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B as a Central Mediator in the Induction of TGF-ß in Monocytes from Patients with Idiopathic Myelofibrosis: An Inflammatory Response Beyond the Realm of Homeostasis1
,
Departments of
*
Medicine-Hematology,
Pathology and Laboratory Medicine, and
Pediatrics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103; and
§
Department of Biological Sciences, Florida Atlantic University, Boca Raton, FL 33431
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B that
is associated with inflammatory responses. We transiently knocked out
RelA from IMF monocytes with antisense oligonucleotides and showed that
RelA is central to IL-1 and TGF-ß production and to the adhesion of
IMF monocytes. Because the NF-B family comprises subunits other than
RelA, we used aspirin and sodium salicylate to inhibit kinases that
activate NF-B and showed effects similar to those of the RelA
knockout system. It is unlikely that RelA could be interacting directly
with the TGF-ß gene. Therefore, we determined its role in TGF-ß
production and showed that exogenous IL-1 could induce TGF-ß and
adherence of IMF monocytes despite the depletion of NF-B. The
results indicate that IL-1 is necessary for TGF-ß production in IMF
monocytes, but NF-B activation is required for the production of
endogenous IL-1. Initial adhesion activates NF-B, which led to IL-1
production. Through autocrine means, IL-1 induces TGF-ß production.
In total, these reactions maintain overactivation of IMF
monocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immune-mediated mechanisms of BM fibrosis have been suggested (4, 10, 11) with up-regulation in the expression of extracellular matrix (ECM) proteins and cytokines such as TGF-ß, basic fibroblast growth factor, platelet-derived growth factor, and thrombopoietin (1, 14, 15, 16, 17, 18). Adhesion molecules are also implicated in the development of BM fibrosis (12), and interactions with ubiquitous ECM proteins in patients with BM fibrosis could probably explain their role in this disease (12, 18, 19, 20, 21, 22, 23).
TGF-ß levels are increased in the BM and peripheral circulation of patients with BM fibrosis (13, 15), and this increase could be relevant to the fibrosis and its accompanying pathophysiology. Regulation of specific receptors for ECM protein by TGF-ß to produce pathological levels of cytokines could be an example of TGF-ß role in the development of BM fibrosis (20, 21, 22). Together, BM fibrosis seems to involve a complex network that includes ECM proteins, cytokines, and their respective receptors. TGF-ß appears to be a central mediator in different forms of tissue fibrosis, and its high levels are not unique to the BM but are found in other tissues (15, 16, 23). Gene chip arrays in an experimental model of lung fibrosis indicate that TGF-ß-inducible genes comprise a major set of genes that are up-regulated (23). This suggests that understanding the induction of TGF-ß in the relevant patients could be important in the treatment of tissue fibrosis.
Activation of monocytes in patients with primary and secondary BM fibrosis is accompanied by increased induction of IL-1 and TGF-ß (8). We studied the induction of TGF-ß and determined the influence of IL-1 in its expression using monocytes from patients with IMF. Despite the low incidence of IMF, we excluded the patients with secondary fibrosis so as to eliminate confounds of the underlying cause, leukemia, lymphoma, AIDS, etc. An understanding of the biology of IMF monocytes would allow future studies to incorporate other cells within the hemopoietic hierarchy. Other cell types may have equal or greater importance than monocytes in the development of BM fibrosis. However, the experimental hurdle of this disease poses limitation in the types of in vitro model that could be developed. Dense fibrosis prevents retrieval of adequate number of BM cells for experimental purposes. Monocytes from patients with BM fibrosis are used in this study because they: 1) could produce fibrogenic and other relevant cytokines (24), 2) are activated in vivo (18, 25, 26), and 3) are increased in patients with BM fibrosis (25, 26).
We examined the role of the proinflammatory transcriptional factor,
NF-B in the activation and dysregulation of IMF monocytes. NF-B
was studied because it is a mediator of IL-1, which is produced at high
levels following adhesion of monocytes from patients with BM fibrosis
(18). Adhesion is similarly required for TGF-ß
production in patients with BM fibrosis (18). In cancer,
tumor growth is regressed if NF-B activation is prevented, and this
is accompanied by prevention of cell adhesion and TGF-ß production
(27, 28). This indicates that NF-B could be central to
three parameters that are associated with the development of BM
fibrosis, IL-1, TGF-ß, and cell adhesion (28, 29, 30).
NF-B is a multigene family of heterodimeric and homodimeric proteins
of the Rel family of transcription factors, p50, p52, c-Rel, RelB, and
RelA (31). The Rel homology domain interacts with the
inhibitory proteins, IBs, to inactivate NF-B and retain the
complex in the cytoplasm (31). Compared with p50, RelA is
more relevant in modulating cell adhesion, a crucial step for the
observed increase in cytokine production by IMF monocytes (27, 32). This underscores the importance of RelA as a potential
target for NF-B in IMF.
We hypothesize that in vivo, overexpression of adhesion molecules in
IMF monocytes has a major role in their overactivation
(18). To investigate adhesion-mediated activation of IMF
monocytes, we used an in vitro model in which adhesion is induced on
polystyrene surface (18). We used antisense RelA to
transiently knock out p65 in IMF monocytes and then determined the
relationships among cell adhesion, IL-1, and TGF-ß. The results
showed that, indeed, initial adhesion of IMF monocytes led to nuclear
translocation of NF-B and that this is required for the induction of
IL-1 and TGF-ß. However, unlike IL-1 (33, 34), it is
unlikely that NF-B has direct interactions with TGF-ß gene
(27). This suggests that NF-B could be inducing a
trans-acting factor to stimulate TGF-ß production through
the production of IL-1. Exogenous IL-1 restored cell adhesion and
TGF-ß production in the RelA knockout monocytes. This indicates that
NF-B activation is an upstream event followed by stepwise production
of IL-1 and TGF-ß. In the presence of exogenous and/or paracrine
IL-1, NF-B activation is not required. The effects of the antisense
knockout cells were similar to experiments with aspirin and sodium
salicylate, inhibitors of kinases that are important for the
degradation of IB. This suggests that RelA is important in the
activation of IMF monocytes (35, 36).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Institutional Review Board of University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School approved the use of blood from each patient diagnosed with IMF (n = 10) or age-matched healthy, normal controls (NC, n = 10). The ages of study subjects ranged between 36 and 71 years. The mean ages for NC and IMF were 48 and 51 years, respectively. To avoid variation by the degree of BM fibrosis, only patients with advance/significant fibrosis were included (1). Briefly, all patients with IMF had a leukoerythroblastic picture with teardrop poikilocytosis in their blood smears. Their peripheral blood counts showed different degrees of cytopenias with two patients having pancytopenia. All patients presented with palpable spleen and a dry tap on BM aspiration. At least 4 mo before participation in this study, subjects were not transfused with blood, showed no sign of infection, and were not taking any medication.
Animals
Mice were purchased from the Frederick Cancer Research and Developmental Center (Frederick, MD), and rats were from Charles River Laboratories (Wilmington, MA). All animals were maintained and handled in the American Association of Laboratory Animal Care-accredited Research Animal Facility at UMDNJ-New Jersey Medical School, Newark, NJ.
Cell lines
Maintenance of mink lung epithelial (CCL 64) and D10.G4.1 cell lines were previously described (18). The following hybridomas were purchased from American Type Culture Collection (Manassas, VA) and maintained according to their instructions: anti-CD14, IgG1k (HB-44); anti-CD3, IgG2a (CRL 8001); anti-CD57 (HNK-1); Lym-1, IgG2a (HB-8612); Lym-2, IgG1 (HB-8613).
Cytokines and Abs
Hoffman-La Roche (Nutley, NJ) provided recombinant human (rh)
IL-1
. Polyclonal rabbit anti-human (h) IL-1 and
anti-hIL-1ß and rhIL-1ß were purchased from Endogen (Boston,
MA). hIL-1 and hIL-1ß mAb were purchased from Collaborative
Biomedical (Bedford, MA). TGF-ß, rabbit polyclonal anti-hTGF-ß
and rhIL-1 receptor antagonist (rhIL-1RA) were purchased from R&D
Systems (Minneapolis, MN). Neutralization studies indicated that the
anti-hTGF-ß was specific for the -ß1 form. Nonimmune IgG was
purchased from Sigma (St. Louis, MO).
Hybridomas were grown in female DBA/2 retired breeders, and the IgG was purified from ascites with protein G-Sepharose CL-4B (Sigma). The working concentration for each mAb was determined as the optimal amount necessary to detect and deplete >98% of the respective cell, based on FACScan: 125 µg/ml for CD14 and 100 µg/ml for the others.
PE-conjugated rat anti-mouse IgG (optimum concentration, 12.5
µg/ml) was purchased from Becton Dickinson (San Jose, CA).
PE-conjugated nonimmune murine IgG1 was purchased from
Immunotech-Coulter (Miami, FL). Rabbit polyclonal anti-RelA and
RelA control blocking peptide were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Biotinylated goat anti-rabbit IgG
and biotinylated horse anti-mouse IgG were purchased from Vector
Laboratories (Burlingame, CA).
Monocyte separation
Two different techniques were used to separate monocytes from PBMC, obtained from heparinized peripheral blood (18): adherence to Falcon 3003 petri dishes (Becton Dickinson Labware, Lincoln Park, NJ); and negative selection. Sera and media for monocyte separation consisted of <0.035 ng/ml endotoxin. The adhesion procedure was performed by incubating PBMC for 1 h at 37°C on autologous fibronectin, mobilized on gelatin-coated petri dishes as described (18). After deadherence, >95% of the cells were positive for nonspecific esterase and CD14, determined by FACS.
Monocytes were negatively selected by depleting T-, B-, and NK cells
from PBMC. This technique resulted in a population that was 
90%
CD14+ and 10% CD3+,
CD19+, or CD56+. Cells were
depleted by initial incubation with a cocktail of Abs for T- (CD3), B-
(Lym-1 and Lym-2), and NK cells (HNK-1). After this, cells were washed
to remove the excess Abs and then resuspended at
107/ml in RPMI 1640 (Sigma) with 2% human serum
albumin (HSA) and 4 ml added to bacteriological grade petri dishes
(Fisher, Springfield, NJ), precoated with sheep anti-murine
F(ab')2 (U.S. Biochemical, Cleveland, OH).
Precoating was performed for 1 h at room temperature in 0.005 M
Tris with 0.15 M NaCl, pH 9.5.
Labeling of cells for immunofluorescence followed standard procedure.
Indirect labeling used PE-conjugated rat anti-mouse IgG.
Nonspecific binding was determined with nonimmune isotype controls.
Labeled cells were immediately analyzed by FACScan with an instrument
that was optimized daily with fluorescent microspheres (Calibrite,
Becton Dickinson).
Antisense oligonucleotides and cDNA probes
Antisense and sense phosphorothioate analogues of
oligonucleotides were designed from the 5' end of RelA cDNA and
included the initiation codon, 5'-G AGG GGG AAC AGT TCG TCC ATG GC-3'
(27, 37). Oligonucleotides were purified with HPLC using
reverse phase ion exchange. cDNA probes for 28S rRNA, IL-1, IL-1ß,
and TGF-ß1 were previously described (18).
RelA knockout monocytes
Adhesion-separated monocytes were incubated in Teflon jars (Savillex, *Minneapolis, MN) overnight at 106/ml in RPMI 1640 with 2% HSA. After this, >99% cells were in suspension and produced baseline levels of IL-1 and TGF-ß (18). Transfer to polystyrene reversed the levels of IL-1 and TGF-ß to 445 ± 36 U/ml and 125 ± 15 ng/ml, respectively.
RelA knockout monocytes were established by transferring 1 ml
Teflon-incubated cells, resuspended at 106/ml in
RPMI 1640 with 2% HSA, to 24-well polystyrene dishes (Falcon 3047,
Becton Dickinson Labware). Monocytes were cultured with media alone
(untreated), 20 µM antisense RelA or 20 µM sense RelA, 2.5 mM
sodium salicylate (Sigma), or 2.5 mM aspirin (Sigma). The cells take up
20% of the single-stranded oligonucleotides (38).
Cultures were analyzed for the following: 1) percentages of nonadherent
cells, determined by viability and cell count after 48 h; 2)
levels of TGF-ß and IL-1 protein after 48 h; 3) steady-state
TGF-ß1, IL-1, and IL-1ß mRNA after 24 h; and 4) subcellular
localization of RelA after 30 min. IL-1 and TGF-ß levels were
determined in cell-free supernatant, aliquoted in siliconized tubes,
and then stored at -70°C (18). Enumeration of the
adherent cell population was performed after deadhering with ice-cold
Ca2+/Mg2+-free
PBS.
Immunofluorescence for intracellular RelA
Monocytes were placed on Superfrost/Plus slides (Fisher Scientific, Fair Lawn, NJ) and then fixed with cold methanol. Cells were incubated with PBS containing 0.2% Tween 20 and 5% nonfat dry milk for 15 min. Monocytes were first incubated with rabbit anti-RelA (1 µg/ml) at 37°C for 1.5 h and then biotinylated goat anti-rabbit IgG (10 µg/ml) at 37°C for 1 h. After this, cells were incubated with rhodamine 600-avidin D, 1/1000 (Vector Laboratories), at 37°C for 15 min and then examined with an Axiovert 135 Zeiss microscope (New Jersey Scientific, Middlebush, NJ). Preincubation of anti-RelA with RelA (Santa Cruz Biotechnology) showed background label in cells that were positive with the untreated Ab. Each parameter in the technique was optimized with LPS-stimulated monocytes.
Preparation of cell extracts
Nuclear, cytoplasmic, or whole cell extracts were isolated from IMF or NC monocytes as described (18). Cell extracts were prepared by sonicating 6 x 106 monocytes in 0.5 ml PBS with the following protease inhibitors: 0.2 mol/L pepstatin; 5 mol/L leupeptin; and 10 mol/L PMSF. Cells were cleared by centrifugation at 4°C and 10,000 x g for 30 min.
Western blot
RelA was immunoprecipitated from cell extracts at 4°C with rabbit anti-RelA (1/100) for 16 h followed by 4 h incubation with 5 µg proteinA-Sepharose CL-4B. Protein A-Sepharose was pelleted (20 min at 10,000 x g) and then analyzed in Western blots for RelA as described (18). Briefly, samples were subjected to electrophoresis through 12.5% SDS-PAGE, and proteins were transferred to nylon membranes (Immobilon-P, Millipore, Bedford, MA). Membranes were incubated overnight first with rabbit anti-RelA (1/10,000) and then with 50 ng/ml alkaline phosphatase-conjugated goat anti-rabbit IgG (R&D Systems). Alkaline phosphatase was detected with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and the Mr of developed bands determined with prestained midrange protein standards (Life Technologies).
EMSA
EMSA was performed as described (39) with
double-stranded DNA, 5'-GTA GGG GAC TTT CCG
AGC TCG AGA TCC TAT G-3'. The binding site for NF-B is underlined.
DNA was synthesized at the Molecular Core Facility, UMDNJ-New Jersey
Medical School. DNA was labeled by reverse transcriptase to fill in 16
nt at the 3' end using 50 Ci [-32P]dATP
(3000 Ci/mmol, NEN, Boston, MA) and 10 mM concentrations of the other
three NTPs. Protein samples (1 µg) were incubated with
104 dpm in the presence or absence of
anti-RelA and then electrophoresed on 6% PAGE. Gel was dried and
then developed by autoradiography after 12 h.
IL-1 determination
Levels of bioactive and immunoreactive IL-1 were determined in a
proliferative assay and a sandwich ELISA, respectively
(40). The bioassay for IL-1 is based on the ability of
supernatants to support the growth of Con A (Sigma)-stimulated
D10.G4.1. Each assay consisted of a standard curve that was established
with serial dilutions of either rhIL-1 or rhIL-1ß starting at 500
U/ml. Half-maximal [3H]TdR incorporation in the
standard curve is equivalent to 1 U bioactive IL-1. ELISA was performed
as described (12) with rabbit anti-hIL-1 and -1ß
as capture Abs and hIL-1 and hIL-1ß mAb as the second Abs. Binding
of the second Ab was detected by consecutive incubation with
biotinylated horse anti-murine IgG, avidin D, 20 µg/ml (Vector
Laboratories), and biotinylated alkaline phosphatase, 0.2 U/ml (Vector
Laboratories). Enzyme activity was detected with Sigma 104 phosphatase
substrate (Sigma), and OD was measured at a wavelength of 405
nm.
TGF-ß quantitation
Growth inhibition of CCL 64 cells forms the basis of the
bioassay used to quantitate active TGF-ß (18). Each
sample was tested in triplicate with 25, 50, or 100 µl supernatants.
TGF-ß levels were determined from a standard curve established with
TGF-ß concentrations ranging from 0.001 to 10 ng/ml vs cell
concentration. Samples with TGF-ß concentrations 
20 ng/ml were
reassayed in the presence of neutralizing anti-hTGF-ß. Direct
ELISA quantitated immunoreactive TGF-ß as described
(12).
RNA extraction and slot blot
Total cytoplasmic RNA was extracted in a one-step procedure with
100 µl 10 mM Tris, 1 mM EDTA, 0.1 mM vanadyl ribonucleoside complex
(Life Technologies), and 0.5% Nonidet P-40 (U.S. Biochemical). RNA was
immediately resuspended in 6x SSC and 7.4% formaldehyde and then
blotted to presoaked (10x SSC) nylon membrane (Nytran, Schleicher and
Schuell, Keene, NH) using a Minifold II Manifold (Schleicher and
Schuell). Membranes were UV cross-linked and then baked for 1 h at
80°C as described (18). Membranes were hybridized with
[-32P]dATP (3000 Ci/mmol)-labeled cDNA
probes for TGF-ß1, IL-1, or IL-1ß. Labeling of probes,
hybridization, and detection have been previously described
(18).
Statistics
Statistical significance was determined by ANOVA followed by Tukey’s multiple comparison at p < 0.001. All results are given as a two-tailed p value.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in circulating IMF monocytes
Circulating IMF monocytes showed phenotypic and functional
evidence of cellular activation (18). Coincident with this
activation is the presence of relatively high levels of cellular IL-1
(18). Because this cytokine is proinflammatory, we
determined whether NF-B associated with this category of cytokines
is activated in IMF monocytes. Isolation of monocytes requires adhesion
to polystyrene, which could cause nonspecific activation. We therefore
used a negative selection procedure and then compared nuclear and
cytoplasmic RelA by Western blots. A representative blot of three
different experiments, each with a different subject, is shown in Fig. 1
A. RelA was not detected in
the nuclei of NC (lane 1) but was present in the
cytoplasm (lane 2). For IMF, no band was detected
with cytoplasmic extract (lane 3), but a bright band
was detected with nuclear extract (lane 4). Gel shift
assay showed that the immunoreactive RelA could bind to NF-B domain
(Fig. 2). The results showed that RelA is
translocated in the nuclei of circulating IMF monocytes but is retained
in the cytoplasm of monocytes from NC. Due to ethical issues, we are
limited by the total volume of blood. Therefore, because intracellular
immunofluorescence required significantly less cells than Western blot,
we used the former technique for seven patients and showed results
similar to those in Fig. 1.
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B in overactivation of IMF monocytes
We next determined whether nuclear translocation of RelA in IMF
monocytes (Fig. 1A
) is relevant to cell adhesion and the increased
production of IL-1 and TGF-ß. We transiently knock out RelA in IMF
monocytes with antisense RelA and then replated them in an
adherence-supporting surface (polystyrene). After this, we determined
the number of cells that adhered to polystyrene by enumerating the
adherent and nonadherent populations and quantitate bioactive and
immunoreactive IL-1 and TGF-ß. Previous studies showed that
incubation of freshly separated monocytes in Teflon could revert their
overactivated state to "quiescence" with background levels of IL-1
and TGF-ß (18). We therefore incubated freshly separated
monocytes in Teflon for 24 h, washed them, and then transferred
them to polystyrene at 106/ml with antisense
RelA, sense RelA, or medium alone. After 48 h, labeling for
intracellular RelA by immunofluorescence indicated that indeed RelA was
transiently depleted. This was confirmed by Western blot (Fig. 1B) with nuclear extracts from IMF and NC. A strong band was
observed for IMF monocytes in medium alone (lane 5)
or with sense RelA (lane 6) and a light band with
antisense RelA (lane 7). No band was observed for NC
regardless of the culture design, medium, or sense or antisense RelA
(lane 8).
Regardless of exposure to oligonucleotides, cell viability was >99%
as determined by trypan blue exclusion. Antisense RelA blunted the
adhesion of IMF monocytes to polystyrene with minimal effect on NC.
Adhesion was determined by enumerating the floating (nonadherent) and
adherent cells. The results, shown in Table I, indicated that antisense blocked
adhesion of 96 ± 3 and 15 ± 5% monocytes from IMF and NC,
respectively, in contrast to <1% nonadherent IMF monocytes cultured
in media or sense RelA. Because sense RelA showed no effect, the
results shown for antisense cannot be attributed to nonspecific effects
by oligonucleotides (41). Complexing of subunits other
than p65 in NF-B led us to the next set of experiments. We used
inhibitors of kinases that are required for phosphorylation of IB
(35, 36), aspirin, and sodium salicylate to repeat the
studies and showed similar results as the RelA knockout cells
(Table I).
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B on IL-1 and TGF-ß were studied at the mRNA and
protein levels. Because the IMF patients had low blood counts, the
number of monocytes retrieved ranged between 4 x
106 and 6 x 106.
Because of the limited number of cells, we studied mRNA levels using
slot blot rather than Northern analysis. In RelA knockout, IMF
monocytes showed reduction in IL-1 protein by 10-fold and TGF-ß
protein to background levels, p < 0.001 (Table II and III). Similar results were observed for
untreated IMF monocytes cultured in the presence of aspirin and sodium
salicylate. Regardless of culture conditions, NC monocytes produced
background IL-1 and TGF-ß (Tables II and III). Furthermore, IL-1 and
TGF-ß proteins correlated with the densities of the respective steady
state mRNA (Fig. 3). The results indicate
that activation of NF-B is required for IL-1 and TGF-ß production
in adherent IMF monocytes.
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We next determined whether knockout of RelA in IMF monocytes is reversible and also, whether nuclear translocation of RelA depends on adhesion. The former was addressed by readhering RelA knockout cells (n = 5) in polystyrene in the absence of oligonucleotides. After 24 h, >99% of IMF monocytes readhered to polystyrene with concomitant production of IL-1 (446 ± 32 U/ml (±SD)) and TGF-ß (130 ± 35 ng/ml (±SD)). These cytokine levels were similar to untreated, adherent IMF monocytes: IL-1, 545 ± 35 U/ml; and TGF-ß, 128 ± 12 ng/ml. The data indicate that RelA depletion is reversible in terms of adherence and IL-1 and TGF-ß production.
To study the relationship between adhesion of IMF monocytes and nuclear
translocation of RelA, we transferred IMF monocytes from Teflon to
polystyrene and after 30 min used Western blots to determine nuclear
RelA. The results, shown in Fig. 4, indicated that initiation of cell adhesion is accompanied by nuclear
translocation of RelA. No band was observed in extracts from NC
incubated in Teflon or polystyrene (lanes 1 and
2) and IMF incubated in Teflon (lane 3).
However, strong bands were observed in extracts when IMF monocytes were
transferred from Teflon to polystyrene (lane 4).
These results show that adhesion initiated early translocation of RelA
to the nuclei and that the functional effects caused by RelA depletion
are reversible.
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Inhibiting the kinases required for NF-B activation or
transient knockout of RelA in IMF monocytes blunted TGF-ß production
and adherence to polystyrene (Tables II and III). Because the TGF-ß
promoter does not have an NF-B binding domain (27), the
results suggest that NF-B could be mediating TGF-ß induction
through a trans-acting transcription factor. Because IL-1 is
sensitive to NF-B activation, we determined whether TGF-ß
production could occur through the induction of IL-1. Because IL-1RA
binds to the type I receptor, expressed on monocytes (42),
we transferred monocytes from Teflon to polystyrene in the presence or
absence of IL-1RA. Enumeration of adherent and nonadherent cells showed
that IL-1RA prevented >99% cell adhesion (Table IV). NC monocytes were not affected.
These results suggest that production of IL-1 by IMF monocytes could be
important in maintaining their activation through auto stimulation.
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to IMF monocytes that were cultured for
48 h with antisense RelA, sense RelA, or media. IL-1 was chosen
due to its higher affinity for IL-1 receptor type I compared with
IL-1ß. Before adding IL-1, we determined that RelA was depleted by
intracellular immunofluorescence. The results, shown in Table V, indicate that addition of IL-1
resulted in readhesion of >99% of the RelA knockout monocytes and
induction of high levels of TGF-ß (Table V, Fig. 5). Despite the addition of IL-1, RelA
was undetected as determined by immunofluorescence. Experiments
performed with aspirin and IL-1 showed results similar to RelA
knockout cells stimulated with IL-1 (Table V). The results indicate
that the restoration of cell adhesion and TGF-ß production require
IL-1, but if the source is exogenous, NF-B activation is not
necessary.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B could have a role in the
development of BM fibrosis in IMF patients, thereby adding to the list
of diseases that implicate this transcription factor in their
pathophysiology (30, 43). Activation of NF-B induces
the production of IL-1 and TGF-ß in IMF monocytes. Because these
cytokines are part of the overactivated state of IMF monocytes, NF-B
could be a key mediator in the pathophysiological dysfunction of IMF
monocytes. The in vitro model used in this study is appropriate because
similar activation seems to be occurring in vivo. This is demonstrated
by the presence of RelA in the nuclei of circulating IMF monocytes
(Fig. 1). Consistent with nuclear RelA in IMF monocytes is the increase
of intracellular IL-1 and TGF-ß (18). Adhesion of IMF
monocytes triggers nuclear translocation of NF-B (Fig. 4), a
transcription factor that is involved in the expression of several
cytokines (31). Therefore, these findings support an
immune-mediated mechanism in the pathophysiology of BM fibrosis and
perhaps fibrosis in other organs.
The absence of NF-B binding site on genomic TGF-ß
(27) indicates that the role of this transcription factor
in TGF-ß production could be indirect. This is supported by exogenous
IL-1 inducing TGF-ß in RelA knockout IMF monocytes (Table V).
These observations indicate that the requirement for NF-B activation
is an upstream step for IL-1 induction, which is necessary for
autocrine and/or paracrine stimulation to produce TGF-ß in IMF
monocytes. However, this report does not prove that the effects of
IL-1 are common to other factors such as LPS, which concomitantly
activates NF-B and induces the expression of TGF-ß and adhesion
molecule. Incubation in Teflon reduced high concentrations of IL-1 to
baseline until readhesion to polystyrene (18). Together,
the results suggest that initial adhesion triggers nuclear
translocation of NF-B, which mediates IL-1 production. IL-1 is then
released and autostimulates the cells to produce TGF-ß. It is likely
that once the initial TGF-ß is produced, it could begin to
autoregulate its own induction, through sensitive sites on its promoter
(44).
The high IL-1 concentrations may be important for the high avidity that
IMF monocytes demonstrate in adherence to polystyrene (our unpublished
observation). Also, because IL-1 could mediate nuclear translocation of
RelA (34, 45), its production could also be important to
retain NF-B in the nuclei. These studies provide new insights into
the mechanism of monocyte activation by adhesion. An understanding of
this biological effect is important because the high levels of ECM
proteins in IMF patients are potential ligands for adhesion molecules
(23). Furthermore, hyaluronic acid, an ECM protein that is
increased in IMF could interact with the adhesion molecule, CD44 to
stimulate IMF monocytes (12). With respect to ECM
protein-adhesion molecule interactions, the ubiquitous distribution of
ECM proteins in IMF patient (20, 21) may partly explain
the in vivo activated state of IMF monocytes (18), shown
in Fig. 1. Cytokines other than IL-1 might be involved. A likely
candidate would be TNF- because it is produced by monocytes.
However, we could not implicate this cytokine in the activation of IMF
monocytes (our unpublished observation). Activation of NF-B in IMF
monocytes could be utilized to induce apoptosis with exogenous TNF-
(46). This could be a therapeutic strategy because
macrophages and M-CSF, a terminal differentiation factor for monocytes,
are increased in patients with BM fibrosis (1, 25, 26).
The high levels of TGF-ß could not blunt the production of IL-1,
suggesting that part of the dysregulation in IMF monocytes could be the
loss of the normal feedback mechanism to maintain biological
homeostasis. Also, the results showed no evidence of the feedback by
IB and RelA on NF-B (47). Distinct signal
transduction pathways to activate NF-B could be dependent on the
type of cell (48). Therefore, a distinct signaling
pathways in NF-B activation in IMF monocytes cannot be discounted
and could very well be unique to those previously described.
Gene chip microarray was used to determine differences in gene
expression in lung fibrosis (23), an organ in which
TGF-ß has been implicated in the pathophysiology of fibrosis
(49). TGF-ß-inducible genes comprise a major category in
this study of lung fibrosis (23). Our study could provide
further leads for candidate genes not only for fibrosis in the BM but
also for other tissues. Because megakaryocytes could be important in
the pathophysiology of BM fibrosis (13, 50), this study
should be taken in the context of the biology of megakaryocytes and
also the role of other transcription factors (51). TGF-ß
could be a target either directly or indirectly in IMF and other
disease with organ fibrosis based on relevant functions. These include
neoangiogenesis and induction of ECM proteins (1). The
fact that IL-1 can restore the adherence and cytokine production in
RelA knockout IMF monocytes indicates that this manipulation is not
toxic to the cells. Furthermore, with reference to cell adhesion,
minimal effect was observed in antisense-treated normal monocytes
(Table I). Therefore, these studies suggest that the activation of IMF
monocytes could be manipulated and possibly lead to potential targets
for therapeutic intervention. The finding shown in Table I is a subject
of ongoing investigation because the evidence suggests that RelA
treatment lead to down-regulation of highly expressed adhesion
molecules.
Limited forms of therapy have been reported for BM fibrosis. Allogeneic BM transplant may be useful in some patients (52, 53). With the advent of combination therapy for AIDS, targeting of BM fibrosis could be contemplated because this is a common clinical feature in AIDS, creating significant disruption of the hemopoietic system (54). Furthermore, with the reduction of viral load by combination drug therapy in AIDS, reversal of BM fibrosis will be important for maintaining immune competence.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pedro Gascon, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Medical Science Building, Room E-579, 185 South Orange Avenue, Newark, NJ 07103.
3 Abbreviations used in this paper: BM, bone marrow; IMF, idiopathic bone marrow fibrosis; ECM, extracellular matrix; NC, normal controls; UMDNJ, University of Medicine and Dentistry of New Jersey; h, human; rh, recombinant human; HSA, human serum albumin; RA, receptor antagonist.
Received for publication March 8, 2000. Accepted for publication May 25, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B Rel A involves transforming growth factor ß. Mol. Cell. Biol. 14:5326.B with a cell adhesion model. Mol. Cell. Biol. 13:3802.B, a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336:1066.B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
B block CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes. Blood 82:625.B puzzle?. Curr. Biol. 8:R19.[Medline]
B activation: the IB kinase revealed?. Cell 91:299.[Medline]
B-responsive genes. J. Immunol. 163:5608.B kinase-ß. Nature 396:77.[Medline]
B and Sp1 is required for HIV-1 enhancer activation. EMBO J. 12:3551.[Medline]
B/Rel DNA-binding motifs: interaction of both subunits of NF-B with DNA is required for transcriptional activation. Mol. Cell. Biol. 12:4412.B without inhibition of NF-B activity and mutations in the IB gene in Reed-Sternberg cells. Blood 94:3129.B activation in T helper 1 and T helper 2 cells. J. Immunol. 156:56.[Abstract]
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B controls expression of inhibitor IBa: evidence for an inducible autoregulatory pathway. Science 259:1912.B induction by IL-1ß in epithelial and lymphoid cells. J. Immunol. 159:5264.[Abstract]
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