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Osteogenic Growth Peptide Increases Blood and Bone Marrow Cellularity
and Enhances Engraftment of Bone Marrow Transplants in Mice
By Olga Gurevitch, Shirnon Slavin, Andras Muhlrad, Arye Shteyer, Dan Gazit, Michael Chorev, Marina Vidson,
Malka Narndar-Attar, Esther Berger, llan Bleiberg, and ltai Bab
The osteogenic growth peptide (OGPI was characterizedrecently in regeneratingbone marrow (BM) and normal serum.
In vitro, the OGP regulates stromal-cellproliferationand differentiated functions. In vivo, an increase in serum OGP accompaniesthe osteogenic phaseof postablation BM regeneration. The present resultsin normal mice show that OGP
induces a balanced increasein WBC counts and overallBM
cellularity. In mice receiving myeloablative irradiation and
syngeneicorsemiallogeneic BM transplants, OGP stimulates hematopoieticreconstruction and doubles
the survival
rate; these effects are dependent on initiating the OGP administrationbefore irradiation.Chimerism measurementsin
semiallogeneicgraft recipients suggest no preferential effect
of OGP on residual host cells. The data implicate
OGP in the
acceleration of hematopoiesis secondary
to expansion ofthe
stromal microenvironmentandlor enhancement of stromaderivedsignals to stem cells.Thelow-doseeffectiveness
of OGP is explained by the demonstration of an autocrine
positive feedback loopthat together with the OGP-binding
protein sustains high serum levels
of the peptide. A potential
OGP-based treatment in combination with chemoradiotherapy isattractive because of the OGP-induced balancedmultilineageenhancement of hematopoiesisandpossiblereplacement of expensive recombinant cytokines
by a readily
synthesized peptide.
0 1996 by The American Society of Hematology.
R
is present physiologically in human and other mammalian
serum, mainly in the form of an OGP-OGP-binding protein
c ~ m p l e x . ’ ~A, ’marked
~
increase in serum boundandunbound OGP accompanies the osteogenic phase of postablationBM regeneration and associated systemic osteogenic
response.”
Because of both the importance of bone as part of the
stromal hematopoietic microenvironment and the OGP stimulation of stromal-cell proliferation and bone formation, the
present in vivo experiments were designed to study the peptide’s regulatory role in hematopoiesis, in particular after
myeloablative irradiation and BMT.
EGULATION OF hematopoiesis remains an important
goal of both clinical and experimental studies. It is
particularly relevant for the enhancement of hematopoietic
reconstruction to reduce morbidity and mortality in patients
subjected to radiotherapy and chemotherapy, as well as after
myeloablative chemoradiotherapy and autologous or allogeneic bone marrow transplantation (BMT).’
Hematopoietic stem cells home to the BM compartment,
suggesting a specific affinity of progenitor cell-surface determinants to the BM stroma.* Physiologically, the formation
of hematopoietic BM follows new bone formation in the
embryo and during repair of bone
In addition,
bone formation precedes hematopoiesis in instances of heterotopic ossification, such as myositis ossificans and ossicles
induced by bone morphogenetic protein^.'.^ Perhaps the most
distinct relationship between the formation of bone and BM
is that observed after BM injury.’,’ In this process, an intense
osteogenic phase precedes the appearance of a new hematopoietic tissue. In humans and experimental animals, the local
osteogenic response is accompanied by a systemic enhancement of bone formation:”’ an activity mediated by factors
released into the circulation by the healing tissue.” Recently,
one of these factors, a 14-amino acid peptide, has been
characterized and named osteogenic growth peptide (OGP).I3
OGP is identical to the C-terminal sequence of histone H4
and shares a five-residue motifwith a T-cell receptor pchain V-region and the Bacillus subtilis outB locus. In vitro,
OGP stimulates the proliferation of osteoblastic and fibroblastic cells. When administered to rats, it stimulates bone
formation and increases bone mass. OGP in high abundance
Table 1. Effect of Grafted Cell Number on 30-Day Survival of Mice
Receiving Syngeneic EMT
Experiment
Cell No.
Live Mice
I
4 X 104
II
4 X 105
4 x 108
4 X 104
4 X 105
4 x 106
0/1 o*
6/10
7/10
1/10
5/10
8/10
* Experiments were performed in groups of 10 mice per condition.
Data are proportion of live animals.
Blood, Vol 88, No 12 (December 15). 1996 pp 4719-4724
MATERIALS AND METHODS
Animals. FemaleC57BL/6(C57), BALB/C (BALB), and firstgeneration (F,) mice weighing 20 to 24 g werepurchased from
Harlan Sprague Dawley (Indianapolis, IN).
Adnzinistration of OGP. Synthetic OGP (sOGP), identical to the
native polypeptide,was prepared asdescribed previou~ly”~’~
according to the standard solid-phase peptide synthesismeth~dology.’~
Forinjection,sOGP was dissolved in phosphate-bufferedsaline
(PBS) and administered subcutaneously (SC) or intravenously (IV).
Effect of OGP on blood and BM cellularity. sOGP, 0.1 nmol in
From the Department of Bone Marrow Transplantation and Cancer/lmmunobiology Research Laboratory, Hadassah-Hebrew University Hospital, Jerusalem; Departments of Oral Biology, Oral and
Maxillofacial Surgery, Oral Pathology and Bone Laboratory, Faculty of Dental Medicine and Department of Pharmaceutical Chemistry, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem; Department of Histology and Cell Biology, Sackler Medical
School, Tel-Aviv University, Tel-Aviv, Israel.
Submitted May 15, 1996: accepted August 6, 1996.
Supported in part byResearch Grant No. 3584-91 fromthe Ministry of Science and Technology, the Government of Israel.
Address reprint requests toShimon Slavin, MD, Professor and
Chairman, Department of Bone Marrow Transplantation and Cancer/Immunobiology Laboratory, PO Box 12000, Jerusalem 91 120,
Israel.
The publication costs of this 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 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8812-0016$3.00/0
A714
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GUREVITCH ET AL
4720
30r
PBS
SOGP
Fig l. Enhancement of normal blood and BM cellularity by OGP.
Data are mean SE obtained in 5 mice per treatment group. *WBC
counts higher inOGP- over PBS-treated mice, Mann-Whitney, P =
.016; **Femoral BM cell counts higher in OGP-over PBS-treated mice,
Mann-Whitney, P = ,004.
1 0 0 pL PBS, was injected SC every second day to C57 mice. The
animals were killed 2 weeks after the onset of treatment. At the time
of death, the BM from both femurs was flushed through the distal
metaphysis to form a cell suspension in PBS. Differential cell counts
were performed in smears stained with polychrome methylene blue/
eosin. Blood samples were obtained from the retroorbital sinus with
heparinized glass capillaries immediately before the animals were
killed. These samples were subjected to differential WBC counts in
a Coulter Counter (Coulter Electronics, Hialeah, FL), as well as
RBC and platelet counts.
Effect of OGP on engrafiment of BM transplants. Mice were
exposed to a single dose of myeloablative total-body irradiation
(TBI) delivered using Clinac 6X Linear Accelerator (Varian Associates, Palo Alto, CA). Twenty-four hours later, the mice were inoculatedwith a limiting number (4 X IO4 to 1 X IO5) (Table l)" of
BM cells. Syngeneic BMT was performed in CS7 mice receiving
900 cGy TBI. The recipients of semiallogeneic BMT were F, hybrids
conditioned with 1200 cGy TB1 and receiving a BM-cell inoculum
from either C57 or BALB mice. The treatment consisted of daily
injections of 0.001 to IO nmol sOGP in 1 0 0 pL PBS commencing
on day 8 before irradiation and until day 4 thereafter. Total BM cell
femora. Some
counts were performed 8 days post-BMTinboth
experiments tested the effect of OGP on survival after syngeneic
BMT. To assess the OGP differential effect on transplanted donor
and residual recipient cells, blood samples from some recipients of
semiallogeneic BMT were subjected to chimerism measurements.
Detection of chimerism. Peripheral blood lymphocytes from
BALB F1 chimeras were separated over a Ficoll-hypaque gradient.
-.
After lysis of residual erythrocytes, the cells were treated with antiCS7 antiserum and complement or complement alone as described
previously'* andthe percentage ofkilled cells determined using
trypan blue dye exclusion.
Effect of sOGP on . w r m OGP /eve/s. To assess the effectof
treatment on the serum OGP levels, normal CS7 mice were administered a single SC dose of 7 X IO-' to 7 X IO' nmolsOGP. The
OGP levels were measured in blood samples obtained fromthe
retroorbital sinus 24 to 96 hours later. Because of a possible effect
of bleeding on the serum OGP
different animals were used
in the various time groups.
Serum OGP meas~trements. OGP levels were determined by
competitive enzyme-linked immunosorbent assay (ELISA) asreported
The assay was performed using rabbit antiOGP antiserum generated against N-Ac[(Cys")]OCPconjugatedwith
maleimide-activated keyhole limpet hemocyanin. Steady-state levels
were measured in fresh samples. To determine the total OGP. the
serum was boiled before the assay. Boiling differentially denatures
the OGP-binding protein and releases the intact OGP."
RESULTS
In normalmice,the sOGP induced a greater than 50%
increase in the WBC counts and approximately 40% enhancement of the BM cellularity (Fig l ) . The proportion of
the different cell types was not altered bythe OGP treatment
(Tables 2 and 3). suggesting a nondiscriminative enhancement of hematopoiesis. When administered SC in combination with syngeneic BMT, OGP stimulated the femoral cellularity dose dependently between 0.01 and IO nmol/dper
mouse. The maximal effect, a twofold stimulation, was triggered by the I - andIO-nmol/d doses (Fig 2). The initial
calibration experiments (data not shown) indicated that this
effect was missing unless the OGP treatment began at least
1 week before the ablative radiotherapy andBMT.When
dosed optimally with sOGP, 20 of 24 (83%) lethally irradiated mice reconstituted with syngeneic BMT survived at 30
days. In the sham-treated group, only I I of 25 (44%) animals
subsisted (Fig 3). In the OGP-treated group, no deaths occurred after day 14; the preceding mortality rate had been
0.07 mice per day. In the sham-treated group, deaths continued until day 20, with an average 10-fold higher mortality
rate (0.7 mice per day) (Fig 3). The 30-day mortality rate
wasmorethan threefold higher in the sham-treated group
as compared with OGP-treated mice. When semiallogeneic
BMT from C57 or BALB donors was administered to lethally irradiated F, hybrids, recipients treatedwith sOGP
(0.01 nmol daily IV) showed respective twofold and 3.6-fold
increases in their total femoral cellularity (Fig 4). Antibodyinduced killing of peripheral blood lymphocytes from the
chimeric mice showed a continuous reduction in the proportion of recipient cells over a 40-day period. This proportion
was similar in the sOGP- and sham-treated controls (Table
Table 2. Differential Cell Counts in Blood of Normal Mice Treated With OGP
X Nucleated Cells Present
Cells
Polymorphonuclear
Lymphocytes Group
PBS
OGP
80.5
13.1 2 0.6
80.8 2 0.1
-c 0.5
12.8
6.4 2 0.7
NOTE. Data are means -c SE. Total counts are shown in Fig. 1.
10.2
6.3 2 0.3
2 0.5
2 0.1
10.9 2 0.1
853 -C 55
805 2 43
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OGP ENHANCES ENGRAFTMENT
IN
OF BMT
MICE
4721
Table 3. Differential Cell Counts in Femoral Bone Marrow of OGP-Treated Normal Mice
Grow
MEL
PMN
EOS
MON
ERC
MKE/MKC
LYM
PLC
PBS
OG P
17.2 2 1.3
17.0 2 0.4
22.3 2 1.0
24.2 t I.?
6.4 t 0.8
7.2 2 1.1
2.8 2 0.3
2.8 2 0.2
30.6 2 0.8
27.8 C 1.4
1.0 t 0.2
0.9 2 0.4
18.0 2 1.1
18.5 2 1.5
I.? t 0.2
1.6 2 0.4
NOTE. Data are mean 2 SE of percent of all cells present. Total counts are shown in Fig. 1.
Abbreviations: MBL, myeloblasts; PMN, neutrophiles; €OS, eosinophiles; MON. monocytes;
MKC, megakaryocytes; LYM, lymphocytes; PLC, plasma cells.
4). These measurements of chimerism suggest that the sOGP
effect does not discriminate between donor and recipient
(possibly residual) hematopoietic cells.
A single SC sOGP dose (0.1 nmol) induced a progressive
linear increase in serum immunoreactive OGP (irOGP) concentration that peaked after 48 hours and declined gradually
thereafter (Fig 5). Considering the 2-mL volume of mouse
blood, the respective overall enhancement of the steady state
and total irOGP content was approximately fourfold and 34fold higher than the amount of injected sOGP. An experiment
designed to further explore theOccurrence of a possible feedback regulationof the serum OGP content, showed that SOGP,
administered once, stimulates the endogenous peptide levels
dose dependently (Fig 6). The totalandsteady-stateirOGP
levels wereincreasedby 7 X
to 7 X 10” and 7 X
to 7 X 10” nmol, respectively, followed by a dose-response
reversal of the stimulatory effect at higher doses (Fig 6).
DISCUSSION
The present results demonstrate that OGP treatment enhances the blood cellularity of normal mice under resting
conditions, as well as the engraftment of BMT in myelo-
ERC, erythroid cells; MKB, rnegakaryoblasts:
ablated recipients. The differential cell counts inboth the
blood andBM show thatOGP induces a multilineage nondiscriminative stimulation of WBCs, RBCs, and platelets. This
is an important feature that distinguishes OGP from many
of the hematopoietic polypeptide factors, such as the colonystimulating factors, some of the interleukins, and erythropoietin, which, unless administered in synergistic combinations,
affect preferentially well-defined hematopoietic
Another recently reported hemoregulatory synthetic peptide,
SK&F107647, also has a multilineage
This effect
is probably mediated by enhancement of cytokine production
by stromal ~ e l l s . ’ ~However,
-~~
it is rather unlikely that the
SK&F107647 increases the production of all the cytokines
to the same extent and therefore differences in the magnitude
of mitogenic stimulation of the different hematopoietic lineages are to be expected. In fact, an effect of SK&F107647
on thenumber of terminally differentiated hematopoietic
cells has not yet been shown. On the other hand, the OGP
increases similarly the number of all morphologically differentiated hematopoietic cells, suggesting a mitogenic effect
on early progenitors or cells expressing hematopoietic regulatory polypeptides.
In addition to its stimulatory effect on hematopoiesis in
normal mice, OGP enhances hematopoietic reconstitution
after syngeneic and semiallogeneic BMT in lethally irradiated mice.Using a limiting number of donor cells, OGP
treatment more than doubled the BM cellularity already 1
-
f 16
0
P&
............. ............ 100 ng sOGP /2 “dday
25
7
11
lb
X
l
v)
J
J
e
10
’
I
1
4 I
c
0.001 0.01
I
0.1
I
1
20
-
15
-
I
10
10
nmol/mouse/day
0
10
20
30
Days after BMT
Fig 2. Effect of OGP on engraftment ofsyngeneic BM. Animals
were administered 4 x 10‘donor cells. Data are mean 2 SE of total
femoral medullarycell countsin 6 to 9 mice per condition.
Correlation
coefficient between log SOGP dose and cell number is significantat
P < .01.
Fig 3. Effect of OGP on survival after syngeneicBMT. Animals
were administered 4 x IO‘ donorcells;30-daysurvivalofsOGPtreated mice was higher than PBS controls at P = .007 (Fisher‘s exact
probability test).
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GUREVITCHET AL
4722
ea
PBS
sOGP
C57BI into
BALB/C into
(BALB/CXC57Bl)Fl
(BALB/CXC57BI)Fl
Fig 4. Effect of OGP on engraftment of semiallogeneic BM. Animals were administered 1 x lo5 donor cells. Data are mean f SE of
total femoral medullarycell countsin 10 mice per condition. *Femoral
BM cell counts higher
in OGP- overPES-treatedmice, Mann-Whitney,
P < .01.
week post-BMT. That the newly established hematopoietic
tissue is indeed functional is shown by our survival experiments in whichthe30-daymortality
rate was threefold
higher in sham-treated control than OGP-treated recipients.
The mechanism of the OGP stimulation of the BMT engraftment appears to differ from that of the hematopoietic
growth factors. These factors are targeted directly to hematopoietic progenitors”’.’’ andtherefore applied to minimize the
pancytopenic risk period by ( I ) expansion and mobilization
in vivo, or ex vivo, of autologous and allogeneic cell inoculi
beforecryopreservationandtransplantation,respectively,3””
or (2) administration to recipients commencing at the time
of transplantation or shortly thereafter.3s.’”’ Onthe other hand,
in the case of the OGP acceleration of BM regeneration postBMT, it seems essential to initiate treatment at least 1 week
before ablative radiotherapy andBMT. The progressive
decline in host-derived WBC counts (percent killed C57marker-bearing cells), which was similar in OGP and
PBS-treated chimeric recipients, suggests that unlike interleukin-l, for example,” preirradiation OGP treatment
does not confer radioprotection to hematopoietic progenitors.
The present data therefore implicate OGP in the acceleration
of hematopoiesis secondary to an expansion of the stromal
microenvironment” and/or enhancement of stroma-derived
signaling to hematopoietic stem cells.’x This conclusion is
supported by the following considerations: The stromal cells
-
Table 4. Proportion of Host Peripheral Blood Lymphocytes in
OGP-Treated BALE F, Chimeras
% Killed Cells
Days
Post-BMT
OGP
PES
20
40
46.2 -C 4.1
9.3 -C 0.7
44.0 2 4.0
9.1 t 0.6
NOTE. Data are means
-C
SE obtained in 5 mice per condition.
are substantially more radioresistant than their hematopoietic
counterparts’”“‘)and their proliferation is accelerated by
OGP. I3.15.J I It is not unlikelythat this is also the in vivo
scenario inasmuch as OGP administration to experimental
animals induces an increase in osteoblast number, bone formation,and trabecular bone mass.” Osteoblasts and other
stromal cells express an array of hematopoietic regulatory
polypeptides’5-’”.‘’ and, therefore, a closely matched multilineage acceleration of hematopoiesis would be anticipated if
the OGP mitogenic upregulation of these cells is well balanced.
To explain the low-dose effectiveness of the OGP treatment we performed postadministration measurements of serum irOGP.Most of theserum OGP is complexed to a
binding proteinr3”’: theboundandmostoftheunbound
irOGP is identical to OGP( l-I4).l4The unbound OGP levels
are represented by the steady-state irOGP, which is a substantial overestimation usefulonly for comparative purposes.“.“ It is the unbound OGP that presumably conveys
the peptide’s biologic activity. A single sOGP dose induces
a vast increase in serum irOGP, in particular bound OGP.
This increase ismaintained
for several days, probably
A
t
n
H
0 . 2 5 y 4 ” “
-o
“””””””
””””~
\
0
E, 0.15-
B
‘
2.0
48
0
I
24
I
I
72
t
96
Time after injection (hours)
Fig 5. Delayed effect of administered OGP on serum OGP levels.
(A) Steady-state OGP; (B) total OGP. Data are mean 2 SE obtained
in 5 mice per condition.
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41 23
OGP ENHANCES ENGRAFTMENT OF BMTIN MICE
1 -
0.4
rence of the peptide in man.I4 Furthermore, OGP can be
readily synthesized and replace or supplement expensive recombinant hematopoietic cytokines.
Steady State
Total
REFERENCES
0.1
L
I I
0 .0007 .007 .07
.7
7
70
'
700
'
1.0
Injected OGP (nmol)
Fig 6. Effect of OGP dose on serum OGP levels. Date are mean ?
24 hours in 5 mice per condition. P values for
total and steady-state curves are .l4 and .01, respectively (analysis
of variance).
SE obtained after
through a tightly controlled release of unbound OGP from
the complex.
This relationship between sOGP administration and endogenous peptide levels is consistent with an autocrine positivehegative feedback loop at low and high doses, respectively. Similar feedback loops have been reported for other
polypeptide factors present in the serum, such as interleukin2, interleukin- 12, platelet-derived growth factor, and transforming growth factor
Clinical trials using currently available preparations for
stimulating hematopoiesis consist mainly of the administration of recombinant cytokines such as granulocyte colonystimulating factor, granulocyte-macrophage colony-stimulating factor, and various i n t e r l e ~ k i n s .Most
~ ~ - ~of~ these
cytokines enhance preferentially proliferation of progenitor
cells already committed to particular WBC lineages. Consequently, the critical time to neutrophil recovery is decreased.49 However, these agents do not elicit an overall
balanced multilineage response and, hence, protracted anemia and thrombocytopenia frequently remain major clinical
problems. Furthermore, there are some major concerns regarding the therapeutic use of hematopoietically active cytokines, because some tumors, and especially leukemic cells,
possess normal receptors for these c y t o k i n e ~ ~and
~ ~their
~'
administration can increase relapse rates by enhancing the
proliferation of residual recipient tumor cells that may escape
chemoradiotherapy. In contrast, OGP may offer a balanced
reconstruction of hematopoiesis after high-dose radiotherapy
and chemotherapy and after BMT. If indeed OGP acceleration of hematopoiesis is secondary to the peptide's effect on
the stromal microenvironment and unaccompanied by direct
stimulation of recipient cells, then OGP treatment would be
especially useful in cases of hematopoietic malignancies.
Indeed, preliminary results in our laboratory show that OGP
does not affect the number of transplanted malignant hematopoietic cells (data not shown). OGP-based therapy is particularly attractive because of the physiologic natural occur-
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1996 88: 4719-4724
Osteogenic growth peptide increases blood and bone marrow
cellularity and enhances engraftment of bone marrow transplants in
mice
O Gurevitch, S Slavin, A Muhlrad, A Shteyer, D Gazit, M Chorev, M Vidson, M Namdar-Attar, E
Berger, I Bleiberg and I Bab
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