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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3096-3105
CCAAT/Enhancer Binding Protein  Is Critical for Effective
Neutrophil-Mediated Response to Inflammatory Challenge
By
Julie Lekstrom-Himes and
Kleanthis G. Xanthopoulos
From the Clinical Gene Therapy Branch, National Human Genome Research
Institute, National Institutes of Health, Bethesda, MD.
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ABSTRACT |
Targeted mutation of CCAAT/enhancer binding protein
(C/EBP)  in mice results in early death, primarily due to
spontaneous infection with Pseudomonas aeruginosa. Functional
analysis of C/EBP-deficient neutrophils, in an in vivo model of
peritoneal inflammation, shows multiple defects. Reduction of
phagocytotic killing by C/EBP-deficient neutrophils is a result of
decreased uptake of opsonized bacteria as well as little to no
expression of secondary granule proteins. Abnormalities in neutrophil
migration detected in a chemical peritonitis model are likely secondary to abnormal CD11b integrin and L-selectin expression on
C/EBP-deficient neutrophils. Alterations in neutrophil cytokine
expression in response to inflammation show decreased levels of
interleukin-1 receptor antagonist (IL-1Ra) and increased levels of
tumor necrosis factor- (TNF-) expression by C/EBP-deficient
neutrophils. Additionally, TNF- expression is increased in
nonactivated, circulating C/EBP-deficient neutrophils. Overall,
C/EBP-deficient neutrophils are severely functionally impaired,
evoking an abnormal microenvironment, which may contribute to the loss
of normal responses to inflammatory stimuli. Similarities between the
C/EBP-deficient mouse model and the human disease, specific granule
deficiency, will be discussed.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
CCAAT/ENHANCER BINDING proteins (C/EBPs)
are basic-leucine zipper (bZIP) transcription factors,
which dimerize, bind cognate DNA sequences, and effect transcriptional
activation or repression via N-terminal domains.1-4
C/EBP , the newest member of the leucine zipper C/EBP family of
transcription factors, plays a critical role in
myelopoiesis.5-9 C/EBP is unusual among C/EBPs due to
its restricted expression in myeloid and T-cell lineages in contrast to
other family members that are more ubiquitously expressed.8
Also, induction of neutrophil differentiation in promyelocytic leukemia
lines is associated with C/EBP expression.8,10 C/EBP's targeted expression suggests a role in myeloid cell
function; however, it is not the only C/EBP with an apparent role in
hematopoiesis. C/EBP -deficient neutrophils develop no further than
the myeloblast stage.11 Additionally, these cells lack
primary and secondary granule proteins and have significant
downregulation of granulocyte colony-stimulating factor (G-CSF)
receptor expression.11 Macrophages deficient for C/EBP
have altered cytokine expression and an impaired immune response
towards challenged pathogens, Listeria monocytogenes and
Candida albicans.12,13
Our initial work showed that mice deficient for C/EBP are severely
immunocompromised, surviving no longer than 5 months of age with 60%
succumbing to systemic infection with Pseudomonas aeruginosa.5 Furthermore, C/EBP-deficient
neutrophils are functionally defective, lacking an oxidative burst in
response to phorbol myristate acetate (PMA)
stimulation.5 This defect, however, does not explain the
pathogenicity of P aeruginosa in C/EBP-deficient mice.
Patients with chronic granulomatous disease (CGD) and mice engineered
with similar genetic defects lack an effective oxidative burst;
however, they do not develop spontaneous infections with P
aeruginosa.14,15 Both X-linked
gp91phox and autosomal recessive
p47phox CGD mice develop spontaneous infections
with Staphylococcus aureus and Aspergillus fumigatus, a
phenotype similar to CGD patients.14,15 These correlations
strongly suggest that the severe immunodeficiency seen in
C/EBP-deficient mice represents multiple defects, intrinsic to
neutrophil function and possibly involving cytokine signaling and
response to inflammatory stimuli.
Finally, C/EBPs have known regulatory effects on other C/EBP family
members. C/EBP, for example, has cognate recognition sequences for
C/EBP dimers in its upstream elements and its hepatic expression is in
part regulated by C/EBP and C/EBP .16,17 Analogous
regulation of C/EBPs in the myeloid lineage is likely and may influence
the phenotype of the C/EBP-deficient neutrophil.
Here we show that C/EBP-deficient neutrophils have both intrinsic
and extrinsic defects. Phagocytotic and migratory function is abnormal
as well as neutrophil cytokine response to an inflammatory challenge.
Finally, neutrophil expression of other C/EBP family members is altered.
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MATERIALS AND METHODS |
Mice.
C/EBP-deficient mice5 and wild-type littermates were
bred in specific pathogen-free conditions. Genotype of all progeny was
verified by Southern blot analysis, as described.18 All mouse experiments complied with the National Human Genome Research Institute (NHGRI) Animal Care and Use Committee and Association for
Accreditation of Laboratory Animal Care (AALAC) regulations.
Thioglycollate challenge and peritoneal lavage.
Mice were injected intraperitoneally with 2 mL of 3% thioglycollate
broth via a 27-gauge needle. Mice were euthanized by CO2 inhalation and peritoneal exudate cells harvested using one
intraperitoneal wash with 8 mL of phosphate-buffered saline (PBS)
containing 0.1% bovine serum albumin (BSA) and 20 µmol/L disodium
EDTA. Peritoneal exudate cells were counted by Coulter Counter
(Coulter, Hialeah, FL) and cytospin stained with
Wright-Giemsa staining. Cells of myeloid lineage comprised the majority
of exudate cells (>95%), including granulocytes, monocytes, and
macrophage cells.
Phagocytosis assays.
Phagotest kit (Orpegen Pharma, Heidelberg, Germany) was used for
quantitation of neutrophil phagocytosis of opsonized FITC-labeled Escherichia coli with the following modifications. Mouse whole blood collected into heparized tubes was incubated with 40 µL of
precooled E coli bacteria (1 × 109/mL).
Additionally, incubation periods were lengthened from 10 minutes to 1 hour at 37°C or 0°C. After incubation and quenching of
external, nonphagocytosed bacteria, the percentage of cells ingesting
bacteria and the fluorescent intensity was determined using FACScaliber
flow cytometer and Cell Quest software (Becton Dickinson,
San Jose, CA). Staphylocidal assay with lysostaphin (L-0761; Sigma, St
Louis, MO) was performed as described,19 using 250 µL of
whole blood. A total of 1.25 × 106 bacteria was added
to each sample. Total cell counts were (mean ± standard error
[SE]) 7.7 ± 1.0 (× 1,000/µL) for wild-type
samples and 9.8 ± 2.4 (× 1,000/µL) for knock-out samples.
Neutrophil counts were 932 ± 82 cells/µL for wild-type samples
and 1,417 ± 649 cells/µL for knock-out samples. Aliquots at 0-, 30-minute, and 60-minute time points were osmotically lysed and
dilutions plated onto trypticase soy agar (TSA) plates.
Colony counts were recorded and used to derive statistical analysis
(Mann-Whitney U test).
Northern blot and ribonuclease protection assays.
Total RNA was harvested from peritoneal lavage or bone marrow using
Ultraspec RNA isolation system (Biotecx, Houston, TX). A total of 10 µg of bone marrow or peritoneal RNA was resolved on 1% agarose gel
under denaturing conditions as described20 and transferred
to nylon membrane. Hybridization was performed as
described20 using probe concentrations of 2 × 106 cpm/mL buffer. Granule protein probes were the generous
gift of Drs Atsushi Iwama and Daniel Tenen (Harvard University).
Peritoneal lavage total RNA (10 µg) from wild-type or knock-out mice
was hybridized with ribonucleotide probes generated from multiprobe
template sets and Riboquant, an in vitro transcription kit (Pharmingen,
San Diego, CA) according to manufacturer's instructions. Multiprobe
template set mCK-2 contained the following templates, with probe and
protected fragment sizes given (probe/protected fragment): interleukin
(IL)-12p35 (388/359), IL-12p40 (349/321), IL-10 (314/287), IL-1
(284/257), IL-1 (255/229), IL-1Ra (231/202), macrophage inhibitory
factor (MIF) (209/180), IL-6 (191/163), interferon
(IFN)- (172/143), L32 (141/112), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (125/97). Multiprobe template set mCK-3 contained: tumor necrosis factor (TNF)- (389/361), lymphotoxin (Lt) (351/322), TNF- (316/287), IL-6 (284/255),
IFN- (257/228), IFN- (232/203), transforming growth factor
(TGF)1 (208/179), TGF2 (191/162), L32 (141/112), and GAPDH
(125/97). Multiprobe template set mCK-4 contained: IL-3 (389/360),
IL-11 (351/322), IL-7 (315/286), granulocyte-macrophage
colony-stimulating factor (GM-CSF) (283/257), M-CSF (257/231), G-CSF
(230/202), leukemia inhibitory factor (LIF) (209/180),
IL-6 (191/163), stem cell factor (SCF) (173/144), L32 (141/112), and
GAPDH (125/97). Control lanes included yeast RNA with or without RNase
digestion. HybSpeed Ribonuclease Protection Assay (RPA)
(Ambion, Austin, TX) was used according to manufacturer's
instructions, hybridizing at 68°C for 10 minutes followed by RNase
digestion at 37°C for 30 minutes. Resultant protected fragments
were resolved on 6% polyacrylamide, 8 mol/L urea, denaturing gels
(Novex, San Diego, CA).
Intracellular cytokine staining and in vitro stimulation of
peritoneal neutrophils.
Protocol, antibodies, and recombinent mouse cytokines described herein
were obtained from Pharmingen. Peritoneal exudate cells collected by lavage with Hanks' balanced salt solution
(HBSS) or peripheral blood cells (after lysis with Ack
lysis buffer) were washed once with RPMI complete and incubated for 4 hours at 37°C with Golgistop (monesin) 4 µL/mL. Cells were washed
twice with staining buffer, labeled with Gr-1, fixed with 4%
paraformaldehyde for 20 minutes at 4°C, permeabilized with 0.1%
saponin in staining buffer at 4°C, and labeled with R-phycoerythrin
(PE)-anti-IL-6, PE-anti-TNF-, or PE-anti-IL-10 (1 µg/106 cells.) Cells were immediately analyzed by flow
cytometry. Neutrophils were gated by morphologic criteria or by double
staining with Gr-1.
Surface antigen labeling.
Cells used for surface labeling experiments were washed twice in
staining buffer (Dulbecco's PBS without magnesium or calcium, 1%
heat-inactivated fetal bovine serum (FBS), 0.1% sodium azide, filtered), resuspended in 100 µL staining buffer, and incubated with
labeled antibody (Pharmingen) for 30 minutes at 4°C in the dark.
Antibody concentrations used were 1 µg/106 cells. After
incubation, cells were washed twice in staining buffer, resuspending in
0.5 mL staining buffer, and analyzed by flow cytometry.
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RESULTS |
Neutrophils deficient for C/EBP phagocytize bacteria poorly.
Mice deficient for C/EBP were recently described.5
Peripheral blood neutrophils from mice deficient for C/EBP were
assessed for uptake of fluorescein isothiocyanate (FITC)-labeled,
opsonized E coli by flow cytometry. Both wild-type and
C/EBP-deficient neutrophils showed no evidence of phagocytosis after
incubation at 0°C. Intracellular FITC-labeled bacteria were
detected in neutrophils after 37°C incubation; however, both the
number of cells harboring bacteria (mean no. phagocytosing neutrophils ± SE: wild-type 54.3% ± 6.2%, knock-out 25.7% ± 3.8%;
P < .03, Mann Whitney U test) and the mean fluorescent
intensity of the samples were significantly decreased in
C/EBP-deficient neutrophils compared with wild-type cells (P < .03; Mann Whitney U test). Figure 1A
(panel a) shows representative histograms of wild-type and
C/EBP-deficient neutrophils, demonstrating decreased uptake of
FITC-labeled bacteria by C/EBP-deficient samples. Figure 1A (panel
b) shows a box and whisker graph indicating mean, SE, and standard
deviation (SD), summarizing the analysis of three separate experiments,
each including nine samples (three wild-type and six knock-out).
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| Fig 1.
(A) Phagocytosis activity of peripheral blood
neutrophils. Whole blood collected from wild-type and
C/EBP-deficient mice was incubated with FITC-labeled, opsonized
E coli at either 0°C or 37°C for 60 minutes, then
accessed by flow cytometry. Panel a shows representative histograms of
wild-type and C/EBP-deficient neutrophil samples: thin lines,
0°C incubation; thick lines, 37°C incubations. Panel b
summarizes results represented graphically by Box and Whisker graph
showing standard deviation, standard error, and mean. ( ) Represent
wild-type neutrophils, gray boxes represent C/EBP-deficient
neutrophils. An asterisk (*) indicates wild-type neutrophil
phagocytosis, as determined by geometric mean of fluorescent intensity,
is significantly greater than C/EBP-deficient neutrophils (P < .03, Mann Whitney U). (B) Phagocidal activity of peripheral blood
neutrophils. Whole blood collected from wild-type and
C/EBP-deficient mice was incubated with titered S aureus,
followed by treatment with lysostaphin. At represented time points,
aliquots were lysed osmotically, samples streaked on TSA plates, and
incubated overnight. Results are represented by Box and Whisker graph
showing standard deviation, standard error, and mean. () Represent
wild-type neutrophils, gray boxes represent C/EBP-deficient
neutrophils. An asterisk (*) indicates wild-type neutrophil bacterial
killing is significantly greater than C/EBP-deficient neutrophil
killing at 60 minutes (P < .02, Mann Whitney U). (C) Granule
protein expression in wild-type and C/EBP-deficient bone marrow.
Northern blot hybridization of total RNA harvested from bone marrow,
resolved by electorphoresis and transferred to Nytran. Blots were
hybridized with 32P--dCTP-labeled granule protein
probes. C/EBP-deficient neutrophil RNA ( /); wild-type
(+/+). Equivalent sample loading is shown by 18S bands.
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In addition, we tested for neutrophil bacteriocidal activity using the
staphylocidal lysostaphin assay. Results are summarized in Fig 1B, from
three separate experiments, each including six animals. Neutrophils
from C/EBP-deficient mice contained significantly more live bacteria
60 minutes after addition of lysostaphin, compared with wild-type
neutrophils (P < .02; Mann Whitney U test). Taken together,
these experiments show that the phagocytosis defect present in
C/EBP-deficient neutrophils includes both uptake of opsonized
bacteria as well as intracellular killing.
Expression of granule protein mRNA was thus addressed to explore other
tiers of bacteriocidal defects in C/EBP-deficient neutrophils. Total
RNA from bone marrow harvested from 3-week-old C/EBP-deficient mice
and wild-type littermates was fractionated by electrophoresis,
transferred to nylon membranes, and hybridized with a variety of
32P deoxycytidine triphosphate
(dCTP)-labeled granule protein cDNAs. Myeloid to
erythroid (M:E) ratios were calculated from Wright-Giemsa-stained cytospins and were similar for all samples, ranging from 1.95 to 2.01. Bone marrow differential for myeloid cells in the wild-type sample was
myeloblast, 7%; promyelocyte, 2%; myelocyte, 3%; metamyelocyte, 8%;
band, 24%; and segmented, 57%. Similarly, the myeloid cell differentials for the two C/EBP-deficient samples were myeloblast, 5% to 13%; promyelocyte, 2% to 10%; myelocyte, 9% to 17%;
metamyelocyte, 22% to 27%; band, 10% to 23%; and segmented, 29% to
34%. Expression of primary granule proteins, myeloperoxidase,
neutrophil elastase, and proteinase-3 was slightly upregulated in bone
marrow mRNA of C/EBP-deficient mice compared with wild-type.
Expression of secondary granule protein lactoferrin and tertiary
granule protein, gelatinase B, however, was severely downregulated in
the C/EBP-deficient neutrophils. Lysozyme M, present in both primary
and secondary granules, was detected equally well in wild-type and
C/EBP-deficient bone marrows (Fig 1C).
C/EBP-deficient neutrophils migrate poorly to site of inflammatory
challenge.
Mice deficient for C/EBP and wild-type control mice received 3%
thioglycollate broth intraperitoneally at time zero and were subsequently killed at specified time points and assessed for neutrophil migration into the peritoneal cavity, surface antigen expression, and cytokine expression. Figure
2A shows that migration of C/EBP-deficient neutrophils at 4 hours
after thioglycollate challenge was significantly reduced (P < .001; Mann Whitney U test). By 24 hours, however, numbers of
neutrophils within the peritoneal cavity are similiar for
C/EBP-deficient and wild-type mice. Additionally, peritoneal
neutrophil numbers are similar at 48 and 72 hours after challenge.
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| Fig 2.
(A) Neutrophil migration into the peritoneal cavity after
thioglycollate challenge. At indicated time points after
intraperitoneal thioglycollate injection, peritoneal cells were
harvested by lavage, counted, and cytospins stained for differential
counting. Results are represented graphically, showing the migration of
neutrophils into the peritoneal cavity over time after thioglycollate
stimulus. ( ) Represent wild-type neutrophils; ( ) represent
C/EBP-deficient neutrophils. An asterisk (*) indicates significantly
increased numbers of wild-type neutrophils at 4 hours, compared with
C/EBP-deficient neutrophils (P < .001, Mann Whitney U).
(B) CD11 expression on peritoneal neutrophils after thioglycollate
challenge. Mice received thioglycollate intraperitoneally, and
neutrophils were harvested at indicated time points. Cells were doubly
stained with Gr-1(FITC) and CD11b(PE) and assessed by flow cytometry.
Results are represented graphically, by geometric mean of fluorescent
intensity. () Represent wild-type neutrophils; () represent
C/EBP-deficient neutrophils. CD11 staining is significantly
decreased on C/EBP-deficient neutrophils compared with wild-type
cells (P < .004, Mann Whitney U) at 4 hours. By 24 hours,
however, CD11 staining is more intense on C/EBP-deficient cells
(P < .004), compared with wild-type. CD11b staining of
peripheral blood neutrophils is shown at time = 0. (C) CD11b
expression on PMA-stimulated peripheral blood neutrophils. Peripheral
blood neutrophils were stimulated with PMA, stained with -Gr-1 (PE)
and -CD11b (FITC), and examined by flow cytometry. Results are
represented by box and whisker graph, showing mean, standard deviation,
and standard error of fluorescent intensity. () Represent wild-type
neutrophils, gray boxes represent C/EBP-deficient neutrophils. (D)
L-selectin (CD62L) expression on peritoneal neutrophils after
thioglycollate challenge. Peritoneal lavage cells were harvested at
indicated time points and doubly stained with Gr-1(FITC) and anti-mouse
CD62L(PE) and assessed by flow cytometry. Results are represented
graphically, by geometric mean of fluorescent intensity. ()
Represents wild-type neutrophils; () represents C/EBP-deficient
neutrophils. Circles graphed at 0 hours represent data obtained from
circulating neutrophils from nonchallenged mice (P < .001, Mann Whitney U). L-selectin staining is significantly increased on
C/EBP-deficient neutrophils compared with wild-type cells (P < .04, Mann Whitney U) at 4 hours. By 24 hours, however, L-selectin
expression is higher on wild-type neutrophils (P < .02, Mann
Whitney U) compared with C/EBPe-deficient neutrophils.
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Next, the presence of surface antigens integral to neutrophil migration
was assessed. Coincident with the delay in neutrophil migration, CD11b
(integrin) expression is significantly decreased on C/EBP-deficient
neutrophils at 4 hours after thioglycollate challenge compared with
wild-type neutrophils (P < .004; Mann Whitney U test) (Fig
2B). By 24 hours after challenge, however, CD11b is more abundant on
C/EBP-deficient cells compared with wild-type neutrophils (P < .004; Mann Whitney U test). CD11b expression on circulating
peripheral blood neutrophils, shown at time zero, is not statistically
different between groups. Further, CD11b expression was examined on
PMA-stimulated, circulating neutrophils to determine if the delay
observed with thioglycollate challenge was due to deficient
intracellular stores. Peripheral blood neutrophils from wild-type and
knock-out animals were stimulated with PMA in vitro, then stained with
-Gr-1 (PE) and -CD11b (FITC), and analyzed by flow cytometry.
Results shown are representative of three experiments using eight
samples each. Both wild-type and C/EBP-deficient neutrophils showed
the expected upregulation of CD11b expression after PMA stimulation, as
shown on Fig 2C, with no statistical differences detectable
(unstimulated, P = .135; stimulated, P = .263, independent t-test). These results suggest that intracellular
stores of CD11b are present in C/EBP-deficient neutrophils; however,
surface expression is delayed with in vivo inflammatory challenge.
In contrast to CD11b integrin expression, L-selectin expression is
higher on C/EBP-deficient neutrophils at 4 hours after challenge
(P < .04; Mann Whitney U test) and higher on wild-type cells
20 hours later (P < .02) (Fig 2D). L-selectin expression on
peripheral, unstimulated neutrophils, shown on the y-axis, is
significantly higher on C/EBP-deficient neutrophils, compared with
wild-type cells (P < .001; Mann Whitney U test).
Neutrophil-mediated cytokine expression is significantly altered in
C/EBP-deficient neutrophils.
Response of neutrophils to chemical peritonitis, in vivo, was measured
by intracellular cytokine fluorescent antibody staining and flow
cytometry. Summarized results comprise three separate experiments each
using 6 to 10 animals. Additionally, total RNA harvested from
peritoneal exudate cells was analyzed by RPA. Results shown are
representative for four separate RPA experiments each using RNA from
two wild-type and two knock-out animals at each time point (4 hours, 24 hours, 48 hours, and 72 hours).
Intracellular cytokine staining of peritoneal exudate cells showed
significantly increased levels of TNF- in C/EBP-deficient neutrophils compared with wild-type cells at 4 hours after
thioglycollate challenge (Fig 3A, panel a).
Neutrophil production of cytokines, IL-6 and IL-10, was also tested at
4 and 24 hours and found to be similarly elevated in activated
peritoneal neutrophils from both C/EBP-deficient and wild-type mice.
(Geometric mean of fluorescent intensity at 4 hours: wild-type IL-6,
8.4 ± 1.1; knock-out IL-6, 6.6 ± 0.4; wild-type IL-10, 3.47 ± 0.32; knock-out IL-10, 3.43 ± 0.13. Geometric mean of
fluorescent intensity at 24 hours: wild-type IL-6, 24.09 ± 2.13; knock-out IL-6, 20.02 ± 1.39; wild-type IL-10, 6.79 ± 0.24; knock-out IL-10, 9.27 ± 0.38.) Figure 3A (panel b) shows representative dot blots derived from fluorescence-activated cell
sorting (FACS) analysis of wild-type and C/EBP-deficient neutrophils.
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| Fig 3.
(A) TNF- expression by peritoneal neutrophils
after thioglycollate challenge. Peritoneal lavage cells were incubated
with monesin for 5 hours to block transport of proteins from the golgi
apparatus, permeabilized, and stained with FITC-labeled Gr-1 and
PE-labeled anti-mouse TNF-. Panel a shows box and whisker plot of
geometric mean of fluoroscent intensity as determined by flow
cytometry. () Represent wild-type neutrophils, gray boxes represent
C/EBP-deficient neutrophils. An asterisk (*) indicates
TNF- staining is significantly increased in
C/EBP-deficient neutrophils at 4 hours compared with wild-type
neutrophils (P < .005, Mann Whitney U). Panel b shows
representative dot blots of wild-type and C/EBP-deficient neutrophil
samples (+/+ wild-type; / C/EBP-deficient). (B) Cytokine
expression by ribonuclease protection assay in peritoneal neutrophils
after thioglycollate challenge. Arrows show indicated cytokine
transcripts as determined by size. L32 and GADPH bands represent
controls for sample size. C/EBP-deficient neutrophil RNA (/);
wild-type (+/+). (C) TNF- expression in circulating,
unstimulated neutrophils as determined by intracellular cytokine
staining. Results are represented graphically by geometric mean of
fluorescent intensity: ( ), unlabeled neutrophils; ( ),
TNF--labeled neutrophils. Fluorescent intensity of labeled and
unlabeled wild-type neutrophils was not statistically different
(P = .999). Asterisk shows indicated statistical
significance of TNF- labeling of peripheral blood C/EBP-deficient
neutrophils (P < .001, Student's t-test).
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RPA analysis of peritoneal exudate neutrophils confirmed these results
showing increased expression of TNF- and LT in C/EBP-deficient neutrophils compared with wild-type cells (Fig 3B). Also, comparison of
cytokine mRNA showed higher levels of IL-1Ra expression in wild-type
neutrophils compared with C/EBP-deficient neutrophils at 4 hours
after thioglycollate challenge (TGC).
Finally, expression of TNF- was assessed by intracellular cytokine
staining in circulating peripheral blood neutrophils from healthy
nonchallenged mice. Results shown are representative of four
experiments each using 15 to 20 samples. Wild-type neutrophils do not
express detectable levels of TNF-. Neutrophils deficient in
C/EBP, however, express detectable amounts of TNF- compared with
wild-type neutrophils (Fig 3C) (P < .001; Student's
t-test). TNF- expression is significantly lower in
nonchallenged circulating neutrophils compared with peritoneal exudate
cells; however, low-level expression was consistently found in
C/EBP-deficient, circulating neutrophils from both young as well as
older mice.
Circulating peripheral blood neutrophils from C/EBP-deficient mice
express decreased levels of Gr-1.
Wild-type peritoneal neutrophils collected at differing time points
after thioglycollate-induced peritonitis showed abundant expression of
Gr-1 at 4 hours, which decreased over time, consistent with local
priming of cells within the peritoneal cavity and shedding of
glycosylphosphatidylinositol (GP-1) anchored epitopes.21 Alternatively, C/EBP-deficient neutrophils maintained poor
expression of Gr-1 throughout the time course of the thioglycollate
challenge, significantly less than wild-type expression at all time
points (P < .004; Mann Whitney U test)
(Fig 4). Circulating peripheral blood
neutrophils in C/EBP-deficient mice also expressed significantly less Gr-1 compared with wild-type cells, as shown on Fig 4, at time
zero (P < .001, Mann Whitney U test).
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| Fig 4.
Gr-1 expression on peritoneal lavage neutrophils. Mice
received thioglycollate intraperitoneally, and neutrophils were
harvested at indicated time points. Cells were stained with Gr-1 (FITC)
and assessed by flow cytometry. Results are represented graphically, by
geometric mean fluorescent intensity. () Represent wild-type
neutrophils; () represent C/EBP-deficient neutrophils.
Significant differences between samples are shown (Mann Whitney U
test).
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Expression of C/EBP is altered in C/EBP-deficient neutrophils.
Northern hybridization of mRNA collected from peritoneal lavage cells
showed differential expression of C/EBP
(Fig 5). Altered morphology of
C/EBP-deficient neutrophils, as previously described,5 did not permit density discrimination from macrophages by Ficol gradient, however, percent mature neutrophils in each sample were similar, ranging from 65.9% to 60.5%, the remainder of
the cells being macrophages. C/EBP and C/EBP are expressed
equally well in C/EBP-deficient neutrophils compared with wild-type
cells. C/EBP expression, however, is downregulated in
C/EBP-deficient neutrophils relative to wild-type cells.
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| Fig 5.
Expression of other C/EBP family members in
C/EBP-deficient neutrophils. RNA harvested from peritoneal lavage
cells (4 hours after thioglycollate challenge) was resolved by
electrophoresis, transferred to Nytran, and hybridized with
C/EBP-specific 32P--dCTP-labeled probes.
C/EBP-deficient neutrophil RNA (/); wild-type (+/+).
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DISCUSSION |
The results described in this study show that C/EBP-deficient
neutrophils have wide-ranging defects relating to phagocytosis and
bacterial killing, migration, and cytokine production. Further, these
neutrophils evoke an abnormal microenvironment, due to increased TNF- production, analogous to disease states of malignancy or chronic inflammation. In its entirety, C/EBP-deficient cells show
the complex signaling network between cellular proliferation and
terminal function and suggest the molecular basis for the severe
immunodeficiency and myelodysplasia seen in C/EBP-deficient mice.5
Northern blot analysis showed equivalent levels of C/EBP and
C/EBP expression in C/EBP-deficient and wild-type neutrophils. Interestingly, C/EBP expression was clearly decreased in
C/EBP-deficient neutrophils compared with wild-type cells. C/EBP
protein expression varies during myelopoiesis. Analysis of temporal
expression of C/EBP factors during myeloid development shows C/EBP
and C/EBP are expressed early, with levels of C/EBP steadily
increasing and C/EBP quickly decreasing with cell
maturation.22 Northern hybridization of peritoneal exudate
neutrophils permits analysis of C/EBP expression in a mature,
homogenous population, unlike bone marrow preparations. C/EBP
recognition sequences are present in the neutrophil elastase promoter
and in the G-CSF receptor promoter.23,24 Also, C/EBP has
functional sites in the regulatory sequences of the genes for IL-1,
IL-8, G-CSF, and GM-CSF.25 Given the varying
transactivating and DNA binding potentials of the different C/EBP
homodimers and heterodimers, the intricacies of transcriptional
regulation within a single family of transcription factors is formidable.
Neutrophils are traditionally viewed as the mediators of nonspecific
cellular immunity, migrating to sites of infection, phagocytizing bacteria, and releasing toxic oxidative products. Recent work, however,
suggests that neutrophils are also metabolically active cells,
expressing inflammatory cytokines and undergoing significant morphological change in response to infection.26-28 Severe
neutropenia, as seen with cancer chemotherapy or ablative therapy
preceding bone marrow transplantation, often precipitates gram-negative bacterial sepsis, the most frequent culprit being P aeruginosa. The appearance of systemic P aeruginosa infection in
C/EBP-deficient mice suggests, at first glance, that these mice have
an early maturation block in myelopoiesis and lack segmented
neutrophils. Analysis of peripheral blood differential smears, however,
shows the presence of numerous, segmented neutrophils.5 The
appearance of this specific organism in unchallenged mice affords the
rare opportunity to assess neutrophil defects secondary to the loss of
C/EBP transcriptional activity.
Multiple components of the antibacterial armaenterium are defective in
the absence of C/EBP transactivation. Our previous work showed the
complete absence of nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase activity in C/EBP-deficient
neutrophils after PMA activation.5 CGD is the human genetic
disease resulting from the lack of NADPH oxidase activity. Mice
engineered to lack various components of the NADPH oxidase protein
complex share phenotypic qualities with the human disease,
specifically, susceptibility to catalase positive organisms, S
aureus, C albicans, and others.14,15 Importantly,
however, neither CGD patients nor these animal models develop
spontaneous infections with P aeruginosa, suggesting that additional functional defects exist in C/EBP-deficient neutrophils.
Phagocytosis is defective on several levels in C/EBP-deficient
neutrophils. Ingestion of opsonized bacteria is significantly decreased
in C/EBP-deficient neutrophils, possibly due to decreased receptor
population. Additionally, intracellular killing of phagocytized bacteria is impaired. Inability to generate an oxidative burst certainly contributes to this defect; however, expression of secondary granule proteins in C/EBP-deficient neutrophils is nearly
undetectable. This is not without precedent, as C/EBP-deficient
neutrophils lack both primary and secondary granule
proteins.11 Likely, the combined lack of toxic oxidative
metabolites and secondary granule proteins renders the
C/EBP-deficient neutrophil ineffective against intracellular bacteria.
Neutrophil migration is also impaired in the absence of C/EBP.
C/EBP-deficient neutrophils show delayed migration into the peritoneal cavity of thioglycollate-challenged mice. Coincident with
this delay is the impaired early expression of neutrophil surface
receptors CD62L (L-selectin) and CD11b on C/EBP-deficient cells,
which are integral to neutrophil adhesion and
migration.29-31
C/EBP-deficient neutrophils display an unusual pattern of selectin
and integrin expression in response to inflammation.
CD11b/CD18-deficient mice show decreased neutrophil adhesion, however,
paradoxically increased numbers of neutrophils in peritoneal exudates
after thioglycollate challenge.32
L-selectin-deficient mice show delayed neutrophil migration
after thioglycollate challenge compared with wild-type
controls.31 The relatively high levels of L-selectin on
circulating knock-out neutrophils may effect the normal response of
selectin shedding with neutrophil activation. Interestingly, loss of
CD11b/CD18 expression is also linked to the absence of neutrophil
phagocytosis, reduction of oxidative burst, and diminished neutrophil
apoptosis.32 Neutrophil phagocytosis and oxidative burst
are blunted in C/EBP-deficient neutrophils, as is neutrophil migration, despite normal or exaggerated neutrophil migration in the
CD11b/CD18 knock-out model.32
CD11a expression is not effected by neutrophil
activation,33 nor was differential expression of CD11a
detected between C/EBP-deficient and wild-type neutrophils (data not
shown). These results suggest that the delay in CD11b upregulation in
response to inflammation lies in an inappropriate response to priming
signals, rather than intrinsic defects in surface receptor expression
levels. Alternatively, an altered microenvironment, due to abnormal
cytokine expression, such as TNF-, may result in aberrant neutrophil responses.
Analysis of neutrophil cytokine expression over the course of an
inflammatory response shows unexpected differences between C/EBP-deficient and wild-type cells. Both intracellular cytokine antibody staining and steady-state mRNA expression show significantly increased expression of TNF- and LT in C/EBP-deficient
neutrophils early in the response to an in vivo inflammatory stimulus.
Additionally, IL-1Ra expression was downregulated in C/EBP-deficient neutrophils.
TNF- is a bifunctional regulator of the inflammatory response. Low
and moderate doses of TNF- prime neutrophils,34 enhance leukocyte recruitment to inflammatory sites,35,36 induce
expression of GM-CSF,37 and prolong neutrophil
survival.38 High-dose TNF-, however, inhibits colony
formation induced by G-CSF, downregulates CSF receptor
expression,39 promotes cellular apoptosis,40 and in the clinical setting, is associated with poor survival from
sepsis or lymphoma.41 Lymphotoxin enhances neutrophil respiratory burst and inhibits locomotion.42 Deciphering
the effects of elevated TNF- levels associated with
thioglycollate-induced peritonitis in C/EBP-deficient mice is
confounded by the associated differences in IL-1Ra expression. One may
speculate that high local levels of TNF- at infectious foci in
C/EBP-deficient mice may set up a cycle of cell recruitment and cell
degradation, with release of cytokines and further cell recruitment.
Interestingly, chronic granuloma formation seen in challenged
gp91phox-deficient (CGD) mice is associated with
elevated levels of TNF- and IL-1.43 Dysregulation of
the mechanisms governing acute inflammation and resolution may result
in the localized accumulation of myeloid cells, seen in a high
proportion of endstage C/EBP-deficient mice.
IL-1Ra, an antagonist of IL-1, is expressed in response to GM-CSF
and TNF- stimulation.44 Yersinia enterocolitica
infection in mice promotes IL-1Ra expression in circulating
neutrophils.45 IL-1Ra completely abrogates the priming
effects on the respiratory burst induced by IL-1 and
IL-1.46 Decreased expression of IL-1Ra in
C/EBP-deficient neutrophils may be due to loss of C/EBP transcriptional activity, particularly because a number of cytokines, including IL-1, IL-8, G-CSF, and GM-CSF have C/EBP cognate sequences within their regulatory regions.25 Interestingly, levels of IL-6, which induce IL-1Ra expression,45 appear normal in
these mice by RPA and intracellular cytokine staining, suggesting
complex signaling pathways of cytokine expression. Decreased IL-1Ra
expression may fail to suppress ongoing acute inflammatory responses.
In conjunction with elevated TNF- levels, the lack of IL-1Ra may be,
in part, responsible for the myeloid cell aggregations seen in
end-stage C/EBP-deficient mice, an aberrant response to low pathogenicity, chronic infections.
TNF- expression in circulating neutrophils preceding thioglycollate
challenge is also increased in C/EBP cells compared with wild-type
cells. Chronically elevated levels of TNF-, even in mice as young as
3 weeks, provides explanation for the preactivated state of
C/EBP-deficient neutrophils, as suggested by decreased Gr-1
staining. Neutrophil priming is reported in human immunodeficiency virus (HIV)-infected patients, associated with increased CD11/CD18 expression, increased actin polymerization, and decreased L-selectin expression.47 Elevated TNF- levels are associated with
adverse prognosis in patients with lymphoma.41 The possible
role of C/EBP in effecting disease states needs to be investigated.
Interestingly, C/EBP-deficient mice share many phenotypic features
with patients with specific granule deficiency (SGD). SGD patients are
severely immunocompromised, developing frequent bacterial infections
with S aureas, P aeruginosa, and
Klebsiella.48,49 SGD neutrophils possess atypical,
bilobed nuclei, similar in appearance to C/EBP-deficient
neutrophils.49 Additionally, SGD neutrophils, like
C/EBP-deficient neutrophils, lack lactoferrin and gelatinase B
expression, display delayed chemotaxis, and decreased bacterial killing
in vitro.48-54 These similarities suggest a regulatory role
for C/EBP transactivation in the disease pathology of SGD.
Analogous to the role of C/EBP in linking hepatocyte and adipocyte
function and differentiation, C/EBP may also regulate both normal
neutrophil function and the terminal steps of myeloid differentiation.
C/EBP-deficient mice have multiple neutrophil defects including
depressed phagocytosis and phagocidal killing, impaired oxidative
burst, deficient secondary and tertiary granule protein mRNAs, and
delayed response to an inflammatory stimulus. The combination of
increased TNF- and decreased IL-1Ra expression further creates an
abnormal microenvironment, likely affecting neutrophil recruitment,
function, and resolution of inflammation. This abnormal
microenvironment, secondary to loss of critical feedback loops
necessary for resolution of inflammatory responses may, in part, result
in uncontrolled myeloid proliferation. Alternatively, intrinsic
neutrophil defects due to loss of C/EBP expression may drive the
myeloproliferation seen in knock-out animals. Work continues to
decipher the processes driving the myeloproliferative phenotype in
C/EBP-deficient mice.
| |
ACKNOWLEDGMENT |
We are grateful to L. Garrett and T. Hernandez for excellent technical
assistance and devoted animal care. We thank Drs D.G. Tenen and D. Bodine for expert advice and reagents, and Dr J.I. Gallin for critical
input. We thank Dr R.M. Blaese for creating an inspiring environment
and providing constant support.
| |
FOOTNOTES |
Submitted June 29, 1998; accepted December 16, 1998.
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.
Address reprint requests to Julie Lekstrom-Himes, MD, 10 Center Drive, 11N120, Bethesda, MD 20892; e-mail:
jlekstrom{at}atlas.niaid.nih.gov.
| |
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TOP
ABSTRACT
INTRODUCTION