ORIGINAL ARTICLES

factor

PETER R. GALBRAITH,* MD, FRCP[C]; FRASER L. BAKER,t PH D. Lois J. COOKE;. DAVID C. MORLEY, JR;§ JANET SINcLAIR;¶ SUSAN PARKER;** DON BRISBINtt

The purpose of this study was to identify factors that influence the production of colony-stimulating factor by leukocytes of humans. The use of nonadherent light-density bone marrow cells in semisolid agar cultures to assay the concentrations of colony-stimulating factor in the supernatant of monocyte and mononuclear leukocyte cultures made it possible to distinguish between colony-stimulating factor, which stimulates colony-forming cells directly, and monocyte-dependent stimulating activity, which acts indirectly, by increasing the monocyte production of colonystimulating factor. Colony-stimulating factor was not detectable in the cytosol of monocytes; that detected in culture must, therefore, have been newly synthesized. Synthesis was enhanced independently by heat-inactivated human serum and by semipurified serum fractions enriched with monocyte-dependent stimulating activity. The kinetics of the production of colony-stimulating factor in the presence and absence of monocyte-dependent stimulating activity indicated that the latter facilitated monocyte production of the former. Factors released from neutrophils were shown to reduce the production of colony-stimulating factor and the proliferation of colony-forming cells and thus may provide a feedback control mechanism limiting the proliferation of neutrophils.

sous dependance monocytaire. La cinetique de production du facteur stimulant Ia formation de colonies en presence et en l'absence d'activite stimulatrice sous dependance monocytaire a indiqu6 que celui-ci facilitait Ia production de celui-la par les monocytes. On a demontre que des facteurs lib6res par les neutrophiles ont reduit Ia production du facteur stimulant Ia formation de colonies et Ia proliferation des cellules formant des colonies; ainsi ces facteurs peuvent offrir un mecanisme de retroaction limitant Ia proliferation des neutrophiles.

Colony-stimulating factor is essential for growth and proliferation of granulocyte- and macrophage-committed colony-forming cells in culture.1 It may be a granulopoietin"3 analogous to erythropoietin. Information concerning its origin, production and action is therefore of interest, particularly since it is not usually detectable in the serum of humans.4 If the term colony-stimulating factor is restricted to factors that directly stimulate colony-forming cells to proliferate, two other classes of factors that enhance clonal growth can be distinguished: factors that enhance the production of colony-stimulating factor by and thus may be termed as having monomonocytes4 Le but de cette etude etait d'identifier les facteurs qui cyte-dependent stimulating activity, and nutritional influencent Ia production du facteur stimulant Ia formation de colonies par les leucocytes des humains. L'emploi de factors that permit clonal growth in the presence of cellules myeloides non adherentes de faible densite culti- colony-stimulating factor.4 The last two classes of vees en gelose semisolide pour mesurer Ia concentration factors are present in the serum of humans; the nu-

du facteur stimulant Ia formation de colonies dans le surnageant de cultures de monocytes et de leucocytes mononucleaires a rendu possible Ia distinction entre le facteur stimulant Ia formation de colonies, qui stimule directement les cellules formant des colonies, et l'activite stimulatrice sous dependance monocytaire, qui agit indirectement par I'augmentation de Ia production du facteur stimulant Ia formation de colonies par les monocytes. Le facteur stimulant Ia formation de colonies n'etait pas decelable dans le cytosol des monocytes; donc il est evident que celui decele en culture cellulaire a ete nouvellement synthetise. La synthese a ete augmentee de fa9on independante par le serum humain inactive a Ia chaleur et par des fractions seriques semipurifiees enrichies d'une activite stimulatrice 172

CMA JOURNAL/JULY 21, 1979/VOL. 121

From

the

department

of

medicine,

Queen's

University,

Kingston, Ont. * Professor of medicine tResearch associate TSenior technician, division of hematology §Third-year medical student, Queen's University ¶Final-year medical student, University of Aberdeen * *5econdyear medical student, Queen's University t tThird-year medical student, Queen's University Reprint requests to: Dr. Peter R. Galbraith, 102 Stuart St., Ste. 113, Kingston, Ont. K7L 3N6

tritional factors remain after the serum has been inactivated by being heated at 580C for 2 hours to destroy the factors having monocyte-dependent stimulating activity and lipoprotein inhibitor.4 Serum is an es.ential ingredient of any culture medium used to support clonal growth, and it facilitates the production of colony-stimulating factor. Heat-inactivated human serum is required in relatively high concentrations (30%) to provide the nutritional factors in sufficient quantity to avoid limiting of the growth of colony-forming cells.4 Although the production of colony-stimulating factor is enhanced by both heat-inactivated serum and monocyte-dependent stimulating activity, the effects are not interdependent but, rather, appear to be additive. In the absence of heatinactivated serum, minute quantities of semipurified serum fractions enriched with monocyte-dependent stimulating activity (0.1 % to 1.0%) increase the monocytes' release of colony-stimulating factor.5 In the study described in this paper we investigated the rate of release of colony-stimulating factor by blood mononuclear leukocytes incubated under different culture conditions. Methods

Culture medium CMRL 1066 tissue culture medium (Grand Island (New York] Biological Company), supplemented with 2% of lOOx nonessential amino acids, 1% of lOOx sodium pyruvate, 1% of 200 mM l-glutamine, 0.05% of 100 x minimum essential medium vitamins, 0.1% of 50 x minimum essential medium amino acids and 0.2% of vitamin B12, 1000 mEq/ml, was used. The medium was supplemented with 30% human serum heated at 58 0C for 150 minutes. Fetal calf serum was excluded throughout. Culture system The cell culture system consisted of a 1 -ml layer of 0.3% agar (Difco Laboratories, Detroit) in plastic Petri dishes 35 mm in diameter (Falcon Plastic Company, Oxnard, California) in which were incorporated target colony-forming cells and the colony-stimulating factor to be assayed. The cultures were incubated for 7 days at 370C in a humidified atmosphere containing 5% carbon dioxide. Colony-forming cells Bone marrow was aspirated from the manubrium sterni of informed healthy male volunteers aged 21 to 28 years; heparin or citrate-phosphate-dextrose solution was used as the anticoagulant.6 After centrifugation of the marrow at 200 x g for 10 minutes the buffy coat was drawn off with a Pasteur pipette and the cells were washed three times with culture medium. The suspension of nucleated cells was then separated by a single-density cut in bovine serum albumin or Ficoll® (specific gravity 1.070 g/ml) (Pharmacia Fine Chemicals, Uppsala, Sweden) to remove the neutrophils.4''7 The light-density fraction (density less than 1.070 g/ml) was recovered and subjected to adherence separation to remove the monocytes,4'8

and the nonadherent light-density marrow cells were thus enriched with colony-forming cells and were seeded in culture (1 x 10. cells per 1-ml culture). Mononuclear leukocytes Venous blood, drawn from informed healthy volunteers into Vacutainers® containing heparin (Becton, Dickinson and Company, Mississauga, Ont.), was allowed to clot at room temperature for 2 hours, then the leukocyte-rich plasma was drawn off with a Pasteur pipette. The leukocytes were centrifuged at 900 x g for 10 minutes, washed three times with culture medium and depleted of neutrophils by the density separation procedure. The light-density fraction, composed of 28 ± 12% monocytes, 71 ± 12% lymphocytes and 1 ± 1 % basophils,t was used to produce colonystimulating factor under a variety of conditions during incubation in culture medium or serum to determine the factors influencing this production. The colonystimulating factor thus produced was then assayed through its ability to stimulate colony formation in cultures of nonadherent light-density bone marrow cells. Adherent mononuclear leukocytes Adherent cells (more than 95% monocytes) were obtained from the light-density fraction of blood mononuclear leukocytes by being allowed to adhere to plastic Petri dishes for 2 to 2½ hours. The culture medium, containing nonadherent cells, was decanted and replaced with fresh culture medium. Colony-stimulating factor This was prepared as specified by incubating blood mononuclear leukocytes or the adherent fraction in culture medium containing serum or heat-treated serum in specified concentrations. Cell suspensions were incubated at 370C in Petri dishes 100 mm in diameter for specified periods, then the supernatant was decanted, filtered (with a Millipore® filter having pores 0.45 .m in diameter), frozen, stored at -200C and later assayed. Collection of serum Venous blood collected in Vacutainers® was allowed to clot at room temperature for 2 hours, then the clot was spun down and the serum drawn off with a Pasteur pipette, filtered (with the same size of Millipore® filter), frozen and stored at -200C until used. Blood was obtained from healthy volunteers and from patients with infantile genetic agranulocytosis of Kostman, leukemia or Felty's syndrome who had severe neutropenia (a neutrophil count of less than 0.06 x 10./l). Neutropenic serum was selected to minimize contamination by inhibitors released from neutrophils.5'6 None of the patients had evidence of sepsis due to gram-negative organisms. Semipurified serum fractions enriched with monocyte-dependent stimulating activity To obtain such fractions normal human serum was fractionated by Sephadex® G200 chromatography (Pharmacia Fine Chemicals).4 Pooled frozen fractions CMA JOURNAL/JULY 21, 1979/VOL. 121

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that had been shown to contain monocyte-dependent to cultures with and without added blood mononuclear stimulating activity but not colony-stimulating factor4 leukocytes (2 x 10. per 1-ml culture). * To determine the rate of production of colonywere thawed and used to investigate the quantitative relation between this activity and the production of stimulating factor by monocytes, blood mononuclear leukocyte fractions were incubated in culture medium colony-stimulating factor. containing 30% neutropenic serum (containing monoInhibitor released from neutrophils cyte-dependent stimulating activity) and heat-inactivNeutrophils (2 x 106/ml) obtained from the non- ated serum (lacking such activity). Aliquots were adherent high-density fraction (density greater than harvested at specified intervals, filtered (through the 1.070 g/ml) of normal blood9 were used to provide same size of Millipore® filter), immediately frozen at inhibitor in a series of experiments. The cells were -200C and later assayed. * To determine the effects of different human used either intact or in the form of a lysate (prepared by freezing and thawing three times in liquid nitrogen). serum on the rate of production of colony-stimulating factor. mononuclear leukocytes (2 x 1 06/ml) were Colony counts incubated in aliquots of 100% neutropenic serum Colonies (aggregates of 20 or more cells) were (neutrophil count less than 0.06 x 10./l), normal counted by means of an inverted transmission micro- serum, and a 50:50 mixture of the two. The aliquots scope (x 40) on triplicate plates after 7 days of in- were harvested after 0, ½, 1, 2, 4 and 8 hours of cubation. The counts were averaged and expressed as incubation, filtered (through the same size of Millithe mean . 1 standard error of the mean (SEM). The pore® filter), immediately frozen and subsequently number of clusters (3 to 19 cells each) was not assayed. recorded. * To determine the effect on the production of colony-stimulating factor of inhibitors released from Rationale neutrophils, a lysate of neutrophils or intact neutroThe experiments were based upon our previous phils was added to aliquots of 100% serum prior to observation' that normal nonadherent light-density conditioning by mononuclear leukocytes, as already bone marrow cells (1 x 10'/ml) suspended in culture described. Since added inhibitor affects the response medium supplemented with 30% heat-inactivated of colony-forming cells in cultures stimulated by serum do not form colonies unless stimulated to do so colony-stimulating factor,6 the net effects of inhibitor by cell-free colony-stimulating factor or by the addi- on the production of colony-stimulating factor were tion of monocytes. This concentration of heat-inac- determined by comparing the effects of inhibitor added tivated serum provides nutritional (or permissive) fac- to aliquots of mononuclear-leukocyte-conditioned setors essential for colony growth in quantities that rum at the beginning of incubation and the effects of do not limit the rate of growth, and the serum is free inhibitor added 5 minutes prior to harvesting of the of inhibitor and of monocyte-dependent stimulating aliquots. activity;4 thus the milieu is appropriate for investigating the effects of different factors on the production of Results colony-stimulating factor. Cultures respond to preparations of this factor with a dose-related increase in Production of colony-stimulating factor by the adherent fraction of blood mononuclear the number of colonies. leukocytes Experimental design Lysed adherent mononuclear leukocytes (more than In all the following experiments colony-stimulating 95% monocytes) added to nonadherent light-density factor was produced by incubating blood mononuclear bone marrow cell cultures under appropriate conditions leukocytes or the adherent fraction under different did not stimulate colony or cluster formation (Fig. 1). conditions, and the amount produced was assessed This suggests that monocytes do not contain detectable from the ability of the supernatant to stimulate colony quantities of preformed colony-stimulating factor in formation by 1 x 10. nonadherent light-density bone their cytosol. marrow cells serving as the source of target colonyThe rate of production of colony-stimulating factor forming cells. In each instance three or more experi- by adherent mononuclear leukocytes is shown in Fig. ments of similar design yielded similar results; there- 2. During a 24-hour period of incubation the producfore, representative data are presented in this report. tion of this factor increased linearly with time and was The following experiments were done: greater when the cells were incubated in the presence * To determine if the monocytes contained de- of neutropenic serum (containing monocyte-dependent tectable amounts of colony-stimulating factor in a stimulating activity) than when they were incubated preformed state, monocytes lysed by freezing and in the presence of heat-inactivated serum (which lacks thawing were added to cultures of colony-forming such activity). The nonadherent mononuclear leukocells. Control cultures contained intact monocytes cyte fraction (not shown) did not produce significant (1 x 10. per 1-ml culture). amounts of colony-stimulating factor during this pe* To determine the effect of monocyte-dependent nod. Therefore, light-density blood mononuclear leukostimulating activity in semipurified serum fractions cytes, which are more readily divided into aliquots of on the production of colony-stimulating factor, these appropriate size, were used to provide monocytes in fractions were added in concentrations of 0 to 1 % the remainder of the experiments. 174 GMA JOURNAL/JULY 21, 1979/VOL. 121

Intact

I. 5

% Serum

Time (hours)

MNL per ml of medium (x gQ6)

the production of colony-stimulating factor by mononuclear leukocytes and the response of colony-forming cells to colony-stimulating factor. Discussion

of colony-stimulating factor by monocytes. Thus, we found that the concentrations of colony-stimulating factor increased linearly with the time of incubation (Figs. 2 and 4). We have shown previously that the presence of heat-inactivated serum is not required for observation of the quantitative relation between added monocyte-dependent stimulating activity (0.1 % to 1.0%) and the content of colony-stimulating factor in supernatants of monocyte cultures.' The effects of heat-inactivated serum and monocyte-dependent stimulating activity appear to be additive rather than interdependent. Heat-inactivated serum may simply enhance monocyte survival. Although not normally detected in human serum,4 colony-stimulating factor appeared briefly when volunteers were given an injection of purified bacterial endotoxin.'7 The concentrations peaked in 1 hour; thereafter the factor was very quickly cleared from the blood. In vivo mechanisms thus exist to provide colony-stimulating factor without delay. Nearly comparable rates of production by monocytes have now been observed in vitro- (Fig. 6). Further studies are required to elucidate the mechanisms involved. Serum stimulatory activity is thought to be due to a glycoprotein with a molecular weight slightly less than that of albumin.'8 Therefore, the possibility that monocyte-dependent stimulating activity is due to a precursor of colony-stimulating factor that is efficiently processed by monocytes needs to be considered. It is not surprising that colony-stimulating factor is not detectable in human serum since it is so rapidly cleared from the blood.'7 However, its production may

The term colony-stimulating factor is used to describe a family of glycoproteins that have in common the ability to stimulate proliferation of colony-forming cells in culture.10 This family is heterogeneous10 with respect to molecular weight,'0-" tissue of origin'3"4 and specificity." Separate factors appear to stimulate the growth of eosinophilic, granulocytic and macrophage colonies. Blood leukocytes are the most accessible source of cells used in the preparation of colony-stimulating factor 16,17 and the central role of the monocyte in the production of colony-stimulating factor is now accepted.'8-'0 Lymphocytes also participate, more so during in vitro immunologic reactions and with stimulation by mitogens."-'4 Lymphocyte participation involves de novo protein synthesis that is not dependent upon the synthesis of deoxyribonucleic acid.'4 It is not clear if stimulatory factors produced by lymphocytes act on colony-forming cells directly or are monocyte-dependent. We have been unable to make this distinction with confidence because of the difficulty of excluding monocytic contamination in preparations of nonadherent lymphocytes or nonadherent light-density bone marrow cells serving as the source of colony-forming cells. Whichever is the case, stimulatory factors are detectable in the supernatant of lymphocyte cultures after 48 hours.'4 Presumably this is the time required for the induction of protein synthesis. In preliminary studies we have not detected the production of colony-stimulating factor by unstimulated nonadherent lymphocytes in cultures maintained for 48 hours or less, but these cells do enhance production in cultures maintained for 7 days (unpublished observations). Liver" and pulmonary alveolar macrophages'6 produce colony-stimulating factor. Thus, in studies of the production of colony-stimulating factor by different tissues and organs the relative contributions of organspecific cells and their macrophages are uncertain. Our experiments have shown that colony-stimulating factor is not detectable in the cytosol of monocytes (Fig. 1), but, rather, appears to be newly synthesized. The rate of synthesis varies greatly under different culture conditions and is influenced by the presence of serum factors. In the absence of serum, little colonystimulating factor is released (Fig. 5), and we cannot exclude the possibility that it is simply released from the cell surface, since "prepackaged" membrane-bound colony-stimulating factor has been described.'7 The rate of synthesis was greatest in our experiments when mononuclear leukocytes were incubated in 100% serum obtained from neutropenic patients (Fig. 6). Two separate serum activities appear to influence the production of colony-stimulating factors. Nutritional factors in concentrations that provide adequate FIG. 7-Pooled results from seven experiments on effect support for the growth of colony-forming cells (30% of inhibitor on rate of production of colony-stimulating heat-inactivated serum) also enhance the production factor. CMA JOURNAL/JULY 21, 1979/VOL. 121

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also be controlled. Rates of production such as we have observed in vitro may not occur spontaneously in vivo. For example, monocytes adhere tightly to the surface of Petri dishes, and this appears to enhance the production of colony-stimulating factor (unpublished observations). Other factors may limit the production of colony-stimulating factor. Lactoferrin may be important in this regard.29 It is contained in neutrophilic granules and is slowly released when neutrophils are incubated in culture medium. The effects of neutrophils on the production of colony-stimulating factor, shown in Figs. 6 and 7 and noted at 8 hours and earlier, appeared to be equivalent in magnitude to the inhibition of the response of colony-forming cells. Neutrophils also release an inhibitor of low molecular weight6 that affects the response of colony-forming cells to colony-stimulating factor. A third inhibitor is present in the lipoprotein fraction of normal human serum.5 Its concentration is a function of the circulating neutrophil count and, as with the inhibitor of low molecular weight, it influences the response of colony-forming cells to colony-stimulating factor. It does not appear to affect the production of colonystimulating factor. The extent to which the production of colonystimulating factor and the growth of colony-forming cells are physiologically relevant remains to be determined. We do not suggest that any of the activities we have observed reflect the activities of singular molecular species; in fact, this is almost certainly not the case. The effects observed o'ccurred under conditions very different from those that exist in vivo, and there are many gaps in our understanding. There is little doubt that lymphocyte subpopulations play a role in the production of colony-stimulating factor in vitro, but the extent of their contribution to the control of myelopoiesis is not yet clear. Finally, factors that influence the levels of monocytedependent stimulating activity in the serum have not been identified. Conditioned neutropenic serum was found to be superior to conditioned normal serum in its ability to stimulate clonal growth, but in view of the other variables this cannot be taken as evidence that the serum levels of this activity are influenced by a feedback control mechanism involving neutrophils. This study was supported by grant 309 from the Ontario Cancer Treatment and Research Foundation. References 1. METCALF D: Studies on colony formation in vitro by mouse bone marrow cells. II. Action of colony stimulating factor. J Cell Physiol 76: 89, 1970 2. MOORE MAS, SPITZER G, METCALF D, et al: Monocyte production of colony stimulating factor in familial cyclic neutropenia. Br J Haematol 27: 47, 1974 3. BERAN M: Serum levels of colony stimulating factor(s) and the growth of bone marrow cells in diffusion chambers. Cell Tissue Kinet 8: 561, 1975 4. BAKER FL, GALBRAITH PR: Nutritional and regulatory roles of human serum in cultures of human granulopoietic cells. Blood 52: 241, 1978 5. Idem: Mechanism of action of serum factors that regulate granulopoiesis in vitro - possible physiological role of serum-inhibiting activity. Blood 53: 304, 1979 178 CMA JOURNAL/JULY 21, 1979/VOL. 121

6. GALBRAITH PR, Coolul U, BAKER FL: Action of inhibitor released from neutrophils and leukemic blast cells. Can Med Assoc J 120: 545, 1979 7. BAKER FL, BROXMEYER HE, GALBRAITH PR: Control of

granulopoiesis in man. III. Inhibition of colony formation by dense leukocytes. J Cell Physiol 86: 337, 1975 8. MESSNER HA, TILL JE, MCCULLOCH EA: Interacting cell populations affecting granulopoietic colony formation by normal and leukemic human marrow cells. Blood 42: 701, 1973 9. BROXMEYER HE, BAKER FL, GALBRAITH PR: In vitro regulation of granulopoiesis in human leukemia: application of an assay for colony-inhibiting cells. Blood 47: 389, 1976 10. MELCALF D: The colony stimulating factor (CSF). Aust J Exp Biol Med Sci 50: 547, 1972 11. PRICE GB, SENN JS, MCCULLOCH EA, et al: The isolation and properties of granulocytic colony-stimulating activities from medium conditioned by human peripheral leukocytes. Biochem J 148: 209, 1975 12. Idem: Heterogeneity of molecules with low molecular weight isolated from media conditioned by human leukocytes and capable of stimulating colony formation by human granulopojetic progenitor cells. J Cell Physiol 84: 383, 1974 13. SHERIDAN JW, STANLEY ER: Tissue sources of bone marrow colony stimulating factor. J Cell Physiol 78: 451, 1971 14. SHERIDAN JW, METCALF D: Studies on the bone marrow colony stimulating factor (CSF): relation of tissue CSF to serum CSF. J Cell Physiol 80: 129, 1972 15. RUSCETTI FW, CYPESS RH, CHERVENICK PA: Specific release of neutrophilic- and eosinophilic-stimulating factors from sensitized lymphocytes. Blood 47: 757, 1976 16. CHERVENICK PA, BooGs DR: Bone marrow colonies: stimulation in vitro by supernatant from incubated human blood cells. Science 169: 691, 1970 17. ISCOvE NN, SENN JS, TILL JE, et al: Colony formation by normal and leukemic human marrow cells in culture: effect of conditioned medium from human leukocytes. Blood 37: 1, 1971 18. MOORE MAS, WILLIAMS N: Characteristics of the colony stimulating cell, in In Vitro Culture of Hemopoietic Cells, VAN BEKKUM DW, DICKE KA (eds), Radiobiological Institute TNO, Rijswijk, Holland, 1972, p 215 19. CHERVENICK PA, LOBUOLIO AF: Human blood monocytes: stimulators of granulocyte and mononuclear colony formation in vitro. Science 178: 164, 1972 20. GOLDE DW, CLINE MJ: Identification of the colonystimulating cell in human peripheral blood. J Clin Invest 51: 2981, 1972 21. MCNEILL TA: Marrow colony stimulating factor release during immunological reactions. Nature 244: 175, 1973 22. CLINE MJ, GOLDE DW: Production of colony-stimulating activity by human lymphocytes. Nature 248: 703, 1974 23. CERNY J: Stimulation of bone marrow haemopoietic stem cells by a factor from activated T cells. Nature 249: 63, 1974 24. RUSCETTI FW, CHERVENICK PA: Regulation of the release of colony-stimulating activity from mitogen-stimulated lymphocytes. J Immunol 144: 1513, 1975 25. JOYCE RA, CHERVENICK PA: Stimulation of granulopoiesis by liver macrophages. J Lab Clin Med 86: 112, 1975 26. GOLDE DW, FINLEY TN, CLINE MJ: Production of colony-stimulating factor by human macrophages. Lancet 2: 1397, 1972 27. PRICE GB, MCCULLOCH EA, TILL JE: Cell membranes as sources of granulocyte colony stimulating activities. Exp Hernatol 3: 227, 1975 28. METCALF D,

MACDONALD HR, CHESTER HM:

Serum

potentiation of granulocyte and macrophage colony formation in vitro. Ibid, p 261 29. BROXMEYER HE, SMITHYMAN A, EGER RR, et al: Identification of lactoferrin as the granulocyte-derived inhibitol of colony-stimulating activity production. Exp Med 148: 1052, 1978