Cell source Mouse Human A Mouse Human A Mouse Human A Human A Human A Human A
Serum source
__
__
Human A Mouse Mouse Human A Human B Human C Human D
CSA levels in conditioned medium 11 * 2 24 1 4 12 +- 2 1811 115 + - 4 45 * 3 68 ? 6 30 * 2 41 + _ 3
a
Conditioned medium was obtained by incubation of described cells and serum for 7 2 hours at 37°C. Mouse cells were derived from the spleen of CS7/bl mice and mouse sera were from a pool of several mice. Human cells were MNC derived as described in Materials and Methods. Sera were added a t 15%final concentration. Cells were used at 1 X lo6 per milliliter for mice and 3 X lo5 per milliliter for humans. CSA from human MNC was assayed using human bone marrow and mouse spleen cells using mouse bone marrow. Sera alone did not produce colonies. The data presented here are from one experiment.
S e r u m Modulation of Granulopoiesis
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TABLE 11. Effect of Enzyme Pretreatment of Serum on Capacity to Stimulate Release of CSA CSA levels in conditioned medium
Enzyme treatmenta
40 * 3 35 c 3 77 i 5 75 + 4 62 f 2
Trypsin Pronase RNase DNase None
aSamples of serum (1 ml) were treated with 50 units of insolubilized trypsin or pronase for 12 hours at 37"C, after which time the enzyme as removed. RNase and DNase (50 units each) were added in similar fashion and no attempt was made to inactivate the enzymes. The treated serum was then added t o cultures of MNC ( 3 X l o 5 cells per milliliter) to 15% and incubated 72 hours at 37°C. CSA was then assayed on human bone marrow.
5a
30
201 10
4b
5b
6b
7b
8b
I
90 TEMPERATURE "C (MAINTAINED 30 MINUTES)
Fig. 2. Effect of temperature on the serum component. Aliquots of serum were treated at each temperature for 30 minutes and then added t o liquid cultures containing 2.5 X lo5 MNC per milliliter. After 7 2 hours of incubation, the conditioned media were assayed for CSA levels on human bone marrow and CSA was expressed as a percentage of activity in a control liquid culture containing untreated serum.
duce 35-65 colonies. Sera were often encountered that would result in neither greater potentiation nor production of CSA. The reasons for this variability are not understood. Partial Characterization of Serum Component
Aliquots of human serum were pretreated with various enzymes t o determine the nature of the component enhancing the release of CSA from MNC (Table 11). Nucleases had no effect on the activity, whereas trypsin and pronase both reduced the activity. That the serum component is heat-labile can be seen in Figure 2. Aliquots of serum were maintained at the indicated temperature for 30 minutes and then incubated with MNC for 72 hours and CSA
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Bolin and Robinson
I
T
30(I)
wz
2 0 0
20(2:
W
m
5z
10-
I
4
I
I
6 8 I b l b pH (MAINTAINED 24 HOURS)
2
Fig. 3. Effect of pH treatment on the serum factor. Samples of serum were maintained at the indicated pH for 24 hours prior to incubation for 72 hours with 2.5 X lo5 MNC per milliliter in liquid culture. CSA was assayed on human bone marrow and expressed as the mean colony count SE .
*
I
0
rc
'$
MNC-DEPENDENT COMPONENT
40-
I
FRACTION NUMBER
Fig. 4.Fractionation of serum on Sephadex G150 to yield CSA MNC-dependent components. A portion (0.3 ml) of each 2.5-ml fraction was assayed for CSA by plating directly on human bone marrow. A second portion (0.4 ml) of each fraction was assayed for MNC-dependent components by incubation with 2.5 X lo5 MNC per milliliter for 48 hours prior to assay of 0.1 ml of the resultant conditioned medium on human bone marrow. The CSA in fractions 8-18 would have little effect on CSA levels in the MNC-dependent assay due to this dilution. Each point is the mean colony count of duplicate plates.
Serum Modulation of Granulopoiesis
x
1
7
DNA SYNTHESIS
P(ROTE NI
SYNTHESIS
24
48
7’2
HOUHS OF CULTURE
Fig. 5. Effect of human serum on the synthesis of DNA and protein by MNC. Cultures containing 1 X lo6 MNC per milliliter were treated with 15% human serum for periods up t o 72 hours and the amount of protein and DNA synthesis that occurred in a four-hour period was periodically determined as described.
was assayed. Temperatures of 50°C had little effect but higher temperatures caused a rapid loss of activity. Figure 3 shows the effect of pH treatment on the serum factor. Samples were maintained at the indicated pH for 24 hours, neutralized, and then incubated with MNC; CSA was assayed as before. The factor was clearly stable from pH 5-1 1 but activity was rapidly lost outside this range. Optimal stability resulted at pH 7-8. The molecular weight of the serum component in comparison to CSA was then determined (Fig. 4). Serum was applied to a column and fractions collected. A portion of each fraction was assayed for CSA directly, and a second portion was incubated with MNC, the resultant conditioned medium being assayed for CSA. As seen in Figure 4 the serum factor enhancing CSA release from MNC appeared t o elute prior t o elution of endogenous serum CSA, indicating an apparently higher molecular weight. Macromolecular Synthesis Requirements for Stimulation
Cultures containing 1 X lo6 MNC/ml, with and without human serum, were cultured for periods up t o 72 hours and the amount of protein and DNA synthesis were periodically determined (Fig. 5). Addition of human serum had no effect on the amount of DNA synthesis. Protein synthesis was variably affected. Stimulation of protein synthesis was usually noted, but instances of no stimulation or little stimulation were also encountered. The reasons for this variability were not determined. In further studies, various inhibitors of macromolecular synthesis were added t o MNC cultures containing human serum t o determine the requirements for the serum-mediated stimulation of CSA production. DNA-RNA synthesis was inhibited with actinomycin D, protein synthesis with puromycin and cycloheximide, and mitosis inhibited with colchicine. The
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TABLE 111. Effect of Various Compounds on Serum-Mediated Release of CSAa Inhibitor added to MNC cultures
Levels of CSA in conditioned medium
Colchicine (0.5 pg/ml) Actinomycin D (1pg/ml) Cycloheximide (50 pg/ml) Puromycin (25 pg/ml) N-Ethylmaleimide (50 pg/ml) None
56+6 1 0 0 0 52 + - 4
"Inhibitors were added to MNC cultures containing 15% human serum and incubated for 96 hours at 37"C, after which time the conditioned medium was dialyzed and subsequently assayed for CSA. Samples treated with inhibitors and then dialyzed did not inhibit growth of bone marrow when stimulated with a known source of CSA.
"1 80
Fig. 6. Effect of puromycin on the serum-stimulated production of CSA and MNC. Puromycin (25 pg/ml) was added to series of MNC cultures containing 15% human serum and 2.5 X lo5 cells per milliliter at times from 0 t o 48 hours after start of incubation. The experiment was terminated 60 hours after start of incubation. Following dialysis, CSA in the resultant conditioned medium was assayed on human bone marrow and expressed as the mean colony count i. SE of triplicate plates. In cultures not treated with serum, puromycin would likewise inhibit CSA production. Refer to Figure 1 for the relative production of CSA with and without addition of human serum.
effect of N-ethylmaleimide, which affects export of proteins among other actions, was also examined. Table 111 shows that all inhibitors except colchicine inhibited the release of CSA. Parallel cultures that did not receive human serum were also treated with inhibitors and the same basic effect on production of CSA was noted (not shown). An experiment was done t o determine if there was a point after which no additional protein synthesis was required, which would be an indication that an event had occurred
Serum Modulation of Granulopoiesis
9
that had resulted in the activation of preformed CSA in the cells or serum. Human serum was added t o cultures at the start of incubation and puromycin was added at increasing intervals throughout the incubation period. Puromycin added prior to 48 hours after start of incubation resulted in decreased CSA release (Figure 6). DISCUSSI ON
These studies have demonstrated that human serum has the potential t o enhance CSA production by mononuclear cells in culture. This does not appear to be the result of potentiation of existing CSA as has been described previously [20,21] , since this effect is dependent on viable cells. The serum factor responsible for this effect does not appear t o be CSA itself, as evidenced by its apparent higher molecular weight. Active cellular processes are required for this serum-mediated stimulation of CSA release by MNC. CSA released in the absence of human serum seems to be subject t o the same controls. RNA and protein synthesis are clearly required for this effect, but mitotic processes do not appear necessary. Colchicine, which inhibits DNA synthesis as well as mitosis, has no effect on this phenomenon, while N-ethylmaleimide, cycloheximide, and puromycin all inhibited the noted effect of serum. Likewise, actinomycin D was effective in stopping the process. This was probably the result of inhibition of RNA and not DNA synthesis, since colchicine had no effect. These data might be interpreted as indicating that the serum factor resulted in increasing CSA production by stimulation at the transcriptional level. The exact mechanism, however, remains t o be determined and appears complex. It should be noted that in most instances, incubation of MNC cultures not receiving human serum eventually resulted in CSA levels reaching a final plateau equivalent t o those receiving human serum. This may be the result of a finite capacity of cells t o produce CSA that is simply reached more rapidly in the presence of human serum, but otherwise can be achieved at a slower rate through the utilization of components present in fetal calf serum. It is also possible that these results represent limitation of the in vitro conditions under which the cells were cultured and the CSA assayed. The importance and physiologic role of the serum factor described here has not been determined. Since MNC are constantly bathed in plasma in vivo, it may simply represent superior nutritive conditions. Another possibility is that the factor represents a humoral signal for CSA production and release by MNC. This view is supported by our findings that certain patients with neutropenia respond t o plasma infusions to produce normal levels of neutrophils [22] . Another possibility is that serum contains a precursor t o CSA that is subsequently activated by MNC. Data presented here would demand that such an activation require continued protein synthesis by MNC. The capacity of serum to potentiate existing sources of CSA, such as MNC-conditioned media, would be compatible with activation of a serum component by an enzyme produced by MNC [20,21]. Previous data from this laboratory have suggested a cooperative interaction between lymphocytes and monocytes t o produce increased amounts of CSA [13] . Serum may play a role in this interaction as well. ACKNOWLEDGMENTS
This study was supported by grants from the National Institutes of Health, National Cancer Institute (lROlCA11305-09 and CA05058-lo), American Cancer Society No. CH8G, National Institutes of Health Basic Oncology (CA13419), and by grant No. RR-51 from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health.
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