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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3223-3231
PHAGOCYTES
The gene for familial Mediterranean fever, MEFV, is
expressed in early leukocyte development and is regulated in response
to inflammatory mediators
Michael Centola,
Geryl Wood,
David M. Frucht,
Jerome Galon,
Martin Aringer,
Christopher Farrell,
Douglas W. Kingma,
Mitchell E. Horwitz,
Elizabeth Mansfield,
Steven M. Holland,
John J. O'Shea,
Helene F. Rosenberg,
Harry L. Malech, and
Daniel L. Kastner
From the Arthritis and Rheumatism Branch, National Institute of
Arthritis and Musculoskeletal and Skin Diseases; Laboratory of Host
Defenses, National Institute of Allergy and Infectious Diseases; and
the Laboratory of Pathology, National Cancer Institute, National
Institutes of Health, Bethesda, MD; and the Department of Rheumatology,
Internal Medicine III, University of Vienna, Austria.
 |
Abstract |
Familial Mediterranean fever (FMF) is a recessive disorder
characterized by episodes of fever and neutrophil-mediated serosal inflammation. We recently identified the gene causing FMF, designated MEFV, and found it to be expressed in mature neutrophils,
suggesting that it functions as an inflammatory regulator. To
facilitate our understanding of the normal function of MEFV, we
extended our previous studies. MEFV messenger RNA was detected
by reverse transcriptase-polymerase chain reaction in bone marrow
leukocytes, with differential expression observed among cells by in
situ hybridization. CD34 hematopoietic stem-cell cultures induced
toward the granulocytic lineage expressed MEFV at the
myelocyte stage, concurrently with lineage commitment. The
prepromyelocytic cell line HL60 expressed MEFV only at
granulocytic and monocytic differentiation. MEFV was also
expressed in the monocytic cell lines U937 and THP-1. Among peripheral
blood leukocytes, MEFV expression was detected in neutrophils,
eosinophils, and to varying degrees, monocytes. Consistent with the
tissue specificity of expression, complete sequencing and analysis of
upstream regulatory regions of MEFV revealed homology to
myeloid-specific promoters and to more broadly expressed inflammatory
promoter elements. In vitro stimulation of monocytes with the
proinflammatory agents interferon (IFN)  , tumor necrosis factor, and
lipopolysaccharide induced MEFV expression, whereas the
antiinflammatory cytokines interleukin (IL) 4, IL-10, and transforming
growth factor  inhibited such expression. Induction by IFN-
occurred rapidly and was resistant to cycloheximide. IFN- also
induced MEFV expression. In granulocytes, MEFV was
up-regulated by IFN- and the combination of IFN- and colchicine.
These results refine understanding of MEFV by placing the
gene in the myelomonocytic-specific proinflammatory pathway and
identifying it as an IFN- immediate early gene.
(Blood. 2000;95:3223-3231)
© 2000 by The American Society of Hematology.
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Introduction |
We and others1,2 identified a novel human
gene, designated MEFV, by positional cloning. Mutations in this
gene cause the autosomal recessive disorder familial Mediterranean
fever (FMF; MIM249100). FMF is characterized by periodic attacks of fever accompanied by serosal, synovial, or cutaneous inflammation. FMF
attacks are self-limited, lasting 1 to 3 days, and include the presence
of purulent neutrophil-rich aseptic exudates at sites of inflammation,
which suggests that the disease-causing alleles of MEFV result
in defects in control of granulocyte-mediated
inflammation.3,4 Seventeen independent mutations in
MEFV have been identified.5-8 These mutations are
limited in scope, causing only single amino acid changes in the
putative protein product. The facts that no null mutations have been
identified and that relatively conservative amino acid substitutions
can result in a disease phenotype suggest that MEFV has an
important physiologic role.
The 3.7-kilobase (kb) MEFV complementary DNA (cDNA) is a member
of a family of highly conserved genes that includes nuclear effector
molecules and nucleic acid binding proteins that regulate inflammation,
hematopoiesis, oncogenesis, and embryonic development.9-11 One member of this family is the ribonuclear protein Ro52, which is a
target of autoantibodies in systemic lupus erythematosus and
Sjögren syndrome. Other family members are transcriptional regulators, including rpt-1, a murine gene that controls
expression of interleukin (IL) 2; Staf50, an interferon
(IF)-regulated gene that attenuates expression of the human
immunodeficiency virus long-terminal-repeat promoter; and PML,
a retinoic acid-dependent transactivator of the p21 WAF1/CIP1
gene that is involved in host defense, myelomonocytic differentiation,
and control of tumor growth. In a survey of normal human tissues,
MEFV messenger RNA (mRNA) was detected only in peripheral blood
leukocytes, and in a preliminary Northern analysis of fractionated
leukocytes, expression was detected specifically in neutrophils, the
principal cell type found in the inflammatory infiltrates
characteristic of FMF.1 Taken together, these data suggest
that MEFV encodes a leukocyte-specific inflammatory regulator,
mutations that cause the autoinflammatory phenotype of FMF.
Although mutations in MEFV lead to illness, the function of the
gene remains a mystery because no gross functional differences have
been observed between neutrophils from families with FMF and those from
healthy volunteers.12,13 Also, compared with normal cells,
FMF neutrophils have neither morphologic changes nor
differences in the release of reactive oxygen products.12 Moreover, no changes in infection rates have been observed in patients
with FMF.3 This is in marked contrast to most
other congenital diseases involving mutations in phagocyte-specific genes, such as chronic granulomatous disease, leukocyte adhesion deficiency, and neutrophil-specific granule deficiency, which are associated with host defense defects and recurrent
infections.14 In fact, in several distinct Mediterranean
populations, FMF carrier frequencies are high, suggesting a selective
advantage in carriers with respect to an unidentified
pathogen.4,5 As a first step in understanding the role of
the MEFV gene in leukocyte biology, we undertook a more
detailed analysis of the expression and regulation of the gene
during hematopoietic differentiation and in response to inflammatory stimuli.
Neutrophil differentiation has multiple discrete stages characterized
by morphologic changes and the acquisition of stage-specific granules.
Primary granules first appear early in differentiation, at the
myeloblast stage, whereas specific granules do not appear until the
myelocyte stage, concurrently with loss of proliferative potential and
granulocytic lineage commitment.15 Elucidation of the
specificity and kinetics of myelomonocytic-specific genes during
differentiation provides insights into their functional roles. Late
gene expression can be studied in the multipotent prepromyelocytic cell
lines HL60 and NB4, which can be further differentiated along the
granulocytic lineage in vitro.16-18 However, differentiation does not proceed normally in these cells, as
illustrated partly by defects in secondary granule-gene expression and
granule formation.19,20 Neutrophil differentiation can be
more accurately recapitulated ex vivo by growth factor-directed
granulocytic differentiation of CD34 hematopoietic precursor cells.
Differentiating cells in these cultures maintain a striking morphologic
and functional similarity to bone marrow precursors.21,22
Increasing evidence from studies of human mutations, functional
knockout experiments, characterization of chromosome breakpoints in
human leukemias, and direct biochemical analysis of
myelomonocytic-specific promoters suggests that key steps in neutrophil
differentiation and activation are transcriptionally
regulated.19,23-26 The promoters of myelomonocytic-specific
genes have common cis-acting elements, as well as
stage-specific and inflammatory mediator-activated promoter
elements,27-31 the identification of which can facilitate understanding of the biologic function of a given gene.32
Although expression studies suggest that MEFV expression is
myeloid specific, no description of the upstream regulatory region of
the gene exists.
Leukocyte-specific mediators of granulocytic inflammation include
reactive oxygen intermediates, eicosanoids, and cytokines, with the
balance of proinflammatory and Th1 mediators (eg, IL-12, tumor necrosis
factor [TNF], and IFN-) and antiinflammatory and Th2 cytokines
(eg, IL-10, IL-4, and transforming growth factor [TGF] ), playing
a key role in regulation of the response.33-37 To begin to
ascertain the normal function of MEFV in the inflammatory cell,
we reexamined the specificity of expression of the gene in fractionated
leukocytes from bone marrow and peripheral blood. We also defined the
kinetics of MEFV induction during differentiation and
inflammatory-mediator activation of these cells.
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Materials and methods |
Purification of peripheral blood leukocytes
Granulocyte purifications from heparin-treated blood from healthy
donors were done as described previously.38 Eosinophils were purified from the granulocyte preparations as described
previously.39 Highly purified populations of lymphocytes
and monocytes were isolated as follows. Healthy volunteers underwent
leukapheresis, and purified lymphocytes and monocytes were obtained by
elutriation.40 Elutriated cells were further purified by
using antibody-conjugated supermagnetic beads (MACS).41 For
lymphocytes, 2 rounds of purification were done with anti-CD3 and
anti-CD19 MACS, according to the manufacturer's instructions (Mitenyi
Biotech, Auburn, CA). For monocytes, cells were further purified with a
combination of anti-CD3, anti-CD7, anti-CD19, anti-CD45RA, anti-CD56,
and anti-IgE MACS, and the mixture was applied to a negative-selection
column to remove residual T cells, natural killer cells, B cells,
dendritic cells, and basophils. The purity of preparations was
determined by fluorescence-activated cell sorting (FACS) and visual
inspection of cytospin slides stained with Wright-Giemsa stain. In
addition, the purity of monocyte preparations was determined by
histologic staining for nonspecific esterase and myeloperoxidase
activity on cytospin preparations.42
Purification and granulocytic differentiation of CD34+
peripheral blood hematopoietic precursors (PBHP)
PBHP were isolated as described previously.21 Purified
CD34+ cells were stored frozen in X-Vivo 10 medium (Bio
Whittaker, Walkersville, MD) supplemented with 1% human serum albumin
(Baxter Healthcare, Deerfield, IL) and 10% dimethyl
sulfoxide (Sigma Chemical, St Louis, MO) until use. Cells
were induced to differentiate along the myeloid lineage in suspension
cultures at 37°C with 7% carbon dioxide (CO2) in
Iscove modified Dulbecco medium supplemented with 10% fetal bovine
serum (FBS) and the following recombinant human growth factors: 50 ng/mL PIXY321 (IL-3 and granulocyte-macrophage colony-stimulating
factor [GM-CSF] fusion protein), 100 ng/mL Flt 3 (gifts from Immunex
Corp, Seattle, WA), 50 ng/mL stem-cell factor, and 100 ng/mL
granulocyte colony-stimulating factor [G-CSF] (R&D Systems,
Minneapolis, MN). Cells maintained viability for approximately 26 days
and appeared to achieve maximal differentiation on or about day 21. Cells were harvested for analysis on days 3, 7, 10, 13, 17, and 21.
Isolation and fractionation of whole bone marrow
After informed consent was given, 20 to 40 mL of bone marrow was
obtained from the posterior superior iliac crest of healthy volunteers.
CD34+ cells from bone marrow were purified by using the
Ceprate immunoaffinity CD34+ cell system (Cellpro, Bothell,
WA). To enrich for leukocytes, erythrocytes were lysed in hypotonic
solution.38 In addition, granulocytes and granulocytic
precursors were isolated from purified bone marrow leukocytes by
standard density-gradient centrifugation on Ficoll gradients, according
to the manufacturer's instructions (Organon Teknika, Durham, NC).
Flow cytometry analysis
Cells were labeled with fluorescein isothiocyanate-conjugated,
phycoerythrin-conjugated, or peridinin chlorophyll protein-conjugated monoclonal antibodies against CD3, CD14, CD16, CD19, CD20, CD56, or
isotype-matched nonspecific control antibodies (Becton Dickinson, San
Jose, CA). Before staining, Fc receptors on monocytes were blocked
by supplementing phosphate-buffered saline-bovine serum albumin
solution with 100 µg/mL human IgG.
Cell lines
HL60, THP-1, and U937 cell lines were obtained from the American
Type Culture Collection (Rockville, MD). The basophilic cell line KU812
and the mast cell line HMC1 were provided by Dr J. Rivera. HMC1 cells
were grown in Iscove media supplemented with  -thioglycerol, and 10%
FBS. All other cell lines were grown in RPMI 1640 (Biofluids,
Rockville, MD) supplemented with 100 U/mL penicillin, 100 µg/mL
streptomycin, 200 mmol/L L-glutamine, and 10% FBS
("complete media"). HL60 cells were induced to differentiate toward the granulocytic lineage with 1 µmol/L or 10 µmol/L retinoic acid for 7 days in cultures containing 2 × 105
cells/mL.43 Cells were induced to differentiate toward the monocytic lineage with 16 nmol/L or 160 nmol/L phorbol ester for 48 hours, followed by 2 washes in RPMI and a subsequent 48-hour incubation, as described previously.44 Adherent cells
were removed by trypsinization and analyzed. Differentiation
toward the eosinophilic lineage by using alkaline culture media was
done as described previously.45 Morphologic assessment of
cells was made on cytospin slides stained with Wright-Giemsa stain.
Leukocyte activation
Cells (107) were seeded with 3 mL of complete media and
left for 2 hours at 37°C in 5% CO2. Monocytes and
granulocytes were stimulated with IL-1 (100 pg/mL), IL-4 (20 ng/mL),
IL-6 (100 units/mL), IL-10 (2.5 ng/mL), TNF- (200 pg/mL), IFN-
(1000 units/mL), IFN- (1000 units/mL), TGF- (10 units/mL), G-CSF
(2 ng/mL), GM-CSF (5 ng/mL; R&D Systems), lipopolysaccharide (LPS; 200 ng/mL), and colchicine (30 ng/mL; Sigma Chemical) alone or in combination.
In situ hybridization
In situ hybridization was done as described
previously.46 For in situ probes, a 435-base-pair (bp)
section of MEFV spanning exons 3 to 5 (nucleotides 986-1420 in
MEFV cDNA; accession no. AF018080) that had no important
similarity to other human DNA sequences in the NR and EST databases
(National Center for Biotechnology Information, National Institutes of
Health [NIH]) was amplified by using the forward primer
5'-CCACGCCCAGGAAGGAGACCCAGTTG-3' and the reverse primer
5'-TGCTTCAGCGCTTCAGTTTGTTTCAG-3'. The polymerase chain
reaction (PCR) product was cloned directly into the pCRII TA-cloning
vector (Invitrogen, Carlsbad, CA), and the resulting plasmid was
designated pV75IE. Sense and antisense digoxigenin-labeled riboprobes
were produced as run-off transcripts from EcoRV-linearized pV75IE and T7 RNA polymerase, and from BamHI-linearized pV75IE and SP6 RNA polymerase, respectively, in the presence of
digoxigenin-labeled uridine triphosphate (UTP).
RNA extraction and reverse transcriptase (RT)-PCR
Total RNA was prepared from cells by using Trizol reagent, and cDNA
was prepared from 2 µg of total RNA with oligo(dT) priming using the
SuperScript Preamplification System (Life Technologies, Gaithersburg,
MD). RT-PCR analyses were performed by using 1/40 of the
reverse transcription reaction (the amount of cDNA derived from 50 ng
of total RNA) as a template to maintain a constant amount of input cDNA
for all samples analyzed. PCR amplification using AmpliTaq Gold (PE
Biosystems, Foster City, CA) was carried out so that
reactions were completed within the exponential cycling phase. The PCR
conditions were 5 minutes at 94°C, followed by cycling for 30 seconds at 94°C, 15 seconds at 61.9°C, and 30 seconds at
68.0°C, then elongation for 10 minutes at 72°C. MEFV,
myeloperoxidase (accession no. J02694), and lactoferrin (accession no.
M83202) were amplified for 45 cycles, and -actin (accession no.
X00351) was amplified for 40 cycles. AmpliTaq Gold requires heat
activation, and accordingly, more cycles for amplification than other
thermal stable polymerases.
PCR products (8 µL) were fractionated on 4% to 20% polyacrylamide
gels (Novex, San Diego, CA) and visualized after ethidium bromide
staining. Because yields of RNA preparations can vary, equal amounts of
RNA were used for cDNA preparations. For all samples, cDNA derived from
50 ng of total RNA was amplified and -actin message levels were
assessed. Oligonucleotide PCR primers (with final product sizes) were
as follows: -actin (650 bp), 5'-CTGGCCGGGACCTGACTGACTACCTC-3' and
5'-AAACAAATAAAGCCATGCCAATCTCA-3'; lactoferrin (422 bp) and
5'-CGGGGCTGGAGACGTGGCTTTTATCA-3',
5'-GCCGGGCAGCCACTTCCTCCTCACTT-3'; myeloperoxidase (728 bp),
5'-GAACCCAACCCCCGTGTCCCCCTCAG-3' and 5'-GGCCAGCCCAGATATACCCCTCACT-3'; and MEFV (351 bp),
5'-GATTGGCGCTC-AGGCACATGCT- GTTA-3' and
5'-GTCGGGGGAACGCTGGACGCCTGGTA-3'.
RNase protection assay
RNA from unstimulated and stimulated cells (107) was
prepared with Trizol (Life Technologies). The MEFV probe used
in this assay was generated from plasmid pV75IE. Labeled RNA probes
were synthesized by using SP6 RNA polymerase and phosphorus 32-labeled UTP. DNA was digested with DNase I (Boehringer Mannheim, Indianapolis, IN), and RNA probes were extracted with phenol and chloroform and
precipitated with ethanol. Labeled RNA probes were hybridized overnight
with target RNA (5 µg) at 56°C) and digested with T1 RNase (Life
Technologies). The protected mRNA fragment was extracted with phenol
and chloroform, precipitated with ethanol, resolved on a 6% denaturing
polyacrylamide gel, and subjected to autoradiography. Gene
transcripts were identified by the length of the protected fragments. Equal loading of RNA was estimated from the amounts of
protected fragments of 2 housekeeping genes, L32 and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Signal
intensities were measured from scanned images by using NIH Image
software. Signals were normalized relative to GAPDH
expression and the relative values reported.
| |
Results |
Kinetics of MEFV expression during granulopoiesis
Previous studies of MEFV expression that used Northern
analysis of panels of human tissues indicated that expression of this gene is limited to peripheral blood leukocytes.1 To gain
more insight into the time course of MEFV expression in
leukocytes, we performed the more sensitive RT-PCR assays. In whole
bone marrow, which is composed predominantly of erythroid cells, a weak
MEFV signal was detected by RT-PCR (Figure
1A). Whole bone marrow was subjected to
hypotonic lysis of red blood cells to produce enrichment for
populations of bone marrow leukocytes and leukocyte precursors. Consistent with the leukocyte-specific expression previously observed, significantly more MEFV mRNA was detected in this population of cells (Figure 1A and B), suggesting that MEFV expression was
not restricted to peripheral, and therefore mature, leukocytes.
Enhanced MEFV expression was also detected in bone marrow
granulocytes and their precursors purified by density-gradient
fractionation (Figure 1A). MEFV expression was
significantly diminished in populations of cells enriched in
CD34+ hematopoietic precursor cells, indicating that
expression is temporally restricted during hematopoiesis. To assess
the possibility that the detection of MEFV message was due to
contamination of bone marrow with peripheral blood, we further analyzed
MEFV expression by using in situ hybridization. This analysis
detected a large population of MEFV-expressing leukocytes in
the bone marrow (Figure 1C).
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| Fig 1.
MEFV expression in bone marrow leukocytes and
precursor cells.
(A). Ethidium bromide (EtBr)-stained polyacrylamide gel
electrophoresis (PAGE) of reverse transcriptase-polymerase chain
reaction (RT-PCR) products of MEFV and -actin messenger RNAs
(mRNAs) from unfractionated bone marrow cells (lane 1), bone marrow
leukocytes (lane 2), bone marrow granulocytic cells (lane 3), and an
enriched population of CD34+ peripheral blood hematopoietic
precursors (PBHP; lane 4). (B) Wright-Giemsa-stained cytospin
preparations of the cells used for these analyses. Original
magnification ×400. (C) Photomicrographs of Wright- Giemsa-stained
in situ hybridizations of bone marrow leukocytes. Results with use of a
gene-specific MEFV antisense riboprobe (right panel) and a
nonspecific MEFV sense strand control riboprobe (left panel)
are shown. Original magnification ×400.
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To define the temporal regulation of MEFV expression during
granulopoiesis and to confirm the results obtained in bone marrow, mobilized CD34+ PBHP were isolated and induced to
differentiate along the granulocytic lineage. Morphologic changes in
the cells in culture were strikingly consistent with those observed in
bone marrow (Figure 2A). Cells maintained a
blast-like morphologic appearance for several days; promyelocytes
(large cells containing small numbers of azurophilic granules) appeared
on or about day 7, and increasing azurophilic granule expression was
observed until about day 10. Day 10 also marked the appearance of
myelocytes, ie, smaller cells containing specific granules. Changes in
nuclear morphologic features and increased expression of specific
granules, characteristic of metamyelocytes, occurred between days 13 and 17. Terminal differentiation of mature neutrophils, characterized
by the appearance of cells with lobate nuclei, occurred on or about day
21 (Figure 2A).
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| Fig 2.
Induction of MEFV expression during
granulopoiesis ex vivo.
(A) Wright-Giemsa-stained cytospin preparations of
cells from PBHP cultures induced to differentiate toward the
granulocytic lineage. Representative images from cultures at 3, 7, 10, 13, 17, and 21 days are shown. Original magnification ×600. (B)
EtBr-stained PAGE of RT-PCR products of the MEFV, lactoferrin
(LF), myeloperoxidase (MPO), and -actin mRNAs from PBHP cells
collected on the days indicated. Positive and negative control
PCR reactions using the MEFV-containing plasmid
pV75-1 and water, respectively, are shown.
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MEFV expression as determined by RT-PCR was first detected
weakly on day 7, with increased expression observed on day 10 and throughout the differentiation process (Figure 2B). Consistent with the
kinetics of differentiation observed morphologically, lactoferrin gene
expression was observed weakly on day 10, with increasing levels
observed for the remainder of the experiment (Figure 2B). Induction of
MEFV expression therefore occurred near the myelocyte stage of
differentiation, at or near lineage commitment.
MEFV expression in cell lines
Specific granule genes are coordinately up-regulated during
granulopoiesis. Because of the similarity of lactoferrin- and MEFV-gene induction in the CD34+ cultures, we
further examined the stage specificity of MEFV expression in
the prepromyelocytic cell line HL60. Although this multipotent cell
line can be induced to differentiate along the granulocytic lineage so
that several functional markers of terminal differentiation appear,
specific granule genes are not expressed during
maturation.19 As expected, lactoferrin was not detected in
HL60 cells induced toward the neutrophilic lineage with retinoic acid
or along the eosinophilic lineage with alkaline pH (Figure
3A). Although MEFV expression was
not detected before differentiation, it was observed after induction
(Figure 3A), thereby confirming the findings on the kinetics of
MEFV expression determined in the CD34+ cultures
and suggesting that MEFV expression is not under the common
regulatory program described for secondary granule genes.
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| Fig 3.
MEFV expression in unstimulated and in vitro
differentiated myelomonocytic cell lines.
(A) EtBr-stained PAGE of RT-PCR products of the MEFV, LF, MPO,
and -actin RNAs from resting untreated HL60 cells
(lane 1) and cultured HL60 cells induced to differentiate toward the
neutrophilic lineage with 1 or 10 µmol/L retinoic acid (lanes 2 and
3), toward the eosinophilic lineage with alkaline pH (pH 7.8; lane 4),
and toward the monocytic lineage with phorbol ester. Positive and
negative control PCR reactions using the MEFV-containing
plasmid pV75-1 and water, respectively, are shown. (B) EtBr-stained
PAGE of RT-PCR products of the MEFV and -actin mRNAs from
the monocytic cell lines U937 and THP-1.
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HL60 cells were also induced to differentiate along the monocytic
lineage with phorbol esters. Because expression of MEFV was not
previously detected in peripheral blood monocytes, we anticipated that
no expression would be observed on commitment of HL60 cells to the
monocytic lineage. Surprisingly, MEFV mRNA was observed in
these cultures (Figure 3A) and in the monocytic cell lines U937 and
THP-1 (Figure 3B). No expression was detected in the human mast cell
line HMC-1 or in the human basophil line KU812 (data not shown).
MEFV expression in purified peripheral blood leukocytes
In purified populations of peripheral blood leukocytes from healthy
donors, MEFV mRNA was observed in neutrophils and in
eosinophils from all samples analyzed (Figure
4A). The purity of the isolated cells was
determined by FACS or histological staining (Figure 4B and C).
Expression was also detected in populations of peripheral blood
mononuclear cells (PBMC) enriched for monocytes by collecting cell
culture plastic-adherent cells in samples from 3 of 4 healthy subjects
(not shown). Expression of mRNA was assessed in highly purified
monocyte preparations (> 98% CD14+ and
nonspecific-esterase positive; Figure 4B) from 3 additional subjects.
MEFV mRNA was detected in all 3 samples; a typical example is
shown in Figure 4A. Message levels among individual subjects appeared
highly variable compared with those observed for the housekeeping gene
-actin.
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| Fig 4.
MEFV expression in peripheral blood leukocytes.
(A) EtBr-stained PAGE of RT-PCR products of the MEFV and
-actin mRNAs from highly purified populations of peripheral blood
neutrophils (lane 1), eosinophils (lane 2), macrophages and monocytes
(lane 3), and lymphocytes (lane 4). Negative and positive control PCR
reactions using water and the MEFV-containing plasmid pV75-1,
respectively, are shown (lanes 5 and 6). (B) Purity of populations
analyzed in A, as determined by fluorescence-activated cell sorting.
Cells were stained for a cell-type-specific marker (red) and
nonspecific isotype control antibody (green). The antibodies used were
monocytes (anti-CD14), neutrophils (anti-CD16), B cells (anti-CD20),
and T cells (anti-CD3). (C) Wright-Giemsa-stained cytospin
preparations of eosinophils used for these analysis. Original
magnification ×400. (D) Photomicrographs of Wright-Giemsa-stained
monocyte in situ hybridizations. Results with use of a gene-specific
MEFV antisense riboprobe (right panel) and a nonspecific
MEFV sense strand control riboprobe (left panel) are shown.
Original magnification ×400.
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To confirm the results obtained with RT-PCR and rule out the
possibility that the signals observed in monocyte preparations were due
to granulocyte contamination, expression of MEFV was assessed
at the single-cell level by in situ hybridization in the highly
purified monocyte preparations shown in Figure 4B. MEFV mRNA
was detected in a large population of monocytes, with individual
cell-expression levels varying widely (Figure 4D), thus confirming the
RT-PCR results. As previously reported,1 no MEFV
mRNA was observed in lymphocytes purified to homogeneity (99%
CD3+ or CD20+; Figure 4B) in samples from 3 healthy subjects (typical example shown in Figure 4A). The purity of
the lymphocyte preparations was essential because signal was detected
in populations of nonadherent PBMC.
The 5' end of the MEFV transcript (accession no.
AF018080) was defined by 5' RACE,1 and the genomic
sequences upstream of the transcription start site in the promoter
region were delineated (accession no. AF111163). Sequence homologies to
transcription factor binding sites in the TRANSFAC database were
identified in the MEFV promotor region by using MatInspector
software.47 The cis-acting elements necessary to
confer myelomonocytic-specific expression were identified and,
consistent with the observed expression profile of MEFV, these
conserved DNA-sequence elements are just upstream of the transcription
start site of MEFV (Table 1). These requirements include a TATA-less sequence containing a PU.1
transcription factor binding site adjacent to the transcription start
site, as well as binding sites for the C/EBP and Runt/PEBP2/CBF
families of transcription factors.32 A putative PU.1 site
was identified 77 bp upstream of the transcription start site
( 77). This sequence is identical to the canonical PU.1 binding
sequence shown to bind PU.1 in vitro.48 In addition, a
C/EBP binding motif is present at position 57, and sites for
several factors shown to mediate myelomonocytic-specific expression,
including AML (acute myelogenous leukemia), c-Myb, MZF (myeloid zinc
finger), and TAL1, were also present within 1 kb of the transcription
start (Table 1). Interestingly, also present in the MEFV
promoter region were DNA-sequence identities to the proinflammatory
mediator-specific cis-acting sites nuclear factor (NF)  b
(164), IF-stimulated response element (105), IF
regulatory factor (496), and -IF activation sequence (GAS; 731), as well as the more ubiquitous AP-1 (647). These
observations are additional indications that MEFV expression is
modulated by inflammatory mediators.
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Table 1.
Sequence similarities to myelomonocytic and
activation-specific cis-acting sites in 1 kilobase of genomic
DNA upstream of MEFV
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Regulation of MEFV mRNA levels by means of soluble
inflammatory mediators
Because of the results described above, we stimulated peripheral
blood monocytes from healthy control subjects with proinflammatory cytokines and mediators, including IL-1, IFN-, TNF-, and LPS; antiinflammatory cytokines IL-4, IL-10, and TGF-; and G-CSF, GM-CSF,
and IL-6. After stimulation, we quantified changes in MEFV expression by using a RNase protection assay.
In vitro stimulation of monocytes with the proinflammatory mediators
IFN-, TNF-, and LPS for 24 hours resulted in increased levels of
MEFV mRNA, whereas treatment with the antiinflammatory cytokines IL-4, IL-10, and TGF- reduced MEFV message levels
(Figure 5A). Moreover, all 3 antiinflammatory cytokines suppressed IFN--induced and LPS-induced
up-regulation of message levels after 24 hours; IL-4 treatment resulted
in an almost complete attenuation of MEFV expression (Figure
5B). No changes in gene expression were observed with the remaining
inflammatory mediators (data not shown).
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| Fig 5.
Quantitation of cytokine- and lipopolysaccharide
(LPS)-mediated regulation of MEFV mRNA levels in peripheral
blood myelomonocytic cells.
Autoradiograms of results obtained with RNase protection assay of total
RNA derived from peripheral blood leukocytes by using an MEFV
gene-specific riboprobe and 2 housekeeping gene-specific riboprobes
(L32 and GAPDH) are shown. Baseline levels of
MEFV mRNA in resting cells before stimulation (untreated) are
shown in each panel. (A) Products from monocytes treated for 24 hours
in vitro with interleukin (IL) 10, transforming growth factor (TGF-), IL-4, interferon (IFN) , LPS, or tumor necrosis factor.
(B) Products from monocytes treated with LPS and IFN- alone and with
LPS and IFN- in combination with either IL-4, IL-10, or TGF-. (C)
Time course of MEFV induction in monocytes treated with IFN-
for 0.5, 1, 4, 12, and 24 hours. (D) Products from untreated monocytes
and monocytes treated with IFN- and both IFN- and cycloheximide.
(E) Time course of MEFV induction in monocytes treated with LPS
for 0.5, 1, 4, 12, and 24 hours. (F) Time course of MEFV
induction in monocytes treated with IFN- for 0.5, 1, 4, and 12 hours. (G) Products from granulocytes treated with colchicine, IFN-,
IFN-, IFN- and colchicine, and IFN- and colchicine.
|
|
Our findings regarding the kinetics of induction revealed that both
IFN- and LPS up-regulated MEFV message levels within 30 minutes, suggesting that these mediators act directly and independently (Figure 5C and 5E). Accordingly, IFN- induction was not inhibited by
cycloheximide (Figure 5D), and MEFV can therefore be classified as an IFN- immediate early gene. IFN- treatment results in
activation of the transcription factor STAT1, which up-regulates gene
expression by binding to a GAS. The presence of the conserved GAS DNA
sequence motif in the promoter region of MEFV further suggests
that IFN- directly induces MEFV expression. Furthermore, the
presence of DNA-sequence identities to NF-B and AP-1 sites suggests
that both LPS and TNF- may also raise message levels by means of
direct induction of transcription and, in this way, independently
regulate MEFV expression (Table 1). Maximal induction of
MEFV mRNA levels by IFN- and LPS was similar both in
magnitude and kinetics, with 5.7- and 6-fold induction, respectively,
compared with results in untreated cells at 1 hour. IFN-, which
functions as an antiinflammatory agent in patients with FMF, was the
most potent inducer in monocytes, increasing MEFV mRNA levels
58-fold in comparison with levels in untreated cells after 4 hours of
stimulation (Figure 5F).
Soluble inflammatory mediators also modulated MEFV message
levels in neutrophils. IFN- increased message levels (Figure 5G). Interestingly, the combination of colchicine and IFN- up-regulated MEFV mRNA levels in neutrophils (Figure 5G), thereby suggesting a role for MEFV in the antiinflammatory effects of both these agents. Colchicine alone did not change MEFV levels in
neutrophils. Similarly, in monocytes, colchicine treatment alone did
not up-regulate MEFV mRNA levels, nor did it act
synergistically with IFN- to up-regulate message levels above those
observed with IFN- alone (data not shown). Although IFN-
treatment caused up-regulation of MEFV message levels in both
monocytes and granulocytes, several inflammatory mediators that
modulated MEFV mRNA levels in monocytes, including LPS, TNF,
IFN-, IL-10, and IL-4, did not change message levels in neutrophils.
These findings indicate that MEFV is differentially regulated
in the 2 cell types. In addition, no change in MEFV mRNA levels
was observed in neutrophils cultured with C5a, GM-CSF, or G-CSF (data
not shown). MEFV mRNA levels were highly variable in
neutrophils and monocytes from healthy donors. Therefore, to detect
cytokine-induced changes in MEFV expression, these analyses used monocytes and neutrophils from donors in whom MEFV mRNA
levels were low relative to the detection limits of the assay.
| |
Discussion |
The data presented in this paper extend understanding of the
expression of a novel gene, mutations in which cause a dramatic inflammatory phenotype. Through studies of in vitro differentiation of
CD34+ hematopoietic precursor cells and leukemic cell
lines, we demonstrated that MEFV expression begins
approximately at lineage commitment, which is earlier in granulocytic
differentiation than previously found.1 In addition to
MEFV expression in neutrophils, we also observed MEFV
expression in monocytes and eosinophils and showed that
monocyte expression is regulated by inflammatory cytokines and LPS.
These findings place MEFV in the context of several important proinflammatory and antiinflammatory mediators and establish it as an
IFN- immediate early gene. In addition, we showed that expression
levels in mature granulocytes are under the control of cytokines and of
pharmacologic agents.
Several lines of evidence strongly indicated that MEFV is
expressed in bone marrow, including direct observation of message in
fractionated bone marrow leukocytes by means of RT-PCR and in situ
hybridization, induction of expression near the myelocyte stage in
CD34+ hematopoietic stem-cell cultures induced to
differentiate along the granulocytic lineage, induction of expression
in the multipotent myelomonocytic cell line HL60 after induction toward
the granulocytic or monocytic lineage, and the appearance of message in
nonterminally differentiated monocytic cell lines. The overlap in
analyses was necessary because collection of bone marrow can result in
contamination by peripheral blood leukocytes, which are known to
express MEFV message. The facts that no MEFV expression
was observed early in the CD34+ cultures and that
expression was induced before full maturation in these cultures and in
the HL60 cell line indicate that MEFV is expressed in
granulocyte precursors.
In the CD34+ cultures, MEFV expression occurred
almost concurrently with the appearance of myelocytes, a stage of
differentiation heralded by the production of specific granules; at
this time, lineage commitment is clearly established.49
Consistent with this observation, expression in the multipotent
prepromyelocytic cell line HL60 was observed only after differentiation
was induced. The myelocyte stage is characterized by the coordinate
induction of specific granule-gene transcription.15 Because
of the kinetics of MEFV expression, it was considered possible
that MEFV is under the same developmental
controls. However, the detection of MEFV mRNA in
HL60 cells indicates that this hypothesis is unlikely to be correct
because, although HL60 cells are capable of attaining a functionally
mature stage of granulocyte differentiation, they do not express
specific granule genes.19 Because MEFV does not share the common program of primary granule-gene expression, multiple independent regulatory controls must operate during this stage of differentiation.
In the periphery, MEFV expression was limited to eosinophils,
monocytes, and neutrophils. Eosinophilia is present in a variety of
pathologic states in which inflammatory lesions are present, including
allergy, helminth infections, and inflammatory bowel disease.50 Although eosinophils have not been observed at
sites of acute inflammation during attacks of FMF, a regulatory role for MEFV in these cells is suggested by a possible reduced
prevalence of asthma in FMF carriers compared with ethnically matched
control subjects and an increase in serum levels of eosinophil cationic protein in patients with FMF.51,52 These observations
indicate that further analysis of the regulation of MEFV
expression in eosinophils is warranted.
The most prevalent inflammatory cell at sites of acute attacks in FMF
is the neutrophil. However, mononuclear cells were observed in the
synovial tissues of arthritic joints of patients with
FMF,53 and monocytes from FMF patients had a decreased
phagocytic and bactericidal capacity54 and increased
spontaneous release of a thromboplastin-like procoagulant factor in
unstimulated cultured cells.55 Thus, the
activity of these cells may be affected by mutations in MEFV.
However, as with neutrophils in FMF, functional analyses have so far
provided no clear clues about the function of MEFV in
monocytes.56
In response to inflammatory stimuli, neutrophils can regulate the
inflammatory response by means of the production of soluble proinflammatory and antiinflammatory mediators, and it is clear that
monocytes play a central role in coordinating inflammatory processes.57,58 Our data suggest that MEFV is a
downstream element in cytokine-induced regulatory cascades.
Proinflammatory activators, including the Th1 cytokine IFN-, TNF,
and LPS, up-regulated monocyte MEFV message levels; and
antiinflammatory cytokines, including the Th2 cytokines IL-4 and IL-10
and TGF-, down-regulated monocytic MEFV expression.
Up-regulation by IFN- occurred rapidly and the effect was not
inhibited by cycloheximide, showing that MEFV is an IFN-
immediate early gene and suggesting that MEFV plays a direct
role in IFN- activation. It is intriguing that of the stimulators
used singly in these studies, only IFN- up-regulated MEFV
levels in both monocytes and granulocytes. These results suggest that
MEFV may mediate common IFN--specific mechanisms of action in these cells.
It should be noted that not all proinflammatory mediators up-regulated
MEFV levels in neutrophils. No changes were observed on
stimulation of cells with C5a, IL-8, or TNF, indicating that MEFV up-regulation is not a general property of activated
cells. IFN--mediated leukocyte activation functions primarily
through the modulation of gene expression. More than 50 IFN--induced genes have been described, including major
histocompatibility complex (MHC) classes I and II transcriptional
regulators (eg, p48, IRF1), apoptosis-related genes, and the 3 MEFV homologs PML-1, acid finger protein, and 52-kd SSA/Ro
autoantigen.59 The fact that IFN-
directly and rapidly induced MEFV expression indicates that
MEFV plays a role in the early stages of
IFN--mediated cell activation, and perhaps, given the
importance of this cytokine in host defense, regulates phagocyte
response in general.
The effects of TNF and LPS on mature monocytes are also mediated by
activation of transcription factors that control inflammatory regulator
and effector genes. IFN- activates STAT-1, which in turn binds to a
conserved cis-acting promoter element (the GAS), whereas LPS
and TNF directly regulate transcription principally by activating
NF-B.60-64 Our examination of the DNA
sequences upstream of the start of MEFV transcription
revealed sequences that match the consensus GAS site at position
731 and the NF-B binding site at position 164.
Laboratory studies to test whether these factors regulate MEFV
expression directly are warranted.
The balance of production of Th1 and Th2 provides both a critical
regulatory circuit for the adaptive immune response and communication
to the innate immune response, as indicated by the profound effects of
these classes of cytokines on monocytes and neutrophils. IFN-
mediates up-regulation of a variety of effector functions, including
phagocytosis, superoxide production, microbicidal activity, and
antitumor activity. Th-2 cytokines (ie, IL-4 and IL-10) can have strong
deactivating effects on these cells and directly antagonize the effects
of proinflammatory agents.65,66 Repression of Th1
cytokine-induced gene expression by Th2 cytokines, as observed by us
for MEFV, is common for several genes of consequence to the
inflammatory response, including MHC class II, iNOS, and IRF-1.67-69 Although no direct data on MEFV
mechanism of action were obtained from the study of purified leukocytes
from patients with FMF, the facts that FMF is a recessive disease and
that no cases in which a carrier has symptoms of the disease have been reported suggest that the role of MEFV is as an inhibitor of
inflammation. If MEFV does play an antiinflammatory role, then
the facts that MEFV message levels are increased by
proinflammatory and Th1 mediators and diminished significantly by
antiinflammatory and Th2 mediators suggest that the gene functions in a
negative-feedback loop that is specific for Th1 and
proinflammatory-mediator activation of myelomonocytic cells.
Given this hypothesis, it is intriguing that both IFN- and
colchicine, the only therapeutic agents known to ameliorate FMF attacks, also up-regulated MEFV message levels. IFN- is
known to have pleiotropic effects, suppressing symptoms in some
autoinflammatory diseases70-72 but exacerbating
inflammation in others.73,74 This agent may exert its
therapeutic effect in FMF by causing overexpression of a functionally
deficient MEFV-gene product by stimulating Th1 or
proinflammatory pathways. Consistent with this idea, administration of
IFN- during an FMF attack increased levels of C-reactive protein and
erythrocyte sedimentation rate while effecting a rapid, complete
alleviation of abdominal pain.75 The effect of colchicine
in suppressing inflammation in granulocyte-mediated diseases, including
FMF, gout, and Behçet disease, has been thought to be due to
either inhibition of leukocyte adhesion and migration or effects on
inflammatory signaling.76-79 Our observation of
up-regulation of MEFV mRNA levels by IFN- and colchicine,
which has not been reported before, suggests that MEFV may play
a previously unrecognized role in the specific physiologic effects of
these agents. In this context, it is of note that the effects of
colchicine on MEFV expression in vitro were observed only in
conjunction with IFN- stimulation. However, when used alone
therapeutically, colchicine blocks FMF attacks, suggesting that IFNs or
similar Th1-inducing agents may be already be present at inflammatory
sites in patients with FMF, although at levels insufficient to inhibit
pathogenesis. If this model of therapeutic effect is correct, then
up-regulation of MEFV mRNA by other agents, specifically
IFN-, may be worth evaluating as an alternative treatment for FMF attacks.
Because mutations in MEFV result in a dysregulation of the
inflammatory response, it was hypothesized before its identification that MEFV was likely to be an inflammatory regulator. The data presented here support this hypothesis. MEFV is expressed
specifically in the primary cellular effectors and regulators of
inflammation, and gene expression is controlled by characterized and
fundamental cytokine-mediated pathways of the inflammatory cascade.
Given the dramatic pathophysiologic consequences of MEFV
mutations, our data suggest that MEFV is an inflammatory
regulator of the phagocyte-mediated inflammatory response. In regard to
etiologic aspects of FMF, defects in several inflammatory regulators
have been hypothesized to underlie the periodic dysregulation of the inflammatory response, including changes in the activities or production of lipocortins,80 TNF,81 and a C5a
inhibitor.82 Our data suggest an alternative
hypothesis that MEFV mediates a Th-1-responsive
negative-feedback loop during proinflammatory activation of
myeloid cells and that the pathophysiologic features of FMF result
from defects in this inhibitory activity. Moreover, these data
clearly establish an expanded context in which to study the function of
MEFV as it pertains to the normal function of the inflammatory
response, perhaps with important implications for infectious,
rheumatic, and autoinflammatory diseases.
| |
Acknowledgments |
We thank the Clinical Pathology Department and the Department of
Transfusion Medicine of the Warren Magnuson Clinical Center, National
Institutes of Health, for supplying cells from healthy donors and for
advice regarding this work.
| |
Footnotes |
Submitted September 30, 1999; accepted January 11, 2000.
Supported by a grant (to M.A.) from the Max Kade Foundation.
Reprints: Michael Centola, Arthritis and Rheumatism Branch,
National Institute of Arthritis and Musculoskeletal and Skin Diseases,
Building 10, Room 9N210, National Institutes of Health, Bethesda, MD
20892-1820; e-mail: centolam{at}arb.niams.nih.gov.
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.
| |
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Abstract
Introduction
