Vol.
174,
January
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
31, 1991
STIMULATION MULTINUCLEATED
CELL
OF MACROPHAGE FORMATION
BY INSULIN-LIKE
GROWTH
IN RAT BONE GROWTH
AND
MARROW
FACTOR
647-653
CULTURES
I
Ben A.A. Scheven* and Nicola J. Hamilton The Rowett Research Institute, Received
December
14,
Bucksburn, Aberdeen Al32 9SB, UK
1990
In this study the effects of rhIGF-I on macrophage differentiation and growth have been studied using liquid suspension cultures of rat bone marrow cells. IGF-I stimulated macrophage growth in a dose-dependent manner, a maximum response was found at a concentration of 20 rig/ml. IGF-I effects could be ascribed to stimulation of both postmitotic and proliferating cells. A remarkable finding was that IGF-I induced formation of multinucleated cells (MNC). The MNC resembled macrophage-like cells (AcP, NSE positive). A monoclonal antibody to rhIGF-I significantly inhibited IGF-stimulated macrophage growth and MNC formation. A specific antibody to mouse CSF-1 reduced IGF-stimulated macrophage growth in mouse bone marrow cultures indicating that IGF-I effects could, at least in part, be ascribed to endogenous production of CSF-1. These findings indicate that IGF-I in concert with locally induced CSF-1 can influence the differentiation and growth of bone marrow-derived macrophages. o 1991Academic press. I”=. Macrophages
are derived from bone marrow progenitors
various tissues where they perform reactions and tissue remodeling
many diverse functions
(1). Though
the knowledge
biology is increasing, the precise cellular and biochemical
and homed in
related
to defence
about macrophage
processes regulating
the
development and growth of macrophages in the haemopoietic and “target” tissues have not been clearly elucidated (1,2). Macrophages are capable to secrete bioactive molecules, paracrine cellular
some of which are considered to be involved in autocrine or growth (3-7). It has been shown that activated alveolar macro-
phages produce insulin-like growth factor (IGF)-type molecules (8). In another study it was reported that macrophages in nasal inflammatory foci displayed a
*Author responsible for correspondence (present address of both authors): Dr. B.A.A. Scheven, University Hospital, Department of Internal Medicine Research Group for Bone Metabolism, P.O. Box 85.500, 3508 GA Utrecht, The Netherlands
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
strong immunocytochemical staining with an anti-IGF-I antibody (9). Very recently, Nagaoka et al., (10) described IGF-I gene expression in human macrophage-like cells. These studies suggest that IGF-I
may play an important
role in inflammatory
reactions. IGFs exert mitogenic
and differentiation
promoting
activities
in various
tissues (11). IGFs have also been suggested to play a role in the regulation haemopoiesis.
It has been shown that IGF-I
of
receptors are present on human
leukemic cells (12) and fetal mouse liver cells (13). Furthermore, it has been demonstrated recently that erythroid colony-forming units require direct interaction with IGF-I
for further development
The present influence
experiments
(14,1.5). were designed
the growth of macrophages.
We report
to study whether here that IGF-I
IGF-I
can
stimulates
macrophage growth in serum-free cultures of rat bone marrow cells and that IGFI induces multinucleated cell formation. MATERIALS
AND METHODS
Cytokines and antibodies. Recombinant human IGF-I (rhIGF-I) was purchased from Amersham (UK) or was obtained from Dr. A. Skottner, KabiVitrum (Stockholm, Sweden); no differences were noted between the different rhIGF-I preparations. The monoclonal mouse antibody to rhIGF-I (sm 1.25) was a gift from Dr. J.J. van Wyk (University of North Carolina, Chapel Hill, USA), and the anti-CFS-1 antibody (anti-L cell CSF-1 goat antiserum) was supplied by Dr. E.R. Stanley (Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA). Bone marrow culture. Bone marrow cells were isolated from the long bones of the hind limbs of adult rats (200 g) by flushing the bones with Hanks balanced salt solution (HBSS) using a fine gauge needle. The cells were first pre-cultured in a culture flask containing MEM (Gibco, Scotland) supplemented with 1% fetal bovine serum (FBS) (Flow Lab., UK) for 4 h at 37°C in a 5% CO, in air atmosphere. This producedure promotes the attachment of fibroblastic cells and mature macrophages. The non-adherent mononuclear cell population which is more or less enriched for immature myeloid cells (granulocyte-macrophage progenitor cells) was used for further incubation in control or test media in 24-well-plates under the same conditions as above (about 20,000 cells in 0.5 ml per well). The culture medium contained 1 mg/ml bovine serum albumin (BSA, Sigma) but no serum. At different time points the cultures were ended and the adherent cells were stained for acid phosphatase (AcP) or nonspecific esterase (NSE) using Sigma histochemical kits. Counterstaining was performed with haematoxylin. To confirm the macrophage-like nature of the enzyme positive cells found in the cultures, an immunocytochemical staining using the monoclonal antibody ED1 was carried out as described previously (16). Autoradiography. Bone marrow cells were incubated in serum-free MEM/BSA in the presence of 0.065 &i/ml tritiated thymidine (3H-TdR; spec. act.: 2 Ci/mmol, Amersham, UK) in tissue chamber slides (Flow Lab.). After 3 days the cultures were stained for AcP and subsequently processed for light microscopic autoradiography. Briefly, the slides were dipped in Kodak K5 emulsion and exposed in a light-tight box for 2 weeks at 4°C; development was carried out in Kodak D19 and fixation in Kodak Unifix solution. 648
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cell counting and statistics. Macrophages were identified by their morphology and by their strong staining for AcP or NSE activity. The number of mononuclear (labelled and non-labelled in the autoradiographs) macrophages were counted in 10 random microscopic fields. The number of multinucleated cells (MNC) was determined by screening the whole well. Statistical differences were assessed by the Student’s t-test. RESULTS After
preculture
of the whole rat bone marrow preparation,
the non-
adherent cells were cultured in serum-free control media or in media containing recombinant
human IGF-I.
After culture the adherent macrophages were identified
by their strong AcP or NSE activity. No other (hemopoietic)
cell types remained
attached in the cultures, except for a few fibroblasts, which could be easily distinguished from the macrophages on the basis of their morphology staining. IGF-I
did not significantly
IGF-I
a dose-dependent
elicited
cultures (Fig. 1). Interestingly,
alter the numbers of fibroblastic increase in the number
nature.
Furthermore,
cells, however,
of macrophages
in the
MNC were frequently observed in the IGF-cultures.
These MNC displayed strong AcP and NSE staining indicating like
and weak enzyme
the cells were positively
labelled
their macrophagewith
the EDl-
monoclonal antibody, which specifically recognizes members of the mononuclear phagocyte system. Addition of sodium tartrate to the AcP substrate solution inhibited the enzyme reaction in most of the MNC. MNC formation occurred after 3 days in vitro and gradually increased thereafter (Fig. 2). Some MNC were also observed in control cultures indicating susceptible to MNC formation
0 1
IGF-I 1020concentration40
(rig/ml) 80
that rat bone marrow cells are especially
in vitro. The average number of nuclei of the MNC
02
0 Culture
time
(d)
dose-response relation for mononuclear macrophage (0) and MNC (0) formation in 5-days’ cultures. Mononuclear macrophages and MNC were identified by positive reaction for AcP or NSE staining. Results are expressed as the number of macrophages counted in 10 random microscopic fields, and the total number of MNC per well. The results are mean + SEM of 3 independent experiments (n = 12). Fig. 2. Time course of MNC formation in control (0) or IGF-I (0) cultures. IGFFie. 1. IGF-I
I concentration was 80 “g/ml. The total number per well was counted. Results are mean +- SEM of 4-6 cultures. 649
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
was 5.1 + 0.4 and 3.7 + 0.1 for the 5-days’ IGF- and control cultures respectively. A maximal response for MNC induction was found at a concentration of 40 rig/ml, whereas a dose of 20 rig/ml
was sufficient for optimal
nuclear macrophages (Fig. 1). To further define the effects of IGF-I graphy experiments presence of tritiated stimulated
were performed thymidine
the formation
on macrophage
of the mono-
growth, autoradio-
on bone marrow cultures incubated
(3H-TdR).
of labelled
stimulation
After 3 days in culture, IGF-I
macrophages indicating
in the greatly
that the IGF-I-stimu-
lated macrophage growth was mainly due to an effect on the replication of progenitor cells (Fig. 3, upper panel). However, there was also a significant increase in the number of non-labelled
macrophages
and this means that IGF-I
also stimulated macrophage differentiation from nonproliferating, postmitotic cells (Fig. 3, upper panel). A similar response can be seen in the distribution of nuclei in the MNC
(Fig. 3, bottom
panel). The labelling
index was 38 f 7.5% for the Mononuclear
a
Mononuclear 60
7
0
Unlabelled
-
Labelled
‘;; G I
Multinuclear
2
IGF-I
Multinuclear
z s 203o I-t
a
w
C
a r-l
a,b
(.-got- Lh Control
C
IGF-I
IGF-I
&,.-& Effect of IGF-I on incorporation of tritiated thymidine into bone marrow macrophages. Cultures were labelled for 3 days followed by AcP histochemistry and LM autoradiography. The average number of labelled (closed columns) and nonlabelled (open columns) mononuclear macrophages in 10 microscopic fields (upper panel) or the average number of labelled (closed columns) of non-labelled nuclei in the MNC (bottom panel) k SEM are shown. a: significantly different from unlabelled controls (p < 0.05); b: significantly different from labelled controls (p < 0.001). EIg9. Effect of monoclonal antibody to IGF-I on mononuclear macrophage growth [top panel) and MNC induction (bottom panel) in control (c) or IGF-I cultures. Open columns are cultures without antibody; closed columns are cultures with antibody (l/4000 dilution; i.e. about 2.5 fig/ml IgG). Normal purified control mouse IgG had no effect on the number of macrophages in control or IGFcultures. IGF-I concentration was 40 “g/ml. Results are mean + SEM of 4 cultures. a: p < 0.01 vs control values; b: p < 0.01 vs IGF-I values. 650
Vol.
174,
No.
2, 1991
BIOCHEMICAL
=”
AND
50
BIOPHYSICAL
a
COMMUNICATIONS
a
u” ;
RESEARCH
a,b
30
z 2
10 i
:
0
Ub ‘/a000
c
‘I4000
anti-CSF-I
Ab
-IGF-I-
Fig. 5. Effect of antibody to CSF-1 on macrophage proliferation in mouse bone marrow cultures. IGF-I concentration was 80 rig/ml. Control, non-immune, goat serum in the same dilution range as the CSF-1 antiserum did not significantly affect macrophage numbers in the control or IGF-cultures. Results are mean + SEM of 4 cultures. a: p < 0.01 vs control values; b: p < 0.005 vs IGF-I values.
MNC nuclei, a figure similar to that found for the mononuclear
macrophages (37.9
+ 2.3%; p < 0.005 vs control values). These results suggest that the MNC were formed from the mononuclear was probably mononuclear
population
implying
caused by the fusion of the postmitotic of a monoclonal
led to a significant inhibition
antibody to rhIGF-I
of the IGF-stimulated
and recently replicated (17) to the culture medium
macrophage growth and MNC
in the bone marrow cultures (Fig. 4). This indicates that the responses
found in the cultures were IGF-I
dependent
and not a non-specific
culture. However, these results do not exclude the possibility stimulated
the cultures by endogenous production
phages and fibroblasts production
that this process
cells.
Addition formation
macrophage
that IGF-I
effect of indirectly
of other cytokines. As macro-
may be a source of CSF-1, we studied whether local
of CSF-1 was responsible for the IGF-effects
found. The effect of a
specific antibody to mouse CSF-1 (18) was therefore investigated in cultures of bone marrow cells isolated from mice. Dilutions of the CSF-1 antiserum were used that either were completely inhibitory (1:4000) or partly inhibitory (+ 50%; 1:8,000) for colony-formation (Stanley and Williams,
induced by CSF-1 in semisolid personal communication).
mouse bone marrow cultures
IGF-I also stimulated
macrophage
growth in all mouse bone marrow cultures (Fig. 5). Though some MNC were found in these cultures, the response was less than that found in the rat bone marrow cultures (not shown). The anti-CSF-1 antiserum inhibited the IGF-I-stimulated macrophage growth in a dose-dependent manner, indicating that endogenous production of CSF-1 was involved in macrophage growth in these cultures (Fig. 5). DISCUSSION
Here we have presented in vitro evidence that IGF-I was able to influence macrophage production from bone marrow. From the autoradiography results it is 651
Vol.
174,
No.
2, 1991
BIOCHEMICAL
clear that the effects of IGF-I postmitotic
macrophages.
specific antibody of IGF-I
BIOPHYSICAL
The results
that endogenously
growth
This could
mononuclear
to IGF-I.
phagocytes,
I. However, were
in the bone marrow
minimally
response
represented
to IGF-I.
indicating
of other factors
directly
the results
and/or
macrophage
haemopoiesis
(12-15),
work,
is required induced
that IGF-I
and that IGF-I
observation
accomplished
by a monoclonal driven by IGF numbers
cells present in the response
cultures
are small (17). Nonethe-
we suggest that IGF-I locally
induced
This supposition
is implied
CSF-1,
antibody
supplemented
of the MNC
indicating
that the MNC
resorption,
the osteoclasts,
to IGF-I.
macrophages
Whether
assays,
did not display were
to the MNC
responsible
IGF-I
cultures
to induce MNC formation
D, (20), interferon (this
in pathogenic
reactions
652
for bone
positive cells was observations).
in vitro, such as 1,25-
(21) and interleukin-4 study)
activity
high levels of this enzyme (20). (unpublished
Other agents have been reported
cell forma-
AcP (TRAP)
enhanced
to control
macrophage
reactions or tissue remodel-
significantly
compared
was
was specifically
of giant multinucleated
as these cells contain
greatly
and greatly inhibited
in mononuclear
tartrate-resistant
not similar
were
This process
this phenomenon
to the increase Generation
MNC
of TRAP
formation
of
(g-10).
and colony-forming
it should be noted here that the number
control MNC
with
biology
Nonetheless,
agents, including
in regulating
role in the regulation
with IGF-I.
associated with defence (inflammatory)
ling. Most
several
is involved,
is in accordance
in macrophage
cell populations
by fusion of postmitotic
needs yet to be clarified.
dihydroxyvitamin
to IGF-
in this study, fibroblasts
was that macrophage-like
or could be ascribed
tion is generally
other cell
this point.
in the bone marrow
apparently
on the
data from the literature
plays an important
for example on purified
An interesting
with
and growth.
to substantiate
with
alone on proliferation
in concert
dilution
they did not show any mitogenic
is consistent
of our study together,
differentiation evidence
Further
(19) and, moreover,
indirectly,
accumulating
of fibroblastic
and/or
role in the macrophage
This latter finding
in the
acted directly
culture system employed
that the effects of IGF-I
less, taken
of an antiserum
mean that IGF-I
but the involvement
could have played a significant
a
induced by CSF-1, there was still a
types cannot be ruled out. In fact, the influence cultures
with
CSF-1 was co-operating
in the presence
colony formation
experiments
and
factor for the macrophage/monocyte
produced
capable of totally preventing
COMMUNICATIONS
of both proliferating
of the neutralization
(Fig. 5). Nonetheless,
response
RESEARCH
were due to a stimulation
to CSF-1, the specific growth
lineage (2,18), showed action
AND
may be involved
(22) suggesting
that
in the regulation
or normal tissue remodelling
sites.
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS
We are grateful to Drs. N. Loveridgde and S.P. Robins for critical reading of the manuscript. We also thank Drs. A. Skottner, J.J. van Wyk and E.R. Stanley for their generous gifts of rhIGF-I, and the anti-IGF-I and CSF-1 antibodies, respectively. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22.
Dougherty, G.J., McBride, W.H. (1984) J. Clin. Lab. Immunol. 14: l-11. Metcalf, D. (1987) Proc. R. Sot. Lond. B. 230: 389-423. Kurland, J., Broxmeyer, H.E., Pelus, L.M., Bockman, R.S., Moore, M.A.S. (1978) Blood 52: 388-407. Nathan, C.F., Murray, H.W., Cohn, Z.A. (1980) N. Engl. J. Med. 303: 622626. Auron, P.E., Webb, A.C., Rosenwasser, L.J., Mucci, S.F., Rich, A., Wolff, S.M.. Dinarello. C.A. (1984) Proc. Natl. Acad. Sci. USA 81: 7907-7911. Old,‘L.J. (1985) Science 23b: 630-632. Sluiter, W., Hulsing-Hesselink, E., Elzenga-Claasen, I., Van HemsbergenOomens, L.W.M., Van der Voort van der Kleij-van Andel, A.,, Van Furth, R. (1987) J. Exp. Med. 166: 909-922. Rom, W.N., Basset, P., Fells, G.A., Nukiwa, T., Trapnell, B.C., Crystal, R.G. (1988) J. Clin. Invest. 82: 1685-1693. Petruson, B., Hansson, H.A., Petruson, K. (1988) Acta Otolaryngol. (Stockh) 106: 156-160. Nagaoka I., Trapnell, B.C., Crystal, R.G. (1990) J. Clin. Invest. 85: 448-455. Zapf, J., Froesch, E.R. (1986) Hormone Res. 24: 121-130. Pepe, M.G., Ginztzon, N.H., Lee, P.D.K., Hintz, R.L., Greenberg, P.L. (1987) J. Cell. Physiol. 133: 219-227. Akahane, K., Tojo, A., Tobe, K., Kasuga, M., Urabe, A., Takaku, F. (1987) Exp. Hematol. 15: 1068-1079. Kurtz, A., Zapf, J., Eckhardt, K.U., Clemmons, G., Froesch, E.R., Bauer, C. (1988) Proc. Natl. Acad. Sci. USA 85: 7825-7829. Sawada, K., Krantz, S.B., Dessypris, E.N., Koury, S.T., Sawyer, S.T. (1989) J. Clin. Invest. 83: 1701-1709. Dijkstra, C.D., Dopp, E.A., Joling, P., Kraal, G. (1985) Immunol. 54: 589-599. Van Wyk, J.J., Russell, W.E., Underwood, L.E., Svoboda, M.J., Gillespie, G.Y., Pledger, W.J., Adashi, E.A., Balk, S.D. (1986) In: Human growth hormone. S. Raiti and R.A. Tolman, editors. Penum Medical Book Company, New York. 585-599. Stanley, E.R. (1985) Meth. Enzymol. 116: 564-587. Hume, D.A., Allan, W., Fabrus, B., Weidemann, M.J., Hapel, A.J., Bartelmez, S. (1987) Lymphokine Res. 6: 127-140. Vaes, G. (1987) Clin. Orthop. Rel. Res. 231: 239-271. Tominaga, S.I. (1988) J. Cell. Physiol. 135: 350-354. McInnes, A., Rennick, D.M. (1988) J. Exp. Med. 167: 598-611.
653
174,
January
No.
BIOCHEMICAL
2, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
31, 1991
STIMULATION MULTINUCLEATED
CELL
OF MACROPHAGE FORMATION
BY INSULIN-LIKE
GROWTH
IN RAT BONE GROWTH
AND
MARROW
FACTOR
647-653
CULTURES
I
Ben A.A. Scheven* and Nicola J. Hamilton The Rowett Research Institute, Received
December
14,
Bucksburn, Aberdeen Al32 9SB, UK
1990
In this study the effects of rhIGF-I on macrophage differentiation and growth have been studied using liquid suspension cultures of rat bone marrow cells. IGF-I stimulated macrophage growth in a dose-dependent manner, a maximum response was found at a concentration of 20 rig/ml. IGF-I effects could be ascribed to stimulation of both postmitotic and proliferating cells. A remarkable finding was that IGF-I induced formation of multinucleated cells (MNC). The MNC resembled macrophage-like cells (AcP, NSE positive). A monoclonal antibody to rhIGF-I significantly inhibited IGF-stimulated macrophage growth and MNC formation. A specific antibody to mouse CSF-1 reduced IGF-stimulated macrophage growth in mouse bone marrow cultures indicating that IGF-I effects could, at least in part, be ascribed to endogenous production of CSF-1. These findings indicate that IGF-I in concert with locally induced CSF-1 can influence the differentiation and growth of bone marrow-derived macrophages. o 1991Academic press. I”=. Macrophages
are derived from bone marrow progenitors
various tissues where they perform reactions and tissue remodeling
many diverse functions
(1). Though
the knowledge
biology is increasing, the precise cellular and biochemical
and homed in
related
to defence
about macrophage
processes regulating
the
development and growth of macrophages in the haemopoietic and “target” tissues have not been clearly elucidated (1,2). Macrophages are capable to secrete bioactive molecules, paracrine cellular
some of which are considered to be involved in autocrine or growth (3-7). It has been shown that activated alveolar macro-
phages produce insulin-like growth factor (IGF)-type molecules (8). In another study it was reported that macrophages in nasal inflammatory foci displayed a
*Author responsible for correspondence (present address of both authors): Dr. B.A.A. Scheven, University Hospital, Department of Internal Medicine Research Group for Bone Metabolism, P.O. Box 85.500, 3508 GA Utrecht, The Netherlands
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
strong immunocytochemical staining with an anti-IGF-I antibody (9). Very recently, Nagaoka et al., (10) described IGF-I gene expression in human macrophage-like cells. These studies suggest that IGF-I
may play an important
role in inflammatory
reactions. IGFs exert mitogenic
and differentiation
promoting
activities
in various
tissues (11). IGFs have also been suggested to play a role in the regulation haemopoiesis.
It has been shown that IGF-I
of
receptors are present on human
leukemic cells (12) and fetal mouse liver cells (13). Furthermore, it has been demonstrated recently that erythroid colony-forming units require direct interaction with IGF-I
for further development
The present influence
experiments
(14,1.5). were designed
the growth of macrophages.
We report
to study whether here that IGF-I
IGF-I
can
stimulates
macrophage growth in serum-free cultures of rat bone marrow cells and that IGFI induces multinucleated cell formation. MATERIALS
AND METHODS
Cytokines and antibodies. Recombinant human IGF-I (rhIGF-I) was purchased from Amersham (UK) or was obtained from Dr. A. Skottner, KabiVitrum (Stockholm, Sweden); no differences were noted between the different rhIGF-I preparations. The monoclonal mouse antibody to rhIGF-I (sm 1.25) was a gift from Dr. J.J. van Wyk (University of North Carolina, Chapel Hill, USA), and the anti-CFS-1 antibody (anti-L cell CSF-1 goat antiserum) was supplied by Dr. E.R. Stanley (Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA). Bone marrow culture. Bone marrow cells were isolated from the long bones of the hind limbs of adult rats (200 g) by flushing the bones with Hanks balanced salt solution (HBSS) using a fine gauge needle. The cells were first pre-cultured in a culture flask containing MEM (Gibco, Scotland) supplemented with 1% fetal bovine serum (FBS) (Flow Lab., UK) for 4 h at 37°C in a 5% CO, in air atmosphere. This producedure promotes the attachment of fibroblastic cells and mature macrophages. The non-adherent mononuclear cell population which is more or less enriched for immature myeloid cells (granulocyte-macrophage progenitor cells) was used for further incubation in control or test media in 24-well-plates under the same conditions as above (about 20,000 cells in 0.5 ml per well). The culture medium contained 1 mg/ml bovine serum albumin (BSA, Sigma) but no serum. At different time points the cultures were ended and the adherent cells were stained for acid phosphatase (AcP) or nonspecific esterase (NSE) using Sigma histochemical kits. Counterstaining was performed with haematoxylin. To confirm the macrophage-like nature of the enzyme positive cells found in the cultures, an immunocytochemical staining using the monoclonal antibody ED1 was carried out as described previously (16). Autoradiography. Bone marrow cells were incubated in serum-free MEM/BSA in the presence of 0.065 &i/ml tritiated thymidine (3H-TdR; spec. act.: 2 Ci/mmol, Amersham, UK) in tissue chamber slides (Flow Lab.). After 3 days the cultures were stained for AcP and subsequently processed for light microscopic autoradiography. Briefly, the slides were dipped in Kodak K5 emulsion and exposed in a light-tight box for 2 weeks at 4°C; development was carried out in Kodak D19 and fixation in Kodak Unifix solution. 648
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Cell counting and statistics. Macrophages were identified by their morphology and by their strong staining for AcP or NSE activity. The number of mononuclear (labelled and non-labelled in the autoradiographs) macrophages were counted in 10 random microscopic fields. The number of multinucleated cells (MNC) was determined by screening the whole well. Statistical differences were assessed by the Student’s t-test. RESULTS After
preculture
of the whole rat bone marrow preparation,
the non-
adherent cells were cultured in serum-free control media or in media containing recombinant
human IGF-I.
After culture the adherent macrophages were identified
by their strong AcP or NSE activity. No other (hemopoietic)
cell types remained
attached in the cultures, except for a few fibroblasts, which could be easily distinguished from the macrophages on the basis of their morphology staining. IGF-I
did not significantly
IGF-I
a dose-dependent
elicited
cultures (Fig. 1). Interestingly,
alter the numbers of fibroblastic increase in the number
nature.
Furthermore,
cells, however,
of macrophages
in the
MNC were frequently observed in the IGF-cultures.
These MNC displayed strong AcP and NSE staining indicating like
and weak enzyme
the cells were positively
labelled
their macrophagewith
the EDl-
monoclonal antibody, which specifically recognizes members of the mononuclear phagocyte system. Addition of sodium tartrate to the AcP substrate solution inhibited the enzyme reaction in most of the MNC. MNC formation occurred after 3 days in vitro and gradually increased thereafter (Fig. 2). Some MNC were also observed in control cultures indicating susceptible to MNC formation
0 1
IGF-I 1020concentration40
(rig/ml) 80
that rat bone marrow cells are especially
in vitro. The average number of nuclei of the MNC
02
0 Culture
time
(d)
dose-response relation for mononuclear macrophage (0) and MNC (0) formation in 5-days’ cultures. Mononuclear macrophages and MNC were identified by positive reaction for AcP or NSE staining. Results are expressed as the number of macrophages counted in 10 random microscopic fields, and the total number of MNC per well. The results are mean + SEM of 3 independent experiments (n = 12). Fig. 2. Time course of MNC formation in control (0) or IGF-I (0) cultures. IGFFie. 1. IGF-I
I concentration was 80 “g/ml. The total number per well was counted. Results are mean +- SEM of 4-6 cultures. 649
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
was 5.1 + 0.4 and 3.7 + 0.1 for the 5-days’ IGF- and control cultures respectively. A maximal response for MNC induction was found at a concentration of 40 rig/ml, whereas a dose of 20 rig/ml
was sufficient for optimal
nuclear macrophages (Fig. 1). To further define the effects of IGF-I graphy experiments presence of tritiated stimulated
were performed thymidine
the formation
on macrophage
of the mono-
growth, autoradio-
on bone marrow cultures incubated
(3H-TdR).
of labelled
stimulation
After 3 days in culture, IGF-I
macrophages indicating
in the greatly
that the IGF-I-stimu-
lated macrophage growth was mainly due to an effect on the replication of progenitor cells (Fig. 3, upper panel). However, there was also a significant increase in the number of non-labelled
macrophages
and this means that IGF-I
also stimulated macrophage differentiation from nonproliferating, postmitotic cells (Fig. 3, upper panel). A similar response can be seen in the distribution of nuclei in the MNC
(Fig. 3, bottom
panel). The labelling
index was 38 f 7.5% for the Mononuclear
a
Mononuclear 60
7
0
Unlabelled
-
Labelled
‘;; G I
Multinuclear
2
IGF-I
Multinuclear
z s 203o I-t
a
w
C
a r-l
a,b
(.-got- Lh Control
C
IGF-I
IGF-I
&,.-& Effect of IGF-I on incorporation of tritiated thymidine into bone marrow macrophages. Cultures were labelled for 3 days followed by AcP histochemistry and LM autoradiography. The average number of labelled (closed columns) and nonlabelled (open columns) mononuclear macrophages in 10 microscopic fields (upper panel) or the average number of labelled (closed columns) of non-labelled nuclei in the MNC (bottom panel) k SEM are shown. a: significantly different from unlabelled controls (p < 0.05); b: significantly different from labelled controls (p < 0.001). EIg9. Effect of monoclonal antibody to IGF-I on mononuclear macrophage growth [top panel) and MNC induction (bottom panel) in control (c) or IGF-I cultures. Open columns are cultures without antibody; closed columns are cultures with antibody (l/4000 dilution; i.e. about 2.5 fig/ml IgG). Normal purified control mouse IgG had no effect on the number of macrophages in control or IGFcultures. IGF-I concentration was 40 “g/ml. Results are mean + SEM of 4 cultures. a: p < 0.01 vs control values; b: p < 0.01 vs IGF-I values. 650
Vol.
174,
No.
2, 1991
BIOCHEMICAL
=”
AND
50
BIOPHYSICAL
a
COMMUNICATIONS
a
u” ;
RESEARCH
a,b
30
z 2
10 i
:
0
Ub ‘/a000
c
‘I4000
anti-CSF-I
Ab
-IGF-I-
Fig. 5. Effect of antibody to CSF-1 on macrophage proliferation in mouse bone marrow cultures. IGF-I concentration was 80 rig/ml. Control, non-immune, goat serum in the same dilution range as the CSF-1 antiserum did not significantly affect macrophage numbers in the control or IGF-cultures. Results are mean + SEM of 4 cultures. a: p < 0.01 vs control values; b: p < 0.005 vs IGF-I values.
MNC nuclei, a figure similar to that found for the mononuclear
macrophages (37.9
+ 2.3%; p < 0.005 vs control values). These results suggest that the MNC were formed from the mononuclear was probably mononuclear
population
implying
caused by the fusion of the postmitotic of a monoclonal
led to a significant inhibition
antibody to rhIGF-I
of the IGF-stimulated
and recently replicated (17) to the culture medium
macrophage growth and MNC
in the bone marrow cultures (Fig. 4). This indicates that the responses
found in the cultures were IGF-I
dependent
and not a non-specific
culture. However, these results do not exclude the possibility stimulated
the cultures by endogenous production
phages and fibroblasts production
that this process
cells.
Addition formation
macrophage
that IGF-I
effect of indirectly
of other cytokines. As macro-
may be a source of CSF-1, we studied whether local
of CSF-1 was responsible for the IGF-effects
found. The effect of a
specific antibody to mouse CSF-1 (18) was therefore investigated in cultures of bone marrow cells isolated from mice. Dilutions of the CSF-1 antiserum were used that either were completely inhibitory (1:4000) or partly inhibitory (+ 50%; 1:8,000) for colony-formation (Stanley and Williams,
induced by CSF-1 in semisolid personal communication).
mouse bone marrow cultures
IGF-I also stimulated
macrophage
growth in all mouse bone marrow cultures (Fig. 5). Though some MNC were found in these cultures, the response was less than that found in the rat bone marrow cultures (not shown). The anti-CSF-1 antiserum inhibited the IGF-I-stimulated macrophage growth in a dose-dependent manner, indicating that endogenous production of CSF-1 was involved in macrophage growth in these cultures (Fig. 5). DISCUSSION
Here we have presented in vitro evidence that IGF-I was able to influence macrophage production from bone marrow. From the autoradiography results it is 651
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174,
No.
2, 1991
BIOCHEMICAL
clear that the effects of IGF-I postmitotic
macrophages.
specific antibody of IGF-I
BIOPHYSICAL
The results
that endogenously
growth
This could
mononuclear
to IGF-I.
phagocytes,
I. However, were
in the bone marrow
minimally
response
represented
to IGF-I.
indicating
of other factors
directly
the results
and/or
macrophage
haemopoiesis
(12-15),
work,
is required induced
that IGF-I
and that IGF-I
observation
accomplished
by a monoclonal driven by IGF numbers
cells present in the response
cultures
are small (17). Nonethe-
we suggest that IGF-I locally
induced
This supposition
is implied
CSF-1,
antibody
supplemented
of the MNC
indicating
that the MNC
resorption,
the osteoclasts,
to IGF-I.
macrophages
Whether
assays,
did not display were
to the MNC
responsible
IGF-I
cultures
to induce MNC formation
D, (20), interferon (this
in pathogenic
reactions
652
for bone
positive cells was observations).
in vitro, such as 1,25-
(21) and interleukin-4 study)
activity
high levels of this enzyme (20). (unpublished
Other agents have been reported
cell forma-
AcP (TRAP)
enhanced
to control
macrophage
reactions or tissue remodel-
significantly
compared
was
was specifically
of giant multinucleated
as these cells contain
greatly
and greatly inhibited
in mononuclear
tartrate-resistant
not similar
were
This process
this phenomenon
to the increase Generation
MNC
of TRAP
formation
of
(g-10).
and colony-forming
it should be noted here that the number
control MNC
with
biology
Nonetheless,
agents, including
in regulating
role in the regulation
with IGF-I.
associated with defence (inflammatory)
ling. Most
several
is involved,
is in accordance
in macrophage
cell populations
by fusion of postmitotic
needs yet to be clarified.
dihydroxyvitamin
to IGF-
in this study, fibroblasts
was that macrophage-like
or could be ascribed
tion is generally
other cell
this point.
in the bone marrow
apparently
on the
data from the literature
plays an important
for example on purified
An interesting
with
and growth.
to substantiate
with
alone on proliferation
in concert
dilution
they did not show any mitogenic
is consistent
of our study together,
differentiation evidence
Further
(19) and, moreover,
indirectly,
accumulating
of fibroblastic
and/or
role in the macrophage
This latter finding
in the
acted directly
culture system employed
that the effects of IGF-I
less, taken
of an antiserum
mean that IGF-I
but the involvement
could have played a significant
a
induced by CSF-1, there was still a
types cannot be ruled out. In fact, the influence cultures
with
CSF-1 was co-operating
in the presence
colony formation
experiments
and
factor for the macrophage/monocyte
produced
capable of totally preventing
COMMUNICATIONS
of both proliferating
of the neutralization
(Fig. 5). Nonetheless,
response
RESEARCH
were due to a stimulation
to CSF-1, the specific growth
lineage (2,18), showed action
AND
may be involved
(22) suggesting
that
in the regulation
or normal tissue remodelling
sites.
Vol.
174,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
ACKNOWLEDGMENTS
We are grateful to Drs. N. Loveridgde and S.P. Robins for critical reading of the manuscript. We also thank Drs. A. Skottner, J.J. van Wyk and E.R. Stanley for their generous gifts of rhIGF-I, and the anti-IGF-I and CSF-1 antibodies, respectively. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22.
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