1973 42: 341-348

G. B. Price, E. A. McCulloch and J. E. Till

ActivityA New Human Low Molecular Weight Granulocyte Colony Stimulating

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Blood, Vol. 42. No. 3 (September). 1973 341

A New Human Low Molecular Weight

Granulocyte Colony Stimulating Activity

By G. B. Price. E. A. McCulloch. and J. E. Till

Fractionation of conditioned medium de- gel filtration on Sephadex G-25. The lowrived from cultures of human peripheral molecular weight colony-stimulating ac-blood leukocytes by uftraflltration yielded tivity (LMW-CSA) was trypsin-sensitive,material of low molecular weight, capable and extractable into chloroform or diethylof stimulating the formation of granulo- ether. Unlike unfractionated leukocyte-poietic colonies by human marrow cells in conditioned medium, the LMW-CSAculture. The molecular weight of the active showed no colony-stimulating activitymaterial was found to be less than that of when tested on mouse, rather than human,vitamin B12 (1330 mol wt) on the basis of marrow cell cultures.

M ATERIALS OBTAINED FROM several human sources, includingurine, ı and conditioned medium from cultures of peripheral leuko-

cytes,2’3 spleen,4 or embryo kidney,5 exhibit “colony stimulating activity,”in that they stimulate colony formation in culture by human or mouse granulo-poietic progenitor cells. In a preliminary investigation of the CSA present inmedia from cultures ofhuman peripheral leukocytes, Austin6detected a macro-molecule with a molecular weight of approximately 35,000, as determined bygel filtration and velocity sedimentation. This high molecular weight CSA hasbeen found to promote colony formation by either mouse or human marrowcells.

We report here the presence, in human leukocyte-conditioned media, of asecond CSA which exhibits several novel properties. It is of low molecularweight, is extractable with hydrophobic solvents, and stimulates colony forma-tion by human but not mouse granulopoietic progenitor cells.

MATERIALS AND METHODS

Bone Marrow

Human bone marrow cells were obtained from three patients during the course of investigationof nonmaIi�nant hematological diseases.. Marrow cell suspensions were prepared as described

previously.

Preparation of Conditioned Medium

Conditioned medium was prepared from suspension cultures of peripheral leukocytes from

normal and polycythemic human heparinized blood. Cells were suspended in serum-free alpha

From the The Ontario Cancer Institute and Departments of Medicine and Medical Biophysics.

University of Toronto, Toronto, Ontario. Canada.Submitted November 20, 1972; revised January 29. /973; accepted February 20, 1973.Supported by the National Cancer Institute of Canada. the Medical Research Council, Canada.

Grant MT-1420, and the Ontario Cancer Treatment and Research Foundation, Grant 236.0. B. Price, Ph.D.: Post-doctoral Fellow of The Damon Runyon Memorial Fund, Ontario Cancer

Institute, Toronto, Canada. E. A. McCulloch, M.D., �.R.C.P. (C): Department of Medicine,University of Toronto, and the Ontario Cancer Institute, Toronto, Canada. J. E. Till, Ph.D.: De-partment of Medical Biophysics, University of Toronto, and the Ontario Cancer Institute, Toronto,Canada.

©1973 by Grune & Stratton. Inc.

342 PRICE, MC CULLOCH. AND TILL

7 at concentrations of 5 x 106 nucleated cells/ml. Suspension cultures were incu-bated 7 days at 37’C in a humidified 10% C02-90% air atmosphere; the supernatant fluid andfloating cells were removed from their tissue culture flasks, and cells or debris removed bycentrifugation at 400 g for 20 mm. All conditioned medium was filter-sterilized through 0.45-itgrid membrane units (Nalgene, Rochester, N.Y.) and stored at 4’C.

Assayfor Colony Stimulating A ctivity

The assay for colony stimulating activity (CSA) was similar to that described by Worton et al.8for mouse cells using a single layer methyl cellulose culture system; it consisted of normal humanbone marrow cells, immobilized in 0.8% (wt/wt) methyl cellulose in alpha medium supplementedwith 20% fetal calf serum (Gibco, Grand Island, N.Y.). Varying sources of CSA, at a concentra-tion of 20% (v/v) unless otherwise specified, were added to the above mixture and incubated at37’C for 14 days in a humidified 10% C02-90% air atmosphere. The number of colonies con-taming more than 20 cells was counted after 14 days incubation. Mouse bone marrow cultureswere plated under similar conditions and number of colonies containing more than 50 cells was

counted as previously described.9

Separation by Adherence

In order to obtain populations of human marrow cells showing a low autostimulated back.ground, a separation by adherence was performed. The method was that of ı modifiedby Messner et al)’ Adherent cells were separated from marrow suspensions by incubating thesuspensions in glass petri dishes for ı hr, then decanting the nonadherent cells. The adher-ence procedure was repeated for the decanted cells for 2 4 hr and the resultant populationof nonadherent cells was tested for colony-forming ability in the usual way.

Characterization Procedure

Enzyme treatment with trypsin (crystalline) (Worthington Biochemical Corp., Freehold, N.J.)was carried out in serum-free alpha medium at pH 7, 37’C for 4 hr at a concentration of 100 agtrypsin/mI of LMW-CSA. Trypsin was inhibited by a 200-fold molar excess of phenylmethanesul-fonyl fluoride (PMSF). 12 PMSF was also added at the same concentration to a preparation ofunfractionated CSA used as a standard in these studies, to determine if it caused any inhibitionofcolony growth.

Chloroform-methanol extraction was carried out at room temperature by mixing equal volumesof conditioned medium and a solution of chloroform methanol in the ratio of 2:1. The aqueousphase was aerated until all traces of methanol were removed; the chloroform phase was aeratedto dryness and then resuspended in alpha medium. Both phases were filter-sterilized before testingfor CSA. Diethyl ether extraction was carried out in a similar manner.

Fractionation Procedures

Gel filtration was carried out using Sephadex 0-25 (Pharmacia, Montreal, Quebec) in 0.15 M

Tris-HC1, pH 6.7 in a 2.5 x 45-cm column using downward flow and a flow rate of 15 to 20 mlper hr. Fractions of 5 ml were collected and filtered through a O.45-ı Millipore filter beforebeing tested for CSA. Insulin (Connaught Medical Research Labs., Toronto, Ontario) andvitamin B,2 (Steinlab, Toronto, Ontario) were used as markers in gel filtration experiments.The optical density of these markers at 280 and 361 nm, respectively, was used to indicate theirposition since there was virtually no contribution of conditioned medium to optical densitiesmeasured at those wavelengths.

Ultrafiltration membranes (Amicon, Lexington, Mass.) having molecular cut-offs at 20,000and 10,000 mol wt were used to fractionate LMW-CSA.

RESULTS

Low Molecular Weight Colony Stimulating A ctivity

Crude CSA from human peripheral leukocytes, prepared as described in the

Materials and Methods, was tested to see if it contained a low molecular weight

,�11)

-ı

cC.

0

,�#{149}iJ)

‘t�SC..

q)0.C,)

.Q)

Fig. 1. (A) Colony formation by various numbers of normal human marrow cells in the pres-ence of 20% (v/v) CSA. A, LMW-CSA1, <10,000 mol wt. v, LMW-CSA2, <20,000 mol wt.

#{149},standard CSA from unfractionated leukocyte-conditioned medium. A, v, LMW-CSA1 andLMW-CSA2 after dialysis against 0.15 M NaCI. o, no added CSA. (B) Sensitivity of LMW-CSA

to trypsin digestion, as measured on the same sample of normal human marrow as in panel A. 0,

trypsin-digested LMW-CSA2, assayed in the presence of trypsin inhibitor. X, standard CSA as-sayed in the presence of the same concentration of trypsin inhibitor. The solid lines are taken from

panel A. Each point represents the mean colony count from three or four dishes. Error bars are

standard errors.

Cells per dish 12044

GRAN U LOCYTE COLONY STIMULATING ACTIVITY 343

colony-stimulating activity (LMW-CSA) in addition to CSA of high molecularweight.6To achieve an initial separation of LMW-CSA from the high molecu-lar weight CSA, filter-sterilized conditioned medium was passed through mem-branes with molecular weight cut-offs near 20,000 or 10,000 molecular weight.High molecular weight CSA was retained by the membrane. A representativeexperiment demonstrating the ability ofcrude LMW-CSA obtained in this wayto stimulate colony formation by normal human marrow cells is shown in Fig.IA (closed triangles). Cultures containing LMW-CSA yielded significantlymore colonies (p<< 0.01) than control cultures containing no added CSA, butless than cultures containing a preparation of unfractionated leukocyte-condi-tioned medium used as a standard in these experiments (closed circles, Fig. 1A).The colonies were predominantly granulocytic, with occasional macrophagecolonies; the distribution of colony types was similar regardless of whetherstimulation was with standard or LMW-CSA. However, the colonies formed inthe presence of the standard unfractionated CSA were often somewhat largerthan those obtained with LMW-CSA, consisting in the experiment of Fig. 1, ofa maximum of about IOı cells per colony in the former case, and about 5 x 102

cells per colony in the latter. The activity of LMW-CSA was lost after dialysisagainst 0.15 M saline (open triangles, Fig. 1A), indicating either molecules withlow molecular weight, or inactivation of the LMW-CSA during dialysis. Equiv-alent findings have been obtained repeatedly although normally only one or twocell concentrations were tested.

0.7

0.6 -...ı

0.5 Li0.4 ı

(\Jfr)

‘c50.2

0.l

1620 30 40 65

Fraction number

Fig. 2. Gel filtration of

crude LMW-CSA on Sephadex

G-25. 0, LMW-CSA. 0, insulin.#{149},vitamin B12. The colony-forming activity of LMW-CSAis expressed relative to theactivity detected on 5 X i04human marrow cells prior togel filtration.

�1i�

.I�5

0.

.4.)

,�,C,)

.ı

4)0.C,)

.4)0

20542.5

Cells per dish

Fig. 3. (A) extraction of LMW-CSA from the aqueous phase by chloroform. A, chloroform extract.v , aqueous phase. A, crude LMW-CSA.., standard unfractionated CSA. 0, control, no added CSA.(B) Extraction of LMW-CSA by diethyl ether. X. ether extract. +, aqueous phase. Other symbolsas in panel A. (Note the change in ordinate scale compared with panel A-see text.)

344 PRICE, MC CULLOCH. AND TILL

-..‘

0

c)

-4

Preliminary Characterization of LM W-CSA

To obtain a better estimate of molecular weight, crude LMW-CSA was sub-jected to gel filtration on Sephadex G-25, using, as markers, insulin (approx-imately 6000 mol wt) and vitamin B,2 (1330 mol wt). LMW-CSA was repro-ducibly eluted from the gel after both markers, indicating a molecular weightfor LMW-CSA of less than 1330 (Fig. 2).

Trypsin digestion of crude LMW-CSA was carried out as described in Ma-terials and Methods, and trypsin inhibitor was added prior to the assay forcolony formation, in order to prevent inhibition of colony formation by thepresence of trypsin in the assay dishes. The results obtained in one of severalexperiments (Fig. 1B, squares) indicate that trypsin digestion destroyed thecolony-stimulating activity of the low molecular weight material. No significantreduction of colony stimulation by the standard CSA preparation was observedat the concentration of trypsin inhibitor used in these experiments (Fig. lB,

GRANULOCYTE COLONY STIMULATING ACTIVITY 345

crosses). Also, the numbers of colonies formed in the presence of trypsindigestion products of LMW-CSA were similar to those found in their absence(Fig. I B, squares). Thus, it seems unlikely that trypsin digestion products in theLMW-CSA exerted inhibitory effects; rather, the observed loss of stimulationmay be attributed to enzymic inactivation of LMW-CSA.

LMW-CSA was found to be extractable from the aqueous phase into chloro-

form (Fig. 3A) or diethyl ether (Fig. 3B). Several repetitions of the experimentshown in Fig. 3 yielded similar results; in no case was significant residualactivity detected in the aqueous phase.

It should be noted that the number of colonies per dish in the experiment

of Fig. 3B was approximately fourfold less than that observed in the experimentof Fig. 3A. Two different human marrow cell suspensions were used in theseparticular experiments, and the differences in colony-forming efficiency weretypical of those observed between different marrow samples.

Test of Purified L M W-CA S on Nonadherent Human Marrow Cells

Interpretation of experiments using whole marrow cell suspensions wascomplicated by high levels of background colony-formation observed withoutadded CSA and presumed to be the result of endogenously-produced CSA.Nonadherent cell populations have greatly reduced levels of background colonyformation and are more suitable for studies of exogenous CSA.” Table 1 givesthe results of an experiment in which LMW-CSA, purified by extracting anultrafiltered preparation with chloroform, was tested on two concentrations ofnonadherent human marrow cells. It is evident that stimulation was observedunder these conditions.

Test of LM W-CSA on Mouse Marrow Cultures

Unfractionated peripheral leukocyte conditioned medium used as a standardsource of CSA stimulated colony formation by mouse marrow cells (Table 2,Experiments 3,5,6). Austin6 found that the material with a molecular weight ofapproximately 35,000 isolated from leukocyte-conditioned medium also stimu-lated colony formation by mouse marrow cells. In contrast, LMW-CSA showedno detectable activity on mouse marrow cells (Table 2). Included in the tableare the results of an experiment in which LMW-CSA was mixed with mouseL-cell CSA (Experiment 4) and with standard CSA (Experiments 5 and 6). Noinhibitory or additive effects were observed.

Table 1. Test of Purified LMW -CSA on Nonadherent Cells

SourceofCSA PerCentCSA

Colonies per Dish’

lxlO5t 2x105t

Control

Std.CSA

LMW-CSA

None

20

5

0. 0. 0. 0 6. 7. 3

122. 124 247.213. 166

40.54 81.93

Counting criterion was 20 cells per colony. Colony size varied from 20 cells to approximately 40

cells per colony in this experiment.

t Number of nucleated nonadherent human marrow cells per dish.

346 PRICE. MC CULLOCH. AND TILL

Table 2. Test for Activity of LMW-CSA on Mouse Marrow Cells

Source ofCSA

%CSA

Colonies per dish -

Expt. 1 Expt. 2 Expt. 3 Expt. 4 Expt. 5 Expt. 6

Control

LMW-CSA

Std.CSA

Mouse L-cell

CSA

Mouse L-cell

CSA

+

LMW-CSA

Std.CSA

+

LMW-CSA

Std.CSA

+

LMW-CSA

none

10

2030

5

10

20

30

2

10

2030

2

10

10

10

10

20

0.0 2.9 0.0 0.0 0.0.0

- - - 0.0 0.0.0.0

1.2 - - - 0.0.0.0- 6.9 - - 0.0,0.0

- - - - 6.6.7.8

- - - - 16.17.18.20

- - - - 23.28.28.31

- - 30,46 - -

- - - 26.36 -

- - 55,62 -

40.46 - - - 80.92.93.109

- 62,65 41.49 - -

- - - 28,36 -

- - - - 14.14.16,18

- - - - 14.17.18.19

0.0.0.0

0.0.0.0

0.0.0.00.0.0.0

4.6.8.9

11.14.16.18

20.21.30.32

-

-

-

66.77,103.131

-

-

14,15.17,17

10.14.19.20

Number of colonies formed by 5-6 x 1 ı nucleated cells per dish.

DISCUSSION

The low molecular weight material described in this paper seems more likelyto be a stimulator of colony-formation (CSA), rather than an enhancer analo-gous to the enhancer from red cells described by Bradley et al. ‘ı First, purifiedLMW-CSA stimulated colony formation by populations of nonadherent mar-row cells; such populations show low levels of background colony formation(Table 1). Second, LMW-CSA made no contribution, either positive or nega-tive, to the number ofcolonies formed by mouse marrow cells in the presence ofeither standard or mouse L-cell CSA (Table 2). Third, removal of LMW-CSAfrom crude active CSA material did not abolish the activity of the materialretained by the filter (data not shown), indicating that LMW-CSA is not anessential co-factor for standard CSA. Finally, the colonies stimulated byLMW-CSA were similar in morphology, although somewhat reduced in size, incomparison with those stimulated by standard preparations.

The low molecular weight CSA described in this paper differs markedly frompreviously reported factors with colony stimulating activity in human or mousemarrow cell cultures. 15,9,I416 The estimated molecular weight of LMW-CSA isless by an order of magnitude than that of the smallest factor described pre-viously. “ A second novel feature of LMW-CSA is that it exhibits significantactivity in human marrow cultures, but not in mouse marrow cultures. In con-trast, preliminary work in our laboratory has indicated that high molecularweight CSA from human peripheral leukocyte conditioned media6 can be active

GRANULOCYTE COLONY STIMULATING ACTIVITY 347

in both human and mouse marrow cultures, while CSA from cultures of mouse

L cells9 or from human urine’ appears to be less active in human marrow than

in mouse marrow cultures.

Although the true identity of LMW-CSA is not yet known, preliminary bio-

chemical studies have shown it to be sensitive to trypsin and extractable with

chloroform or diethyl ether. It therefore appears to be a hydrophobic molecule,

probably containing lysyl or arginyl peptide-like bonds (-NH-CO-). 11,18 Al-

though other explanations exist, one possibility is that LMW-CSA consists of

one or more peptides having cyclic structures, as many of the known cyclic

peptides are soluble in organic solvents. ‘� Examples of cyclic peptides known

to have biological activity are the nonapeptidyl hormones of the posterior

pituitary, such as oxytocin, which is a monocyclic disulfide with a molecular

weight of about 1000, and is extractable in a hydrophobic solvent (secondary

butyl alcohol),2#{176} and the cyclic peptide antibiotics, such as valinomycin, which

have been used as models to study the selective transport of ions across mem-

branes.21’22 Work is presently under way to characterize more extensively the

biochemical properties of LMW-CSA, and to determine the extent to which it

acts, either directly or indirectly, as a physiological regulator of the growth and

differentiation of granulopoietic cells.

ACKNOWLEDGM ENT

We are grateful to Dr. J. S. Senn for supplying the samples of human marrow used in these

studies.

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