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The Journal of Immunology, 2000, 165: 2271-2277.
Copyright © 00 by The American Association of Immunologists

NF-{kappa}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

Pranela Rameshwar*, Ramaswamy Narayanan{ddagger}, Jing Qian*, Thomas N. Denny{dagger},{ddagger}, Cristina Colon* and Pedro Gascon2,*

Departments of * Medicine-Hematology, {dagger} Pathology and Laboratory Medicine, and {ddagger} 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Immune-mediated mechanisms have been implicated in the etiology of idiopathic bone marrow fibrosis (IMF). However, the mechanism remains poorly defined. Compared with healthy controls, IMF monocytes are overactivated, with increased production of TGF-ß and IL-1. TGF-ß is central to the progression of fibrosis in different organs. In the lung, fibrosis is associated with up-regulation of TGF-ß-inducible genes. Because IL-1 and TGF-ß have pro- and antiinflammatory properties and neither appears to regulate the high levels of each other in IMF, we studied the mechanism of this paradigm. We focused on the role of RelA, a subunit of the transcription factor, NF-{kappa}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-{kappa}B family comprises subunits other than RelA, we used aspirin and sodium salicylate to inhibit kinases that activate NF-{kappa}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-{kappa}B. The results indicate that IL-1 is necessary for TGF-ß production in IMF monocytes, but NF-{kappa}B activation is required for the production of endogenous IL-1. Initial adhesion activates NF-{kappa}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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myelofibrosis is a hematological disorder characterized by fibrosis, hypercellularity, excessive deposits of extracellular matrix proteins, and neoangiogenesis in the bone marrow (BM)3 (1, 2). Myelofibrosis could be idiopathic (IMF) or secondary to several diseases (1, 3, 4, 5). The mechanism of BM fibrosis remains undefined. It is believed that the fibrosis, caused by excessive proliferation of fibroblastic mesenchymal cells (6), is a reactive process secondary to dysfunctions caused by the clonal hemopoietic stem cell (7). This premise is derived from studies that showed clonality of the stem cells and heterogeneity among BM fibroblasts. The underlying cause of IMF remains undefined because patients are presented with BM fibrosis in the absence of any other pathology. Progression of BM fibrosis leads to compensatory hemopoiesis in extramedullary tissues (8), and in general hemopoiesis is impaired with different degrees of cytopenias (1). Despite similar clinical presentation in the BM of patients with idiopathic and secondary BM fibrosis, there are inconsistencies in the correlation between peripheral hemopoietic progenitors and the degree of BM fibrosis between the two major subsets of patients (9). Therefore, to understand the pathophysiology of this disease, the functions of particular candidate cells need to be dissected.

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-{kappa}B in the activation and dysregulation of IMF monocytes. NF-{kappa}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-{kappa}B activation is prevented, and this is accompanied by prevention of cell adhesion and TGF-ß production (27, 28). This indicates that NF-{kappa}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-{kappa}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, I{kappa}Bs, to inactivate NF-{kappa}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-{kappa}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-{kappa}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-{kappa}B has direct interactions with TGF-ß gene (27). This suggests that NF-{kappa}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-{kappa}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-{kappa}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 I{kappa}B. This suggests that RelA is important in the activation of IMF monocytes (35, 36).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study subjects

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{alpha}. Polyclonal rabbit anti-human (h) IL-1{alpha} and anti-hIL-1ß and rhIL-1ß were purchased from Endogen (Boston, MA). hIL-1{alpha} 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 {kappa} 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 {kappa} 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{alpha}, 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{alpha}, 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-{kappa}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 [{alpha}-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{alpha} 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{alpha} and -1ß as capture Abs and hIL-1{alpha} 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 [{alpha}-32P]dATP (3000 Ci/mmol)-labeled cDNA probes for TGF-ß1, IL-1{alpha}, 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nuclear translocation of NF-{kappa}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-{kappa}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. 1GoA. 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-{kappa}B domain (Fig. 2Go). 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. 1Go.



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  		FIGURE 1. Nuclear and cytoplasmic RelA in IMF and NC monocytes. A, RelA was immunoprecipitated in nuclear and cytoplasmic extracts from NC or IMF monocytes, isolated by negative selection, and then analyzed by Western blots for RelA. Lane 1, NC-nuclear; lane 2, NC-cytoplasmic; lane 3, IMF-cytoplasmic; lane 4, IMF-nuclear. B, IMF or NC monocytes were transferred from Teflon to polystyrene in the presence of medium, sense RelA, or antisense RelA, and nuclear extracts were analyzed for RelA by Western blot. Lane 5, IMF-medium; lane 6, IMF-sense; lane 7, IMF-anti-sense; lane 8, NC-medium, NC-sense, or NC-antisense. Values represent one of three experiments, each with a different patient.

 


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  		FIGURE 2. Binding of nuclear extracts from IMF or NC to DNA with NF-{kappa}B domain. IMF and NC monocytes were isolated as for Fig. 1Go. Nuclear extracts were analyzed in gel shift and super shift with 32P-labeled DNA. Representative gel shift of three experiments is shown. A, Lane 1, Probe alone; lane 2, IMF; lane 3, IMF with excess cold DNA; lane 4, NC. B, Lane 1, IMF; lane 2, supershift.

 
Role of NF-{kappa}B in overactivation of IMF monocytes

We next determined whether nuclear translocation of RelA in IMF monocytes (Fig. 1AGo) 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. 1GoB) 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 IGo, 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-{kappa}B led us to the next set of experiments. We used inhibitors of kinases that are required for phosphorylation of I{kappa}B (35, 36), aspirin, and sodium salicylate to repeat the studies and showed similar results as the RelA knockout cells (Table IGo).


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  		Table I. Adherence vs nonadherence of IMF and NC monocytes1

 
The effects of NF-{kappa}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 IIGo and IIIGo). 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 IIGo and IIIGo). Furthermore, IL-1 and TGF-ß proteins correlated with the densities of the respective steady state mRNA (Fig. 3Go). The results indicate that activation of NF-{kappa}B is required for IL-1 and TGF-ß production in adherent IMF monocytes.


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  		Table II. Bioactive IL-1 and TGF-ß by IMF or NC monocytes1

 

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  		Table III. Immunoreactive IL-1 and TGF-ß by IMF or NC monocytes1

 


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  		FIGURE 3. TGF-ß1, IL-1{alpha}, and IL-1ß mRNA in RelA knockout monocytes. Cells, 2 x 106/ml, from IMF or NC were transferred from Teflon to polystyrene and then cultured with antisense RelA, sense RelA, or media alone. After 24 h, cytoplasmic RNA was analyzed for TGF-ß1, IL-1{alpha}, or IL-1ß in slot blot. Representative result is shown from three experiments.

 
Effects of adhesion on nuclear translocation of RelA

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. 4Go, 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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  		FIGURE 4. Effect of adhesion on nuclear translocation of RelA in IMF monocytes. Freshly isolated monocytes, 6 x 106, were incubated in Teflon. After 24 h, 50% cells were transferred to polystyrene; 24 h later, nuclear extracts were isolated and then determined for RelA by immunoprecipitation followed by Western blots. Lane 1, NC-Teflon; lane 2, NC-polystyrene; lane 3, IMF-Teflon; lane 4, IMF-polystyrene.

 
Role of IL-1 in adhesion and TGF-ß production by IMF monocytes

Inhibiting the kinases required for NF-{kappa}B activation or transient knockout of RelA in IMF monocytes blunted TGF-ß production and adherence to polystyrene (Tables IIGo and IIIGo). Because the TGF-ß promoter does not have an NF-{kappa}B binding domain (27), the results suggest that NF-{kappa}B could be mediating TGF-ß induction through a trans-acting transcription factor. Because IL-1 is sensitive to NF-{kappa}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 IVGo). 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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  		Table IV. Effects of IL-1RA on the adhesion of IMF and NC monocytes1

 
The importance of IL-1 in TGF-ß production and adhesion was studied by adding exogenous IL-1{alpha} to IMF monocytes that were cultured for 48 h with antisense RelA, sense RelA, or media. IL-1{alpha} was chosen due to its higher affinity for IL-1 receptor type I compared with IL-1ß. Before adding IL-1{alpha}, we determined that RelA was depleted by intracellular immunofluorescence. The results, shown in Table VGo, indicate that addition of IL-1{alpha} resulted in readhesion of >99% of the RelA knockout monocytes and induction of high levels of TGF-ß (Table VGo, Fig. 5Go). Despite the addition of IL-1{alpha}, RelA was undetected as determined by immunofluorescence. Experiments performed with aspirin and IL-1{alpha} showed results similar to RelA knockout cells stimulated with IL-1{alpha} (Table VGo). The results indicate that the restoration of cell adhesion and TGF-ß production require IL-1, but if the source is exogenous, NF-{kappa}B activation is not necessary.


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  		Table V. Effect of IL-1{alpha} on adhesion of IMF monocytes and TGF-ß production1

 


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  		FIGURE 5. Effects of IL-1{alpha} on TGF-ß1 mRNA in RelA knockout monocytes. Cells, 2 x 106/ml, from NC or IMF were transferred from Teflon to polystyrene and then incubated with 20 µM antisense RelA or media alone. After 48 h, IL-1{alpha} and fresh anti-sense RelA were added to the antisense cultures. Cytoplasmic RNA was extracted and then probed with TGF-ß1 cDNA. Representative results are shown from three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that NF-{kappa}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-{kappa}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-{kappa}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. 1Go). 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-{kappa}B (Fig. 4Go), 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-{kappa}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{alpha} inducing TGF-ß in RelA knockout IMF monocytes (Table VGo). These observations indicate that the requirement for NF-{kappa}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{alpha} are common to other factors such as LPS, which concomitantly activates NF-{kappa}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-{kappa}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-{kappa}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. 1Go. Cytokines other than IL-1 might be involved. A likely candidate would be TNF-{alpha} 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-{kappa}B in IMF monocytes could be utilized to induce apoptosis with exogenous TNF-{alpha} (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 I{kappa}B{alpha} and RelA on NF-{kappa}B (47). Distinct signal transduction pathways to activate NF-{kappa}B could be dependent on the type of cell (48). Therefore, a distinct signaling pathways in NF-{kappa}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{alpha} 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 IGo). 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 IGo 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.


   Footnotes
 
1 This research was supported in by National Institutes of Health Grants HL-54973 and HL-57675, Foundation of University of Medicine and Dentistry of New Jersey, and Ruth Estrin Goldberg Memorial for Cancer Research. Back

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. Back

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. Back

Received for publication March 8, 2000. Accepted for publication May 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Barosi, B.. 1999. Myelofibrosis with myeloid metaplasia: diagnostic definition and prognostic classification for clinical studies and treatment guidelines. J. Clin. Oncol. 17:2954.[Abstract/Free Full Text]
  2. Tefferi, A., M. N. Silverstein. 1996. Current perspective in agnogenic myeloid metaplasia. Leuk. Lymphoma 22:169.
  3. Spivak, J. L., B. S. Bender, T. C. Quinn. 1984. Hematologic abnormalities in AIDS. Am. J. Med. 77:224.[Medline]
  4. Kaelin, W. G., J. L. Spivak. 1986. Systemic lupus erythematosus and myelofibrosis. Am. J. Med. 81:935.[Medline]
  5. Taube, T., K. Franssila, L. Risteli, J. Risteli, I. Elomaa. 1992. Monitoring of multiple myeloma and bone marrow fibrosis with aminoterminal propeptide of type III collagen. Br. J. Haematol. 82:32.[Medline]
  6. Greenberg, B. R., L. Woo, I. C. Veomett, C. M. Payne, F. R. Ahmann. 1987. Cytogenetics of bone marrow fibroblastic cells in idiopathic chronic myelofibrosis. Br. J. Haematol. 66:487.[Medline]
  7. Sato, Y., T. Sada, J. Suda, A. Ohsaka, K. Kubota, M. Saito, Y. Miura. 1986. Multilineage expression of haematopoietic precursors with an abnormal clone in idiopathic myelofibrosis. Br. J. Haematol. 64:657.[Medline]
  8. Ward, H. P., M. H. Block. 1991. The natural history of agnogenic myeloid metaplasia (AMM) and a critical evaluation of its relationship with the myeloproliferative syndrome. Medicine 50:357.
  9. Colovic, M. D., P. H. Wiernik, G. M. Jankovic, A. D. Vidovic, S. Janosevic, N. M. Basara. 1999. Circulating haemopoietic progenitor cells in primary and secondary myelofibrosis: relation to collagen and reticulin fibrosis. Eur. J. Haematol. 62:155.[Medline]
  10. Hasselbalch, H., H. Jans, P. L. Nielsen. 1987. A distinct subtype of idiopathic myelofibrosis with bone marrow features mimicking hairy cell leukemia: evidence of an autoimmune pathogenesis. Am. J. Hematol. 25:225.[Medline]
  11. Cappio, F. C., R. Vigliani, A. Novarino, G. Caamussi, D. Campana, F. Gavosto. 1981. Idiopathic myelofibrosis: a possible role for immune complexes in the pathogenesis of bone marrow fibrosis. Br. J. Haematol. 49:17.[Medline]
  12. Rameshwar, P., V. T. Chang, P. Gascón. 1996. Implication of CD44 in adhesion-mediated overproduction of TGF-ß and IL-1 in monocytes from patients with bone marrow fibrosis. Br. J. Haematol. 93:22.[Medline]
  13. Martyré, M. C.. 1995. TGF-ß and megakaryocytes in the pathogenesis of myelofibrosis in myeloproliferative disorders. Leuk. Lymphoma 20:39.[Medline]
  14. Martyré, M. C., M. C. Le Bousse-Kerdiles, N. Romquin, S. Chevillard, V. Pravloran, J. L. Demory, B. Dupriez. 1997. Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis. Br. J. Haematol. 97:441.[Medline]
  15. Rameshwar, P., V. T. Chang, U. F. Thacker, P. Gascón. 1998. Systemic transforming growth factor-ß in patients with bone marrow fibrosis: pathological implications. Am. J. Hematol. 59:133.[Medline]
  16. Johnston, J. B., B. I. Dalal, S. J. Israels, S. Oh, E. McMillan, A. Begleiter, G. Michaud, L. G. Isreals, A. H. Greenberg. 1995. Deposition of transforming growth factor-ß in the marrow in myelofibrosis, and the intracellular localization and secretion of TGF-ß by leukemic cells. Am. J. Clin. Pathol. 103:574.[Medline]
  17. Usuki, K., T. Tahara, S. Iki, M. Endo, M. Osawa, K. Kitazume, T. Kato, H. Miyazaki, A. Urabe. 1996. Serum thrombopoietic level in various hematological diseases. Stem Cells 14:558.[Abstract]
  18. Rameshwar, P., T. Denny, D. Stein, P. Gascón. 1994. Adhesion of monocytes from patients with bone marrow fibrosis is required for the production of fibrogenic cytokines: potential role for IL-1 and TGF-ß. J. Immunol. 153:2819.[Abstract]
  19. Ignotz, R. A., J. Massagué. 1987. Cell adhesion protein receptors as targets for transforming growth factor-ß action. Cell 51:189.[Medline]
  20. Hasselbalch, H., P. Junker, I. Lisse, U. Lindqvist, A. E. Laurent. 1991. Circulating hyaluronan in the myelofibrosis/osteomyelosclerosis syndrome and other myeloproliferative disorders. Am. J. Hematol. 36:1.[Medline]
  21. Hasselbalch, H., P. Junker. 1994. Serum laminin P1 in idiopathic myelofibrosis and related diseases. Leuk. Res. 18:623.[Medline]
  22. Taipale, J., J. Keski-Oja. 1997. Growth factors in the extracellular matrix. FASEB J. 11:51.[Abstract]
  23. Kaminski, N., J. D. Allard, J. F. Pittet, F. Zuo, M. J. D. Griffiths, D. Morris, X. Huang, D. Sheppard, R. A. Heller. 2000. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proc. Natl. Acad. Sci. USA 97:1778.[Abstract/Free Full Text]
  24. Bosque, F., Y. Belkaid, E. Briend, B. Hevin, M. Lebastard, N. Soussi, G. Milon. 1997. The biology of macrophages. Pathol. Biol. 45:103.[Medline]
  25. Thiele, J., C. Braechel, S. Wagner, B. Falini, D. Dienenmann, H. Stein, R. Fischer. 1992. Macrophages in normal human bone marrow and in chronic myeloproliferative disorders: an immunohistochemical and morphometric study by a new monoclonal antibody (PG-M1) on trephine biopsies. Virchows Arch. A Pathol. Anat. Histopathol. 421:33.[Medline]
  26. Titius, B. R., J. Thiele, H. Schaefer, H. Kreipe, R. Fischer. 1994. Ki-S1 and proliferating cell nuclear antigen expression of bone marrow macrophages. Acta Haematol. 91:144.[Medline]
  27. Perez, J. R., K. A. Higgins-Sochaski, J. Y. Maltese, R. Narayanan. 1994. Regulation of adhesion and growth of fibrosarcoma cells by NF-{kappa}B Rel A involves transforming growth factor ß. Mol. Cell. Biol. 14:5326.[Abstract/Free Full Text]
  28. Narayanan, R., K. A. Higgins, J. R. Perez, T. A. Coleman, C. A. Rosen. 1993. Evidence for differential functions of the p50 and p65 subunits of NF-{kappa}B with a cell adhesion model. Mol. Cell. Biol. 13:3802.[Abstract/Free Full Text]
  29. Kopp, E. B., S. Ghosh. 1995. NF-?B and rel proteins in innate immunity. Adv. Immunol. 58:1.[Medline]
  30. Barnes, P. J., M. Karin. 1997. Nuclear factor-{kappa}B, a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336:1066.[Free Full Text]
  31. Jr Baldwin, A. S.. 1996. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
  32. Sokoloski, J. A., A. C. Sartorelli, C. A. Rosen, R. Narayanan. 1993. Antisense oligonucleotides to the p65 subunit of NF-{kappa}B block CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes. Blood 82:625.[Abstract/Free Full Text]
  33. Baeuerle, P. A.. 1998. Pro-inflammatory signaling: last pieces in the NF-{kappa}B puzzle?. Curr. Biol. 8:R19.[Medline]
  34. Stancovski, I., D. Baltimore. 1997. NF-{kappa}B activation: the I{kappa}B kinase revealed?. Cell 91:299.[Medline]
  35. Stevenson, M. A., M.-J. Zhao, A. Asea, C. N. Coleman, S. K. Calderwood. 1999. Salicylic acid and aspirin inhibit the activity of RSK2 kinase and repress RSK2-dependent transcription of cyclic AMP response element binding protein- and NF-{kappa}B-responsive genes. J. Immunol. 163:5608.[Abstract/Free Full Text]
  36. Yin, M-J., Y. Yamamoto, R. B. Gaynor. 1998. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I{kappa}B kinase-ß. Nature 396:77.[Medline]
  37. Perkins, N. D., N. L. Edwards, C. S. Duckett, A. B. Agranoff, R. M. Schmid, G. J. Nabel. 1993. A cooperative interaction between NF-{kappa}B and Sp1 is required for HIV-1 enhancer activation. EMBO J. 12:3551.[Medline]
  38. Sarmiento, U. M., J. R. Perez, J. M. Becker, R. Narayanan. 1994. In vivo toxicological effects of rel A antisense phosphorothioates in CD-1 mice. Antisense Res. Dev. 4:99.[Medline]
  39. 39. Rameshwar, P., P. Gascon. 1992. Release of interleukin-1 and interleukin-6 from human monocytes by antithymocyte globulin: requirement for de novo synthesis. Blood 80:2531.[Abstract/Free Full Text]
  40. Kunsch, C., S. M. Ruben, C. A. Rosen. 1992. Selection of optimal {kappa}B/Rel DNA-binding motifs: interaction of both subunits of NF-{kappa}B with DNA is required for transcriptional activation. Mol. Cell. Biol. 12:4412.[Abstract/Free Full Text]
  41. Khaled, Z., L. Benimetskaya, R. Zeltser, T. Khan, H. W. Sharma, R. Narayanan, C. A. Stein. 1996. Multiple mechanisms may contribute to the cellular anti-adhesive effects of phosphorothioate oligodeoxynuceotides. Nucleic Acids Res. 24:737.[Abstract/Free Full Text]
  42. Sims, J. E., M. A. Gayle, J. C. Slack, M. R. Alderson, T. A. Birol, J. G. Giri, F. Collotta, F. Re, A. Mantovani, K. Shanebeck. 1993. Interleukin-1 signaling occurs exclusively via type I receptor. Proc. Natl. Acad. Sci. USA 90:6155.[Abstract/Free Full Text]
  43. Emmerich, F., M. Meiser, M. Hummel, G. Dernel, H. D. Foss, F. Jundt, S. Mathas, D. Krappmann, C. Scheidereit, H. Stein, B. Dorken. 1999. Overexpression of I{kappa}B{alpha} without inhibition of NF-{kappa}B activity and mutations in the I{kappa}B{alpha} gene in Reed-Sternberg cells. Blood 94:3129.[Abstract/Free Full Text]
  44. Kim, S. J., K. T. Jeang, A. B. Glick, M. B. Sporn, A. B. Roberts. 1989. Promoter sequences of the human transforming growth factor-ß1 autoinduction. J. Biol. Chem. 264:7041.[Abstract/Free Full Text]
  45. Lederer, J. A., J. S. Liou, S. Kim, N. Rice, A. H. Lichtman. 1996. Regulation of NF-{kappa}B activation in T helper 1 and T helper 2 cells. J. Immunol. 156:56.[Abstract]
  46. Chang, N.-S.. 2000. TGF-ß-induced matrix proteins inhibit p2/44 MAPK and JNK activation and suppress TNF-mediated I{kappa}B{alpha} degradation and NF-{kappa}B nuclear translocation in L929 fibroblasts. Biochem. Biophy. Res. Commun. 267:194.[Medline]
  47. Sun, S. C., P. A. Ganchi, D. W. Ballard, W. C. Green. 1993. NF-{kappa}B controls expression of inhibitor I{kappa}Ba: evidence for an inducible autoregulatory pathway. Science 259:1912.[Abstract/Free Full Text]
  48. Bonizzi, G., J. Piette, M. P. Merville, V. Bours. 1997. Distinct signal transduction pathways mediate nuclear factor-{kappa}B induction by IL-1ß in epithelial and lymphoid cells. J. Immunol. 159:5264.[Abstract]
  49. Taylor, M. L., P. W. Noble, B. White, R. Wise, M. C. Liu, B. S. Bochner. 2000. Extensive surface phenotyping of alveolar macrophages in interstitial lung disease. Clin. Immunol. 94:33.[Medline]
  50. Taksin, A. L., J. P. LeCouedic, I. Dusanter-Fourt, A. Masse, S. Giraudier, A. Katz, F. Wendling, W. Vainchenker, N. Casadevall, N. Debili. 1999. Autonomous megakaryocyte growth in essential thrombocythemia and idiopathic autocrine stimulation by Mpl-L. Blood 93:125.[Abstract/Free Full Text]
  51. Sortechini, A. R., S. Rupoli, G. Piccinini, M. M. Luchetti, I. Cantori, A. Gabrielli, P. Leoni. 1999. Expression of c-myb and B-myb oncogenes on myelofibrotic marrow fibroblasts. Leuk. Lymphoma 33:295.[Medline]
  52. Guardiola, P., H. Esperou, D. Cazals-Hatem, N. Ifrah, J. P. Jouet, A. Buzyn, L. Sutton, N. Gratecos, H. Tilly, B. Lioure, E. Gluckman. 1997. Allogeneic bone marrow transplantation for agnogenic myeloid metaplasia. Br. J. Haematol. 98:1004.[Medline]
  53. Anderson, J. E., G. Sale, F. R. Appelbaum, T. R. Chauncey, R. Storb. 1997. Allogeneic marrow transplantation for primary myelofibrosis and myelofibrosis secondary to polycythaemia vera or essential thrombocytosis. Br. J. Haematol. 98:1010.[Medline]
  54. Harris, C. E., J. C. Biggs, A. J. Concannon, A. J. Dodds. 1990. Peripheral blood and bone marrow findings in patients with acquired immunodeficiency syndrome. Pathology 22:206.[Medline]



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