Promotion of maturation of hematopoietic progenitor cells

A method for promoting maturation of a hematopoietic precursor cell of an animal, which method includes the step of contacting the cell with a maturation-promoting amount of GRO, a polypeptide growth factor.

GRO is a polypeptide growth factor encoded by a gene termed gro (Anisowicz 
et al., Proc. Natl. Acad. Sci. USA 84:7188-7192, 1987; Anisowicz et al. 
Proc. Natl. Acad. Sci. USA 85:9645-9649, 1988). The amino acid sequence of 
ma human GRO (i.e., the sequence deduced by Anisowicz et al., 1987, from 
the gro cDNA sequence, but minus the 34-amino acyl residue leader peptide) 
is identical to a 73-amino acyl residue-long protein present in the 
conditioned medium of a human malignant melanoma cell line, and which has 
been termed melanoma growth stimulation activity, or "MGSA" (Richmond et 
al., Cancer Research 43:2106-2112, 1983; Richmond et al., EMBO J. 
7:2025-2033, 1988). Likewise, a protein secreted by activated neutrophils 
(neutrophil-activating peptide-3 or NAP-3) has an amino-terminal sequence 
identical to that of GRO/MGSA, at least up to the 31st residue of each 
(Schroder et al., J. Exp. Med. 171:1091-1100, 1990). GRO/MGSA acts as a 
growth factor for melanoma cells, lung carcinoma cells, nevus cells, and 
some immortalized fibroblast cell lines (Richmond and Thomas, J. Cell 
Biol. 107:40A #203, 1988). 
The gro genes exhibit DNA sequence similarities to a family of genes 
encoding secretory proteins associated with the inflammatory response. The 
expression of each gene of this family of genes, including gro, is rapidly 
induced in susceptible cells by such agents as phorbol esters, 
interleukin-1, and tumor necrosis factor (TNF), as well as other cytokines 
and growth factors (Anisowicz et al., 1987; Anisowicz et al., 1988). 
Members of this family encode proteins which exhibit a high degree of 
sequence homology, including four cysteine residues present in analogous 
positions; such proteins include platelet factor 4 (Deuel et al., Proc. 
Natl. Acad. Sci. USA 74:2256, 1977); platelet basic protein and its 
cleavage products: .beta.-thromboglobulin (Begg et al., Biochemistry 
17:1739, 1978) and connective tissue activity peptide III (CTAP III) 
(Castor et al., Proc. Natl. Acad. Sci USA 80:765, 1983); interferon 
inducible protein 10 (IP10) (Luster et al., Nature 315:672, 1985); 
macrophage inflammatory protein-2 (MIP-2) (Wolpe et al., Proc. Natl. Sci. 
USA 86:612, 1989); and neutrophil activating peptide-1/interleukin-8 
(NAP-1/IL-8) (Walz et al., Biochem. Biophys. Res. Commun. 149:755, 1987) 
[also known as NAF (Walz et al.; Lindley et al., Proc. Natl. Acad. Sci. 
USA 85:9199, 1988), MDNCF (Yoshimura et al., Proc. Natl. Acad. Sci. USA 
84:9233, 1987), MONAP (Schroder et al., J. Immunol. 139:3474, 1987), and 
GCP (Van Damme et al., J. Exp. Med. 167:1364, 1988)]. This latter compound 
is a neutrophil-specific chemotactic factor (Walz et al.; Lindley et al.; 
Yoshimura et al.; Schroder et al.; and Van Damme et al.) and cellular 
activator (Wolpe et al.; Lindley et al.), as well as an inhibitor (at low 
concentrations) (Gimbrone et al., Science 246:1601, 1989) and activator 
(at high concentrations) (Carveth et al., Biochem. Biophys. Res. Commun. 
162:387, 1989) of neutrophil adhesion to endothelial cells. 
Based on what is said to be largely indirect evidence, Anisowicz et al. 
(1988) suggest that the gro gene may play a role "in a variety of 
important cellular functions: as a putative [positive] early response gene 
in cell growth, as a mediator of the IL-1-induced inflammatory response in 
fibroblasts, and as a negative regulatory factor in epithelial cells." 
SUMMARY OF THE INVENTION 
In general, the invention features a method for promoting maturation of a 
hematopoietic precursor cell (preferably a CFU-GEMM cell or a CFU-GM cell) 
of an animal (e.g., a human), which method includes the step of contacting 
the cell with a maturation-promoting amount of GRO. In preferred 
embodiments, the cell is first removed from the animal (e.g., by 
withdrawing bone marrow from a bone of the animal), and is contacted with 
GRO in vitro; the cell and/or its descendants are then preferably 
reinserted into the animal, or alternatively into a second animal (most 
preferably a human). 
Also featured is a method for promoting the maturation of a hematopoietic 
precursor cell (preferably a CFU-GEMM cell or a CFU-GM cell) within an 
animal (e.g., a human) by treating the animal with a maturation-promoting 
amount of GRO. The form of GRO utilized in any method of the invention is 
preferably a recombinant GRO (i.e., produced by expression of a 
recombinant DNA molecule encoding GRO), and may, for example, have the 
amino acid sequence of a mature human GRO, or may be an analog or fragment 
of a naturally-occurring GRO. 
Another method of promoting the maturation of a hematopoietic precursor 
cell within an animal is to introduce into the animal one or more cells 
capable of excreting GRO (i.e., secreting or otherwise causing GRO to be 
exported out of the cell); alternatively, a gene encoding and capable of 
expressing GRO can be introduced into one or more cells of the animal, to 
form a cell (herein termed a "transgenic cell") capable of excreting GRO 
within the animal at a level sufficient to promote maturation of the 
hematopoietic percursor cell. 
The term "hematopoietic cells" herein refers to fully differentiated 
myeloid cells such as erythrocytes or red blood cells, megakaryocytes, 
monocytes, granulocytes, and eosinophils, as well as fully differentiated 
lymphoid cells such as B lymphocytes and T lymphocytes; it also 
encompasses the various hematopoietic precursor cells from which these 
differentiated cells develop, such as BFU-E (burst-forming 
units-erythroid), CFU-E (colony forming unit-erythroid), CFU-Meg (colony 
forming unit-megakaryocyte), CFU-GM (colony forming 
unit-granulocyte-monocyte), CFU-Eo (colony forming unit-eosinophil), and 
CFU-GEMM (colony forming 
unit-granulocyte-erythrocyte-megakaryocyte-monocyte). The 
interrelationships among these hematopoietic cells and their positions 
along the various paths of differentiation are illustrated in FIG. 1. 
"Maturation" of a hematopoietic precursor cell is used herein to mean the 
generation of descendents of such precursor cell which are either 
identical to or more differentiated than such precursor cell, or a mixture 
of both. For example, a CFU-GEMM would be induced by the method of the 
invention to generate multiple cells, some of which are CFU-GEMMs and 
others of which are further along the paths of differentiation, such as 
CFU-GM, CFU-Eo, and megakaryocytes, or are fully differentiated, end-stage 
cells such as monocytes/ macrophages, platelets, or granulocytes (e.g., 
neutrophils, basophils, or eosinophils). A maturation-promoting amount of 
GRO is that amount of protein which is sufficient to cause maturation of a 
significant number of hematopoietic precursor cells present in a bone 
marrow or taken from a bone marrow. For example, out of a population of 
approximately 10.sup.6 light-density bone marrow cells (i.e., bone marrow 
cells which do not pellet in the Ficoll-Hypaque method described below) 
plated on a semi-solid substrate, at least one hematopoietic precursor 
cell would be induced by this amount of GRO to proliferate until the cell 
had formed a colony (a group of cells all of which are descended from a 
single cell) of at least 8 cells (after 7 days incubation at 37.degree. 
C.) or at least 40 cells (after 14 days incubation at 37.degree. C.), 
which cells are identical to or more differentiated than the precursor 
cell from which they were derived. For in vivo treatment with GRO, a 
maturation-promoting amount of GRO would be an amount capable of 
increasing the number of hematopoietic cells in the treated patient. 
The ability of a given form or amount of GRO to promote maturation of cells 
can be measured by any standard procedure. For example, this biological 
activity can be measured in vitro by measuring the colony-promoting 
activity of GRO on cells taken from a bone marrow and grown on a 
semi-solid substrate as described below. 
By GRO is meant not only the MGSA protein of Richmond et al. (1983, 1988) 
and the gro-encoded protein of Anisowicz et al. (1987, 1988), but also any 
comparably active GRO endogenous to any animal species (particularly 
mammals or other vertebrate species). Three different human gro cDNAs have 
been cloned. Anisowicz et al. (1987) identified the first (now termed gro 
.alpha.) from a human bladder carcinoma cell line (T24) cDNA library. An 
adherent monocyte cDNA library probed with gro .alpha. cDNA yielded an 880 
bp partial cDNA clone, the sequence of which differed somewhat from that 
of gro .alpha. cDNA; this partial cDNA was used to probe a second cDNA 
library, producing positively-hydridizing clones representing gro .alpha. 
cDNA and two variants termed gro .beta. and gro .gamma.. Partial sequence 
analysis of genomic clones obtained by probing a human genomic DNA library 
with gro .alpha. cDNA confirmed that the three forms are derived from 
related but different genes, all three of which appear to map to the same 
region of chromosome 4q. The nucleotide sequences and predicted 
translation sequences of the three cDNAs are compared in FIG. 2. 
Genes or cDNAs encoding such alternative naturally-occurring GROs may be 
identified and cloned using human or other gro cDNA as a hybridization 
probe, in a manner similar to that employed by Anisowicz et al. (1987). 
The cDNA sequences and corresponding amino acid sequences for human GRO 
.alpha. and for Chinese hamster GRO, as published by Anisowicz et al. 
(1987), are set forth in FIG. 3. The term GRO also encompasses any analog 
or fragment of any naturally-occurring GRO, which analog or fragment is 
stable in solution and exhibits a maturation-promoting biological activity 
comparable to that of the naturally-occurring GRO/MGSA of which it is an 
analog or fragment. It is critical only that the maturation promoting 
portion of GRO/MGSA be provided. The critical portion of GRO/MGSA can be 
determined by any of a number of standard techniques. For example, the 
cDNA or cloned gene encoding GRO may be modified by standard in vitro 
mutagenesis techniques to cause expression of a GRO analog with amino acid 
substitutions, additions, and/or deletions of one or more amino acids at 
one or more locations. The amino acid substitutions may be either 
conservative or non-conservative, and may be designed, for example, to 
remove proteolytically sensitive sites from the GRO protein. [By 
conservative is meant that the substituted amino acyl residue is 
chemically similar (e.g., acidic, basic, hydrophobic, aromatic) to the 
residue for which it is substituted: for example, substitution of a valine 
for a leucine.] Examples of GRO proteins with potentially useful additions 
would include a GRO with a short peptide added to either terminus, such as 
a leader peptide to facilitate secretion of the protein out of the cell, 
or a peptide added by means of genetic engineering to provide an antigenic 
site to permit immunoaffinity-based purification of the protein product; 
and chimeric GRO proteins covalently bound to polypeptide ligands capable 
of binding to particular receptors. Forms of GRO with internal amino acid 
additions which do not destroy the maturation promoting activity are also 
within the definition of GRO. Once generated, any such analogs can then be 
tested for the desired biological activity, i.e. maturation promoting 
activity. In this way, the maturation promoting portion of GRO can be 
specifically determined, and those amino acyl residues not critical to 
that function removed or replaced with other residues. Alternatively, the 
naturally-occurring or recombinant protein may be digested with a variety 
of proteases, for example, trypsin, to provide fragments which can then be 
tested for maturation promoting activity. Those fragments or analogs which 
have the maturation promoting portion of naturally-occurring GRO can be 
readily determined using simple in vitro techniques. 
Also included by the term GRO is a protein or polypeptide having an amino 
acid sequence of between 70 and 100 amino acyl residues with either (a) a 
contiguous 20-residue segment thereof having at least 80% sequence 
homology with a portion of a naturally-occurring mature GRO (i.e., when 
the 20-residue segment is lined up with such portion of a 
naturally-occurring mature GRO, at least 80% of the residues of the former 
will be identical to the corresponding residues of the latter), or (b) a 
contiguous 10-residue segment thereof having at least 90% sequence 
homology with a portion of a naturally-occurring mature GRO. If the GRO 
polypeptide is of lesser size than 70 amino acids (for example 20 to 30 
amino acids), such polypeptide will have at least 80% sequence homology 
with some portion of the naturally-occurring GRO. GRO can be produced by 
any standard technique, including by extraction from animal tissues or 
cells which naturally produce the protein or can be induced to do so, by 
chemical synthesis, and by recombinant DNA technology. As discussed above, 
the DNA encoding the desired GRO can be modified by standard techniques to 
encode a GRO having a different amino acid sequence from that described by 
Richmond et al., 1988, and Anisowicz et al., 1987, and may be expressed in 
any desired cell type. It is not necessary that GRO be produced in a 
glycosylated state, since the naturally-occurring protein is not 
glycosylated. 
Applicants have surprisingly discovered that GRO is useful for promoting 
maturation of certain hematopoietic precursor cells. Previously, factors 
which were useful for promoting hematopoietic precursor cell maturation 
included colony-stimulating factors (CSFs) such as multi-CSF (also termed 
interleukin-3 or IL-3), granulocyte colony stimulating factor (G-CSF), 
macrophage colony stimulating factor (M-CSF), and granulocyte-macrophage 
colony stimulating factor (GM-CSF). All of these are glycoproteins of 
molecular weight 14-45 kD that are synthesized by multiple cell types 
including endothelial cells, fibroblasts, macrophages, and lymphocytes. In 
contrast, GRO is not a glycosylated protein and has a molecular weight of 
only 7 kD. Further, its amino acid sequence is very different from the 
respective sequences of the known CSFs. Other factors which have been 
found to work in synergy with the CSFs include IL-1 and IL-6, neither of 
which has an amino acid sequence similar to that of GRO. 
The method of the invention provides a way to boost a patient's level of 
fully differentiated hematopoietic cells (such as granulocytes and 
macrophages) by inducing the proliferation and maturation of hematopoietic 
precursor cells. Therapy with GRO can be accomplished either in vivo or ex 
vivo, and can utilize the patient's own bone marrow cells or cells 
provided by a donor. GRO may be used alone or in combination with other 
growth factors/cytokines such as the interleukins (particularly IL-1, 
IL-3, IL-6, and IL-8), the colony stimulating factors (e.g., GM-CSF, 
G-CSF, and M-CSF), and erythropoietin, in order to achieve optimal 
clinical results. 
Other features and advantages of the invention will be apparent from the 
following description of the preferred embodiments thereof, and from the 
claims.

PREATION OF GRO 
As stated above, GRO may be prepared by any standard 10 method, including 
but not limited to those utilizing recombinant DNA techniques. Two 
independent preparative methods are described below in Examples 1 and 2, 
but alternative methods will be apparent to those of ordinary skill in the 
art of protein production. 
EXAMPLE 1 
COS-1 cells (ATCC No.CRL1650) were transiently transfected with a pXM 
expression vector (available from Genetics Institute, Cambridge, Mass.) 
containing human gro cDNA (Anisowicz et al., 1987). Cells were maintained 
for one day in Alpha medium (Gibco) containing 10% fetal calf serum (FCS), 
and then were washed with serum-free medium and maintained for two days in 
Alpha salts plus 100 U/ml penicillin and 100 .mu.g/ml streptomycin. 
Culture medium was harvested and subjected to low-speed centrifugation 
(500.times.g for 5 min) to pellet cellular debris. The culture supernatant 
was dialyzed against 10 mM sodium phosphate, pH 6.5, and applied directly 
to a cation exchange column (CM-Sephadex, Pharmacia, Uppsala, Sweden). The 
bound proteins were eluted with a linear salt gradient of 0 to 0.7M NaCl 
in 10 mM sodium phosphate, pH 6.5. GRO-containing fractions were 
determined by analyzing aliquots of each fraction on 18% 
SDS-polyacrylamide gels, with a distinct silver-staining band at 6 kD 
indicative of the presence of mature recombinant GRO. The identity of the 
GRO-containing band was confirmed by positive cross-reactivity with an 
antibody prepared against a 10-amino acyl residue carboxy-terminal 
fragment of GRO. GRO-containing fractions were pooled and the protein was 
concentrated by differential filtration through a membrane with a 
molecular weight cut-off of 3,000 daltons. 
EXAMPLE 2 
An analog of mature human GRO [differing from naturally-occurring mature 
human GRO .alpha. by an amino-terminal octapeptide tag: 
AspTyrLysAspAspAspAspLys (Hopp et al., Biotechnology 6:1204-1210, 1988)] 
was produced in yeast by recombinant DNA techniques. A segment of gro cDNA 
encoding mature human GRO .alpha. was inserted into the yeast expression 
plasmid p.alpha.ADH2 (Price et al., Gene 55:287-293, 1987) and expressed 
in S. cerevisiae strain XV218/(a/.alpha.-trp-1). The resulting GRO analog 
was purified in a one-step immunoaffinity procedure utilizing an antibody 
specific for the first four residues of the octapeptide tag, according to 
the method of Hopp et al. 
Biolooical Assays for GRO 
GRO (including fragments and analogs of a naturally-occurring GRO) may be 
assayed for biological activity by a method such as one described in 
Examples 3-5. 
EXAMPLE 3: 
Assay for Maturation Promotion Activity 
The ability of GRO to promote maturation of hematopoietic precursor cells 
can be conveniently assayed in vitro by an assay such as the one herein 
described. Functional variations on this assay, and alternative assays, 
will be apparent to those of ordinary skill in the art. 
Bone marrow from a normal human or other animal is harvested by standard 
sterile procedures, heparinized, and either frozen or used immediately. 
Light-density bone marrow cells are isolated by density gradient 
centrifugation on Ficoll-Hypaque (LSM, Organon Technica, Durham, N.C.) 
according to standard methods, and then washed and adjusted to 
1.times.10.sup.6 cell/ml in RPMI 1640 medium (Gibco) containing 100 U/ml 
penicillin, 100 .mu.g/ml streptomycin, 12.5% FCS, and 12.5% horse serum. 
The cells (1.times.10.sup.5) are plated in duplicate on Lux 35-mm gridded 
culture dishes (Nunc, Inc., Naperville, Ill.) in 1.0 ml of the same 
culture medium containing methylcellulose (1500 centipoise) at a final 
concentration of 0.9% (w/v). The GRO preparation to be tested is added to 
a final concentration of, for example, 25, 50, or 100 ng/ml. The dishes 
are then placed in a 150 mm dish with water for humidification, and 
incubated in 5% CO.sub.2 in air at 37.degree. C. After 14 days of 
incubation, plates are scored for hematopoietic cell colonies (.gtoreq.40 
cells/aggregate). Each colony generally contains a mixture of 
hematopoietic cell types, with the particular combination of types present 
in a given colony indicative of the identity of the hematopoietic 
precursor cell from which the colony descended. For example, a colony 
which contains only granulocytes, monocytes, and/or CFU-GM cells arises by 
the action of a maturation-promoting activity on a single CFU-GM cell and 
would be scored as a CFU-GM colony, while a second colony which contains 
those three cell types plus eosinophils, CFU-Eo, megakaryocytes, 
erythrocytes, CFU-Meg, CFU-E, BFU-E, and/or CFU-GEMM is descended from a 
more primitive precursor cell, a CFU-GEMM, and so would be scored as a 
CFU-GEMM colony. Methods of distinguishing one hematopoietic cell type 
from another are well known to those of ordinary skill in the field of 
hematology. For example, the cellular morphology of individual colonies 
may be evaluated by differential counts performed on cytocentrifuge 
preparations of cells stained with the Diff-Quick modification (Sigma 
Chemical Co., St. Louis, Mo.) of the Wright's Giemsa technique. 
As a negative control, an equivalent aliquot of buffer lacking GRO (or any 
cytokine) is added to similarly prepared bone marrow cells. Positive 
controls vary with the specific type of hematopoietic precursor cell being 
analyzed for colony formation. In the experiments set forth in Table I 
below, recombinant human GM-CSF at 5 U/ml (Genetics Institute, Cambridge, 
Mass.) was used as a positive control for induction of colony formation by 
CFU-GM cells, while a combination of 2 U/ml recombinant human 
erythropoietin (Amgen, Thousand Oaks, Calif.), 0.5 mM 2-mercaptoethanol, 
and conditioned culture medium from the human bladder carcinoma cell line 
5637 (Welte et al., Proc. Natl. Acad. Sci. USA 82:1526, 1985) was used as 
a positive control for induction of colony formation by both CFU-GEMM and 
burst forming unit-erythroid (BFU-E) cells. The choice of other cytokines 
as positive controls for induction of formation of colonies by other types 
of hematopoietic cells is within the skills of those in the field to which 
the invention pertains. 
The results of six separate experiments using the above-described in vitro 
assay to test the bioactivity of recombinant human GRO .alpha. are shown 
in Table I below. The number of colonies formed after incubation for 14 
days in the presence of each concentration of GRO tested (100 ng/ml, 50 
ng/ml and 25 ng/ml) is expressed as a percentage of the colonies formed 
after incubation for a similar period in the presence of the applicable 
positive control cytokine (as described above). Under these culture 
conditions, the positive controls generate approximately 100-250 CFU-GM 
colonies, 24-60 CFU-GEMM colonies, and 40-200 BFU-E colonies per 10.sup.6 
bone marrow cells plated. Values for negative control plates are shown 
immediately below each corresponding GRO test result. The results show 
that recombinant human GRO is capable of stimulating generation of 
colonies of CFU-GM cells, CFU-GEMM cells, and possibly BFU-E cells in a 
bone marrow cell preparation, in some cases to a extent greater than that 
seen in the positive control. 
TABLE I 
______________________________________ 
COLONY FORMATION BY BONE MARROW 
CELLS TREATED WITH GRO 
Number of colonies/10.sup.6 light-density 
bone marrow cells expressed as a % 
of (+)control 
CFU-GM CFU-GEMM BFU-E 
______________________________________ 
Experiment #1 
GRO (100 ng/ml) 
87% 103% 70% 
Buffer (1:15) 
76 32 34 
GRO (50 ng/ml) 
98 64.5 64 
Buffer (1:30) 
67 26 27 
GRO (25 ng/ml) 
76 48.4 56 
Buffer (1:60) 
70 16 15 
Experiment #2 
GRO (100 ng/ml) 
69 82 82 
Buffer (1:15) 
35 35 64 
GRO (50 ng/ml) 
ND ND ND 
Buffer (1:30) 
18 41 9 
GRO (25 ng/ml) 
102 88 86 
Buffer (1:60) 
18 24 18 
Experiment #3 
GRO (100 ng/ml) 
117 116 99 
Buffer (1:15) 
69 37 84 
GRO (50 ng/ml) 
172 130 74 
Buffer (1:30) 
74 63 63 
GRO (25 ng/ml) 
109 137 93 
Buffer (1:60) 
87 87 28 
Experiment #4 
GRO (100 ng/ml) 
115% 125% 65% 
Buffer (1:15) 
38 25 10 
GRO (50 ng/ml) 
91 108 45 
Buffer (1:30) 
35 17 15 
GRO (25 ng/ml) 
62 75 15 
Buffer (1:60) 
29 33 10 
Experiment #5 
GRO (100 ng/ml) 
72 68 9 
Buffer (1:15) 
58 19 5 
GRO (50 ng/ml) 
66 50 14 
Buffer (1:30) 
38 19 2 
GRO (25 ng/ml) 
57 69 5 
Buffer (1:60) 
35 31 2 
Experiment #6 
GRO (100 ng/ml) 
75 7 40 
Buffer (1:15) 
14 0 5 
GRO (50 ng/ml) 
83 4 15 
Buffer (1:30) 
18 1 0 
GRO (25 ng/ml) 
98 12 25 
Buffer (1:60) 
7 1 0 
______________________________________ 
EXAMPLE 4: 
Assay Following Depletion of Accessory Cells 
In order to determine whether or not GRO stimulates proliferation by direct 
action on the proliferating cells, or by inducing production of an 
undetermined cytokine by other "accessory" cells present in the bone 
marrow preparation, an accessory cell depletion experiment was carried 
out. Light-density bone marrow cells were prepared as described above and 
depleted of monocytes by exposing the preparation to a plastic Petri dish 
for 3 hours to permit adherence of monocytes. Non-adherent cells were then 
removed from the dish, washed, and subjected to further analysis. 
Depletion of NK (natural killer) and T lymphocytes was accomplished by E 
rosetting by the method of Elliott and Pross (Methods in Enzymology 
108:49-64, 1984). The results of treating such depleted bone marrow cell 
populations with recombinant GRO, shown in Table II, indicate that GRO 
retains most or all of its ability to stimulate maturation of CFU-GM cells 
even in the absence of monocytes and/or NK and T cells, suggesting that 
GRO may act directly on the proliferating CFU-GM cells. In contrast, the 
ability of GRO to stimulate formation of colonies by CFU-GEMM appears to 
be decreased in the absence of such accessory cells. This result suggests 
that GRO affects CFU-GEMM cells at least in part by an indirect route that 
involves other cells, perhaps by inducing accessory cells to produce a 
different growth-stimulating cytokine. 
TABLE II 
______________________________________ 
COLONY FORMATION BY ACCESSORY 
CELL-DEPLETED BONE MARROW CELLS 
TREATED WITH GRO 
Number of colonies/10.sup.6 (pre- 
depletion) bone marrow cells, 
expressed as a % of (+)control 
CFU-GM CFU-GEMM BFU-E 
______________________________________ 
Experiment #7 
Monocyte-depleted 
GRO (100 ng/ml) 
121% 18% 16% 
GRO (50 ng/ml) 
80 11 16 
GRO (25 ng/ml) 
78 46 8 
(-)control (buffer) 
26 7 0 
NK + T cell-depleted 
GRO (100 ng/ml) 
93 79 20 
GRO (50 ng/ml) 
93 114 17 
GRO (25 ng/ml) 
101 129 34 
(-)control (buffer) 
31 7 2 
Experiment #8 
Monocyte-depleted 
GRO (100 ng/ml) 
128 41 40 
GRO (50 ng/ml) 
124 72 27 
GRO (25 ng/ml) 
131 45 19 
(-)control (buffer) 
56 24 6 
NK + T cell-depleted 
GRO (100 ng/ml) 
115 71 38 
GRO (50 ng/ml) 
142 71 23 
GRO (25 ng/ml) 
104 150 54 
(-)control (buffer) 
51 21 0 
Experiment #9 
NK + T cell-depleted 
GRO (100 ng/ml) 
112% 400% 13% 
GRO (50 ng/ml) 
75 300 17 
GRO (25 ng/ml) 
46 400 20 
(-)control (buffer) 
31 0 0 
Monocyte + NK + T 
cell-depleted 
GRO (100 ng/ml) 
82 100 5 
GRO (50 ng/ml) 
71 250 5 
GRO (25 ng/ml) 
87 100 5 
(-)control (buffer) 
23 0 5 
Experiment #10 
NK + T cell-depleted 
GRO (100 ng/ml) 
89 0 0 
GRO (50 ng/ml) 
83 0 0 
GRO (25 ng/ml) 
58 0 0 
(-)control (buffer) 
20 0 0 
Monocyte + NK + T 
cell-depleted 
GRO (100 ng/ml) 
58 11 3 
GRO (50 ng/ml) 
26 11 3 
GRO (25 ng/ml) 
26 0 0 
(-)control (buffer) 
9 0 0 
______________________________________ 
EXAMPLE 5: 
Chemotaxis Assay 
Certain members of the gene family to which the gro genes are related 
encode proteins which are chemotactic for neutrophils. In order to 
determine whether or not recombinant GRO is also chemotactic for such 
cells, the following assay was carried out: 
Human peripheral venous blood taken from normal volunteers was subjected to 
dextran sedimentation (Pharmacia, Uppsala, Sweden) followed by 
centrifugation on Ficoll-Hypaque (Lympho-prep, Organon Technica, Durham, 
N.C.) as described by Williams et al. (Proc. Natl. Acad. Sci. USA 74:1204, 
1977). Pellets containing polymorphonuclear leukocytes (PMNs, a type of 
neutrophil) were subjected to hypotonic lysis (x2); they contained greater 
than 95% PMNs as judged by microscopic examination of Wright's stained 
specimens. The buffy coat containing mononuclear cells was washed twice 
with Hank's balanced salt solution (Gibco, Grand Island, N.Y.) containing 
0.01M HEPES, pH 7.0; 4.3 mM NaHCO.sub.3 (HBSS) and 2% bovine serum albumin 
(HBSS-BSA). Mononuclear cell preparations contained 25-35% monocytes as 
determined by nonspecific esterase staining. Chemotaxis was quantified 
using 48-well microchambers (Neuroprobe, Inc., Cabin John, MD) (Harvath et 
al., J. Immunol. Methods 37:39, 1980; Falk et al., J. Immunol. Methods 
33:239, 1980). PMNs (0.05 ml, 1.times.10.sup.6 /ml) suspended in HBSS, pH 
7.2, were placed in the upper wells of the microchamber. HBSS alone or 
HBSS containing GRO or another chemotactic stimulant (0.03 ml) was placed 
in each of the lower chambers and was separated from the cells by a 3.0 
.mu.m pore diameter polyvinyl pyrrolidine (PVP)-free polycarbonate filter 
(25 mm.times.80 mm). Following incubation at 37.degree. C. in humidified 
air for 60 min, the non-migrated cells were removed from the top of the 
filter, as counted and the migrated cells were stained with a 
leukocyte-specific stain (Leuko Stat, Fisher Scientific, Orangeberg, 
N.Y.). PMN chemotaxis was quantified as the average number of cells/field 
which had migrated completely through the filter, as counted in ten oil 
immersion (.times.1000) fields. Assays were performed in triplicate and 
expressed as the mean cells/field .+-.S.D. As shown in FIG. 4, recombinant 
human GRO increased the number of migrated PMNs approximately 2.8-fold 
when compared to HBSS alone. GRO induced significant migration of PMNs in 
concentrations ranging from 0.05 nM to 5.0 nM. Maximal neutrophil 
chemotactic activity was obtained at a GRO concentration of 0.7.+-.0.2 nM, 
with an effective concentration which produced 50% of maximal migration 
(EC.sub.50) of 0.07.+-.0.05 nM. The total number of cells migrating in 
response to GRO ranged from 53% to 83% of the number of cells responding 
to 100 nM f-Met-Leu-Phe, a known chemoattractant. Monocyte chemotaxis was 
similarly quantified in the 48-well microchambers except that cells were 
suspended to 1.5.times.10.sup.6 monocytes/ml in HBSS-BSA, pH 7.0; 5.0 
.mu.m PVP-coated polycarbonate filters were used; and incubations were 
allowed to proceed for 90 min in 37.degree. C. humidified air. Unlike 
human PMNs, human monocytes did not respond chemotactically to 
concentrations of GRO ranging from 0.01 nM to 10 nM (data not shown). 
These results, analogous to those produced by NAP-3 and NAP-1/IL-8, suggest 
a role for GRO in acute inflammation. 
Use 
The maturation promoting ability of GRO is useful for treatment of a 
variety of diseases and conditions. For example, it can be used to promote 
regeneration of hematopoietic cells between cycles of myelotoxic 
chemotherapy, permitting use of increased doses of chemotherapeutic 
agents. The protein may also be used during or after autologous, 
allogeneic, or even xenogeneic bone marrow transplantation to promote 
accelerated engraftment. In addition, a number of genetic diseases 
characterized by neutropenia and thrombocytopenia can be treated by 
promoting the maturation of appropriate hematopoietic precursor cells. 
Continuous low-dose therapy will result in an increase in the patient's 
level of durable neutrophils and platelets. Azidothymidine (AZT) treatment 
of acquired immunodeficiency syndrome (AIDS) patients can induce severe 
neutropenia and anemia, which can be combatted by GRO therapy. Thus, GRO 
is broadly useful for treatment of hematopoietic cell deficiencies, 
whether congenital or therapy-induced. 
GRO therapy can be accomplished by treating the patient systemically with 
an intravenous bolus or infusion of GRO (e.g. 1-100 .mu.g/kg body weight 
per day) in a pharmaceutically effective carrier, or by any other 
effective means (such as an oral dose of a GRO analog that retains its 
potency when so administered, or by localizing the GRO injection directly 
to the in vivo site of the bone marrow to be treated). Alternatively, a 
preparation of the patient's bone marrow (or the bone marrow of a donor) 
can be treated ex vivo with GRO, cultured for a period to permit 
generation of hematopoietic cells, and then implanted in the patient. 
Other embodiments are within the following claims.