(Received for publication, April 20, 1995; and in revised form, July 26, 1995)
From the
The proliferation of human myeloid progenitor cells is
negatively regulated in the presence of certain members of the
chemokine family of molecules. This includes interleukin 8 (IL-8) and
platelet factor 4 (PF4), which in combination are able to synergize,
resulting in cell suppression at very low concentrations of these
molecules. A series of PF4 and IL-8 mutant proteins were analyzed in an in vitro colony formation assay for myeloid progenitor cells
to assess domains of these proteins that are required for activity.
Mutation of either of the two DLQ motifs within PF4 resulted in an
inactive protein. Perturbations within the IL-8 dimer interface region
also resulted in mutants that were incapable of suppressing colony
formation. A class of chimeric mutants consisting of domains of either
PF4 and IL-8, Gro- and PF4, or Gro-
and PF4 were observed to
inhibit myeloid cell proliferation at concentrations which were between
500- and 5000-fold lower than either the IL-8 or PF4 wild-type proteins
alone. These chimeric mutants possessed activities that were comparable
to or better than the activity observed when IL-8 and PF4 were added
together in vitro. One of these highly active chimeric
proteins was observed to be 1000-fold more active than either IL-8 or
PF4 alone in suppressing not only the proliferation but also the cell
cycling of myeloid progenitor cells following intravenous injection of
the mutant into mice. Examination of additional IL-8-based mutants in
the colony formation assay, which centered on the perturbation of the
amino-terminal ``ELR'' motif, resulted in the observation
that the highly active IL-8 mutant required both aspartic acid at amino
acid residue 4 and either glutamine or asparagine at residue 6. Single
mutations at either of these positions resulted in mutants with
myelosuppressive activity equivalent to wild-type IL-8. Mutants such as
IL-8M1 and IL-8M10 were observed to be significantly reduced in their
ability to activate isolated human neutrophils, suggesting that
separate mechanisms may exist by which myeloid progenitor cells and
neutrophils are affected by chemokines.
Myelopoiesis is a complex, highly regulated process, which is
dependent on the action of both positive and negative growth factors to
control the proliferation of primitive morphologically indistinct cells
from hematopoietic organs to supply functional end-stage blood cells.
Factors that stimulate cell growth and differentiation have been well
characterized and include the colony-stimulating factors (GM-CSF), ()granulocyte colony-stimulating factor, and macrophage
colony-stimulating factor), erythropoietin, some of the interleukin
family members (e.g. IL-1, IL-3, IL-4, IL-6, IL-9, IL-11) as
well as other cytokines including Steel factor 1-3). A number of
suppressor molecules have also been identified. These include E-type
prostaglandins, H-ferritin, lactoferrin, interferons, tumor necrosis
factors, and transforming growth factor-
(1, 2, 3) . More recently, several members of
the chemokine family of proteins including macrophage inflammatory
protein-1
(MIP-1
), MIP-2
(Gro-
), interleukin 8
(IL-8), platelet factor 4 (PF4), monocyte chemotactic and activating
peptide (MCAF/MCP-1), and
interferon-inducible protein, molecular
weight 10,000 (
IP10), have been demonstrated to possess inhibitory
activity toward the proliferation of immature stem/progenitor cells in vitro and in
vivo(4, 5, 6, 7, 8, 9, 10, 11, 12, 13) .
Chemokines are a family of small inducible proteins possessing
structural similarities and high amino acid
identities(14, 15, 16) . Although activity
differences exist between the proteins, all are believed to possess
chemoattractant properties for various cell types. The family is
subdivided into two groups based on positioning of cysteine residues
within the amino-terminal domain. The CXC group (2 cysteines
with an intervening amino acid) includes IL-8, Gro-, Gro-
,
NAP-2, PF4, ENA78, and
IP10. The three-dimensional structures of
IL-8 and PF4 have been solved and show general structural
identity(17, 18) . Protein family members that possess
the amino acid motif ``ELR'' within the amino terminus have
all been observed to elicit potent neutrophil chemoattractant and
stimulatory activities. This motif has also been shown to be required
for specific interaction with either of the two IL-8 receptor proteins
on the surface of neutrophils(19, 20, 21) .
The remaining members of the CXC subgroup display a more
diverse activity profile, weak or no neutrophil chemoattracting
activity, and less sequence homology to the ELR motif containing
subgroup. Neither PF4 nor
IP10 have demonstrated significant
neutrophil-related
activities(22, 23, 24, 25) .
The
other half of the chemokine family is characterized by the CC motif
(two adjacent cysteine residues located within the amino terminus) and
displays a much more diverse sequence homology and activity profile. CC
chemokines act predominantly on monocytes, although basophils,
lymphocytes, and eosinophils have also been reported to be target cells
for various CC proteins including RANTES, MIP-1, and
MCP-1(26, 27, 28, 29) . Compared to
the CXC family, less is understood regarding domains within
the proteins which are required for biological activity. However,
recent structural information on MIP-1
should facilitate this
understanding(30) .
Activated platelets have been observed
to release high concentrations of a high molecular weight proteoglycan
complex consisting of chondroitin sulfate and PF4(31) . In
addition to high affinity binding and neutralization of heparin, PF4
also has been observed to inhibit angiogenesis, inhibit bone
resorption, and reverse the immunosuppressive effect of lymphoma
cells(32, 33, 34, 35, 36) .
IL-8 has been observed to possess potent chemotactic and stimulating
properties toward human neutrophils in vitro and has been
shown to bind with high affinity to either of the two cloned human IL-8
receptors in vitro. In addition to these activities, IL-8 and
PF4, as well as MIP-1, MCP-1, Gro-
, and
IP10, were all
observed to inhibit early myeloid progenitor cell proliferation at
equivalent concentrations >25 ng/ml(10, 11) .
Several members of the chemokine family, including NAP-2, Gro-
,
Gro-
, RANTES, and MIP-1
did not possess any inhibitory
activities in this assay. A third group of chemokines including
Gro-
and Gro-
(MIP-2
) blocked the inhibitory activity of
IL-8 and PF4(10) . Similarly, MIP-1
was observed to
inhibit the activity of MIP-1
(6, 10) .
Combinations of any two of the six active chemokines resulted in a
synergistic decrease in the amount of each chemokine needed to inhibit
proliferation (0.1 ng/ml of each chemokine), suggesting the possibility
of a novel mechanism of action on the
progenitors(10, 11) . The low concentrations of PF4
and IL-8 required to elicit inhibition suggest the presence of
protein-based receptors on the progenitor cells. To address this issue,
a series of chimeric IL-8/PF4 mutants were expressed, purified, and
tested for inhibitory activity toward immature subsets of myeloid
progenitor cells.
Figure 1: Amino acid sequences of IL-8, PF4, and related chemokine mutants. Sites of mutation are highlighted for each protein.
Figure 2:
A, summary of ability of
chemokine mutant proteins to elicit elastase release from human
neutrophils in vitro. Chemokines at concentrations of either
10M or 10
M (0.1 µg/ml or 1 µg/ml) were added to isolated neutrophils
while gently mixing. Extent of elastase release was monitored
spectrofluorometrically following cleavage of the fluorogenic substrate
as described under ``Materials and Methods.'' Relative
potency of each mutant was compared to the amount of cleaved product
generated by neutrophil stimulation with equivalent concentrations of
IL-8. B, dose-dependent release of elastase from isolated
human neutrophils by chemokine mutants. Samples include IL-8 WT,
; IL-8M8,
; IL-8M9,
; IL-8M10,
.
Additional IL-8 mutants, designated IL-8M8 (ELQ), IL-8M9
(DLR), and IL-8M10 (DLN), which were developed to examine in greater
detail the requirements surrounding the NH-terminal ELR
motif, were also tested in the elastase release assay in a
concentration-dependent manner (Fig. 2B). As
anticipated, all three displayed either significantly reduced activity
or no ability to elicit degranulation of the isolated human
neutrophils. Neither IL-8M8 or IL-8M10 elicited any release of elastase
at concentrations as high as 4
10
M (40 µg/ml) and 1.25
10
M (100 µg/ml), respectively. IL-8M9 demonstrated the ability to
release elastase, although at concentrations approximately 200-fold
greater than for the native sequence IL-8. Perturbation of the ELR
motif resulted in profound effects on the ability of these chemokine
mutants to function on the neutrophil.
The ability of each of these mutant proteins to elicit chemotaxis of neutrophils was also examined. Each chemokine mutant was tested in a concentration-dependent manner for ability to elicit chemotaxis of isolated human neutrophils in a boyden chamber. Each concentration of each mutant was tested in triplicate in two separate experiments. Each data point was read in triplicate as well, for a total of 18 data points/concentration/chemokine. The results obtained demonstrate a direct correlation between the ability of the chemokine mutants to elicit chemotaxis of neutrophils and the ability to cause neutrophil degranulation as exhibited in the elastase release assay results (Fig. 3, A-D). With the exception of PF4M2, none of the PF4-derived mutants displayed any chemotactic activity toward neutrophils. Similarly, with the exception of IL-8M1, which showed substantially reduced activity, all of the IL-8-derived mutants exhibited potent neutrophil chemotactic activity, although some reduction in activity was also observed for IL-8M4, IL-8M64, and IL-8M7. However, this decrease also correlated with the data obtained in the elastase release assay.
Figure 3:
Concentration dependence of chemokine
mutants on neutrophil chemotaxis activity in vitro.
Experiments were performed as described under ``Materials and
Methods.'' A, samples include IL-8 WT, ; IL-8M3,
; PF4 WT,
; IL-8M1,
. B, IL-8 WT,
;
IL-8M64,
; PF4M1,
; PF4M2,
. C, IL-8 WT,
; IL-8M6,
; IL-8M4,
; PF4-426,
. D,
IL-8 WT,
; IL-8M7,
; PF4-413,
; PF4-421,
.
Binding of the IL-8-derived mutants
to CHO cells containing the stably transfected IL-8 receptor subtype B
was also performed. The B subtype receptor is able to bind with high
affinity to IL-8 as well as other ``ELR''-containing
chemokines including Gro-, Gro-
, and NAP-2. As shown in Table 1, competition binding experiments utilizing
I-labeled IL-8 and unlabeled mutant chemokine competitors
demonstrated that each of the proteins that was able to activate the
neutrophils was also able to bind to the neutrophil receptors. IL-8M1
and PF4M1, which displayed decreased ability to elicit elastase release
from the neutrophils, showed a similarly decreased ability to compete
with the labeled IL-8 for receptor binding.
Several
mutants were no longer able to inhibit the proliferation of the myeloid
progenitors. Even at concentrations up to 100 ng/ml (1
10
M), no activity could be detected. These
proteins include PF4M1, PF4-412, PF4-413, IL-8M3, IL-8M4, and IL-8M6.
Of this group, three distinct types of mutations resulted in loss of
activity. IL-8M3, IL-8M4, and IL-8M6 result from changes within the
dimer interface region of IL-8. PF4M1 results from a point mutation
within the DLQ motif located within the amino-terminal domain. Finally,
PF4-412 and PF4-413, result from domain swaps with IL-8 and NAP-2,
respectively, at the COOH-terminal DLQ region of PF4.
The third
phenotype of chemokine mutant includes proteins possessing enhanced in vitro inhibitory activity toward proliferation of the
CFU-GM population. The activities of these proteins, shown in Fig. 4(A and B), are compared to the
activities of either IL-8 or PF4 alone. The results are expressed as
mean percent change from control ± 1 S.E. These proteins include
PF4M2, IL-8M1, IL-8M10, PF4-414, and PF4-426. PF4M2 and PF4-414 were
active down to a concentration of 0.01 ng/ml (19 ± 8% and 34
± 8% inhibition, respectively). The other highly active proteins
displayed suppressive activity down to concentrations of 0.001 ng/ml
(1
10
M). Previously, it was
observed that combining two individual active chemokines such as PF4
and IL-8 produced inhibitory activity at concentrations of each protein
as low as 0.1 ng/ml (
1
10
M)(10) . The results obtained with these
proteins suggest that specific mutations produce effects comparable to
or better than the synergistic activity previously demonstrated.
Figure 4:
Summary of activities of chemokine mutants
for in vitro inhibition of CFU-GM. A, comparison of
highly active IL-8-derived mutants compared to IL-8 and PF4. Samples
include IL-8 WT, ; PF4 WT,
; IL-8M1,
; IL-8M10,
. B, comparison of highly active PF4-derived mutants
compared to IL-8 and PF4. Samples include IL-8 WT,
; PF4 WT,
; PF4M2,
; PF4-426,
; PF4-414,
.
A series of chemokine mutants have been cloned, expressed,
purified, and evaluated for in vitro myelosuppressive
activity. Of the proteins examined, one group of proteins have been
identified that are able to inhibit myeloid progenitor cell
proliferation at very low concentrations. The activities of these
individual mutants appeared comparable to or greater than the activity
observed previously when low concentrations of IL-8 and PF4 were added
together(10) . These proteins include PF4M2, PF4-414, PF4-426,
IL-8M1, and IL-8M10. These synergistic mutants were found to be active
at concentrations as low as 0.001 ng/ml (1
10
M monomer concentration). PF4M2
contains the NH
-terminal ELR motif from IL-8 with the
remaining COOH-terminal domains from PF4. It has been shown to possess
both neutrophil-related activities as well as an ability to bind
heparin and inhibit the proliferation of cultured endothelial cells.
Conversely, IL-8M1 contains the amino-terminal DLQ motif from PF4 with
the remaining COOH-terminal domains from IL-8. This potent mutant
displayed significantly reduced neutrophil binding, chemotaxis and
activation activities. Comparable to native IL-8, IL-8M1 binds to
heparin with an affinity that is significantly reduced relative to PF4.
Because this protein is inactive on neutrophils, but highly active on
progenitor cells, it is likely that progenitor-related activity occurs
via a different mechanism than that which occurs on neutrophils.
IL-8M10 was another highly active mutant in the myeloid progenitor
proliferation assay. This mutant contains the sequence DLN as a
replacement for ELR in wild-type IL-8. The activity of this mutant is
similar to IL-8M1 and demonstrates that either glutamine or asparagine
in this position is well tolerated on the progenitor cell. IL-8M9,
which contains DLR, displayed an equivalent activity to the wild-type
IL-8 and suggests that specific amino acids replacing the arginine
residue are likely to result in the highly active phenotype. Similarly,
IL-8M8 containing ELQ possessed activity comparable to wild-type IL-8.
The results obtained from this mutant would suggest that the aspartic
acid at amino acid position 4 is critical for the highly active
phenotype. The dramatic difference in activity imposed by the
difference of a CH moiety at amino acid 4 between IL-8M1
and IL-8M8, suggests a highly specific interaction must be occurring on
the progenitor cell. The conclusion obtained from these mutants
suggests that a double mutation of the ELR motif is critical for the
highly active phenotype.
PF4-414 contains a sequence from Gro-
replacing the second, COOH-terminal DLQ domain from PF4. Unlike similar
inactive mutants, that contain domains of IL-8 (PF4-412) or NAP-2
(PF4-413), PF4-414 displayed enhanced activity, comparable to IL-8M1.
The region of PF4 encompassing this second DLQ motif is likely to play
a role in either maintaining the appropriate protein conformation or in
direct interaction with the progenitor cell. The latter hypothesis is
currently favored since peptides containing this domain have been
previously observed to be active in suppression of progenitor cell
proliferation in vitro(13, 40) . Furthermore,
analysis of the crystal structure of PF4 predicts that this region of
the protein assumes a reverse
-turn conformation, which is
solvent-accessible (17) . It is not currently understood why
this mutation would result in a highly active chemokine in the
progenitor proliferation assay since Gro-
alone was inactive in
suppression of progenitor cell proliferation(10) . However,
Gro-
was observed to block PF4- and IL-8-dependent inhibition of
proliferation(10) , suggesting that Gro-
is able to bind
to the progenitor cell. It is possible that the combination of the DLQ
motif from PF4 with the sequence ACLNPASPIVK is sufficient to generate
a molecule that possesses an activity analogous to the combination of
two of the active chemokine proteins(10) . It is suspected that
correct combinations of domains from various chemokine proteins elicit
a synergistic activity on myeloid progenitor cells.
This hypothesis
is supported by mutant PF4-426. This mutant contains three point
mutations, each of which replaces an arginine residue with glutamine.
The result is a highly active protein, which on first glance is a
simple PF4 mutant. A closer analysis, however, reveals that
substitution of the third arginine residue at position 49 with
glutamine results in the generation of the sequence IATLKNGQK, which is
identical to a sequence within Gro-. Gro-
has been
demonstrated previously to be able to synergize with PF4 in the
progenitor proliferation assay(10) . The results demonstrate
that correctly placed domains that result in chimeric chemokines are
able to elicit an enhanced suppressive activity on myeloid progenitor
cells.
Another class of mutants appeared either inactive or
significantly reduced in inhibitory activity in the assay. All of the
IL-8 mutants which contain mutations within the dimerization domain of
IL-8 were either inactive or significantly reduced in activity,
suggesting that this region and perhaps more specifically the sequence
ELRV plays a role in suppression of proliferation of progenitor cells.
These three IL-8 mutants all elicited elastase release and were able to
chemoattract neutrophils, demonstrating that they are likely to be
correctly refolded in a manner analogous to the native sequence IL-8.
Although IL-8M3, IL-8M4, and IL-8M6 contain mutations within the dimer
interface of IL-8, only IL-8M4 appears to be monomeric in solution at
0.1 mg/ml (1 10
M) concentration
(data not shown). (
)Another monomeric IL-8 mutant, IL-8M64,
possessed equivalent activity as wild-type IL-8 on both neutrophils and
progenitor cells, suggesting that oligomeric state may not be a
critical factor for activity on myeloid progenitor cells. Furthermore,
at concentrations in the range of 10
M,
wild-type IL-8 is likely to exist predominantly as a monomeric species
in solution(42) . The data from this class of mutant suggest
that activity may not be oligomeric state-dependent but rather a result
of a specific amino acid sequence within the protein or correct protein
folding, which is required for myeloid suppression.
Another mutant that lacked the ability to inhibit progenitor proliferation was PF4M1. This protein contains a single point mutation (DLQ to DLR) within the amino-terminal domain. It is unclear whether this amino acid change results in a direct effect on the interaction with the progenitor cells. However, crystal structure data demonstrate that spacially, the DLQ motifs of two PF4 monomers (the A and D subunits) lie adjacent to each other(17) . The glutamine residues in particular are situated side by side in the intact tetramer. Replacement of these glutamine residues with the positively charged arginine groups may result in charge repulsion and an altered oligomeric conformation of the protein. The DLQ motif of PF4 appears to be highly important with regard to activity on the progenitor cells. The region surrounding the COOH-terminal DLQ motif of PF4 also appears important for myeloid cell growth suppression. Two mutants, PF4-412 and PF4-413 were generated, which replace this domain with the analogous regions of either IL-8 or NAP-2, respectively. The resulting loss of activity suggests that this region also is involved either in direct interaction with progenitor cells or is required for proper folding of the protein. Both of these mutants were observed, however, to inhibit endothelial cell proliferation in vitro and to bind heparin at concentrations comparable to wild-type PF4.
Fig. 5is a summary of the
domains that have been identified to be involved in this activity. In
IL-8, the amino-terminal ELR motif appears to be required for activity,
especially if inserted into PF4. The dimer interface region of IL-8
also appears critical for suppressive activity. In PF4 both of the DLQ
motifs appear necessary for this protein to inhibit myeloid progenitor
proliferation. Loss of either of these domains results in loss of
activity of PF4. Combinations of IL-8 and PF4, PF4 and Gro-, as
well as PF4 and Gro-
result in synergistic effects. It is
anticipated that other combinations of chemokines may also generate
additional synergistic mutants.
Figure 5:
Schematic representation of a summary of
the chemokine domains necessary for myelosuppression. IL-8-derived
domains are depicted in black with PF4-derived domains in gray. Domain 1 is the NH-terminal ELR domain from
IL-8. Domain 2 is the dimer interface domain from IL-8. Domain 3 is the
amino-terminal DLQ sequence from PF4. Domain 4 is the COOH-terminal
domain surrounding the second DLQ domain from
PF4.
A number of chemokine receptors have been identified to date, including the two human neutrophil IL-8 receptors and the Duffy antigen on erythrocytes(43, 44, 45) . Recent work by Cacalano et al.(46) has shown that deletion of a murine gene with high homology to the two human IL-8 receptors results in a mouse with a phenotype of elevated levels of B cells, metamyelocytes, band, and mature neutrophils, suggesting that the receptor plays a role in the negative control of development of blood cell components. However, neither of the two identified human IL-8 receptors displays the activity profile with either the mutant chemokines or native sequence chemokine family members that has been observed with the progenitor cells(46) . This would suggest that the neutrophil receptors may not be involved in the regulation of progenitor cell proliferation. Furthermore, no receptor has as yet been identified as being specific for platelet factor 4. Since the activity pattern of the IL-8 and PF4 mutants does not correlate with the activity profile observed on neutrophils, it is possible that a new family of receptors may exist on the progenitors. Furthermore, the observation that IL-8M3 was able to inhibit the suppressive activity of IL-8 but not PF4 suggests that it is not a single receptor, but possibly a family of receptors that are responsible for interaction with each chemokine. One potential model for the mechanism of action of the chemokines on progenitor cells that is supported by our data, in conjunction with work by Broxmeyer et al.(10) , suggests that two or more different occupied receptors, each with a high affinity for a specific chemokine (such as IL-8 or PF4) and a weaker affinity for each of the other active chemokines, interact with each other, leading to signal transduction and suppression of progenitor cell cycling. Since a minimum of two bound receptors with different specificities would be required for this synergistic suppression, it is likely that chimeric chemokines such as IL-8M1 are simultaneously interacting with two distinct receptors with different specificities with high affinity, leading to the synergistic phenotype observed. The apparent weaker activity observed with a single chemokine such as IL-8, may result from specific binding to the high affinity IL-8 receptor and nonspecific binding to a much lower affinity PF4 (or other chemokine) receptor. Further work in this area will provide insight into the mechanism of chemokine-dependent myeloid progenitor cell regulation.