1989 73: 700-705  

CA Dahl and C Lindqvist marrow cellsEffects of normal mouse serum on the IL-3-induced proliferation of bone 

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700 Blood, Vol 73, No 3 (February 15), 1989: 700-705

Effects of Normal Mouse Serum on the IL-3-Induced Proliferation of Bone

Marrow Cells

By Carol A. DahI and Christer Lindqvist

Normal mouse serum (NMS) devoid of colony-stimulating

factor (CSF) was found to enhance the interleukin 3

(11-3)-driven colony formation of bone marrow in vitro.

Inclusion of NMS in bone marrow colony-forming assays

resulted in greatly increased numbers of colonies and

clusters following seven days incubation; however. incuba-

tion of bone marrow with NMS before the colony-forming

assay had no effect on resultant colony number. The levels

of serum-enhancing activity (SEA) did not appear to varysignificantly with age and in part was species restricted, in

that human and guinea pig serum did not enhance mouse

bone marrow colony formation. Conversely. NMS had no

effect on human bone marrow colony formation. Levels of

SEA were found to vary between strains. as did the degree

A VARIETY OF FACTORS have been found to

influence the in vitro proliferation and colony forma-

tion of hemopoietic progenitor cells. Essential to the prolifer-

ation and differentiation of hemopoietic progenitors are

colony-stimulating factors, such as interleukin 3, which

selectively stimulate the formation of colonies of particular

committed progenitor cell types.’�’ Additionally, there are

factors such as interferon and acidic isoferritins�7 that can

reduce the colony formation of CSF-stimulated progenitors

(eg, colony-forming unit-granulocyte-macrophage [CFU-

GM]), and other factors such as hemin and hemoglobin that

enhance the colony formation by CSF-stimulated progenitor

cells.8’9 It has also been shown that certain interleukins

and/or CSFs can have synergistic effects on bone marrow

colony formation.’#{176}�’4 We report here the presence of a

factor(s) in normal mouse serum which greatly enhances the

IL-3-stimulated in vitro colony formation of murine bone

marrow cells. This activity is present in sera containing no

detectable CSF and has no effects on cells during preincuba-

tion in the absence of IL-3, yet it greatly enhances colony

formation even when it is added late in the course of the

assay.

MATERIALS AND METHODS

Mice. Mice were obtained from Gl. Bomhabtgard, Ry, Den-mark; Department of Immunology, University of Uppsala; Depart-

From the Department of Immunology. Karolinska Institute,Stockholm, Sweden.

Supported in part by grants from the research foundation of

Karolinska Institute. Sigurd and Elsa Goijes Foundation. the4hlen Foundation, the Vilhelm Johansson Foundationfor Medical

Research, and by NC! grant 2R01 CA26782-04, and grants from

theSwedish Cancer Societ�’ awarded to R. Kiessling.Address reprint requests � � Carol Dahi, PhD, Pittsburgh Cancer

Institute, 3343 Forbes Avenue, Pittsburgh. PA 15213.

Submitted January 1, 1988; accepted October 26, 1988.The publication costs ofthis article were defrayed in part by page

charge payment. This article must therefore be hereby marked

“advertisement” in accordance with 18 U.S.C. section 1 734 solely to

indicate this fact.

© 1989 by Grune & Stratton. Inc.

0�%/6-4971/89/7303-lYfl8$3.00/0

to which bone marrow from various strains was enhanced

by the serum. Serum fractionation studies indicated three

active fractions with molecular weights of 800-900 Kd,

60-70 Kd. and 20-30 Kd. The fraction at 800-900 Kd

inhibited colony formation at high concentrations and

enhanced colony formation on dilution. whereas the two

other active fractions contained enhancing activity at all

concentrations tested. These results would indicate that

normal serum can play a greater role in colony-forming

assays than nutritional supplements. The relationship of

the SEA factors to other factors that have been reported to

modulate bone marrow colony formation is discussed.

C 1989 by Grune & Stratton, Inc.

ment of Tumor Biology, Karolinska Institute; and Department ofImmunology, Karolinska Institute.

CFU-C assay. Bone marrow cells were obtained from mice byaspiration of the femurs and tibias. Cells were washed twice inRPMI 1640 (Gibco) before counting. Bone marrow cells (105/mL)were seeded in 1 mL of 0.3% agarose (Sea Plaque, FMC Corp.

Rockland, ME) containing an exogenous source of CSF in 30 mmgridded culture dishes. RPMI 1640 supplemented with antibiotics,

L-glutamine (2 mM), sodium pyruvate (1 mM) (Gibco, Middlesex,

England), B2-mercaptoethanob (5 x i0� M) and 20% heat-inactivated fetal bovine serum (FBS) (Gibco) was used in theagarose cultures. IL-3 was obtained (as indicated in the results) from

media conditioned by the murine myebomonocytic leukemic WEHI-

3 (a gift from Dr Norman Iscove), or from 48-hour supernatants ofCon A-stimulated rat spleen cells. Alpha methyl mannoside was

included in the cultures to preclude any effects that Con A remain-ing in the rat spleen cell supernatants might have on colony

formation. The levels of alpha methyl mannoside added weresufficient to eliminate any residual Con A mitogenic activity asassayed on responsive lymphocyte populations. Alpha methyl man-noside alone had no effect on colony formation. In assays for

inhibition of bone marrow colony formation, test supernatants wereeither (as indicated in results) added directly to the bone marrowagarose cultures or bone marrow cells were incubated in the test

supernatants for the indicated time, washed, and then resuspendedin agarose containing IL-3. When NMS was added after the

initiation of the colony-forming assay, the bone marrow cells were

originally plated in 0.35% agarose. An overlay of 0.3% agarosecontaining the appropriate concentrations of NMS, FBS, and IL-3

to give the indicated final concentrations was added at the appropri-

ate day. Control plates were given overlays containing comparablequantities of FBS and IL-3, without the addition of NMS. Coloniesof greater than 50 cells were scored after seven days of incubation at37#{176}Cin 5% CO2. All samples were run in duplicate.

Serum. Serum was obtained by retro-orbitab bleeding. Afterclotting, the serum was cleared by centrifugation and used immedi-

ately for assay.Columnfractionation ofserum. The chromatography was done

according to the Pharmacia Fine Chemicals Inc instruction manual

(Uppsala, Sweden). Fractionation was � performed at 4#{176}CwithSepharose CL-6b equilibrated with 0.9% NaCl and 10 mM HEPES.Column size was 95.5 x 1.5 cm, with sample volume being 2.5mL ofNMS. The flow rate was 0.24 mL/minute, with a void volume (asdetermined from the elution volume of blue dextran 2000) of 63 mL.The eluate was collected in fractions, sterile filtered, and tested for

SEA activity.

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ual strains tended to be consistent across multiple experi-

NORMAL SERUM EFFECTS ON COLONY FORMATION 701

RESULTS

Serum enhancement of bone marrow colony forma-

tion. Mouse bone marrow cells were cultured in agarose

cultures containing optimal concentrations of both fetal calf

serum and WEHI-3 conditioned medium (a potent source of

IL-3).”2 When normal mouse serum or plasma having no

detectable colony-stimulating activity (data not shown) was

included in the cultures, colony counts following seven days

of culture were increased greatly (Table 1), as was the size of

the individual colonies. Serum concentrations as low as 0.6%

were found to increase colony counts significantly (data not

shown). Serum concentrations that were found to enhance

colony formation (groups of greater than 50 cells) were also

found to enhance cluster number (groups of 5-50 cells). The

serum-enhancing activity on bone marrow colony formation

was not restricted to cultures using WEHI-3-conditioned

medium-derived CSF but also was apparent in cultures

stimulated with CSFs obtained in supernatants from Con

A-stimulated rat spleen cells (data not shown).

Addition of NMS directly to IL-3-containing bone mar-

row cultures resulted in a significant increase in colony

formation. Preincubation of bone marrow cells with NMS

alone, followed by culture in IL-3 containing agarose did not

result in substantial increases in colony number (Table 1).

Addition of NMS after initiation of bone marrow cultures

resulted in large increases in final colony number (Fig 1),

with small increases in colony number resulting from the

addition of NMS as late as six days into the seven-day

culture period in some experiments.

Effects of age, strain, and species on production of

SEA. SEA did not appear to be related to the age of the

mouse from which the serum was obtained. Serum samples

obtained from C57B1/6 (B6) mice varying from 5 to 31

weeks of age were all found to stimulate roughly similar

levels of colony number enhancement when bone marrow

was from the same strain (Table 2).

Experiments were done testing the ability of sera derived

from a variety of strains to enhance the colony formation of

(B6 X DBA/2)F1 (BDF1) or B6 bone marrow. These results

Table 1 . Effects of Preincubation in NMS on Colony Formation

Colmrns Obtained with Tr.atment

Strain of Sera Du.cttSerum Donor Concen�ation Addition Pr.IncL�atIOn�

- 0% 71.0±2.0 73.0±7.0

B6 2% 225.0 ± 8.0 83.5 ± 6.5

B6 5% 227.5 ± 16.5 90.5 ± 7.5

B6 10% 254.0 ± 8.0 85.5 ± 7.5

BDF, 2% 91.0 ± 8.0 73.5 ± 2.5

BDF, 5% 232.5 ± 10.5 65.5 ± 5.5

BDF, 10% 257� 94.0 ± 4.0

#{149}Coloniesp& 10� bone marrow cells.

tIn this experiment, B6 bone marrow cells were mixed directly withindicated concentrations of NMS and 25% WEHI 3 conditioned superna-

tants and culti.wed as described in agarose for seven days.

�In this experiment, B6 bone marrow cells were mixed with the

indicated concentrations of NMS for seven hours before culture in

ageroso containin9 WEHI 3 conditioned supernatant (preincubation).

§One replicate.

C,)Ui 150z0

� 100

50

DAY OF ADDITION

Fig 1 . B6 bone marrow cells were plated in 0.35% agarose

cultures containing WEHI-3 conditioned media at day 0. At dayO orthe indicated day following initiation of culture. cultures wereoverlaid with 1 mL ofO.3% agarose (A) or 0.3% agarose containingNMS. such that the final culture concentration was 5% (0).Colonies of greater than 50 cells were counted following seven

days of incubation. Colony counts are indicated as colonies per 10’cells. For further Information, see Materials and Methods. Similarresults were obtained in two additional experiments of identical

design.

are shown in Fig 2. While no dramatic separation of high and

bow enhancers is apparent, when tested on B6 bone marrow,

individual strains do appear to fall either in an upper or lower

range of enhancement. On this basis, two groupings can be

distinguished: the BDF1, DBA, A/Sn, and MRL/lpr sera all

tend to have moderate to high levels of SEA, whereas the

BALB/c, AKR, A/JAX, CBA/H, and C3H sera all tend to

have moderate to low bevels ofSEA. B6 sara does not fall into

any consistent placement. When tested on BDF1 bone mar-

row, most of the strains remain in the same categories as

tested above; however, the BDF1, AKR, and A/JAX sera all

switch categories to become low and high enhancers respec-

tively.

Data from experiments done testing the effects of BDF1 or

B6 sera on colony formation by bone marrow from various

strains obtained similar results (data not shown). Once

again, there was no dramatic separation of high and low

responders. However, as with the above experiments, individ-

Table 2. Relationship of Donor Age to SEA Level

Don�Ags’ NMSCOnCSnSFSIIOnt

5% 1.25%

5 60.0 ± 3.5 43.5 ± 2.5

15 49.0 ± 3.0 42.5 ± 2.5

21 49.0 ± 4.0 38.5 ± 1.5

27 41.0 ± 6.0 47.5 ± 0.5

31 47.5 ± 5.5 44.0 ± 0.0

Age of B6 serum donor in weeks.

tme indicated NMS concentrations were mixed with B6 bone marrowcells and 25% WEHI 3 conditioned me�um. Colony counts are given as

colonies per 10’ bone marrow cells. Cuftures containing 25% WEHI 3

supernatants in the absence of NMS contained 30 +1- 0.5 colonies

and cultures with media alone, 0 colonies. For further information, see

Materials and Methods.

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400

350

300

250

200

150

100

50

(I)uJz0-J00

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702 DAHL AND LINDQVIST

Fig 2. Bone marrow cellsfrom either BDF, (experiment A)

or B6 (experiments B. C. and D)animals were plated in culturescontaining WEHI-3 conditionedmedia and 5% NMS from theindicated strain. Colonies ofgreater than 50 cells were

counted following seven days ofincubation. Colony counts areindicated as colonies per 10’cells.

ments in giving relatively high or lower responses within the

group of strains compared.

Human, guinea pig, and rat sera were also assayed for

their ability to enhance mouse bone marrow colony forma-

tion. While Lewis rat serum appeared to have low levels of

SEA for B6 bone marrow colony formation, guinea pig was

somewhat inhibitory, and various preparations of human

sera when added to the in vitro colony-forming assay resulted

in greatly reduced numbers of colonies relative to controls

(data not shown). Reciprocally, when normal BDF1 sera

containing potent mouse bone marrow colony-enhancing

activity was added to human bone marrow cultures (stimu-

lated by either CSF containing placenta-conditioned media

or GCT-conditioned media [Gibco]), no enhancing activity

was seen, and in some cases colony numbers were slightly

reduced (data not shown).

Production of SEA is resistant to long-term irradia-

tion. The effects of long-term irradiation of bone marrow

cells on the levels of colony-enhancing activity and potential

radiosensitivity of the producing cells were examined. In one

series of experiments (shown in Fig 3), mice were given

0.4-0.8 Ci of 89Sr. Sera were collected two to three months

later and assayed for colony-enhancing activity. Levels of

colony-enhancing activity in the sera from 89Sr-irradiated

animals were found to be similar to the levels in the control

C,)

z0-J

00

1””

A B C D E F

Fig 3. Bone marrow cells from CBA/H mice were cultured inthe presence of 5% NMS from normal CBA/H animals (column A)or from animals that had been given 0.4-0.8 �sCi of “Sr two tothree months earlier (columns B-F). Colonies of greater than 50

cells were counted following seven days of incubation.

animals. In contrast, the levels of natural killer (NK) cell

activity was greatly reduced in these animals (data not

shown), as is normally found in animals treated with 89Sr.’5

Characterization of SEA. Whole sera containing SEA

were tested for heat lability. SEA was found to be stable at

temperatures up to 90#{176}Cfollowing one hour of treatment

(data not shown).

Serum fractionation studies using Sepharose CL-6B col-

umns demonstrated three active fractions with molecular

weights (MW) of 800-900 Kd, 60-70 Kd, and 20-30 Kd (see

Fig 4). While the active fractions from 60-70 Kd and 20-30

Kd consistently enhanced bone marrow colony formation,

the active material in the 800-900 Kd fractions varied

between batches in either enhancing or inhibiting bone

marrow colony formation. Dilution of batches of high-MW

fractions that were inhibitory showed that the inhibitory

effect was seen only at high concentrations of those fractions.

When lower concentrations of the same batches were

included in the colony-forming assay, there was a profound

enhancement of bone marrow colony formation. Active

fractions of 60-70 Kd and 20-30 Kd were never found to

inhibit colony formation.

DISCUSSION

We describe here an enhancing activity for IL-3-stimu-

lated bone marrow colony formation associated with normal

mouse serum. The sera tested were found to have no colony-

stimulating activity on their own, but could enhance bone

marrow colony formation stimulated by WEHI-3-condi-

tioned media (even when saturating levels of various prepa-

rations of IL-3 and fetal bovine serum were used). No

enhancing activity was detected when bone marrow cells

were preincubated in serum and then assayed in the presence

of IL-3; however, increased numbers of colonies were seen

when the serum was added directly to the assay containing

IL-3, even if the addition was multiple days into the seven-

day assay. This would seem to suggest that either the

presence of NMS is required for a longer period of time

(such as the seven-day culture period) or that the effect of

the NMS occurs only in the presence of the IL-3.

Additionally, we found that SEA can be derived from all

strains tested so far, as well as from mice of various ages. The

results in Fig 2 indicate that the major histocompatability

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12 22 32 42

NORMAL SERUM EFFECTS ON COLONY FORMATION 703

(I)

LU

z0-J00

NMS FRACTION TESTED

Fig 4. Bone marrow cells from BDF1 mice were cultured in the presence of a constant amount of both IL-3 (25% WEHI-3 conditionedmedia) and the indicated NMS fraction number (25%). Colonies of greater than 50 cells were counted following seven days of incubation.Positive control of 25% WEHI-3 conditioned media ( . . . ) gave 138 colonies per 10’ cells. Relative O.D. (- - -). and colonies per 10’ bone

marrow cells (-) are given. Arrows indicate. from left to right. void volume and elution volumes for 1gM. lgG, and albumin. In thisfractionation. fraction 1 9 contains the 800-900 Kd enhancing/inhibitory peak. fraction 34 contains the 60-70 Kd enhancing peak. andfraction 46 contains the 20-30 Kd enhancing peak. Similar results were obtained in six additional fractionations. For further details. seeMaterials and Methods.

complex (MHC) compatability of the sera and bone marrow

donors are unrelated to SEA levels. Although the cell source

of the activity has not yet been identified, our data suggest

that either the producer cell is a bone marrow-dependent

radiation-resistant cell, such as the radio-resistant stromal

cells,’6 or resides elsewhere in the body.

Serum fractionation studies have indicated at least three

components that can alter levels of bone marrow colony

formation, with MW of 800-900 Kd, 60-70 Kd, and 20-30

Kd. While only the two lower MW active fractions have been

found to have enhancing activity, the highest MW active

fractions have been found to eliminate colony formation at

high concentrations while enhancing colony formation at low

concentrations.

There is much confusion in the literature to date as to

whether inclusion of serum in in vitro assays of granulopoie-

sis inhibits,17’9 enhances,8’9’20’2’ or has no effect22’23 on bone

marrow colony formation. While we consistently found

enhancement of colony formation with whole serum, it was

clear from fractionation studies that at least one of the active

serum components could also inhibit at high concentrations.

It is possible therefore to speculate that the conflicting

previous reports may represent differences between the van-

ous samples in terms of the amounts of the three active

components we have identified. Whether such changes in

composition can occur due to differences in environmental

conditions, endogenous pathogens, or disease has not been

shown and would provide an interesting area for further

investigation.

Primarily, serum-associated inhibitory activity has been

attributed to serum lipoproteins, although whether the inhi-

bition is due to very-light-density, light-density, or high-

density lipoproteins is still a matter of controversy)719 The

inhibitory activity described by Metcalf and RusselPt would

not seem to correspond with the high-MW inhibitor we have

described, as it has a MW of around 250 Kd and is effective

as an inhibitor even when bone marrow cells are preincu-

bated in the active fractions. The results of Mortensen et al’7

more closely resemble ours, in that preincubation in the

active fractions has no effect on subsequent colony-forming

assay; they show no evidence, however, for associated

enhancing activity upon dilution.

Enhancement of colony formation by serum has been

attributed to both hemin and hemoglobin8’9’2’ as well as to an

unidentified factor described by Metcalf et a12#{176}that elutes

from a G- 100 column in the region of serum albumin. While

it is possible that hemin and/or hemoglobin contributes in

part to the SEA described here, it is unlikely they are

responsible for a major portion of the activity in that the

enhancing effects of hemoglobin and hemin seemed to be

relatively species unrestricted. The factor described by Met-

calf et al,2#{176}in addition to being relatively species unrestricted

in its effects, is heat labile, which contrasts with our prelimi-

nary data for SEA. Therefore, although these previously

described serum components with colony-enhancing activity

may be responsible in part for the enhancing activity we see,

their activities do not correlate well with the characteristics

for whole serum enhancing activity.

In addition to the previous reports on serum effects on

colony-forming activity, a number of other factors have been

found to function as enhancers on inhibitors of bone marrow

colony formation. Inhibitors of bone marrow colony forma-

tion which have been described include interferon,5’24’25 acidic

isoferritins,6’7’26’27 and a T cell-derived lymphokine of approx-

imately 85,000 Kd.28 It is unlikely that the serum-associated

factor we have identified which can inhibit colony formation

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REFERENCES

704 DAHL AND LINDQVIST

correlates with these previously described factors, as they

have not been described at any concentration to have enhanc-

ing activity and do not fall into the same molecular weight

range. Supernatants from long-term bone marrow cultures

have been found to enhance bone marrow colony formation

even in the presence of plateau (optimal) levels of CSF.293’

This colony-promoting activity (CPA) would appear to

enhance by a different mechanism than serum, as it will

promote cluster formation in the absence of CSF, which is

not seen with serum (C. Lindqvist and C. Dahl, unpublished

observations). Additionally, Izumi et al have found that bone

marrow colony formation can be enhanced by preincubation

of bone marrow cells in CPA, but that the addition of CPA

subsequent to the initiation of culture in CSF results in no

increase in colony number above controls.

More recently, it has been found that certain combinations

of interleukins and CSFs or of multiple CSFs can have

synergistic effects on colony formation by bone marrow.’#{176}

14,33 Included in these are IL-6, GCSF, IL- 1 , and accessory

activity (AA),’2”3’33’34 which have all been shown to have

synergistic effects on multi-potential hemopoietic progeni-

tons when combined with IL-3. All of these factors have sizes

near or within the range of the lowest MW fraction we have

identified of around 20-30 Kd. GCSF and IL-6 are able to

stimulate colony formation in the absence of other CSFs,

while AA and IL- 1 cannot maintain colony formation. As we

have been unable to detect any colony-sustaining factors in

1. Nicola NA, Vadas M: Hemopoietic colony-stimulating fac-tors. Immunol Today 5:76, 1984

2. Iscove NN, Roitsch CA, Williams N, Guilbert U: Molecules

stimulating early red cell granulocyte, macrophage and megakaryo-cyte precursors in culture: Similarity in size, hydrophobicity and

charge. J Cell Physiol (Supple) 1:65, 1982

3. Gough NM, Gough J, Metcalf D, Kelso A, Grail D, NicolaNA, Burgess AW, Dunn AR: Molecular cloning of cDNA encodinga murine haematopoietic growth regulator, granulocyte-macro-phage colony stimulating factor. Nature 309:763, 1984

4. Fung MC, Hapel AJ, Ymer A, Cohen DR. Johnson RM,

Campbell HD, Youn� IG: Molecular cloning of cDNA for munineinterleukin-3. Nature 307:233, 1984

5. Klimpel GR, Fleischmann WR, Klimpel KD: Gamma inter-

feron and IFN suppress murine myeloid colony formation (CFU-C):Magnitude of suppression is dependent upon level of colony-stimulating factor (CSF). J Immunol 129:76, 1982

6. Broxmeyer HE, Bognacki J, Dorner MH, Dc Sousa M: Identi-fication of leukemia-associated inhibitory activity as acidic isoferri-tins. J Exp Med 153:1426, 1981

7. Broxmeyer HE, Bognacki J, Ralph P. Dorner MH, Lu L,

Castro-Malaspina H: Monocyte-macrophage-denived acidic isofer-ritins: Normal feedback regulator of granulocyte-macrophage pro-genitor cells in vitro. Blood 60:595, 1982

8. Rothmann J, Hertogs CF, Malik Z, Pluznik DH: Heminstimulating effect on colony formation of leukemic and bone marrowcells. Exp Hematol 1 1:147, 1983

9. Kriegler AB, Bradley TR, Hodgson GS, McNiece 1K: Identifi-cation of the “factor” in erythrocyte bysates which enhances colony

growth in agar cultures. Exp Hematol 9:11, 198110. Rennick D, Yang G, Muller-Sieburg C, Smith C, Arai N,

Takabe Y, Gemmell L: Interleukin 4 (B-cell stimulatory factor 1)

the NMS samples we have used, it strongly suggests that

GCSF or IL-6 is not responsible for the 20-30 Kd active

fraction we have detected but does not rule out the possibility

of very low levels of IL-6 or GCSF which are undetectable in

bone marrow colony assays. The possibility remains, how-

ever, that low levels of either IL-i or AA may be present in

serum and account for the enhancing activity we see in the

20-30 Kd size range.

Therefore, at least two of the components of the SEA

described here appear to be novel. The means by which the

size and number of colonies are enhanced is the subject of

further investigation. Many previously described modulators

of hematopoiesis have been shown to be cell cycle associated

and are dependent on the expression of class II antigens of

the major histocompatability complex.26’27’32 The potential

association of SEA and class II antigen expression should

prove an interesting area to pursue. The demonstration of

serum factors that regulate CSF-driven bone marrow colony

formation adds another layer of complexity to the hemo-

poietic regulatory pathway. In addition, it emphasizes that

hemopoietic precursor assays performed in the presence of

serum should be interpreted with extreme care.

ACKNOWLEDGMENT

We would like to thank Drs H. Wigzell, R. Kiessling, and S.Cairns for constructive comments.

can enhance or antagonize the factor-dependent growth of hemo-

poietic progenitor cells. Proc Natl Acad Sci USA 84:6889, 19871 1 . Peschel C, Paul WE, Ohara J, Green I: Effects of B cell

stimulatory factor-1/Interleukin 4 on hematopoietic progenitor

cells. Blood 70:254, 198712. Ikebuchi K, Wong GG, Clark SC, Ihle iN, Hirai Y, Ogawa

M: Interbeukin 6 enhancement of interleukin 3-dependent prolifera-

tion of multipotential hemopoietic progenitors. Proc Natl Acad Sci

USA 84:9035, 1987

13. Ikebuchi K, Clark SC, Ihle JN, Souza LM, Ogawa M:

Granulocyte colony-stimulating factor enhances interleukin 3-

dependent proliferation of multipotential hemopoietic progenitors.Proc Natl Acad Sci USA 85:3445, 1988

14. Zsebo KM. Wypych J, Yuschenkoff VN, Lu H, Hunt P.Dukes PP, Langley KE: Effects of hematopoietin-1 and interleukin 1activities on early hematopoietic cells of the bone marrow. Blood

71:962, 1988

15. Meblen PF, Lust JA, Bennett M, Kumanr V: Analysis of lownatural killer cell activity in 89Sr-treated mice. Eur J Immunol

12:442, 1982

16. Song ZX, Quesenberry PJ: Radioresistant murine marrow

stromal cells: A morphologic and functional characterization. ExpHematol 12:523, 1984

17. Mortensen BT, Knudtzon S. Schultz H: Serum lipoproteininhibitors of granubopoiesis in human bone marrow cultures. ExpHematol 7:1, 1979

18. Metcalf D, Russell S: Inhibition by mouse serum of hemo-

poietic colony formation in vitro: Exp Hematol 4:339, 1976

19. Douay L, Barbu V. Baillou C, Najman A, Gorin N-C,

Polonovski J, Duhamel G: The role of serum lipoproteins on the in

vitro proliferative potential of human hematopoietic progenitors

CFU� and CFU,. Exp Hematol 10:499, 1983

For personal use only.on October 31, 2014. by guest www.bloodjournal.orgFrom

NORMAL SERUM EFFECTS ON COLONY FORMATION 705

20. Metcalf D, MacDonald HR. Chester HM: Serum potentia-tion of granulocyte and macrophage colony formation in vitro. Exp

Hematol 3:261, 1975

21 . Rothman J, Hertogs CF, Pluznik DH: Growth enhancement

and serum replacement in cloning of murine mastocytoma andgranubocyte/macrophage precursor cells: Two distinct activities

present in hemolysates. Exp Hematol 7:352, 1979

22. Metcalf D: Stimulation by human urine or plasma of granubo-poiesis by human marrow cells in agar. Exp Hematol 2:157, 1974

23. Blanchet JP, Arnaud 5, Samarut J, Bouabdelli M: A factorpresent in normal mouse serum stimulates late erythroid precursorproliferation. Exp Hematol 12:595, 1984

24. Broxmeyer HE, Lu L, Platzer E, Feit C, Juliano L, RubinBY: Comparative analysis of the influences of human gamma, alpha

and beta interferons of human multipotential (CFU-GEMM), cry-throid (BFU-E), and granulocyte-macrophage (CFU-GM) progeni-torcells. J Immunob 131:1300, 1983

25. Verma DS, Spitzer G, Zander AR, Gutterman JU, McCredie

KB, Dicke KA, Johnston DA: Human leukocyte interferon prepara-lion-mediated block of granulopoietic differentiation in vitro. Exp

Hematol 9:63, 1981

26. Broxmeyer HE: Relationship of cell-cycle expression of Ia-like antigenic determinants on normal and leukemia human granulo-cyte-macrophage progenitor cells to regulation in vitro by acidic

isoferritins. J Clin Invest 69:632, 198227. Broxmeyer HE: Association of the sensitivity of mouse granu-

locyte-macrophage progenitor cells to inhibition by acidic isoferri-tins with expression of Ia antigens for I-A and I-E/C subregionsduring DNA synthesis. J Immunol 129:1002, 1982

28. Trucco M, Rovera G, Ferero D: A novel human lymphokinethat inhibits haematopoietic progenitor cell proliferation. Nature

309:166, 198429. Tsurusawa M, Miyanomae T, Izumi H, Mon KJ: Presence of

colony promoting activity (CPA) in the supernatant of the long-termbone marrow cultures. Leukemia Res 7:167, 1983

30. Izumi H, Oshima H, Mon KJ, Frindel E: Characterization ofpossible receptor ofcolony-promoting activity (CPA) by comparisonwith that of colony-stimulating factor (CSF). Exp Hematol 12:231,

198431. Izumi H, Tsurusawa M, Miyanomae T, Kumagai K, Mon

KJ: Role of humoral factors in granulopoiesis: colony promotingfactor (CPF) and its target “pre-CFU-C” in long-term bone marrowculture. Leukemia Res 7:155, 1983

32. Broxmeyer HE, Dupont B: A role for class-Il major histocom-

patability complex antigens in the regulation of myelopoiesis. Prog

Allergy 36:203, 198533. Iscove NN, Fagg B, Keller G: A soluble activity from

adherent marrow cells cooperates with IL-3 in stimulating growth ofplunipotential hematopoictic precursors. Blood 71 :953, 1988

34. Stanley ER, Bartocci A, Patinkin D, Roscndaal M, Bradley

TR: Regulation of very primitive multipotent hemopoietic cells byhemopoietin-l . Cell 45:667, 1986

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