Differential Effects of the Various Isoforms of Platelet-derived
on Chemotaxis of Fibroblasts, Monocytes, and Granulocytes
Agneta Siegbahn,* Annet Hammacher,t Bengt Westermark,l and Carl-Henrik Heldint
Department of*Clinical Chemistry and Pathology, University Hospital, S-751 85 Uppsala; and
tLudwig Institutefor Cancer Research, Biomedical Center, S-751 23 Uppsala, Sweden
The chemotactic activities of three different isoforms of plate-
let-derived growth factor (PDGF) on fibroblasts, monocytes,
and granulocytes of human origin were investigated. PDGF-
AB and PDGF-BB induced strong, dose-dependent responses
in both fibroblasts and monocytes, whereas PDGF-AA did not
stimulate chemotaxis of these cell types. Instead, PDGF-AA
inhibited the chemotactic activity ofPDGF-AB and PDGF-BB
on fibroblasts and monocytes. However, PDGF-AA was not
able to block monocyte chemotaxis induced by FMLP. In con-
trast, in granulocytes, dose-dependent chemotactic responses
were obtained with all three isoforms of PDGF. All isoforms
gave maximal responses at concentrations between 5 and 20
ng/ml. At higher concentrations the migration was reduced.
Reduction and alkylation of the PDGF molecule, which leads
to loss of the mitogenic activity, also caused a loss of the
chemotactic activities for all three cell types.
The data suggest that the various isoforms ofPDGF stimu-
late and inhibit chemotaxis in an isoform- and cell type-spe-
cific manner. (J. Clin. Invest. 1990. 85:916-920.) chemotaxis.
fibroblasts-phagocytes* PDGF * FMLP
Chemotactic factors are released by various cell types during
wound healing and inflammation, and play an important role
in the recruitment of different types of cells to these sites. A
number of chemoattractants, especially for granulocytes and
monocytes, have been identified. These factors bind to specific
cell-surface receptors, whereby the molecular events of the
chemotactic response are initiated (1, 2). Interestingly, certain
growth factors have been found to have chemotactic activity
for various cell types. Thus, platelet-derived growth factor
(PDGF),I a release product ofplatelet a-granula, is not only a
major mitogen for connective tissue cells in vitro (3, 4), but
also a potent chemotactic agent for fibroblasts (5, 6) and
smooth muscle cells (7).
PDGF is made up as dimers ofA and B polypeptide chains
that are linked by disulphide bonds. All three possible dimeric
Address reprint requests to Dr. Agneta Siegbahn, Department ofClin-
ical Chemistry, University Hospital, S-751 85 Uppsala, Sweden.
Receivedfor publication 26 June 1989 and in revisedform 20 Oc-
1. Abbreviations used in thispaper: NCS, newborn calfserum; PDGF,
platelet-derived growth factor.
forms ofPDGF have been identified and purified from plate-
lets and transformed cells. Human platelets contain PDGF-AB
and PDGF-BB (8, 9), whereas porcine platelets contain mainly
PDGF-BB (10). PDGF-AA is produced, e.g., by human osteo-
sarcoma cells (1 1). The A chain occurs as two variants due to
differential splicing; the COOH-terminal three amino acids in
the shorter variant are replaced by 18 different amino acids in
the longer variant (12, 13). All isoforms ofPDGF are available
in recombinant form (14-16).
PDGF binds to two structurally related but distinct recep-
tor types: PDGF a-receptors, also called A-type receptors, bind
all three isoforms with high affinities, whereas (3-receptors, also
called B-type receptors, bind PDGF-BB with high affinity and
PDGF-AB with lower affinity, but do not bind PDGF-AA with
any appreciable affinity (17, 18).
Previous studies have shown that, in contrast to PDGF
purified from human platelets, PDGF-AA has no chemotactic
activity or ability to induce actin reorganization and mem-
brane ruffling in human foreskin fibroblasts (19); these effects
are therefore likely to be mediated by the (-receptor in these
cells. With regard to the chemotactic activity of PDGF on
granulocytes and monocytes, conflicting data have been pub-
lished (20, 21), and no attempts have been made to discrimi-
nate between the different isoforms ofPDGF. The aim of the
present investigation was therefore to analyze the chemotactic
response of phagocytic cells and fibroblasts to the different
PDGF. PDGF was purified from human platelets, and PDGF-AB and
PDGF-BB were separated by immobilized metal-ion affinity chroma-
tography (8). Recombinant PDGF-AA (long and short variant) and
PDGF-BB were purified to apparent homogeneity from supernatants
ofyeast cells transfected with PDGF A and B chain cDNA constructs,
Purified recombinant PDGF-AA (short variant) and PDGF-BB
were reduced by 25 mM dithiothreitol in 4 M guanidine-HCI, I M
Tris, pH 8.0, and 10 mM EDTA for 2 h at 37°C or 25 min at 680C
(PDGF-BB), followed by alkylation with 100 mM iodoacetamide for 1
h at room temperature. The reduced and alkylated factors were then
dialyzed extensively against 1 M acetic acid at 40C in the presenceof
small amounts ofHSA.
FMLP was purchased from Sigma Chemical Co., St. Louis, MO.
Cell culture. Human foreskin fibroblasts, AG 1523,weregrownto
confluence in Eagle's MEM supplemented with 10% newborn calf
serum (NCS). Before use, the cells were detached by trypsinization (2.5
mg/ml for 10 min at 37°C), washed in Hank's balanced salt solution,
and resuspended in Eagle's MEM with 10% NCS to a cell concentra-
tion of2 X 108 cells/liter.
Isolation ofhuman monocytes and granulocytes. Blood from ap-
parently healthy donors was collected inglasstubes containing heparin
(Venoject, Terumo, Belgium). The blood samples were mixed with an
equal volume of dextran solution (Pharmacia AB, Uppsala, Sweden)
A. Siegbahn, A. Hammacher, B. Westermark, and C.-H. Heldin
J. Clin. Invest.
© The American Society for Clinical Investigation, Inc.
Volume 85, March 1990,916-920
(20 g/liter; dissolved in 0.15 M NaCI); the cells were then allowed to
sediment at room temperature for 30 min. The leukocyte-rich plasma
was removed, centrifuged, and the cell pellet suspended in 2 ml 0.15 M
NaCI. Purification of monocytes was then done according to the
method described by Vadas et al. (22), using a stepwise gradient of 16,
18, 20, 22, and 23% (wt/vol) metrizamide (Nycomed, Oslo, Norway).
The leukocytes were applied on top of the gradient and centrifuged
1,200 g for 45 min at 20'C. The cells from the 16% metrizamide
fractions were used. The purity of monocytes in these fractions was
> 80%. Lymphocytes were the only contaminating cells as estimated
by light microscopy and staining for unspecific esterase (23). Finally,
the monocytes were suspended in Gey's solution (24), supplemented
with 0.2% HSA, and adjusted to a cell concentration of I X 109 cells/
Granulocytes were obtained as previously described (25) and sus-
pended in Gey's solution with 0.2% HSA to a concentration of 1.5
X 109cells/liter. The preparation consisted of- 90% granulocytesand
10% mononuclear cells.
Chemotaxis assay. The migration of fibroblasts, monocytes, and
granulocytes was assayed by means of the leading front technique
using a modified Boyden chamber (26, 27).
For monocyte and granulocyte migration, micropore filters (Milli-
pore/Continental Water Systems, Bedford, MA) with pore sizes of 5
and 3 ,um, respectively, were used. 100 1l of the cell suspension was
added above the filter ofthe Boyden chamber; attractants to be tested
were diluted in Gey's solution with 0.2% HSA and added below the
filter. After 60 min at 370C, the filters were fixed, stained, and
mounted. Cell migration was assayed as the migration distance ofthe
two furthest migrating granulocytes or monocytes visible in focus of
one high-power field (12.5 X 24). The migration distance in each filter
was calculated as the mean of the readings of three to five different
areas of the filter. All experiments werepKrformedwith duplicate or
quadruplicate filters for each concentration ofthe different isoforms of
PDGF. For each set ofexperiments, the migration ofgranulocytes or
monocytes in Gey's solution with 0.2% HSA and with the same me-
dium below the filter was used as control, and referred to as 100%16
For fibroblast migration, micropore filters (pore size 8 um) were
coated with a solution of type-I collagen (100 gg/ml; a kind gift of
Kristofer Rubin, Dept. ofMedical and Physiological Chemistry, Uni-
versity of Uppsala, Uppsala, Sweden) at room temperature overnight.
The filters were air dried for 30 min immediately before use. The assay
was performed as previously described (19). The migration of fibro-
blasts suspended in Eagle's MEM with 10% NCS, and with the same
medium below the filter in the Boyden chamber, served as control, and
was referred to as 100% migration.
Chemotactic response ofhumanforeskinfibroblasts to the var-
ious isoforms ofPDGF. A strong dose-dependent chemotactic
response for fibroblasts was obtained with PDGF-AB, as well
as with PDGF-BB from two different sources, i.e., human
platelets and recombinant material produced by transfected
yeast cells (Fig. 1). The maximal response was obtained with 5
ng/ml of PDGF-AB or 10-20 ng/ml of PDGF-BB. At higher
concentrations, the chemotactic response was reduced. In
contrast, recombinant PDGF-AA did not induce migration of
human foreskin fibroblasts (Fig. 1).
We recently found that PDGF-AA secreted by the clonal
malignant glioma cell line U-343 MGa C12:6 inhibited the
chemotactic activity ofPDGF purified from human platelets
on human fibroblasts (19). We therefore investigated recombi-
nant forms of the two variants of PDGF-AA, with regard to
their ability to reduce the chemotactic activities of optimal
concentrations of PDGF-AB (5 ng/ml) or PDGF-BB (10
ng/ml). Both the short (Fig. 2) and the long (data not shown)
10 20 50 100
Figure 1. Chemotactic response of fibroblasts to PDGF-AB (A),
platelet PDGF-BB (v), recombinant PDGF-BB (v), and the short
variant ofPDGF-AA (a). The results are means and SEM ofthree
experiments. A significant (P < 0.001, t test) chemotactic response
was obtained with 1-50 ng/ml ofPDGF-AB, 5-50 ng/ml of platelet
PDGF-BB, and 1-100 ng/ml ofrecombinant PDGF-BB.
variant of PDGF-AA at concentrations > 10 ng/ml signifi-
cantly (P < 0.05) reduced the chemotactic activity of PDGF-
AB as well as PDGF-BB.
Stimulation of monocyte chemotaxis by the various iso-
forms ofPDGF. Monocytes were found to migrate towards a
concentration gradient of PDGF-AB, as well as platelet or
recombinant PDGF-BB (Fig. 3). A significant (P < 0.01) re-
sponse was noticed at concentrations above
PDGF-AB or recombinant PDGF-BB. PDGF-BB purified
from human platelets induced monocyte chemotaxis at a
somewhat higher concentration (5 ng/ml). The maximal che-
motactic activity was obtained at concentrations between 10
and 20 ng/ml. Higher concentrations gave reduced response of
1 ng/ml of
0.515 10 20
Figure2. Inhibition by PDGF-AA ofchemotaxis induced by PDGF-
AB(v) and PDGF-BB (-). Fibroblasts migrated toward a solution of
5ng/ml ofPDGF-AB or 10 ng/ml PDGF-BB, and various concen-
trations of the short variant ofPDGF-AA. Results are means and
SEM oftwo experiments. A significant reduction ofPDGF-AB- or
PDGF-BB-induced chemotaxis was obtained with> 5 ng/ml or
> 10 ng/ml ofPDGF-AA, respectively.
Platelet-derived Growth Factor-induced Chemotaxis
Figure 3. Chemotactic response of monocytes to PDGF-AB (A),
platelet PDGF-BB (.), recombinant PDGF-BB (a), and the short
variant ofPDGF-AA (o). A significant (P < 0.01, t test) response was
obtained with 1-100 ng/ml ofPDGF-AB, 5-100 ng/ml ofplatelet
PDGF-BB, and 0.5-100 ng/ml ofrecombinant PDGF-BB (means
and SEM ofthree experiments).
the cells. There was no chemotactic response to the short (Fig.
3) or long (data not shown) variant ofPDGF-AA.
The short variant of PDGF-AA inhibited monocyte che-
motaxis induced by 10 ng/ml of PDGF-AB or 20 ng/ml of
PDGF-BB (Fig. 4). PDGF-AA at concentrations > 0.5 ng/ml
significantly (P < 0.01) decreased the chemotactic activity of
PDGF-AB and ofrecombinant PDGF-BB. We also tested the
ability of PDGF-AA to block monocyte chemotaxis induced
by an unrelated chemotactic agent. The short variant of
PDGF-AA was not able to inhibit monocyte chemotaxis to 10
nM ofFMLP (Fig. 4).
Chemotactic response ofgranulocytes to the various iso-
forms ofPDGF. In contrast to the results obtained with fibro-
blasts and monocytes, granulocytes were attracted in a dose-
dependent way to all three isoforms ofPDGF (Figs. 5 and 6).
Figure 4. The influence ofPDWF-AA on chemotaxis induced by
PDGF-AB (A), recombinant PDGF-BB (in), and FMLP (o). Mono-
cytes migrated toward a solution of 10 ng/ml PDGF-AB, 20 ng/ml
PDGF-BB, or 10 nM FMLP, and various concentrations of the short
variant of PDWF-AA. Results are means and SEM of four experi-
ments with PDGF-BB, three with PDGF-AB, and two with FMLP.
A significant (P < 0.01) reduction of PDGF-AB- or PDGF-BB-in-
duced chemotaxis was obtained with > 0.5
ng/ml of PDGF-AA.
FMLP-induced chemotaxis was not inhibited by PDGF-AA.
PDGF-AB or PDGF-BB
Figure5. Chemotactic responseofgranulocytesto PDGF-AB(A),
platelet PDGF-BB(-),and recombinant PDGF-BB(i). Asignificant
chemotacticresponsewas achieved with 0.5-50ng/mlofPDGF-AB
or recombinant PDGF-BB. 5-50ng/ml ofPDGF-BBpurified from
platelets gave asignificant response (means and SEM ofthreeexperi-
PDGF-AB and -BB induced chemotaxis over a broad concen-
tration intervalrangingfrom 0.5 to 50ng/ml (Fig. 5); PDGF-
AA induced asignificant responseat 10-50ng/ml (Fig. 6). The
responses to the PDGF preparations were somewhat lower
than theresponseobtained with 10 nM ofFMLP(130-135%;
Reduced andalkylatedPDGF has no chemotacticactivity.
Themitogenic activity of PDGF is lost when thedisulphide
bonds in the molecule are reduced. Similarly, reduced and
alkylated PDGF-AA wag without chemotactic effect ongran-
ulocytes (Fig. 6),as well as on fibroblasts and monocytes (data
notshown). PDGF-BB was also found to lose its chemotactic
activity for all three cell types after reduction andalkylation
In the present study we show that PDGF-AB and -BB are
strong chemotactic factors for fibroblasts and forphagocytic
cells. Incontrast, PDGF-AA was found to be chemotacticonly
for granulocytes. Some of our findings are at variance with
previous reports. Graves et al.(21) reported that PDGFpun-
Figure6. Chemotactic response ofgranulocytes to the short (n) and
long (A)variant ofPDGF-AA and to reduced and alkylated PDGF-
(e) induced a significant (P < 0.001) response. Concentrations be-
cantresponse (P < 0.01). The results are means and SEM ofthree
.). > 10 ng/ml ofthe short variant ofPDGF-AA
1 and 50 ng/ml ofthe long version ofPDGF-AA gave signifi-
A. Siegbahn, A. Hammacher, B. Westermark, and C.-H. Heldin
fied from human platelets did not induce chemotaxis ofmono-
cytes, whereas we (Fig. 3), as well as Deuel et al. (20), found a
potent effect. The explanation for the discrepancy is not
known, but it should be noted that we observed chemotaxis in
response to both PDGF-AB and -BB, purified from human
platelets as well as from recombinant sources; any possible
interference by contaminating platelet factors can therefore be
excluded. Williams et al. (28) reported that reduced PDGF
retained its chemotactic activity for human neutrophils and
monocytes, and suggested that PDGF mediated mitogenesis
and chemotaxis through different structural determinants. In
contrast, we found that reduced and alkylated recombinant
PDGF-AA and -BB had no chemotactic effect on any of the
cell types tested.
It was recently reported that fibroblasts display two distinct
PDGF receptor classes, denoted a-receptors and 3-receptors
(17, 18). The human foreskin fibroblasts used in this study
have about fourfold more PDGF /-receptors than a-receptors
(15); the amounts of a- and /-receptors on monocytes and
granulocytes have not been determined. Cells that lack PDGF
/-receptors, e.g., endothelial cells isolated from large vessels
(29, 30), do not respond chemotactically to PDGF (31, 32).
This, and the finding that PDGF-AA, which does not bind to
PDGF /-receptors, does not stimulate chemotaxis of human
fibroblasts (Fig. 1), suggests the chemotactic activity ofPDGF
is mediated by the /-receptor class. The recent finding that
after transfection ofPDGF /-receptor cDNA, endothelial cells
become able to migrate toward a concentration gradient of
PDGF-BB (33), supports this conclusion.
It cannot be excluded that the lack ofchemotactic effect of
PDGF-AA in human fibroblasts and monocytes is due to too
few a-receptors on the target cells. However, the fact that
human foreskin fibroblasts have a fairly large number of a-re-
ceptors (- 30,000/cell,) which is sufficient to induce a mito-
genic response (15), is consistent with the conclusion that the
a-receptor does not transduce a chemotactic signal in human
fibroblasts. Likewise, the a-receptor seems incapable of me-
diating actin reorganization and induction of circular mem-
brane ruffles in human fibroblasts (19). The data presented in
this communication thus extend previous studies and indicate
that there are functional differences between the two PDGF
That different signals arise from activation of the two
PDGF receptor classes is furthermore illustrated by the finding
that PDGF-AA inhibited the chemotaxis induced by PDGF-
AB or -BB in human fibroblasts (Fig. 2) and monocytes (Fig.
4). Inasmuch as PDGF-AA did not block FMLP-induced
monocyte chemotaxis, the inhibitory effect is not explained by
a general deactivation process or by a toxic effect. It has been
suggested that the FMLP receptor, after binding ofthe ligand,
interacts with a G protein and the chemotactic signal is me-
diated by phospatidylinositol turnover (1, 2). Our results do
not exclude the possibility that chemotaxis induced by FMLP
or PDGF-BB is transmitted via the same signal system. In such
case, however, the PDGF route seems to be blocked by
PDGF-AA at a point proximal to the convergence of the two
In contrast to the inhibitory effects on fibroblasts and
monocytes, PDGF-AA stimulated chemotaxis ofgranulocytes
(Fig. 6). The explanation for the different effects ofPDGF-AA
on the different cell types remains to be elucidated. The possi-
bility that they are mediated by as yet unidentified receptor
classes has not been excluded. Alternatively, the two cell types
might differ in the intracellular signalling systems that are ac-
tivated after stimulation by PDGF-AA. This possibility is il-
lustrated by the fact that leukocytes differ from fibroblasts in
that they can respond to chemoattractants and migrate in the
presence ofRNA and protein synthesis inhibitors (34, 35).
The present work shows that PDGF-AB and -BB stimulate
chemotaxis of fibroblasts and monocytes, as well as granulo-
cytes, and that PDGF-AA has agonistic or antagonistic effects
depending on cell type. These findings are significant for the
understanding of the role of PDGF in stimulation ofchemo-
taxis in vivo. Thus, depending on which isoform ofPDGF that
is secreted by cells at a site ofinjury or inflammation, different
cell types will be recruited to the area. The possible involve-
ment of PDGF in pathological growth reactions such as ath-
erosclerosis and fibrosis (3), which are associated with the ac-
cumulation ofinflammatory cells, warrants studies on PDGF-
induced chemotaxis in vivo.
We thank Matilda Johnell for skillful technical assistance and Ingegard
Schiller for valuable secretarial assistance.
These studies were supported in part by grants from the Swedish
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