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By
From the Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results and Discussion
References
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Neutrophil-specific granule deficiency (SGD) is a rare disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins. CCAAT/enhancer binding protein (C/EBP)
, a member of
the leucine zipper family of transcription factors, is expressed primarily in myeloid cells, and its
knockout mouse model possesses distinctive defects, including a lack of neutrophil secondary
granule proteins. Sequence analysis of the genomic DNA of a patient with SGD revealed a
five-basepair deletion in the second exon of the C/EBP locus. The predicted frame shift results in a truncation of the 32-kD major C/EBP isoform, with loss of the dimerization domain, DNA binding region, and transcriptional activity. The multiple functional defects observed in these early neutrophil progenitor cells, a consequence of C/EBP deficiency, define
SGD as a defect in myelopoiesis and establish the requirement for C/EBP for the promyelocyte-myelocyte transition in myeloid differentiation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Neutrophil-specific granule deficiency (SGD) is a rare
congenital disorder marked by frequent and severe
bacterial infections. The five reported cases consistently
describe pleiotropic characteristics, including lack of secondary granule proteins and defensins, abnormalities in
neutrophil migration and disaggregation, atypical nuclear
morphology, and impaired bactericidal activity (1).
More recent work has revealed additional granule abnormalities in the eosinophils of SGD patients, with absence of
eosinophil-specific granule contents, including eosinophil
cationic protein, eosinophil-derived neurotoxin, and major
basic protein (12). Platelet disorders and associated bleeding
diatheses, including the neutrophilic phagocytosis of platelets (13) and the absence of platelet-high-molecular-mass von Willebrand factor multimers stored in platelet 
granules (14), have also been reported in SGD patients. In contrast to these seemingly genetically unrelated manifestations,
these patients express normal levels of salivary lactoferrin (8,
15, 16), a characteristic specific granule marker absent in
neutrophils in SGD, suggesting that the responsible defect
involves myeloid-specific transcriptional regulation.
CCAAT/enhancer binding proteins (C/EBPs) comprise a family of transcription factors that are key regulators of cellular differentiation and function in a variety of tissues (17). The prototypic C/EBP is a modular protein consisting of one or more activation domains, a dimerization basic zipper domain and a DNA binding region (18). C/EBPs are least conserved in their activation domains and vary from dominant negative repressors to strong activators.
C/EBP, the newest member of the family, is expressed
exclusively in cells of myeloid and T cell lineage (19).
The human C/EBP gene encodes four mRNA isoforms
with varying splice patterns, driven from two alternative
promoters, and from which are translated three protein isoforms (23). Analogous to what has been shown for C/EBP
and C/EBP
(24, 25), in vitro transfection data suggest
that the full length, 32-kD isoform of C/EBP (C/EBP32)
possesses the fully active transcriptional activation domain, whereas the short, 14.2-kD isoform (C/EBP14) lacks transcriptional activity (23).
Nearly 60% of C/EBP knockout mice (26) succumb to
low pathogenicity bacterial infections by 4-6 mo of age.
Neutrophils from C/EBP knockout mice have morphological features similar to human SGD neutrophils, including bilobed nuclei, absent specific and tertiary granule contents, and defective chemotaxis and bactericidal activity
(27). The striking phenotypic similarities between SGD
defects and the C/EBP knockout model prompted a
search for a C/EBP knockout mutation in an SGD patient's genomic locus.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Patient.
Material from a previously described (5, 6) male patient lacking neutrophil-specific granules was studied. Research was conducted with informed consent under the guidelines of a National Institutes of Health (NIH) Internal Review Board- approved protocol, no. 92-I-99. The patient died from complications of pneumonia at age 20.DNA, RNA, and Protein Extraction.
Peripheral blood neutrophils were isolated as described (28), cryopreserved with dimethylformamide (Sigma Chemical Co.), and maintained at
140°C.
Cell proteins were extracted as described (29). DNA extraction
from cryopreserved fibroblasts proceeded as described (30). RNA
was extracted from patient bone marrow aspirate using RNAzol
reagent (Teltest) as per manufacturer's protocol. Normal human
bone marrow RNA was purchased from Clontech.
PCR Amplification of Genomic Sequence.
PCR reaction was performed using Platinum taq DNA polymerase (Life Technologies) per manufacturer's instructions and cycled as follows: 96°C for 12 min, followed by a three-step cycle
94°C for 30 s, 60°C
for 30 s, and 72°C for 2 minfor 35-40 cycles. PCR products
were gel purified and recovered using Gene Clean (Bio101).
Products were sequenced with an ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp.).
Primers were chosen from a published sequence available from
EMBL/GenBank/DDBJ under accession no. U48865. Primer
sets (upstream, downstream): B, 5'-AGC GGC CAT GCA AAA
GGA AAG ACA, 5'-TCC ACC TAC CCC CAA GAG AAA
GTT (bp 667-1186); C, 5'-CCC ACG GGA CCT ACT ACG
A, 5'-GGG CTG GCC TGC TCT TAC (bp 1818-2343); F, 5'-CTC CCC GGC TGG CCC CTT ACA C, 5'-GCC AAC
AGT CCC AAC ACC CAG TCA (bp 3133-3615); G, 5'-GGA
GGT GGG GCT ACA AAA GAA ACT, 5'-TCA GGG AGG
GGC AGG ACA (bp 1143-1553); H, 5'-ACA GGA GTG GGT
GAC AGA GGA GAC, 5'-GGG CCG AAG GTA TGT GGA
GGG TAG (bp 1563-2104); I, 5'-CCA TGC CCC CTC CTC
TTG TTT CTC, 5'-ACT GCC TTC TTG CCC TTG TGT
AA (bp 2594-3171); K, 5'-AAC TTT CTC TTG GGG GTA
GGT GGA, 5'-TCG TAG TAG GTC CCG TGG (bp 1163-
1837). Homozygosity was determined by hybridization of the
PCR product fragment C to a [32P]
dATP-endlabeled internal
oligonucleotide (H. downstream; sequence above). Labeled oligo
was mixed with hybridization buffer (75 mM NaCl, 5 mM EDTA)
in a ratio of 1:10, and 10 µl was added to 30 µl PCR product.
Hybridization was cycled in a thermal cycler: 95°C for 5 min and
55°C for 10 min. Reactions were immediately placed on ice.
Products were resolved on 4-20% Tris/borate/EDTA polyacrylamide gel (Novex) at 250 V.
RNA Blotting Assay.
10 µg of total RNA isolated from the patient's bone marrow and 0.25, 0.5, and 1 µg of control polyadenylated (pA)-mRNA was electrophoretically separated, blotted, hybridized, and washed as described (27). The membrane was stripped by boiling and stored at20°C.
Immunoblotting.
Protein quantitation was performed using a BCA Protein Assay kit (Pierce Chemical Co.) according to the manufacturer's instructions. 10-100 µg protein extracts were electrophoretically separated, transferred to nitrocellulose, and incubated with primary antibody as described (29). Primary antibody was generated in rabbits by Research Genetics, Inc., using a synthetic peptide encoded in exon 2 of C/EBP, downstream of
the SGD deletion (DPRAVAVKEEPRGPEGSR). The membranes were washed, incubated with anti-rabbit horseradish peroxidase conjugate antibody (ECL Western blot kit; Amersham Pharmacia Biotech, Inc.), and developed according to the manufacturer's instructions. Membranes were stripped and reblotted
with anti-mouse human actin antibody (Boehringer Mannheim) to control for protein loading.
In Vitro Mutagenesis Assay.
The patient's mutation was introduced into the pCMV-C/EBP32 expression vector using a Stratagene QuikChange site-directed mutagenesis kit per manufacturer's
instructions using a complementary oligonucleotide (PAGE purified;
purchased from Genosys Biotech) containing the deletion (5'-CCA
CTA CTT GCC GCC CTC GGC CCT TTG CCT ACC). Presence of the mutation and maintenance of the vector sequences was
verified by sequencing and restriction enzyme digestion, respectively.
Transient Transfections.
HeLa cells were maintained in DMEM (BioWhittaker) supplemented with 10% heat-inactivated FBS (Life Technologies, Inc.) and penicillin/streptomycin at 37°C and 5% CO2. Cells were plated in 6-well plates and transfected within 24 h, at 30-50% confluency. Transfections, using the Mammalian Transfection System (Stratagene), were performed using 5 µg reporter plasmid (G-CSF receptor promoter-luc); 1, 2, or 5 µg inducer plasmid (pCMV-C/EBP, pCMV-C/EBP32 isoform,
pCMV-C/EBP14 isoform, or pCMV-C/EBP32-SGD, described above); and 0.5 µg pCMV, as described (23, 31). The
DNA content of transfections was normalized, and transfection was performed according to the manufacturer's instructions, with 300 µl transfection solution applied to the cells. Samples were harvested 24 h after transfection.
Luciferase and -galactosidase activities were measured using a
Dual-Light kit (Tropix, Inc.), according to the manufacturer's protocol, on a Turner 20/20 Luminometer. Samples were read
for 15 s after a 3-s delay.
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Results and Discussion |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Sequencing of PCR products from genomic DNA detected a 5-bp deletion, TGACC, in exon 2 of the patient's
C/EBP sequence. Fig. 1 A shows sequence data from one
normal control (top sequence) and the SGD patient (bottom sequence). The mutation predicts a frameshift and a
premature termination of the encoded C/EBP32 isoform
(Fig. 1 B). The missense code after the frameshift results in
the loss of the critical DNA binding domain and leucine
zipper region required for C/EBP dimerization and function. C/EBP transcripts encoding the shorter 27- and 14-kD isoforms are predicted to be unaffected, based upon the
splice donor and acceptor and translational start sites (23).
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Homozygosity of the deletion was determined by PCR amplification of the affected region and resolution of the DNA fragments on a 4-20% polyacrylamide gel (Fig. 1 C). DNA from one normal control and the SGD patient were mixed before amplification and electrophoresis (lane 3), showing bands from both affected and normal alleles. In comparison, PCR products from the SGD patient (lane 4) and normal controls (lanes 1 and 2) show only one fragment, indicating homozygosity for their respective alleles.
RNA blot analysis of the SGD patient's bone marrow
total RNA showed decreased amounts of C/EBP transcripts in comparison with control human bone marrow
pA-RNA (Fig. 2 A). Hybridization with a [32P]dCTP-
labeled actin probe (provided by L. Perera, National Cancer Institute, NIH) showed that 10 µg of SGD patient bone
marrow total RNA was equivalent to 1 µg of normal bone
marrow pA-mRNA and verified the stability and quality of
the patient's RNA preparation. Specific loss of C/EBP
transcripts in the SGD patient is likely due to mRNA instability secondary to the frameshift and the premature termination codon, as seen in other similar gene mutations (32, 33).
Residual C/EBP message is likely comprised by C/EBP14
and C/EBP27 transcripts, which are unaffected by the
5-bp deletion and similar in size to the C/EBP32 transcript. Transcripts of C/EBP were present in normal
amounts. C/EBP has a more proximal role in the myelopoietic pathway and specifically induces expression of
C/EBP (31, 34, 35). As expected, message for lactoferrin
was not detected in the SGD patient's bone marrow RNA.
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As predicted from the C/EBP transcript maps (Fig. 1
B), immunoblotting detected C/EBP27 and C/EBP14
isoforms, but not C/EBP32, in neutrophils from the SGD
patient (Fig. 2 B). All three isoforms were seen in the normal control. The antibody used is specific for a peptide sequence immediately downstream of the 5-bp mutation and
should not bind the C/EBP32-SGD protein.
Transient transfection assays in HeLa cells, using the G-CSF
receptor promoter driving the luciferase gene (31), compared the transactivation potentials of the inducer genes
C/EBP, C/EBP32, C/EBP14, and C/EBP32-SGD
(Fig. 3). C/EBP32 has been shown to transactivate the
G-CSF receptor promoter, whereas the C/EBP14 isoform
lacks transactivating function (23). Transient transfection of
these plasmid constructs showed a significant loss of transactivation with the C/EBP32-SGD isoform (P = 0.02, Mann-Whitney U test). The demonstrated in vitro data, as
well as the in vivo SGD phenotype, mark the full length,
32-kD isoform as the major transactivator encoded in the
C/EBP locus.
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The temporal link between granule protein production and myeloid lineage differentiation is well described: primary granule proteins are synthesized in myeloblasts and promyelocytes, secondary granules are produced in myelocytes and metamyelocytes, and tertiary granule proteins are generated in band and segmented neutrophils (36).
Previous work suggested that C/EBP functions at the
terminal stages of myeloid differentiation (23, 26). However, the total absence of patient neutrophil secondary
granules and the selective loss of primary granule defensins
marks an early myelopoietic block at the promyelocyte
transition (Fig. 4). Further evidence for this conclusion comes
from in vitro differentiation experiments using C/EBP-deficient stem cells, which do not proceed beyond the promyelocyte stage (26). Other functional defects seen in mouse and human C/EBP-deficient neutrophils, such as
loss of tertiary granule gelatinase (27) and abnormalities in
chemotaxis and cytokine expression (6, 27), may occur secondary to the block at the promyelocyte or later stage.
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Functional analysis of the previously developed C/EBP
knockout mouse model (26, 27) was critical for the interpretation of the C/EBP mutation in SGD. The apparent
multiplicity of C/EBP target genes at different cell stages
suggests that C/EBP transactivates a set of early cell stage-
specific genes, inducing normal promyelocyte differentiation
and granule development. Additional evidence supporting these conclusions comes from recent observations suggesting
that C/EBP is induced by and transduces the G-CSF signal
in neutrophils early in myelopoiesis (37). Absence of secondary granules, defensins, eosinophil cationic protein,
eosinophil-derived neurotoxin (12), and platelet granule
high-molecular-mass von Willebrand factor (14) in SGD
demonstrates a critical role for C/EBP in the development of granules and their contents in multiple myeloid lineages.
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Footnotes |
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Address correspondence to John I. Gallin, Bldg. 10, Rm. 2C146, 10 Center Dr. MSC 1504, Bethesda, MD 20892-1504. Phone: 301-496-4114; Fax: 301-402-0244; E-mail: jgallin{at}cc.nih.gov
Received for publication 9 March 1999 and in revised form 1 April 1999.
We are grateful to Dr. Helene Rosenberg for providing SGD patient bone marrow RNA and Dr. Mitchell Horwitz for providing normal peripheral blood CD34+ selected cells and expertise.
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References |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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