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(Journal of Leukocyte Biology. 2002;72:347-352.)
© 2002 by Society for Leukocyte Biology

Potent induction of activin A secretion from monocytes and bone marrow stromal fibroblasts by cognate interaction with activated T cells

Masahiro Abe*, Yasumi Shintani*, Yuzuru Eto{dagger}, Kazuyo Harada*, Masaaki Kosaka* and Toshio Matsumoto*

First Department of Internal Medicine, School of Medicine, University of Tokushima, Japan; and
{dagger} Ajinomoto Central Research Institute, Kanagawa, Japan

Correspondence: Masahiro Abe, First Department of Internal Medicine, School of Medicine, University of Tokushima, 3-18-15, Kuramoto, Tokushima, 770-8503, Japan. E-mail: masabe{at}clin.med.tokushima-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activin A is a multifunctional cytokine essential for cell differentiation and apoptosis including erythroid cell differentiation in the bone marrow. In addition, activin A is induced by inflammation and exerts anti-inflammatory effects. However, the mechanism of activin A induction is still unclear, especially by inflammatory processes. Here we show that activin A secretion from monocytes and bone marrow stromal fibroblasts, its major sources in the bone marrow, is markedly enhanced by cognate interaction with activated T cells. This process is mediated by CD40/CD40 ligand interaction as well as concomitantly secreted T cell-derived cytokines, granulocyte macrophage-colony stimulating factor, and interferon-{gamma}. Furthermore, stromal fibroblasts as well as monocytes provide a costimulatory signal to anti-CD3-treated T cells via CD80 and CD86 to maintain the enhanced activin A production. These findings suggest that activin A is potently induced in the bone marrow and may play a role in the suppression of inflammatory or immune processes.

Key Words: CD40 • CD28 • costimulation • GM-CSF • IFN-{gamma}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activin A has been shown to exert a wide range of bioactivities for fundamental physiologic phenomena including embryonic development, cell differentiation, and apoptosis in various tissues. Activin A was originally reported as a protein that enhances the release of follicle-stimulating hormone from the pituitary [1 , 2 ]. It is a covalently linked homodimer of the inhibinß A chain and belongs to the transforming growth factor-ß (TGF-ß) superfamily [3 ]. Subsequent molecular genetic studies showed that activin A is identical to erythroid differentiation factor defined as a protein that induces the differentiation of mouse Friend cells and human K562 cells into hemoglobin-containing cells [4 ]. Its biological activities are neutralized by an activin binding protein, follistatin (FS) [5 , 6 ]. FS has been found in a variety of tissues [7 , 8 ] and acts as a local modulator for activin function [9 ]. Bone marrow tissues express mRNA of the ßA chain and FS [7 ]. Among the bone marrow cells, stromal cells and monocytes are a major source of activin A production [10 11 12 13 ]. Therefore, activin A is secreted and plays as a local regulator for hematopoiesis in the bone marrow milieu.

Several lines of evidence have suggested a role for activin A in inflammatory responses. Activin A expression is strongly induced in the skin after cutaneous injury [14 ], in the gut of patients with inflammatory bowel diseases [15 ], and in the synovial tissue of patients with inflammatory arthropathies [16 ]. Furthermore, activin A suppresses major interleukin-6 (IL-6)-induced inflammatory processes, namely B cell proliferation, phagocytic activities of monocytic cells, and fibrinogen production in HepG2 cells [16 ]. Activin A also inhibits the production of IL-1ß and enhances that of IL-1 receptor antagonist by monocytes [17 ]. These findings suggest that activin A is induced in inflammatory processes and provides anti-inflammatory effects.

Although the importance of this protein has been increasingly emphasized in relation to inflammation as well as hematopoiesis, the regulatory mechanism of activin A and FS secretion has not been fully clarified. T cells in the bone marrow have been demonstrated as being activated in various pathological conditions including autoimmune diseases such as rheumatoid arthritis [18 ], anemia of chronic disorder [19 ], viral infections [20 ], and bone marrow failures such as aplastic anemia [21 ]. The activated T cells in these clinical settings are considered as arising in the bone marrow rather than migrating into the bone marrow, because they are preferentially localized in the bone marrow but not in the peripheral blood or circulation [18 , 19 , 21 , 22 ]. More recently, CD40 ligand expressed on activated T cells has been suggested to enhance the production of hematopoietic growth factors in the bone marrow [23 ]. Therefore, the present study was aimed at defining the mechanism of activin A induction by T cell interaction in monocytes and bone marrow stromal fibroblasts, major sources of this protein in the bone marrow. We found that activin A is markedly induced in monocytes as well as stromal fibroblasts by a cognate interaction with activated T cells, and the induction of activin A is mediated by the interaction of CD40 with CD40 ligand and by concomitantly secreted T cell-derived cytokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Recombinant human (rh)IL-1ß, rhIL-2, and rh-interferon-{gamma} (IFN-{gamma}) were provided by Otsuka Pharmaceutical Co. (Tokushima, Japan). Specific activity of rhIL-1ß and rhIFN-{gamma} was defined as 2 x 108 U/mg by the cell proliferation assay for D 10 cells and 2 x 107 U/mg by the inhibition assay for the infection to WISH cells by vesicular stomatitis virus, respectively. The following reagents were purchased from the indicated manufacturers: rh IL-2, rh IL-3, rh IL-4, rh-granulocyte macrophage-colony stimulating factor (GM-CSF), and rhM-CSF, from Genzyme Techne (Cambridge, MA); lipopolysaccharide (LPS) and phytohemoagglutinin (PHA), from Sigma Chemical Co. (St. Louis, MO); mouse anti-human CD3, CD4, CD5, CD8, CD11b, CD14, CD19, and CD33 monoclonal antibodies (mAb) from Nichirei (Tokyo, Japan); neutralizing mouse anti-human IL-1ß, IFN-{gamma}, GM-CSF, CD80 (clone 12G5), and CD86 (clone 1G10) mAb, from Genzyme Techne, and CD40 ligand mAb (clone M90), from Serotec (Oxford, England); mouse anti-human CD28, CD80 (clone BB-1), and CD86 (clone BU63) and fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD28 and CD40 mAb, from Serotec; and mouse anti-human CD40 (clone 5C3) and CD40 ligand (clone TRAP1), FITC-conjugated mouse anti-human CD80 and CD86, phycoerythrin-conjugated mouse anti-human CD40 ligand mAb, and mouse immunoglobulin (Ig) isotype controls, from Pharmingen (San Diego, CA). Rabbit anti-human activin A and FS polyclonal antibodies and mouse anti-human FS mAb were raised in our laboratory as previously described [24 , 25 ].

Cell preparations and cultures
Peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC) were isolated by Ficoll-Hypaque density-gradient centrifugation (Pharmasia LKB Biotechnology, Upsala, Sweden) from heparinized blood drawn from healthy volunteers after informed consent had been received and were used immediately. All procedures involving human specimens were performed according to the protocol approved by the institutional review board for human protection. Monocytes were obtained from PBMC by removal of nonadherent cells and subsequent depletion of T and B cells using Dynabeads M-450 CD4, CD8, and CD19 (Dynal, Great Neck, NY). Adherent cells were prepared according to the adherence technique as previously described [26 ]. T cells were obtained by immunomagnetical depletion of monocytes, and B cells from nonadherent PBMC and CD4+ cells were further isolated by positive selection. BMMC were resuspended in 75 cm2 tissue flasks in Iscove’s modified Dulbecco’s medium supplemented with 12.5% fetal calf serum (BioWhittaker, Walkersville, MD), 12.5% horse serum (BioWhittaker), 50 U/ml penicillin, and 50 µg/ml streptomycin (Gibco-BRL, Grand Island, NY), after depleting monocytes and myeloid cells by incubation with anti-CD11b, CD14, and CD33 mAb and subsequent addition of Dynabeads M-450 goat anti-mouse IgG (Dynal), according to the manufacturer’s instructions. At weekly intervals, cultures were fed by replacing culture medium. The adherent cells were serially passed at confluency using 0.05% trypsin/0.53 mM ethylenediaminetetraacetate (Gibco-BRL) to achieve a homogeneous population of spindle-shaped cells. These cells were defined to be fibroblasts by their expression of the parenchymal cell antigens including vimentin and collagen types I and III, but neither endothelial cell antigens including collagen type IV and factor VIII antigen nor the hematopoietic cell antigens of various cell lineages. The cells were further passed at 2 x 105 cells/ml onto 24-well culture plates. After being expanded, the culture medium was changed to a serum-free medium containing a 1:1 mixture of Ham’s F-12 medium and Dulbecco’s modified Eagle’s medium (GIT medium, Wako Pure Chemicals, Osaka, Japan). The surface expression of CD28, CD40, CD40 ligand, CD80, and CD86 was analyzed on T cells, monocytes, and stromal fibroblasts by flow cytometry (FCM) using EPICS-Profile (Coulter Electronics, Hialeah, FL).

Monocytes and stromal fibroblasts were cultured in serum-free medium with addition of various cytokines and LPS. Various combinations among monocytes, T cells, and stromal fibroblasts were cocultured. Culture supernatants (CSNs) were harvested after 2 days. Nonadherent cocultures were performed using a transmembrane filter (Intercell TP, Kurabo, Osaka, Japan) to avoid a cell-to-cell contact to activated T cells. For the preparation of activated T cells, T cells were incubated for 2 days with 10 µg/ml PHA or in culture plates coated with 5 µg/ml anti-CD3 mAb. Stimulation with PHA and anti-CD3 mAb induced CD40 ligand expression on CD4+ T cells at 37.1% and 11.1%, respectively, and less than 3% was observed on quiescent CD4+ and CD4- T cells.

Activin A and FS measurement
Immunoreactive activin A protein in CSNs was measured with radioimmunoassay as described previously [18 ]. Briefly, CSNs or standards were incubated in tubes with rabbit anti-human activin A polyclonal antibody before adding 125I-labeled activin A. After overnight incubation of the mixtures, goat anti-rabbit IgG was added. The tubes were centrifuged at 2000 g for 30 min at 4 °C, followed by decanting supernatants. Radioactivity was counted in a well-type {gamma} counter. Immunoreactive FS in CSNs was measured using an enzyme-linked immunosorbent assay (ELISA) with some modifications of our previously described method [19 ]. In brief, mouse anti-human FS mAb was used to coat round-bottomed, 96-well ELISA plates before loading samples or standards. Biotin-labeled goat anti-rabbit IgG and an avidin-biotin peroxidase system (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) were used to detect the second-stage rabbit anti-human FS polyclonal antibody. The detection limit of this assay was 0.1 ng/ml. Intra- and interassay coefficient of variations were 3.4% and 8.7%, respectively.

CD40 ligand transfection
The human CD40 ligand cDNA was generated by reverse transcriptase-polymerase chain reaction from PHA-activated T cells and was subsequently subcloned by an original TA-cloning kit (Invitrogen, Carlsbad, CA). The two primers used to generate a full-length CD40 ligand cDNA were 5'-AAAGCGGCCGCCACCATGATCGAAACATACAACC-3', containing a Kozak consensus sequence and NotI restriction site, and 5'-AAAGCGGCCGCTCATCAGAGTTTGAGTAAGCCAAA-3', containing a NotI restriction site. The human CD40 ligand cDNA was sequenced and digested with restriction endonuclease NotI. The resulting DNA fragment was subcloned under the cytomegalovirus (CMV) enhancer-promoter of mammalian expression vector, pTracer-CMV (Invitrogen). COS-7 cells were transfected with CD40 ligand cDNA-containing plasmid expression vectors or control vectors using Lipofectamin Reagent (Gibco-BRL). CD40 ligand expression on the cells was analyzed in FCM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PHA-activated T cells on activin A secretion from monocytes
Monocytes constitutively secreted activin A but not FS. IL-1ß, INF-{gamma}, and GM-CSF increased activin A production three-, three-, and fivefold, respectively (Fig. 1 ), and IL-2, IL-3, IL-4, and M-CSF showed no effect on activin A production. LPS potently increased activin A secretion sevenfold (Fig. 1) . As activin A production by monocytes was enhanced by activated T cell-derived cytokines such as GM-CSF and IFN-{gamma}, we next investigated the cellular interaction with activated T cells on activin A secretion from monocytes. PHA-activated T cells secreted neither activin A nor FS. However, the activated T cells augmented activin A secretion from monocytes by tenfold while quiescent T cells enhanced only twofold (Fig. 2 ). Blockade of interaction via CD40 ligand by a specific mAb diminished the enhancement of activin A secretion to two-thirds. As expected, neutralization of GM-CSF or IFN-{gamma} partially reduced the increase of activin A secretion. Blocking CD40-CD40 ligand interaction together with neutralizing GM-CSF and IFN-{gamma} mostly abrogated the activated T cell-mediated enhancement. When the adhesion between activated T cells and monocytes was prevented, the increase in activin A production was reduced to fivefold. Neutralization of GM-CSF and IFN-{gamma} in this culture condition further reduced the enhanced activin A secretion. These findings indicate that the up-regulation of activin A secretion from monocytes by activated T cells is largely mediated by their interaction via the CD40 ligand and T cell-derived cytokines, GM-CSF, and IFN-{gamma}.



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  		Figure 1. Effect of cytokines on activin A production from monocytes. The cells (4x105 cells/ml) were cultured in triplicate for 2 days alone or with 100 U/ml IL-1ß, 100 U/ml GM-CSF, 1000 U/ml IFN-{gamma}, or 10 µg/ml LPS. Activin A levels in the CSNs were measured. Results are expressed as ng/ml (mean±SD, n=4). *, Significantly different from the values in nonstimulated cultures according to the Mann-Whitney U test (P<0.05).

 


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  		Figure 2. Effect of T cells on activin A production from monocytes. Monocytes (M) were cocultured in triplicate for 2 days with autologous quiescent (qT) or PHA-activated T cells (aT; final concentrations of monocytes and T cells, 4x105 and 1x106 cells /ml, respectively). To prevent cell-to-cell contact between monocytes and activated T cells, a transmembrane filter (IC) was used. Anti-CD40 ligand ({alpha}CD40L; 20 µg/ml), anti-GM-CSF ({alpha}GM), or anti-IFN-{gamma} mAb ({alpha}IFN) alone or in combination were added in the indicated cultures. Activin A levels in the CSNs were measured. Results are expressed as ng/ml (mean±SD, n=4). *, Significantly different from the values cocultured with activated T cells (P<0.05); **, significantly different (P<0.05).

 
Activin A and FS production in the cocultures of stromal fibroblasts and monocytes with T cells
Bone marrow stromal fibroblasts constitutively secreted activin A and FS. PHA-activated T cells increased activin A and FS secretion from stromal fibroblasts, three- and twofold, respectively, compared with the levels of these proteins secreted from stromal fibroblasts alone (Fig. 3 ). As shown in Figure 3 , activin A was potently secreted from monocytes in cocultures with the activated T cells. The levels of activin A secreted from monocytes were much higher than those from stromal fibroblasts under the stimulation by the activated T cells.



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  		Figure 3. Activin A and FS production in the cocultures of stromal fibroblasts and monocytes with T cells. Confluent, autologous stromal fibroblasts (ST) and monocytes (M) were cocultured in triplicate for 2 days with quiescent (qT) or PHA-activated T cells (aT; final concentrations of monocytes and T cells, 4x105 and 1x106 cells/ml, respectively). To prevent a cell-to-cell contact of monocytes and stromal fibroblasts with activated T cells, a transmembrane filter (IC) was used. Anti-CD40 ligand ({alpha}CD40L) or control mouse IgG was added at 20 µg/ml in the indicated cultures. Activin A and FS levels in CSNs were measured. The data are representative of three independent experiments. Results are expressed as ng/ml (mean±SD, n=4). *, Significant difference according to the Mann-Whitney U test (P<0.05).

 
Cocultures of stromal fibroblasts and monocytes with PHA-activated T cells induced a 23- and 2-fold increase in activin A and FS production, respectively, compared with those with quiescent T cells (Fig. 3) . This activin A induction was, however, substantially decreased to one-half by preventing adhesion of the activated T cells with a membrane filter. Blockade of interaction via CD40 ligand with a specific mAb also inhibited the enhanced activin A production, and control IgG showed no effect. These findings suggest that the CD40 ligand-mediated interaction plays a crucial role in this marked enhancement of activin A production by activated T cells.

Effect of cross-linking CD40 on activin A and FS production by monocytes and stromal fibroblasts
CD40 was found to be constitutively expressed on the surface of bone marrow stromal fibroblasts (Fig. 4 ) as reported on dermal, synovial, and lung fibroblasts [27 , 28 ]. Cross-linking CD 40 molecules with anti-CD40 mAb or CD40 ligand-transfected COS-7 cells augmented activin A production from monocytes and stromal fibroblasts and suppressed FS production from stromal fibroblasts (Fig. 5 ), which confirmed a crucial role for the CD40 ligand-CD40 interaction in the modulation of activin A and FS production from monocytes and stromal fibroblasts.



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  		Figure 4. Surface expression of CD40 and CD80 on bone marrow stromal fibroblasts. Cultured stromal fibroblasts obtained from normal donors were stained with FITC-labeled anti-CD40 or anti-CD80 mAb. The results were representative of four different flow cytometric analyses. NC, Negative control.

 


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  		Figure 5. Effect of CD40 cross-linking on activin A and FS secretion from monocytes and stromal fibroblasts. Monocytes (M; 4x105 cells/ml) or confluent stromal fibroblasts (ST) were cultured in the presence of 10 µg/ml anti-CD40 mAb ({alpha}CD40; a, b) or were cocultured with 4x105/ml CD40 ligand-transfected or control COS-7 cells (c, d). Activin A and FS levels in CSNs were measured at day 2. Results are expressed as ng/ml (mean±SD, n=4). *, Significantly different from the values in control cultures according to the Mann-Whitney U test (P<0.05).

 
Effect of anti-CD3 mAb-treated T cells on activin A and FS secretion
Activation of antigen-specific T cells requires costimulatory signals alone with a stimulation through T cell receptor. Monocytes constitutively express CD80 and CD86, which provide costimulatory signals for T cell activation via CD28, a receptor for CD80/CD86 [29 , 30 ]. To clarify a role of the costimulatory signals, monocytes were cocultured with autologous T cells treated with anti-CD3 mAb. Activin A secretion from monocytes was enhanced in the cocultures compared with the cultures of monocytes alone (Fig. 6 ). Addition of anti-CD80 and anti-CD86 mAb in combination reduced the enhanced activin A secretion in the cocultures. Furthermore, we observed that stromal fibroblasts expressed CD80 constitutively but not CD86 in flow cytometry (Fig. 4) , which agreed with the recent report regarding their expression on murine fibroblasts [31 ]. Activin A and FS secretion from stromal fibroblasts were increased in the cocultures with anti-CD3 mAb-treated T cells compared with the cultures of stromal fibroblasts alone (Fig. 6) . Anti-CD80 and anti-CD86 mAb in combination reduced activin A secretion from stromal fibroblasts in the cocultures and increased FS secretion. These results strongly suggest that the interaction of antigen-specific T cells through costimulatory molecules, CD80 and CD86, as well as the CD40 ligand plays crucial roles in the stimulation of activin A secretion from monocytes and stromal fibroblasts as well as the suppression of FS secretion from stromal fibroblasts.



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  		Figure 6. Effect of anti-CD80 and anti-CD86 antibodies on activin A and FS induction by activated T cells. Monocytes (4x105 cells/ml; a) or confluent stromal fibroblasts (b) were cocultured with anti-CD3 ({alpha}CD3)-treated T cells (4x105 cells/ml) in the presence of 10 µg/ml anti-CD80 and anti-CD86 mAb in combination ({alpha} CD80+86). *, Significantly different according to the Mann-Whitney U test (P<0.05).

 
Reconstitution assays
To investigate the role of CD40 ligand in combination with GM-CSF and IFN-{gamma}, monocytes (4x105/ml) were cocultured with CD40 ligand-transfected COS-7 cells (4x105/ml) in the presence of 100 U/ml GM-CSF and 1000 U/ml IFN-{gamma}. Activin A secretion from monocytes was potently enhanced by addition of GM-CSF and IFN-{gamma} to CD40 ligand-transfected COS-7 cells. However, the levels of activin A in the CSNs were 70–80% of those obtained by cocultures with PHA-activated T cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cytokine bioactivity is dynamically regulated during pathophysiological processes by a network of interacting cells and cell-derived biomolecules. Our present study demonstrates that activin A secretion from monocytes and stromal fibroblasts is markedly enhanced by cognate interaction of activated T cells in which CD40-CD40 ligand interaction plays a critical role. Monocytes and stromal fibroblasts appear to be among predominant sources of activin A in the bone marrow in activated T cell-mediated inflammatory processes.

The natural CD40 ligand is a type 2 transmembrane glycoprotein predominantly expressed on activated T cells and stimulates CD40. The CD40-mediated stimulation by cognate interaction with activated T cells exerts potent biological activities such as cytokine production, expression of adhesion molecules, and cell proliferation in other cell types as well as B cells. The present results on CD40 cross-linking by anti-CD40 antibody or CD40 ligand transfectants demonstrate that CD40 activation alone is sufficient to enhance activin A secretion from monocytes and stromal fibroblasts and suppress the secretion of its inhibitor, FS, from stromal fibroblasts. In addition, concomitantly secreted, activated T cell-derived cytokines such as GM-CSF and IFN-{gamma} further enhance activin A secretion from monocytes. These results suggest that the skewed activin A and FS secretion is associated with activated T cell-mediated inflammation such as infection or immune disorders.

Signaling via CD40 ultimately leads to nuclear factor (NF)-{kappa}B activation in monocytes and fibroblasts [32 , 33 ]. CD40 signaling has also been shown to induce activating protein (AP)-1 in B cells [34 , 35 ]. As an AP-1 binding site at the 3'-flanking sequence is considered to be important in activinß A chain gene expression [36 ], CD40-mediated activin A up-regulation may involve AP-1 as well as NF-{kappa}B activation in monocytes and stromal fibroblasts.

T cells activated by antigen-presenting cells (APC), namely antigen-specific T cells, play a major role in pathological settings such as infection and immunological disorders. Costimulatory signals to antigen-specific T cells are required for their proliferation and cytokine production [37 38 39 ]. Signaling via CD28, a receptor for CD80 or CD86, is considered the most important among the costimulatory signals for antigen-specific T cell activation. Monocytes express CD80 and CD86 [29 , 30 ]. We found that human bone marrow stromal fibroblasts also constitutively expressed CD80. Therefore, monocytes and stromal fibroblasts can provide the costimulatory signal to antigen-specific T cells via CD28. Indeed, this signal was required to maintain the enhanced activin A production from monocytes and stromal fibroblasts by anti-CD3-stimulated T cells. Furthermore, stimulation of CD40 on APC is known to enhance a requisite costimulatory pathway, which includes CD80 and CD86, resulting in further activation of antigen-specific T cells [40 , 41 ]. A contribution of CD80/86 is also suggested by the reconstitution assays. Activin A secretion from monocytes was potently enhanced by addition of GM-CSF and IFN-{gamma} to CD40 ligand-transfected COS-7 cells in which CD80/86-mediated costimulation was not involved. This culture condition, however, did not completely reproduce the effect of activated T cells. This shortage was assumed at least partly by a lack of the costimulatory signal in the T cells. Therefore, CD40-mediated stimulation by antigen-specific T cells is suggested to form a positive feedback circuit to increase activin A production from monocytes and stromal fibroblasts.

Activin A is a molecule with pleiotropic functions. In addition to its defined role in erythroid cell differentiation in bone marrow, activin A has been shown to exert anti-inflammatory effects [14 15 16 17 ]. We demonstrate that activin A secretion is potently up-regulated in monocytes as well as stromal fibroblasts by cognate interaction with activated T cells. Taken together, activin A is suggested to be induced by activated T cells in the bone marrow milieu and plays a functional role in the suppression of inflammation as well as in hematopoiesis. A clinical relevance of activin A in inflammatory processes remains to be clarified.


   ACKNOWLEDGEMENTS
 
We are indebted to Mr. Yoshihito Okamura for flow cytometry and grateful to Dr. Yuji Wakahara for generating CD40 ligand transfectants.

Received July 28, 2001; revised March 17, 2002; accepted March 29, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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