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Prepublished online as a Blood First Edition Paper on September 19, 2002; DOI 10.1182/blood-2002-03-0680.
HEMATOPOIESIS
From the Leukocyte Biology and Inflammation Program,
Renal Unit, Massachusetts General Hospital, Harvard Medical School,
Boston, MA.
Integrin CD11b is a differentiation marker of the myelomonocytic
lineage and an important mediator of inflammation. Expression of the
CD11b gene is transcriptionally induced as myeloid
precursors differentiate into mature cells, then drops as monocytes
further differentiate into macrophages. Previous studies have
identified elements and factors involved in the transcriptional
activation of the CD11b gene during myeloid
differentiation, but no data exist regarding potential down-regulatory
factors, especially in the later stages of differentiation. Using 2 copies of a GC-rich element ( CD11b is the CD11b is a myeloid-specific marker. During hematopoiesis, it is first
detected at the myelocytic and monoblastic stages,7,8 and
its expression is significantly increased as cells differentiate into
monocytes and granulocytes.9 Subsequently, CD11b
gene expression is down-regulated in cells proceeding toward later stages of monocytic differentiation.9 The drop in
expression of CD11b can be reproduced in vitro in human peripheral
blood monocytes as they differentiate into
macrophages.10-13 Although some of the elements and
factors underlying the induction of the CD11b gene are
known, those contributing to its down-regulation thereafter remain to
be defined.
Transcriptional regulation of CD11b is an important
determinant in expression of the receptor.14 The proximal
promoter of CD11b spanning nucleotides Cells
Cloning of ZBP-89 by yeast one-hybrid screening
Plasmid construction
Nuclear extract and total protein lysate preparation Nuclear extracts were prepared using a method modified from that described by Dignam et al.25 Briefly, cultured cells (1.2 × 107) were harvested and washed twice with cold phosphate-buffered saline (PBS). Cell pellets were resuspended with 2 mL buffer A (20 mM Tris [tris(hydroxymethyl)aminomethane]--HCl, pH 7.8, 50 mM KCl, 80 mM NaCl, 0.2 mM EDTA [ethylenediaminetetraacetic acid], 0.1 mM EGTA [ethyleneglycoltetraacetic acid], 0.2 mM phenylmethylsulfonyl fluoride [PMSF], 4% glycerol, and the protease inhibitor mixture "Complete"; Roche Molecular Biochemicals, Indianapolis, IN) and incubated on ice for 10 minutes. After addition of 10 µL 10% Nonidet P-40, the cell suspension was vortexed. Disruption of the cell membranes was monitored under a light microscope by Trypan blue staining. When more than 90% of cells were Trypan blue positive, they were spun at 6000 rpm at 4°C for 5 minutes to pellet the nuclei. The pellet was resuspended in 500 µL buffer B (20 mM Tris-HCl, pH 7.8, containing 300 mM KCl, 0.5 mM dithiothreitol [DTT], 0.2 mM EDTA, 0.1 mM EGTA, 0.2 mM PMSF, 25% glycerol, and the protease inhibitor mixture "Complete") and mixed gently at 4°C for 30 minutes. After spinning the suspension in a microcentrifuge at 16 000g for 15 minutes at 4°C, the supernatant was aliquoted into 20 µL fractions, snap-frozen in liquid nitrogen, and stored at 80°C. For the preparation of the total
protein lysate from human monocytes/macrophages, cells were harvested
and washed with cold PBS. Cell pellets were incubated with the lysis
buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% triton X-100, 0.5%
NP-40, 1 mM DTT, and the protease inhibitor mixture "Complete";
Roche Molecular Biochemicals) for 10 minutes at room temperature. After
spinning the suspension in a microcentrifuge at 13 000 rpm for 15 minutes at 4°C, the supernatant was aliquoted into 20-µL fractions,
snap-frozen in liquid nitrogen, and stored at 80°C.
EMSA and immunoblot analysis Electrophoretic mobility shift assays (EMSAs) were conducted as previously described,18 using the double-stranded oligonucleotides GC (sense strand: 5'-GGGTCAGGAAGCTGGGGAGGAAGGGTGGGCAGGCTGT-3') or GC1 (sense strand: 5'-AGCTGGGGAGGAAGGGTGGGCAGGCTGT-3'). For electrophoretic mobility supershift assay (EMSSA), 1 µL of either anti-ZBP-89 antibody (provided by Dr J. L. Merchant26) or anti-Sp1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was preincubated with nuclear extract for 20 minutes at 4°C, followed by the addition of the DNA probe and further incubation for 20 minutes at 4°C. The oligonucleotides used in the analyses are shown in Figure 3A. The other double-stranded oligonucleotides used were: Sp1, 5'-ATTCGATCGGGGCGGGGCGAG-3'; OCT-1, 5'-TGTCGAATGCAAATCACTAGAA-3'; and ht , 5'-GATCTGGGGGTGGGGTGGGGGTGGGGTGGGGGTGGGG-3' (containing 4 copies
of the CACCC recognition sequence of ZBP-89).27 For immunoblot analysis, nuclear protein (30 µg) was electrophoretically separated by 6% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and electroblotted onto a nitrocellulose
membrane (Bio-Rad, Hercules, CA). A chemiluminescence Western blot
detection kit and protocol (Roche Molecular Biochemicals) were used for
antigen detection. The anti-ZBP-89 antibody was diluted 1000-fold and used as the primary antibody.
Transfection U937 cells were transfected by electroporation in incomplete medium at 280 V and 960 µF using Gene Pulser Cuvettes (Bio-Rad). Cells in the early log phase of growth were harvested by centrifugation, washed once with medium, and resuspended at room temperature. About 4 × 107 cells were transfected with 25 µg luciferase test plasmid together with 5 µg of the plasmid pRSV- (Promega, Madison, WI), which contains the lacZ
gene. Each transfection of pALbWt and pALbM4a was performed in parallel
with a transfection of the promoterless luciferase plasmid pATLuc.
Induction of promoter activity was assessed by treating the
electroporated cells for 16 hours with PMA at 100 ng/mL prior to
harvesting. Cells were subsequently pelleted, washed twice with PBS,
and lysed in 150 µL reporter lysis buffer (Promega). Luciferase and
-galactosidase activities were assessed using assay reagents
purchased from Promega and 100 µL and 20 µL lysate, respectively.
Luciferase activity was measured using a Monolight 2010 Luminometer,
which integrated peak luminescence 10 seconds after injection of assay
buffer, and the values were normalized against -galactosidase
activity. Fold above background for each transfection was calculated by dividing the luciferase activity of each test plasmid by that of
pATLuc. The fold above background derived from transfection of pALbWt
was assigned an arbitrary value of 100%.
Transactivation by ZBP-89 was assessed in cotransfections in
which 8 µg pATLuc or pALbWt or pALbM4a was mixed with 1 µg pRSV- sU937-11b-luc cells were transfected with 25 µg of either
pCMVSport3 or pCMVSport3-ZBP-89 with 1 µg of the plasmid pRSV- For transfection of the Drosophila cell line SL2, cells were
plated at a density of 3 × 106/60-mm dish for 18 hours
and washed with PBS twice before transfection. A total of 2.6 µg DNA
and 10 µL Superfect (Qiagen, Valencia, CA) were mixed in 150 µL
incomplete Schneider Drosophila medium. After 10 minutes at
room temperature, 0.6 mL medium supplemented with 10% heat-inactivated
FCS (tested for use with insect cells; Sigma) was added to the
DNA-Superfect complexes. The suspension was then added drop-wise to the
culture dishes. After a 3-hour incubation at room temperature, cells
were washed with PBS twice and 2 mL complete medium was added. Cells
were harvested 24 hours later, washed 2 times with PBS, and lysed in
150 µL reporter lysis buffer (Promega). Generally, 10 µL cell
lysate was used in the Real-time RT-PCR Total RNA from U937 cells (1.0 × 107), which were treated with PMA (100 ng/mL) for 0 hour, 24 hours, 48 hours and 72 hours, was isolated using the Trizol reagent (Gibco BRL) followed by DNase I treatment (Ambion, Austin, TX) as described by the manufacturer. First-strand cDNA was prepared by extension of an oligo-d(T)16 primer with Moloney-murine leukemia virus reverse transcriptase (RT, Clontech). After incubation at 42°C for 1 hour, the reaction was stopped by heating to 95°C for 5 minutes. Equal amounts of cDNA were subjected to PCR to detect CD11b, ZBP-89, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts.28 PCR primers and TaqMan probes for CD11b and ZBP-89 were designed using Primer Express 1.0 Software program and purchased from Applied Biosystems (Foster City, CA). TaqMan probes for CD11b and ZBP-89 were labeled with a reporter fluorescent dye, FAM (6-carboxyfluorescein), at the 5' end and a fluorescent dye quencher TAMRA (6-carboxy-tetramethyl-rhodamine) at the 3' end. For GAPDH, the predeveloped TaqMan assay reagent (PDAR), which has forward and reverse primers, and a probe labeled with a fluorescent dye VIC at the 5' end and a fluorescent dye quencher TAMRA at the 3' end, was used (Applied Biosystems). PCR conditions were 2 minutes at 50°C (for AmpErase UNG incubation to remove any uracil incorporated into the cDNA), 10 minutes at 95°C (for AmpliTaq gold activation), followed by 40 cycles at 95°C for 15 seconds and at 60°C for 1 minute. Primers and probes for detecting ZBP-89 and CD11b are as follows: ZBP89-forward, 5'-CCGAGCCTTAACTTTGTGACTGAT-3'; ZBP89-reverse, 5'-ATGGTGGCATAGACCTGCTTGT-3'; ZBP89-probe, 5'-6FAM-CCCAAATCAGCCAGCATTCTCTTCC-TAMRA-3'; CD11b-forward, 5'-AGTTGCCGAATTGCATCGA-3'; CD11b-reverse, 5'-GGCGTTCCCACCAGAGAGA-3'; CD11b probe, 5'-6FAM-AGCCCCATTGTGCTGCGCCT-TAMRA-3'.Plasmid DNAs pCDNACD11b29 and pCMVSport3-ZBP-89 were used to generate a standard curve for the determination of the copy number of CD11b and ZBP-89, respectively. To construct the GAPDH-plasmid, a 604-bp fragment was amplified using the forward primer 5'-TCTGCTCCTCCTGTTCGACA-3' and reverse primer 5'-GACGTACTCAGCGCCAGCAT-3', and a cDNA from U937 cell total RNA as a template. The PCR reaction was performed with the following conditions: 45 seconds at 94°C, 45 seconds at 60°C, and 1 minute at 72°C for 30 cycles. This fragment was cloned using TA cloning kits (Invitrogen) and the resulting plasmid sequenced to confirm its identity. All reactions were performed in the ABI Prism 7700 Sequence Detection System (Applied Biosystems). Test samples, standards, and no template controls were run in triplicate and the data were analyzed using the Sequence Detector V 1.6 program. A standard curve was plotted in cycle threshold (Ct) versus the known copy numbers of the templates from each plasmid. The final copy number of CD11b or ZBP-89 was presented, respectively, as a CD11b/GAPDH or ZBP-89/GAPDH ratio.
Identification of ZBP-89 using yeast one-hybrid screens The proximal GC-rich element spanning nucleotides141 to 105
(with respect to the transcriptional start site, Figure 1A, underlined), is required for expression of the CD11b gene in
differentiating U937 cells.18 Two copies of this GC-rich
element were used as bait in a yeast one-hybrid screen to identify
interacting factors. A mutant form of the GC-rich element (Figure 1B)
was used as a negative control to assess specificity. After
7.1 × 106 cells in a first and 2 × 106
cells in a second screen of a HeLa cell cDNA library were
analyzed, 174 positive colonies grew on restreaking onto 45-mM 3-AT
SD-H plates. Among these, 43 did not show PCR products, and 19 were redundant (based on the digestion pattern of DNA with HaeIII
and AluI). The rest of the candidates were retransformed
into the yeast reporter strain and 22 grew on the selection medium. Of these, only 3 grew in the selection medium after transformation into
the wild-type reporter strain but not into a background strain (which
does not carry the bait), or the mutant reporter strain. Sequence
analysis revealed that each of these 3 clones encodes a 206-amino-acid
protein of about 25 kDa. This we named p25. Blast searches demonstrated
that p25 has 100% amino acid sequence identity, except for the last 2 amino acids, with the N-terminal half of the zinc finger transcription
factor, ZBP-89. As shown in Figure 1C, a single nucleotide insertion in
the cDNA encoding p25 caused an in-frame stop codon immediately after
the 4 zinc finger motifs, resulting in premature termination. To test
the possibility that this truncated form of ZBP-89 is a physiologic
product generated by alternative splicing in vivo, RT-PCR was performed
using total RNA extracted from HeLa and U937 cells and U937
cells treated with PMA for 24 hours. Sequencing of the resulting PCR
products demonstrated that none contained the nucleotide insertion,
suggesting that the premature termination in p25 represents an in vitro
artifact in the cDNA library (data not shown).
ZBP-89 binds to the GC-rich element of the CD11b promoter To confirm that p25 actually binds to the bait sequence, EMSA analyses were carried out with a radiolabeled probe (GC, the double-stranded GC-rich element used for the yeast one-hybrid screen) and GST-Z, which represents the protein p25 fused to GST. The fusion proteins were expressed in soluble form in Escherichia coli BL21 (DE3) and used for EMSA. As shown in Figure 2A, the GST protein alone does not bind to GC (lane 2), whereas GST-Z formed a complex with GC (lane 3). This complex was competed with unlabeled GC (lane 4) or with the oligonucleotide ht (lane 5), which represents the ZBP-89-binding
site in the T-cell receptor promoter.27 A
Sp1 consensus oligonucleotide did not compete (lane 6). We also
performed EMSA with the fusion protein GST-ZBP-89, which represents the
full-length ZBP-89 protein fused to GST and obtained similar results
(Figure 2B). This suggests that ZBP-89 binds to the double-stranded
GC-rich element of the CD11b promoter.
To investigate the binding characteristics of ZBP-89 on the
CD11b promoter during myeloid differentiation, EMSA was
carried out by incubating labeled GC with nuclear extracts prepared
from untreated U937 cells or U937 cells treated with PMA for 24 hours to induce their differentiation into monocytelike cells. Figure 2C
shows that a DNA-protein complex was formed using extracts prepared
from induced cells (lane 4) but not from uninduced U937 cells (lane 2).
Unlabeled GC or ht Identification of the residues in the CD11b promoter that are critical for ZBP-89 binding To identify the minimal region required for p25 binding, competitive gel shift assays were performed using the oligonucleotide GC1, which is 9 nucleotides shorter than GC (Figure 3A). As shown in Figure 3B, a GST-Z-containing complex was formed with radiolabeled GC1 and this was competed efficiently with its unlabeled equivalent (lane 3). Next, a series of oligonucleotides (M1-M6) representing sequential mutation of 3 or 5 nucleotides in GC1 were used as competing oligonucleotides. The oligonucleotides M2-M4 lost their ability to compete for binding to GST-Z (lanes 5-7), whereas oligonucleotides M1, M5, and M6 effectively competed (lanes 4, 8, and 9, respectively). We also performed the same competition experiment with the fusion protein GST-ZBP-89 and obtained similar results (Figure 3C). These results demonstrate that p25 and ZBP-89 bind to the double-stranded oligonucleotide spanning nucleotides127 to 113 of the CD11b promoter, thus delineating the
DNA-binding segment for ZBP-89.
MS-2, a putative transcription factor, binds to the GC-rich element of
the CD11b promoter and might be involved in induction of the
CD11b gene during myeloid cell
differentiation.18 In Figure 3E (lane 2), we show that
MS-2 is a purine-rich ssDNA-binding factor. Because the binding sites
for p25/ZBP-89 and MS-2 overlap, it was necessary to identify a
mutation that disrupts ZBP-89 but not MS-2 binding to assess the
specific function of ZBP-89. A competitive gel shift assay was
performed using GC1 as a probe, and a series of mutant double-stranded
oligonucleotides (M4a-e) were used as competitors, each of which
represented a different point mutation within GC1 (Figure 3A). Figure
3D shows that the M4a mutation, which has a transversion of
G>T at nucleotide Overexpression of ZBP-89 represses CD11b gene expression To study the role of ZBP-89 in regulating CD11b promoter activity, increasing amounts of pCMVSport3-ZBP-89 were transfected into U937 in which the wild-type CD11b promoter fused to the luciferase gene was stably incorporated. Transfection with pCMVSport3 served as a negative control. As can be seen in Figure 4A, ZBP-89 induced repression of PMA-induced CD11b in a dose-dependent manner, reaching 75% at the maximal amount of transfected DNA tested (Figure 4A). The effect was also observed in U937 in which pCMVSport3-ZBP-89 was cotransfected with a reporter plasmid containing the CD11b promoter fused to the luciferase gene (Figure 4B, left panel); ZBP-89 decreased PMA-induced wild-type CD11b (CD11b-wt) gene expression by 55%; the reduced repression seen may be a reflection of the lower amount of plasmid DNA allowable in the cotransfection studies. The effect of repression by ZBP-89 was specific because no such repression occurred in U937 cells cotransfected with pCMVSport3-ZBP-89 and CD11b-M4a promoter plasmids (Figure 4B, right panel).
Loss of repression of the M4a-CD11b promoter by endogenous ZBP-89 It remains possible that exogenous ZBP-89 overexpressed in differentiating U937 cells may act indirectly in repressing the CD11b promoter. To establish whether endogenous ZBP-89 also acts as a repressor, we compared the promoter activities of the CD11b-wt and CD11b-M4a in PMA-induced U937 cells (Figure 4C). Expression of the mutant CD11b-M4a reporter in differentiating U937 increased to 160% of the wild-type reporter (Figure 4C), reflecting loss of repression by endogenous ZBP-89. Thus, the level of CD11b gene expression in differentiating U937 cells reflects the activity of endogenous ZBP-89.Reconstitution of ZBP-89 repression in a heterologous expression system Expression of CD11b is normally restricted to differentiating myeloid cells. The CD11b Drosophila Schneider
L2 cell line is deficient in Sp1-related proteins,24,30,31
but can express a CD11b promoter-directed reporter when supplemented
with Sp1.32 We found this cell line to be also lacking
ZBP-89 by Western blotting as well as by EMSA (data not shown). We
cotransfected Schneider L2 cells with 0.2 µg of the construct pPacSp1
(which expresses Sp1), together with increasing amounts of pPacZBP-89
expressing ZBP-89, along with either CD11b-wt or CD11b-M4a-luciferase
plasmids. In the presence of Sp1, ZBP-89 repressed wild-type
CD11b promoter activity by up to 50% (Figure 4D). In
contrast, CD11b-M4a promoter activity was not affected by
expression of ZBP-89 (Figure 4E). Expression of ZBP-89 alone without
Sp1 did not activate the CD11b promoter (data not shown).
Therefore, repression of CD11b by ZBP-89 can also be
achieved in a nonmyeloid cellular context and independently of the
confounding effects of treatment with phorbol esters.
Expression of CD11b does not correlate with endogenous ZBP-89 protein during phorbol ester-induced differentiation of U937 cells We next determined the correlation between the endogenous levels of CD11b mRNA and ZBP-89 protein in U937 cells differentiated by treatment with PMA for 72 hours, a treatment that alters homotypic adhesion in these cells (Figure 5A. Both ZBP-89 protein and CD11b mRNA increased in parallel during the differentiation of PMA-treated U937 cells into monocytelike cells (Figure 5B). Thus whereas overexpressed ZBP-89 represses the CD11b gene, we find no inverse correlation between endogenous ZBP-89 and expression of the CD11b gene in this system. These data argue against a role for endogenous ZBP-89 in repressing CD11b gene during the early stage of monocytic differentiation. Because overexpression of ZBP-89 represses the CD11b promoter-driven reporter in these cells, qualitative or quantitative changes in ZBP-89 protein levels together with the potential inaccessibility of the native CD11b promoter to ZBP-89 during early monopoiesis may account for the lack of correlation between endogenous CD11b mRNA and ZBP-89 protein at this stage.
Down-regulation of CD11b during monocytes to macrophage differentiation correlates with ZBP-89 protein expression Because the level of CD11b protein is known to drop as blood monocytes differentiate into macrophages,10,13 we next assessed the impact of ZBP89 on CD11b expression in these cells. CD11b mRNA and ZBP-89 protein levels were determined during the in vitro differentiation of normal human peripheral blood monocytes into macrophages. Normal blood monocytes assumed the typical features of macrophages after 12 days in culture (Figure 6A, compare left and right panels) as described.21,33 As shown in Figure 6B, CD11b mRNA dropped significantly (by ~75%) in macrophages compared to monocytes. Importantly, this reduction coincided with an increase of protein expression of ZBP-89 (Figure 6C, compare lanes 3 and 4) that has an identical mobility to the form induced in differentiating U937 cells (Figure 6C, lanes 1 and 2; compare lanes 2 and 4). These data suggest that ZBP-89 exerts its repressor activity on CD11b during the late stages of monocytic differentiation.
The major finding in this paper is that ZBP-89 is a repressor of the CD11b gene. Repression is dose-dependent, sequence-specific, and demonstrable in nonmyeloid insect cells. In the U937 model of early-stage monocytic differentiation, no inverse correlation was observed between ZBP-89 levels and CD11b gene expression. However, in the model of late-stage monocyte-to-macrophage differentiation, such an inverse correlation was readily detected. Consequently, these findings suggest that the effect of ZBP-89 on CD11b gene expression occurs primarily at the later rather than the early stages of monocytic differentiation. This observation may underlie the observed drop in the levels of CD11b as monocytes differentiate into tissue macrophages in vivo. ZBP-89 is a 794-amino-acid ubiquitously expressed
factor,26,34 originally cloned in a truncated form, ht The structural basis for ZBP-89-mediated repression of the
CD11b gene remains to be determined. Five to 10% of zinc
finger DNA-binding motifs share a conserved 120-amino-acid domain
termed the POZ or BTB domain.43 Many POZ domain-containing
proteins are transcriptional repressors, although some, such as the
GAGA factor, can counteract repression by chromatin
remodeling.44 Charged, alanine-, alanine/proline-, and
alanine/glutamine-rich motifs have also been found in transcriptional
repressor domains.45-47 However, homology searches reveal
that ZBP-89 contains no such domains. A previous study demonstrated
that a transferable region in ZBP-89 spanning amino acids 136 to 184 repressed Functionally, 4 general mechanisms may give rise to selective
transcriptional repression of genes. First, a repressor protein can
mask a transcriptional activator domain. Second, the repressor may
block interaction of an activator with other components of the
transcription machinery.48 Third, it can displace an
activator from its DNA element by competition,49 and
fourth, the DNA element itself may exert allosteric effects on
transcriptional regulators, such that regulators may activate
transcription in the context of one gene, yet repress transcription in
another.50,51 In regulating expression of the
The ZBP-89 element overlaps with but is distinct from that of MS-2, an
ssDNA-binding factor of the CD11b gene whose identity remains to be determined18 (Figure 3). ZBP-89 may repress
by competing with MS-2 for DNA binding.48 Supporting this
notion is our observation that the activity of the mutant promoter
CD11b-M4a is higher than that of CD11b-wt (Figure
4B). This increase may also be due to the blocked binding of endogenous
ZBP-89. However, the binding of adjacent proteins such as MS-2 may also
affect ZBP-89 activity by modifying its association with comodulators or the basal transcription machinery. There are examples of dsDNA- and
ssDNA-binding proteins binding to overlapping DNA segments and thus
regulating transcription. Within the vascular smooth muscle
The CD11b/CD18 heterodimer is expressed on peripheral blood monocytes, neutrophilic polymorphonuclear leukocytes (PMNs), and natural killer cells, but is generally expressed at reduced amounts on tissue macrophages such as Kupffer cells, bone marrow stromal macrophages, and alveolar macrophages.10,13,57-59 The mechanisms that underlie this reduction are not known. In vitro differentiation of freshly isolated human monocytes into macrophages was accompanied by a significant drop in CD11b mRNA and induction of the ZBP-89 protein. These findings suggest that ZBP-89 acts at a later stage in monocytic differentiation as monocytes further transform into macrophages. CD11b/CD18 is known to be important not only in leukocyte recruitment into tissues but also in the many proinflammatory adhesion-dependent functions mediated by these cells in tissues. Quantitative as well as qualitative down-regulation of this receptor once monocytes transmigrate into tissues as part of their normal differentiation and maturation may be necessary to avert harmful inflammation. The data presented in this work suggest that ZBP-89 may act at the transcriptional level to elicit such a protective effect during the resolution of an inflammatory response.
We thank Dr J. L. Merchant for the anti-ZBP-89 antibody and ZBP-89 expression plasmid, Dr R. Tjian for the pPacO and pPacSp1 plasmids, Dr D. T. Scadden, Dr K. Ferenczi, Dr Boris Nikolic, and Mr D. Olson for helpful discussion and assistance with FACS analysis, Dr M. Xue for helpful suggestions on the nature of MS-2 and Miss G. Rouiffe for technical support.
Submitted March 4, 2002; accepted September 11, 2002.
Prepublished online as Blood First Edition Paper, September 19, 2002; DOI 10.1182/blood-2002-03-0680.
Supported by National Institutes of Health grants DK50305 and DK50779.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: M. Amin Arnaout, Renal Unit, Massachusetts General Hospital, 149 13th St, Charlestown, MA 02129; e-mail: arnaout{at}receptor.mgh.harvard.edu.
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2-integrin CD11b gene
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Abstract
Introduction
subunit of the integrin 
512, gal80-
