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Expression Profile of Active Genes in Granulocytes
By Koichi Itoh, Kousaku Okubo, Hiroyasu Utiyama, Tetsuo Hirano, Junji Yoshii, and Kenichi Matsubara
A number of genes active in granulocytes have been intensively studied as to the function of their products and their
expression controls. However, the intensities and relative
order of these gene activities have not been studied. This
report describes an expression profile of 748 different species of active genes in human peripheral granulocytes obtained by analyzing a 38-directed cDNA library that faithfully
represents the mRNA population in the source cells. A
significant fraction (20.3% of the total) of the expressed
genes in granulocytes consisted of nuclear proteins such as
DNA binding proteins, of secretory proteins such as cyto-
kines, and of membrane proteins such as major histocompatibility complex (MHC) proteins and receptors. By comparing
this expression profile with 11 profiles similarly obtained
with unrelated human cells/tissues, we discovered 10 novel
genes that are likely to act specifically in granulocytes.
Comparison of this expression profile with that obtained
with granulocytoids widely used as a granulocyte model by
inducing a cultured promyelocytic leukemia cell line HL60
showed similarities and dissimilarities of gene expressions.
r 1998 by The American Society of Hematology.
G
cells stopped proliferating, the nucleus became pyknotic and
polymorphic, and the cells expressed several genes unique to
granulocytes. Thus, because they are morphologically and
functionally similar to granulocytes, the cells are referred to as
granulocytoids and are widely used as a model system for
studies of granulocytes.7-10 We monitored the expression profile
of these cells. Comparison of this profile with that of the
peripheral granulocytes showed advantages and disadvantages
in using the granulocytoids as a model system.
RANULOCYTES ARE short-lived cells for body defense,
with a half-life of 6 to 7 hours in blood. Because of this
short life span, their protein synthetic apparatus is poorly
developed,1,2 but they maintain active genes that are responsible
for their unique activities. These genes have been the focus of
intensive study.
We have initiated a systematic survey of active genes, as well
as the relative abundance of mRNA expression, in granulocytes
using an expression profiling method that is based on quantitative analysis of mRNA populations.3 This is performed by using
38-directed cDNA libraries that faithfully represent the mRNA
population and by obtaining short base sequences just upstream
of polyA, called gene signatures (GSs), by single-pass sequencing of randomly selected clones from such libraries.3-5 Active
genes are identified by sequences and the gene activities are
identified by their recurrences. The resulting list showing the
expressed gene species and the abundance of their transcript is
called an expression profile, which illuminates the gene-product–
based cellular phenotype.
In the profile are represented several known genes as well as
novel genes. These genes can be categorized as those that are
commonly expressed in different types of cells (candidate genes
for housekeeping functions) and those that are expressed
uniquely in granulocytes (candidate genes for granulocytespecific functions). We used 11 expression profiles obtained
with different human cells/tissues for the gene categorization
and discovered some genes that are likely to be granulocytespecific, even though we do not yet know their functions.
A promyelocyte cell line, HL60,6 is converted into granulocytoid cells by treatment with dimethylsulfoxide (DMSO). The
From the Institute for Molecular and Cellular Biology, Osaka
University, Yamada-oka, Suita, Osaka, Japan; the Life Science Group,
Faculty of Integrated Arts and Sciences, Hiroshima University, Kagamiyama, Higashihiroshima, Hiroshima, Japan; and Hitachi Software
Engineering, Co, Ltd, Onoe-chou, Naka-ku, Yokohama, Japan.
Submitted December 29, 1997; accepted April 22, 1998.
Address reprint requests to Kenichi Matsubara, PhD, Nara Institute
of Science and Technology 8916-5, Takayama, Ikoma, Nara 630-01,
Japan; e-mail: [email protected].
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9204-0013$3.00/0
1432
MATERIALS AND METHODS
Preparation of human granulocytes. Freshly obtained venous blood
was diluted with the same volume of phosphate-buffered saline (PBS),
and the suspension was centrifuged on a ficoll step gradient (upper
density, 1.077; lower density, 1.119) for 20 minutes at 3,000g.11,12
Granulocytes collected from the interface were washed twice with PBS
and resuspended in the same buffer. The purity of the preparation was
examined under a microscope upon Giemsa staining. At least 99% of
total cells were mature granulocytes.
Induction of granulocytoid cells. HL60 cells (Japanese Cancer
Research Resource Bank, Osaka, Japan) were grown in RPMI 1640
medium (Nissui, Tokyo, Japan) supplemented with 10% (vol/vol) bovine
fetal serum (Hyclone, Logan, UT) and harvested in the logarithmic
phase (106 cells/mL). Granulocytoid cells were prepared by seeding
HL60 in a plate at a concentration of 2 3 105 cells/mL and incubating in
the presence of 1.3% (vol/vol) DMSO (Sigma, St Louis, MO) for 72 hours.
cDNA library construction and sequencing. RNAs were prepared
from the cytoplasmic fraction of DMSO-induced HL60 cells as
described3 and from the total cell lysate13 of granulocytes. Purification
of polyA RNA was not attempted, because of the presence of RNase.
Construction of the 38-directed cDNA libraries and transformation into
Escherichia coli were performed by synthesizing cDNA using pUC19based vector primer, digesting with dam-sensitive four-base cutter Mbo
I, followed by circularization and transformation into E coli.4 The
transformant colonies were randomly selected and cultured in 96-well
plates, and the inserted cDNAs were amplified with flanking primers
and subjected to cycle sequencing.
Data analysis. The polyA tail was removed from the sequencing
data after checking the electropherograms, leaving 3 As as a marker.
From the resulting sequence data, those having inserts shorter than 20
bp or those having more than 5 ambiguous bases (N) within the initial
100 bases were discarded. The sequences of the remaining clones were
truncated where the N content exceeded 5%, and the final N was
replaced by an X to mark the point of truncation. The resulting
sequences are referred to as GSs.
The GSs were compared using the FastA program.14 Identical
signatures were lumped together, and a representative sequence that had
the lowest content of ambiguous bases was selected to represent the
group and deposited in the DDBJ, wherein the locus name corresponds
Blood, Vol 92, No 4 (August 15), 1998: pp 1432-1441
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GENE EXPRESSION PROFILE OF GRANULOCYTES
1433
Table 1. Expression Profile of Active Genes in Human Peripheral Granulocytes (A) and Granulocytoids by Inducing HL60 With DMSO (B)
(A)
GS
lib
PM
GR
HL
MO
00565
00402
00254
01823
02821
00937
08362
03341
01476
08339
01267
01811
08347
01367
00155
02490
04345
05209
01724
00816
00994
01809
04290
02015
j
j
j
>
>
j
h
>
>
h
>
>
h
j
j
j
h
h
>
>
j
>
h
>
24
19
18
18
15
14
13
11
10
9
8
8
8
7
6
6
6
6
6
5
5
5
5
5
3
4
5
1
2
2
07795
08383
00244
01371
02161
05014
05242
03554
08325
08395
08438
01087
00685
00273
01228
00304
01473
01187
01334
01406
01585
01325
00239
01304
01594
01919
03575
01877
00729
06701
04074
04103
01826
03216
08336
05103
08389
08375
08424
08548
Total
>
h
j
h
>
h
h
h
h
h
h
>
>
j
>
j
>
>
>
>
>
j
>
>
h
j
>
>
>
>
>
>
>
>
h
h
h
h
h
h
5
5
4
4
4
4
4
4
4
4
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
370
4
HE
1
1
FL
IL
AL
AP
1
1
1
1
1
2
1
3
1
1
2
VF
LG
CM
KC
4
1
1
1
1
2
2
1
1
5
2
11
2
CO
RE
1
2
3
1
1
2
1
1
1
3
3
1
8
2
1
3
1
1
2
1
1
1
3
2
2
1
1
4
1
4
2
Gene Name
b 2-microglobulin*
Spermidine/spermine N1-acetyltransferase
MHC class I HLA-Cw1
Pre-B cell enhancing factor (PBEF)
CL 100 protein tyrosine phosphatase
HLA-E heavy chain (38 untranslated region)
Granulocyte colony-stimulating factor receptor*
B94 protein
IL-8*
1-8D
H3.3 histone
hnRNP-E1
Thymosin b-4
1
B4-2 protein
5
2
1
2
1
1
3
1
1
1
1
6
1
11
1
14
1
1
2
1
3
4
1
1
1
6
5
4
4
2
2
2
2
2
2
1
1
1
1
2
6
4
11
1
2
2
1
1
1
3
9
2
2
1
Pleckstrin (P47)
Lymphocyte-specific protein 1 (LSP1)
Glutamine synthetase
Lysosomal membrane glycoprotein CD63
LD78 a/macrophage inflammatory protein
(GOS19-1)*
B-cell lymphoma 3-encoded protein (bcl-3)
Zinc-finger protein (bc1-6)
b-actin
AMP deaminase isoform L (AMPD2)
Histone H3.3
11
1
2
1
1
2
1
12
2
1
3
4
1
1
1
1
5
1
3
3
3
5
3
Nramp
Brain-expressed HHCPA78 homolog
L-plastin
Translationally controlled tumor protein
Anonymous mRNA
Ubiquitin
Monocyte activation antigen (Mo3)
1
2
1
Tumor necrosis factor receptor*
sui1iso1
1
2
1
5
1
5
6
1
3
2
1
1
2
5
2
1
1
1
1
1
Transmembrane glycoprotein (CD53)
Leukocyte common antigen T200 (CD45)*
Calcyclin
Histone H3.3
Paxillin
CLP mRNA
19
1
Transcription factor ETR101
HLA-B27 antigen
1
1
74
17
75
17
6
16
8
32
Long-chain acyl-CoA synthetase
Secretory granule proteoglycan peptide
ICAM-3*
E4BP4 gene
MAD-3
30
to the GS number (such as HUMGS01234 to GS01234). All the
representative GSs were searched against GenBank (Re95) using the
FastA program,14 and those that had greater than 90% similarity to the
38 end of the mRNA entries or to the reported terminal exon of genes
were regarded as representing the corresponding genes.
33
37
21
43
12
RESULTS
Expression profiles of active genes in granulocytes. From
the 38-directed cDNA library constructed from human peripheral granulocytes, we randomly selected 1,142 independent
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1434
ITOH ET AL
Table 1. Expression Profile of Active Genes in Human Peripheral Granulocytes (A) and Granulocytoids by Inducing HL60
With DMSO (B) (Cont’d)
(B)
GS
lib
GR
PM
HL
MO
00565
00937
01476
01367
00155
00816
00244
01087
00685
01228
00273
01394
01106
01321
01007
01609
01603
01224
01075
00543
01365
00995
00783
00383
total
00583
00249
00114
01572
01345
01445
01024
00096
01478
01372
j
j
>
j
j
>
j
>
>
>
j
h
>
j
j
>
h
>
>
j
h
j
>
j
h
j
>
j
h
h
>
>
j
>
>
3
4
3
3
3
5
14
6
5
4
4
4
4
3
3
4
4
4
4
4
3
3
3
3
6
5
5
5
4
4
4
4
4
3
3
24
14
10
7
6
5
4
3
3
3
3
2
2
2
2
1
1
1
1
1
1
1
1
1
2
4
1
5
01244
01141
01091
00997
00875
00839
j
>
>
h
j
>
3
3
3
3
3
3
00732
00716
00697
00687
00335
00290
00162
00071
j
j
>
>
j
j
j
>
3
3
3
3
3
3
3
3
1
2
1
8
1
1
2
4
6
11
HE
FL
1
IL
AL
AP
1
1
1
1
1
2
3
2
9
3
2
2
11
1
2
1
1
2
VF
5
1
1
2
1
1
12
2
LG
CM
KC
1
11
4
2
1
4
1
4
3
4
1
1
1
1
5
3
3
5
1
1
1
2
1
2
1
2
2
3
2
1
CO
RE
b-2-microglobulin
HLA-E heavy chain (38 untranslated region)
1
hnRNP-E1
Thymosin b-4
Lymphocyte-specific protein 1 (LSP1)
b-actin
Brain-expressed HHCPA78 homolog
L-plastin
Anonymous mRNA
Translationally controlled tumor protein
Lysozyme
1
1
1
2
Stimulatory GTP-binding protein alpha subunit
g-interferon-inducible protein (IP-30)
1
1
9
5
2
1
1
1
2
2
3
3
3
1
3
31
1
1
1
Gene Name
1
14
1
25
1
1
2
1
1
16
1
2
56
1
1
1
2
30
2
1
1
KIAAO184 protein
2
1
2
42
3
1
49
2
3
1
2
2
34
1
1
2
3
23-kD highly basic protein
1
1
Eukaryotic initiation factor 4AII
37
29
4
Ribosomal protein L3
Liver glycogen phosphorylase type IV
Cytoskeletal gamma-actin
Bactericidal permeability increasing protein (BPI)
1
5
2
2
6
1
6
1
2
2
3
3
2
2
1
1
2
2
1
1
2
1
1
6
5
2
1
15
9
4
4
3
2
5
3
8
2
1
1
5
1
1
1
4
1
2
1
2
1
2
4
1
1
1
1
7
1
5
1
1
7
2
1
1
1
1
3
2
2
1
2
3
1
2
1
1
6
1
4
Leukocyte adhesion protein/LFA-1/Mac-1
a NAC
hnRNP-E2
Antigen migration inhibitory factor-related
protein 14
Mitochondrial ubiquinone-binding protein
Transcriptional coactivator PC4
1
1
1
2
2
1
1
5
1
5
2
1
1
1
1
4
Cercopithecus aethiops UV-damaged DNA-binding
protein
Ribosomal protein L38
Plasminogen activator inhibitor-2 (PAI-2)
Glycoprotein
Ribosomal protein L7a
Ribosomal protein L37a
Acidic ribosomal phosphoprotein P2
Genes are registered according to their order of abundance. Numbers represent the frequency among 1142 cDNA clones analyzed in (A) and 1042 cDNA analyzed in (B).
For simplicity’s sake, only those genes that appeared three times or more are listed. The expression profile of low abundant genes can be accessed electronically in www
bodymapper server (http://www.imcb.osaka-u.ac.jp/bodymap). Abundances of the GSs that appeared in other libraries are also shown for comparison’s sake. Numbers
represent abundance among 1142 cDNA clones. Number of libraries in which a given GS has been detected is shown in the column ‘lib’ by the shading pattern: (solid area)
the GS has been detected in 6 libraries or more representing ubiquitous expression; (open area) only in the PM and GR libraries, possibly representing granulocyte-specific
expression; (hatched area) in 2 through 5 libraries, possibly representing common expression. Genes that have so far been reported in GenBank are listed in the column
‘‘Gene Name.’’
Abbreviations: PM, peripheral granulocytes; GR, granulocytoid cells induced by DMSO; HL, HL60 cells; MO, monocytoid cells obtained by inducing HL60 with
tetradecanoylphorbol-13 acetate (TPA); HE, HepG2 cells; FL, fetal liver; IL, infant liver; AL, adult liver; AP, subcutaneous fat; VF, visceral fat; LG, lung; CM, colonic mucosa;
KC, kerationcyte; CO, cornea; RE, retina.
*Active in granulocytes (only in A).
clones and sequenced them. Among the resulting short sequences called GSs, representing just upstream of the polyA,
sequences that were considered essentially identical were
lumped together to represent the same gene species. After this
treatment, 748 independent GS species resulted. Among them,
216 (28.9%) represented by 493 clones were identified in
GenBank (Re95), and the remaining 532 (71.1%), represented
by 649 clones, were from novel genes. Table 1A shows an
expression profile of active genes as represented by their GSs
and their activities with their relative abundance. We listed here
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Table 2. Identified Genes Detected in Peripheral Granulocytes
GS
PM
GR
HL
MO
HE
FL
IL
AL
AP
VF
LG
CM
KC
CO
RE
Gene Name
A. Energy production
00943
02200
Total
1
1
2
1
1
3
1
1
3
Phosphoglycerate kinase 1
Transaldolase
1
1
B. Miscellaneous
00402
02161
00304
03216
01202
00483
03596
07377
08337
08513
01263
00253
01328
02147
00154
00533
01766
07546
08483
08328
08418
08504
08643
08690
Total
19
4
3
3
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
55
2
1
1
1
2
2
2
1
1
1
3
1
1
1
4
1
1
3
2
3
1
1
1
2
2
1
1
2
1
2
1
2
1
1
2
6
8
3
9
7
15
2
4
20
3
5
6
1
2
5
3
10
9
4
10
14
1
6
12
1
1
Spermidine/spermine N1-acetyltransferase
AMP deaminase isoform L (AMPD2)
Ubiquitin
Long-chain acyl-CoA synthetase
Neutrophil oxidase factor (NCF2)/p67-phox
IMP dehydrogenase type 1
Spermidine/spermine N1-acetyltransferase
Inosine monophosphate dehydrogenase type II
Grancalcin
Proteasome subunit p40 (Mov34)
Vacuolar H1-ATPase Mr 56,000 subunit (HO57)
Importin beta subunit
Calpastatin5glycoprotein BS-17 component
Protein disulfide isomerase related protein (ERp72)
Glutaredoxin
Ferritin H chain
Metallothionein from cadmium-treated cells
Type 1 inositol 1,4,5-trisphosphate receptor
Ubiquitin conjugating enzyme (UBC4)
Ribonuclease A
Erythrocyte-type AMP deaminase
Lysyl hydroxylase (PLOD)
Histidase
Ferritin H chain
2
C. Lysosomal
01809
01106
01533
08663
08679
08396
08464
08664
Total
5
2
1
1
1
1
1
1
13
4
1
3
3
2
6
1
2
3
1
1
1
5
8
2
3
7
2
1
1
2
2
1
6
1
3
2
Lysosomal membrane glycoprotein CD63
Lysozyme
Lysosomal membrane glycoprotein-1
Arylsulphatase A
26S protease (S4) regulatory subunit
Giantin/golgi antigen gcp372
Iduronate sulphate sulphatase (IDS)
Iduronate sulphate sulphatase (IDS)
D. Signal transduction
02821
01724
00816
01919
00651
01089
07614
01524
00124
00286
00637
01687
02144
03705
04134
06319
08680
Total
15
6
5
3
2
2
2
1
1
1
1
1
1
1
1
1
1
45
5
1
1
1
2
1
5
1
5
4
1
5
1
CL 100 protein tyrosine phosphatase
Pleckstrin (P47)
Lymphocyte-specific protein 1 (LSP1)
Calcyclin
Rho GDP-dissociation Inhibitor 2
Tyrosine kinase (HCK)
Cyclin G2
TSE15protein kinase A regulatory subunit
Ras-related protein (Krev-1)
ERK activator kinase (MEK1)
56K autoantigen annexin XI
c-raf-1 proto-oncogene
Protein kinase C z
a-subunit of Gi2
Rapamycin- and FK506-binding protein
Protein kinase
5-lipoxygenase
19
1
1
2
2
1
1
1
1
1
1
1
1
8
7
9
4
2
1
1
5
8
6
1
1
3
1
1
1
4
5
1
1
1
1
1
10
5
5
6
22
E. Ribosomal component and translation factor
01325
00314
00919
00305
00995
00650
00418
00795
00818
00889
00356
03424
Total
3
2
2
2
1
1
1
1
1
1
1
1
17
1
1
1
3
2
1
1
1
11
3
1
6
14
22
1
4
1
16
68
3
1
2
1
5
10
2
1
1
2
1
5
1
2
1
1
2
2
5
1
2
1
1
26
8
7
17
1
2
1
1
2
10
2
2
1
3
1
2
1
1
2
2
6
2
3
4
1
1
1
2
7
6
2
1
1
19
14
10
13
2
1
1
9
22
1
2
1
2
9
3
2
20
sui1iso1
Ribosomal protein S11
Splicing factor (CC1.4)
Ribosomal protein L28
Eukaryotic initiation factor 4AII
Ribosomal protein S18
Ribosomal protein L9
Ribosomal protein L30
Ribosomal protein L5
EF-1 d
L23 putative ribosomal protein
Translational initiation factor 2 b subunit
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Table 2. Identified Genes Detected in Peripheral Granulocytes (Cont’d)
GS
PM
GR
HL
MO
HE
8
1
4
1
1
2
FL
IL
AL
AP
VF
LG
CM
KC
CO
RE
2
4
3
1
4
4
1
1
1
1
1
1
1
9
1
9
5
1
1
1
Gene Name
F. Cytoskeltal
00155
00244
00685
01877
04080
01030
01507
00258
01702
01726
02833
Total
6
4
3
3
2
1
1
1
1
1
1
24
3
14
5
2
2
1
3
2
1
1
1
1
2
1
1
1
1
2
25
2
18
1
1
4
3
2
1
5
3
1
5
Thymosin b-4
b-actin
L-plastin
Paxillin
Cofilin
Myosin regulatory light chain
a-actinin
2.5-kb cytoskeletal tropomyosin TM30 (nm)
Alkali myosin light chain
Striated muscle contraction regulatory protein
Nonmuscle myosin heavy chain (NMHC)
G. Mitochondrial
00994
00313
00360
00934
08345
08434
08667
01995
08536
04986
Total
5
2
1
1
1
1
1
1
1
1
15
2
1
1
1
5
1
2
3
5
1
1
2
1
1
1
3
1
5
3
1
2
3
3
1
1
1
1
5
1
1
4
6
2
1
1
1
3
1
2
1
4
1
6
Glutamine synthetase
SOD-2 manganese superoxide dismutase
Mitochondrial phosphate carrier protein
coxVIb
ATP synthase c subunit
Manganese superoxide dismutase
Glutamine synthetase
Glutathione peroxidase
SOD-2 gene for manganese superoxide dismutase
Catalase
3
H. Nuclear
01811
01367
07795
08383
05014
03575
04074
08375
08389
01431
01719
06259
08044
01566
00827
08621
04003
01772
01912
02730
00487
00648
01803
01832
03413
06299
08013
08366
08411
08454
08546
08647
Total
8
7
5
5
4
3
3
3
3
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
68
1
1
3
2
1
1
1
1
1
2
1
3
1
1
1
1
2
1
1
1
1
2
1
1
1
1
2
1
2
1
1
2
1
1
1
2
1
1
1
1
6
2
7
1
3
1
1
7
7
7
5
6
H3.3 histone
hnRNP-E1
B-cell lymphoma 3-encoded protein (bc1-3)
Zinc-finger protein (bc1-6)
H3.3 histone
H3.3 histone
Transcription factor ETR101
MAD-3
E4BP4 gene
jun-D
Myeloid cell nuclear differentiation antigen
spi-1 proto-oncogene
Zinc finger transcriptional regulator
fus
dUTP pyrophosphatase (hdut)
hnRNP A2/hnRNP B1
Receptor of retinoic acid
Interferon-g induced protein (IFI 16)
CDEI binding protein/amyloid protein homologue
fos proto-oncogene
Transformation upregulated nuclear protein
Proliferating cell nucleolar protein P120
BAT1 nuclear RNA helicase (DEAD family)
jun-B
tpr
Arginine-rich nuclear protein
Replication protein A 70 kD subunit
Pre-mRNA splicing factor SR p75
Transcription factor SUPT4H
Nuclear factor erythroid 2 isoform f
Transcription factor
Putative RNA helicase HRH1
5
I. Secretory and granular
01823
08339
02015
08336
08638
01820
00592
01616
08342
08553
08318
00111
02007
08501
08530
08644
08674
Total
18
9
5
3
2
2
1
1
1
1
1
1
1
1
1
1
1
50
2
11
1
1
2
1
1
1
1
3
1
1
1
1
1
7
20
7
9
10
1
13
14
6
6
1
1
1
1
2
2
Pre-B cell enhancing factor (PBEF)
IL-8
LD78 a/macrophage inflammatory protein
Secretory granule proteoglycan peptide
N-formylpeptide receptor (fMLP-R26)
Antisecretory factor-1
ATL-derived factor/thioredoxin
Cystatin B
Neutrophil peptide/defensin1/HP-1
Cathepsin S
N-formylpeptide receptor (fMLP-R98)
a 1-antitrypsin
Globin
Metalloproteinase-2 inhibitor (TIMP-2)
Platelet-derived endothelial cell growth factor
Platelet-derived endothelial cell growth factor
Monocyte/neutrophil elastase inhibitor
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Table 2. Identified Genes Detected in Peripheral Granulocytes (Cont’d)
GS
PM
GR
HL
MO
HE
4
5
2
1
FL
IL
AL
AP
1
1
1
1
1
VF
LG
CM
KC
1
2
11
4
2
1
5
CO
RE
Gene Name
J. Surface membrane
00565
00254
00937
08362
08438
01187
01585
01304
01594
04103
05103
01624
00303
08355
08594
01048
01395
01229
00474
02079
02156
06689
08349
08524
08563
08564
08592
08661
08678
08686
Total
24
18
14
13
4
3
3
3
3
3
3
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
114
3
4
1
2
2
1
1
1
3
2
1
3
1
1
1
1
2
2
1
1
1
2
1
1
1
2
1
1
1
1
1
1
18
7
14
4
4
1
3
2
7
1
2
2
12
1
2
16
7
4
3
b-2-microglobulin
MHC class I HLA-Cw1
HLA-E heavy chain
Granulocyte colony-stimulating factor receptor
Nramp
Monocyte activation antigen (Mo3)
Tumor necrosis factor receptor
Transmembrane glycoprotein (CD53)
Leukocyte common antigen T200 (CD45)
HLA-B27 antigen
ICAM-3
Aminopeptidase N/CD13 encoding aminopeptidase N
(chromosome 3p25) membrane protein
C5a anaphylatoxin receptor
TAP1
MHC antigen (HLA-B)
Transmembrane receptor protein
Migration inhibitory factor-related protein 8
Transmembrane carcinoembryonic antigen BGPb
Palmitoylated erythrocyte membrane protein (MPP1)
CD14 myelid cell-specific leucine-rich glycoprotein
Leukocyte antigen CD97
Leukocyte adhesion molecule-1 (LAM-1)
Interferon-g receptor
Low-affinity IgG Fc receptor
Complement receptor type 1
Common acute lymphoblastic leukemia antigen (CALLA)
Putative voltage-gated potassium channel (KVLQT1)
MHC class I-related protein
IL-8 receptor b (IL8RB)
1
K. Unknown Function
03341
01267
04345
01087
00273
01228
00729
01288
00080
00600
01926
08056
08235
08444
00543
01224
01609
00185
01032
01608
01665
00751
00936
01000
00129
00241
00363
00434
02011
02135
02443
02662
02820
02835
03864
04100
05241
06150
07841
08011
08353
08372
08393
08400
08410
11
8
6
3
3
3
3
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
4
4
6
11
9
2
11
2
1
1
5
1
2
3
3
5
1
1
1
2
2
4
4
4
2
2
2
2
1
1
1
9
1
5
2
1
2
2
1
2
1
1
1
2
1
2
2
1
1
1
1
1
1
1
1
2
1
2
1
1
1
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
2
1
2
2
2
4
1
2
1
2
1
2
1
1
1
1
1
1
3
1
1
1
1
1
1
1
1
1
2
3
5
1
1
1
1
1
1
1
B94 protein
1-8D
B4-2 protein
Brain-expressed HHCPA78 homolog
Translationally controlled tumor protein
Anonymous mRNA
CLP
Bcl-2 related (Bfl-1) mRNA
Myeloid cell differentiation protein (MCL1)
CIRP
ras-related C3 botulinum toxin substrate (rac) mRNA
Helix-loop-helix basic phosphoprotein (GOS8)
ERF-1
GLUDP2
23-kD highly basic protein
KIAA0184 protein
g-interferon-inducible protein (IP-30)
Putative p64 CLCP protein
polyA binding protein
APPH-amyloid precursor protein homolog
KIAAO168 protein
(clone SAA7C) sequence
MLN51
EVI2B3P
fau
Acute myeloid leukemia associated protein (AML1/EAP)
ORF (complete cds) and HepG2 identical sequence
MLN50
SH3 domain-containing proline-rich kinase (sprk)
KIAA0183 protein
HVLCAD gene
Anonymous mRNA
1-8U
Transformer-2 a (htra-2 a) mRNA
(clone 05) liver expressed protein fragment
Anonymous mRNA
ZFM1 protein
OS-9 precursor mRNA
Brain-expressed HHCPA78 homolog
Phorbolin I
H sapiens (clone pS6) Alu repeat
DNA sequence from cosmid U61B11
fus-chop fusion protein
MG44
Putative 32-kD heart protein PHP32 mRNA
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1438
ITOH ET AL
Table 2. Identified Genes Detected in Peripheral Granulocytes (Cont’d)
GS
PM
08472
08492
08529
08558
08600
08627
08696
12544
Total
1
1
1
1
1
1
1
1
90
GR
HL
MO
HE
FL
IL
AL
AP
VF
LG
CM
KC
CO
RE
Gene Name
Retinoic acid-inducible E3 protein
Fetal brain cDNA 58-end
Bcl-2 related (Bfl-1) mRNA
Anonymous mRNA
ftp-3
Fragile 3 mental retardation protein (FMR-1)
Transcript ch138
Sp17 gene
39
23
25
16
6
13
7
20
23
12
16
19
17
16
Two hundred sixteen gene species matched to known genes were categorized according to their functions and subcellular localizations. Numbers represent frequency
of appearance of the gene transcripts among 1,000 cDNA analyzed. Abundances of the GSs that appeared in other libraries are also shown for comparison’s sake. For some
cells, see legend to Table 1. The bottom line (total) shows the total count of the collection. Numbers of the expressed genes shown in columns PM through RE are from
about 1,000 mRNA analyzed in each library.
Abbreviation: PM, peripheral granulocytes.
only those 64 GSs that appeared 3 times or more in descending
order of appearance. Those genes that appeared twice or less
can be seen in www bodymapper server (http://www.imcb.osakau.ac.jp/bodymap). We believe that this is the first publication
describing relative activities of genes in granulocytes that are
expressed abundantly. The profile reflects several unique features of the granulocytes physiology. First, it includes several
genes that have been well known in peripheral neutrophils, such
as genes for b2-microglobulin,15 granulocyte colony-stimulating factor receptor,16-18 major histocompatibility complex (MHC)
class I, and so on. Genes whose activity has been detected in
granulocytes15-36 are marked with asterisks. High activities of
genes for spermidine/spermine N1-acetyltransferase, pre-B–
cell enhancing factor (PBEF), and B94 protein are also noted.
The purity of the source material guarantees that this result
reflects the relative activities of those genes.
We categorized the 493 known genes active in the granulocytes into subgroups according to their function and subcellular
localization. The results are collectively shown in Table 2. The
most prominent feature of granulocytes is the high activity with
genes for cell surface membrane components. Thirty GS species
represented by 114 clones (Table 2J), amounting to 10% of the
mRNA population, were of this category. Genes for nuclear
DNA binding protein, components for secretory protein, and
components for signal transduction were also noticeably active.
Genes for energy production, lysosomal proteins, protein synthesis machinery, and cytoskeleton are not so active in the
granulocytes, as had been expected.
Comparison of gene activities in granulocytes and DMSOinduced granulocytoids. An expression profile of the granulocytoid cells is represented in Table 1B (column GR). In the
same table are collectively displayed the relevant gene activities
with HL60 cells (HL) and the monocytoids derived from HL60
by tetradecanoylphorbol-13 acetate (TPA)10 treatment (MO).
Comparison of Table 1B with Table 1A or column PM versus
GR in Table 1A with column GR versus PM in Table 1B shows
that about 50% (24/48) of the abundantly expressed genes in the
DMSO-induced HL60 are also present in peripheral granulocytes, although the abundances differ. Scarcity of highly
abundant transcripts is characteristic of the mRNA population
in granulocytoid cells. Generally, genes for cytoskeleton and
protein synthesis machinery are moderately active, unlike genes
for cell surface membrane components in granulocytoid cells.
Further discussions will be presented in the Discussion.
Identification of granulocyte-specific genes. We have prepared expression profiles of active genes in 11 other human
cells/tissues.3,10,37 Genes listed in Table 1A and B were extracted
from each of these profiles, and their activities (abundance of
the transcripts among 1,000 mRNA molecules) were compiled.
The resulting Table 1A, although incomplete, allows us to
categorize genes into those whose expression is peripheral
granulocyte-specific, limited to certain types of cells, or ubiquitous. When the genes have been detected in 6 libraries or more,
we categorized them as ubiquitous (solid area in column ‘‘lib’’).
Genes known to perform house-keeping functions, such as
ubiquitin or ribosomal proteins, are seen in this category. A gene
expressed only in granulocytes or in granulocytes and/or
granulocytoid cells may be categorized as unique to this type of
cell (open area). The rest were categorized as common or
intermediate (hatched area). We categorized 22 GSs as unique,
among which 12 were identified in GenBank and 10 represent
novel genes. Among the 12 known genes are granulocyte
colony-stimulating factor receptor, interleukin-8 (IL-8), leukocyte common antigen T200 (CD45), and ICAM-3, which have
been known to act mainly in granulocytes. No data have been
reported so far as to the cell type specificity of the remaining 8
genes. Thus, at least one third of the genes in the unique
category were indeed those that represent specific functions of
the granulocytes. We argue that we can extrapolate this finding
to the novel genes.
dbEST and the granulocyte GS. As a result of a rapid
expansion in the collection of expressed sequence tags (ESTs),
more than 400,000 fragmentary human cDNA sequences from
more than 20 tissues have been collected in dbEST. This
database can be readily compared with the expression profiles
as described here, because the quality of the source cells for this
library construction has not been biased and because the cDNA
libraries that have been subjected to normalizing protocol do
not reflect the composition of the mRNA population in the
original source cells/tissues. Nevertheless, they can provide
some information as to what RNA species are present in tissues
so far examined. We queried the 22 GS sequences that were
categorized as unique with dbEST. The results, shown in Table
3, demonstrate that 5 of them, GS08339 (IL-8), GS05242,
GS01594 (leukocyte common antigen T200), GS08389 (E4BP4
gene), and GS08424 have not been registered in dbEST.
Considering that no granulocytes or related tissues were used
for the EST collection, it is not surprising that these sequences
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GENE EXPRESSION PROFILE OF GRANULOCYTES
1439
Table 3. Representation of Peripheral Granulocyte GS in dbEST
GS
PM
GR
EST
Lib
08339
05242
01594
9
4
3
08389
08424
08362
3
3
13
3
1
08347
08438
08336
8
4
3
2
1
1
1
1
1
08375
01267
04345
05209
08383
3
8
6
6
5
2
31
7
14
5
1
13
6
7
3
04290
01371
03554
08325
08395
05014
05103
08548
5
4
4
4
4
4
3
3
9
3
2
6
10
11
2
6
3
2
2
4
6
7
2
4
Fetal
Liver
Spleen
Fetal
Spleen
Ovary
Breast
Placenta
Liver
Lung
Infant
Brain
Adult
Brain
Retina
Others
Gene Name
IL-8
1
1
1
Leukocyte
common
antigen T200
(CD45)
E4BP4 gene
3
Granulocyte
colony-stimulating factor
receptor
2
1
1
1
1
2
2
1
1
5
1
3
10
1
2
5
2
1
1
5
3
1
2
4
2
2
2
1
4
1
1
1
2
1
1
1
2
1
1
1
4
1
1
1
Zinc-finger protein (bcl-6)
5
1
2
3
4
1
1
1
1
Nramp
Secretory granule
proteoglycan
peptide
MAD-3
1-8D
B4-2 protein
1
Histone H3.3
ICAM-3
3
The 22 GSs categorized as unique (see text) are regrouped according to their presence or absence in dbEST. BLAST N program was used for this
purpose. Abundance values for our libraries are listed in the columns PM and GR. ESTs matched to these GSs are listed with their frequency of
appearance and the source tissues. The tissue names show the source organ from which ESTs were collected. Notice that recurrence of EST
reflects an abundance of mRNA, but incompleteness of the cDNA library normalization.
Abbreviations: PM, peripheral granulocytes; GR, the granulocytoids; EST, dbEST; Lib, libraries from dbEST.
failed to appear in dbEST. From an opposite point of view, the
absence in dbEST strengthens the idea that such genes are
unique to the granulocytes. Five GSs, GS08362 (granulocyte
colony-stimulating factor receptor), GS08347, GS08438
(Nramp), GS08336 (secretory granule proteoglycan peptide),
and GS08375 (MAD-3) matched ESTs from fetal liver spleen,
placenta, lung, and other tissues. This observation shows the
limit of the application of dbEST for the categorization under
discussion: it may simply show that tissues used in the dbEST
data construction contained some granulocytes. As with novel
genes, one GS (GS08347) is highly likely to represent a gene
that is unique to granulocytes. The other 9 genes were subjected
to further examination, because they were found recurrently in
tissues not related to granulocytes in the dbEST.
DISCUSSION
The cell physiology reflected in the expression profile of
active genes. Granulocytes are a major player in the defense
of the body against foreign materials. About 90% to 95% of
granulocytes are neutrophils, with the remainder being eosinophils and basophils in circulating human blood. Hence, periph-
eral granulocytes represent the activities of neutrophils.1 The
cytoplasm of these cells has highly developed cytomatrixes, as
well as granules that contain microbicidal proteins and digestive
enzymes. The plasma membranes carry a number of receptors
and other structures needed for recognition and disposal of
invading pathogens.2
Although gene activities are not necessarily reflected by the
abundance of mRNA, other methods being not available (except
for quantitizing two-dimensionally separated protein bands),
gene expression profiling3 leads to the best approximation.
Table 1A shows several genes well studied in conjunction with
the functions in granulocytes. The quantitative ratios should
help us understand the regulatory systems acting in the granulocytes. An abundant expression of genes for cell surface membrane proteins drew our attention; eg, genes for b2-microglobulin, MHC class I HLA-Cw, and HLA-E heavy chain, which are
components of cell surface receptors. That a lot of genes for cell
surface membrane proteins are active in granulocytes supports
the notion that granulocyte responses can be evoked by a variety
of stimuli caused by particulate and soluble materials. On the
other hand, most of the genes for cytoskeleton were not so
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1440
active, except those for thymosin b4 and b-actin, in accord with
the notion that these cells do not maintain a rigid shape.
The list has shown several genes not known to be active in
granulocytes. This study points out the importance of elucidating the role of gene products in granulocytes. B94 protein and
B4-2 protein are good examples. Expected changes in the
expression of genes in association with inflammation or changes
in adhesive properties during chemotaxis and phagocytosis
remain to be examined. Fibronectins, b2-integrins, and the
L-selectins, which are notably associated with adhesiveness as
mediators,2 were not detected in our expression profile. Actins,
which play important roles in production of pseudopodium for
locomotion, were not expressed strongly. Activation of these
genes is yet another feature of the activation of granulocytes
worth investigating. Genes for chemotactic factor receptors
were moderately expressed, including tumor necrosis factor
receptor, N-formylpeptide receptor, C5a anaphylatoxin receptor, and IL-8 receptor. On the other hand, expression of
receptors for C3b and C3bi were not detected, in line with the
fact that our granulocytes were in a resting stage.38 Here again,
examination of the profile in induced cells, including the time
course of activation and their relative order of activities, is of
utmost interest.
Genes for secretory proteins, such as cytokines, are not
particularly active in circulating granulocytes and, indeed, only
2 genes, for pre-B–cell colony-enhancing factor and IL-8, were
detected. Thus, the relative activities of these important granulocyte-specific genes in resting cells have been determined. As
with secretable bactericidal components, there were cathepsin
S, neutrophil oxidase factor (NCF2)/p67-phox, proteasome
subunit p40, and defensin, in addition to lysosomal proteins.
Thus, these proteins are constitutively produced at a level of
4.0% or more of total protein synthesis. Bactericidal/permeability-increasing protein (BPI) was found in granulocytoid cells,
but not in the peripheral granulocytes, probably because its
expression level is just at the border of the level of detection.
Whereas active expressions of genes for cell surface membrane proteins, including receptors for chemotactic factors as
well as genes for bactericidal proteins such as lysosomes, are
characteristic to our granulocytes, so is poor expression of
components for protein synthesis machinery, as it is for cells in
the resting stage.
Among the mRNAs in granulocytes identified in GenBank
(Table 1A) are genes for granulocyte colony-stimulating factor
receptor, tumor necrosis factor receptor, and T200. These gene
products are related to neutrophilic granulopoiesis and their
maturation. Thus, these findings strongly suggest that granulocytes in circulating blood wait for stimuli exposing granulopoietic receptors. The list also included IL-8, a neutrophilic
chemoattractant and activator. In contrast to IL-8 receptor
(Table 2J), IL-8 is highly and specifically expressed. This
mRNA has been known to be induced in neutrophils in response
to granulocyte/macrophage colony-stimulating factor.31,39 Although granulocyte colony-stimulating factor regulates the
expression of IL-8 receptor,40 whether granulocyte colonystimulating factor can induce the expression of IL-8 is not clear.
Our results indicate that granulocyte colony-stimulating factor
induces the expression of IL-8 mRNA.
ITOH ET AL
The uniquely active genes in granulocytes. Among the 22
genes categorized as unique in Table 1A, 10 were novel genes.
It is of utmost importance to characterize these genes, although
such categorization can be performed only with abundantly
expressed genes, and yet some misleading categorizations are
unavoidable due to the limited number of GS collections.
Comparison of expression profiles between granulocytes and
granulocytoid cells induced from HL60. b2-microglobulin
and HLA-E heavy chain were commonly expressed in granulocytes and granulocytoid cells. Proteins in lysosomes, BPI, and
leukocyte adhesion protein (Mac-1), which are known to be
expressed in neutrophils, were also expressed in granulocytoid
cells. In total, 20% or more of the mRNAs are commonly
expressed in both types of cells (data not shown).
However, their relative proportions in granulocytoids differ
from those in granulocytes. In general terms, expression profiles
of genes in granulocytes and granulocytoid cells differ from
each other qualitatively and quantitatively. In Table 1A, 24 gene
species represented by 74 clones were commonly expressed in
granulocytes and granulocytoid cells. These observations demonstrate the capacity and limitations of this model system.
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1998 92: 1432-1441
Expression Profile of Active Genes in Granulocytes
Koichi Itoh, Kousaku Okubo, Hiroyasu Utiyama, Tetsuo Hirano, Junji Yoshii and Kenichi Matsubara
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