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(Journal of Leukocyte Biology. 2002;71:987-995.)
© 2002 by Society for Leukocyte Biology

Bone marrow CD34+ progenitor cells stimulated with stem cell factor and GM-CSF have the capacity to activate IgD- B cells through direct cellular interaction

Shunsei Hirohata*, Tamiko Yanagida*, Tetsuya Tomita{dagger}, Hideki Yoshikawa{dagger} and Takahiro Ochi{dagger}

* Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan; and
{dagger} Department of Orthopedic Surgery, Osaka University Medical School, Japan

Correspondence: Shunsei Hirohata, M.D., Department of Internal Medicine, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan. E-mail: shunsei{at}med.teikyo-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have suggested the involvement of bone marrow in the pathogenesis of rheumatoid arthritis (RA), in which proliferation of monocyte-lineage cells (MLC) as well as local B cell activation in the synovium play an important role. Here, we show that bone marrow-derived MLC have the capacity to activate human peripheral blood IgD- B cells. Bone marrow CD34+ cells from RA patients that had been stimulated with stem cell factor and GM-CSF for 3–4 weeks (>90% CD14+ HLA-DR+ cells, <0.5% CD19+ B cells, and <0.5% CD3+ T cells; MLC) induced the production of IgG much more effectively than that of IgM by highly purified B cells from healthy donors in the presence of IL-2 and IL-10. CD34+ cells from cord blood or from bone marrow of osteoarthritis patients also displayed the capacity to induce IgG production. The induction of IgG production by the bone marrow-derived MLC was markedly decreased when they were separated from B cells by a membrane filter. The bone marrow-derived MLC interacted preferentially with IgD- B cells to induce IgG production. These results indicate that upon stimulation with stem cell factor and GM-CSF, CD34+ progenitor cells differentiate into MLC that activate preferentially IgD- B cells through direct cellular interactions to produce IgG. Therefore, the data suggest that the accelerated recruitment of MLC from the bone marrow to the synovium might play a role in the local B cell activation in RA.

Key Words: human • B lymphocytes • cell-to-cell interactions


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rheumatoid arthritis (RA) is characterized by chronic inflammation with infiltration of a variety of inflammatory cells, such as those of myeloid origin as well as T and B lymphocytes into the synovium. One of the characteristic features in RA is local B cell activation, leading to the production of large amounts of immunoglobulin (Ig) and autoantibodies [1 ]. Previous studies have suggested that extralymphoid B cell activation in the rheumatoid synovium may be promoted by fibroblast-like synoviocytes. Thus, B lymphocytes, some of which expressed proliferating cell nuclear antigen, were shown to be in intimate contact with synoviocytes in the subliming layers of the rheumatoid synovium [2 ], suggesting that such direct interactions might be important in promoting B cell responses in the rheumatoid synovium. Moreover, synoviocytes have been shown to support the terminal differentiation of activated B cells into Ig-secreting plasma cells [3 ] as well as the survival of B cells [4 ]. These results suggest a role for synoviocytes in facilitating local B cell responses in RA synovium.

It has been well known that synovial lining cells consist of macrophage-like type A synoviocytes and fibroblast-like type B synoviocytes. Recent studies have suggested that type A synoviocytes are derived from monocyte precursors in the bone marrow [5 ]. Moreover, it has been shown that the spontaneous generation of CD14+ monocyte-lineage cells (MLC) from bone marrow CD14- precursor cells is accelerated in RA, resulting in the facilitated entry of such CD14+ cells into the synovium [6 ]. More importantly, previous studies have disclosed that CD14+ cells derived from the bone marrow of RA patients have various influences on B cell activation. Thus, CD14+ cells generated from bone marrow CD14- precursors of RA patients have the capacity to stimulate the production of the- IgM rheumatoid factor selectively [7 ]. In addition, it has also been shown that CD14+ human leukocyte antigen (HLA)-DR+ cells generated from bone marrow CD34+ progenitor cells of RA patients have the capacity to support survival of B cells, leading to spontaneous transformation of Epstein-Barr virus positive B cell lines [8 ]. These results also suggest that bone marrow-derived synoviocytes might play a role in facilitating local B cell responses in the RA synovium. However, the precise mechanisms of B cell activation by bone marrow-derived cells have not been delineated completely.

The current studies were therefore undertaken in order to explore in detail the capacity of MLC induced by stimulation with stem cell factor (SCF) and granulocyte macrophage-colony stimulating factor (GM-CSF) from bone marrow CD34+ progenitor cells to activate and promote B cell responses. The results indicate that the GM-CSF-stimulated bone marrow CD34+ cells induce IgG production much more effectively than IgM production through direct interactions with IgD- B cells in the presence of interleukin (IL)-2 and IL-10. Therefore, the data support the conclusion that bone marrow-derived type A synoviocytes may also play a role in the local and systemic stimulation of memory B cell responses characteristic of RA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Bone marrow samples were obtained from 31 patients with active RA (4 males and 27 females; mean age, 59.7 years; range, 42–73 years) and from 11 patients with osteoarthritis (OA; 2 males and 9 females; mean age, 71.0 years; range, 57–81 years), who gave informed consent, during joint operations through intramedullary reaming by aspiration from a distal femoral canal prepared for implantation of the artificial femoral head or through aspiration from iliac crest. All 31 RA patients fulfilled the American College of Rheumatology (formerly the American Rheumatism Association) 1987 revised criteria for the disease [9 ]. Cord blood samples were obtained at nondiseased, normal deliveries upon informed consent for research use by parents.

Culture medium and reagents
RPMI-1640 medium (Life Technologies, Grand Island, NY), supplemented with penicillin G (100 units/ml), streptomycin (100 µg/ml), L-glutamine (0.3 mg/ml), and 10% fetal bovine serum (Life Technologies), was used for all cultures. Recombinant human GM-CSF, human IL-10, human SCF, and human BAFF [B cell activating factor belonging to the tumor necrosis factor (TNF) family] were purchased from PeproTech EC (London, UK). Recombinant human IL-2 (TGP-3) was a gift of Takeda Chemical Industries, Ltd. (Osaka, Japan), whose unit activity was determined by the providers (4.2x104 U/mg protein). A variety of monoclonal antibodies (mAb) were used, including anti-CD154 (a murine IgG1 mAb, clone 24–31; Ancell, Bayport, MN), anti-CD70 (a murine IgG1 mAb, clone BU69; Ancell), anti-CD106 (a murine IgG1 mAb, clone 1G11; Immunotech, Marseille, France), and a murine IgG1 control mAb MOPC 21 (Cappel Labs, West Chester, PA).

Preparation and culture of bone marrow CD34+ cells
Bone marrow or cord blood mononuclear cells were isolated by centrifugation of heparinized bone marrow aspirates over sodium diatrizoate-Ficoll gradients (Histopaque; Sigma Chemical Co., St. Louis, MO). CD34+ cells were purified from the mononuclear cells through positive selection using magnetic beads (Dynal CD34 progenitor cell-selection system; Dynal, Oslo, Norway). CD34+ cells thus prepared were approximately 95% CD34+ cells and <0.5% CD19+ B cells and were not found to have the altered phenotypic or functional features as described previously [10 ]. CD34+ cells were incubated in a 24-well microtiter plate with flat-bottomed wells (no. 3524, Costar, Cambridge, MA; 5.0–10.0x104/well) with the presence of SCF (10 ng/ml) and GM-CSF (1 ng/ml) for 3–4 weeks, at which time most of the cells become CD14+ HLA-DR+ [11 ]. RA bone marrow nurse-like cell clones (RA87 and RA91) were a generous gift of Dr. Ryuji Suzuki (Shionogi Co., Osaka, Japan).

Preparation of T cells and B cells
Peripheral blood mononuclear cells obtained from venous blood of healthy adult volunteers were depleted of monocytes and natural killer (NK) cells by treating them with 5 mM L-leucine methyl ester HCl (Sigma Chemical Co.) in serum-free RPMI 1640 (Life Technologies) [12 ]. Highly purified B cells and T cells were obtained from the treated cell population by rosetting with neuraminidase-treated sheep red blood cells, and the CD4+ T cell population was prepared further by a panning technique as described previously [13 ]. The B cells thus prepared contained <1% CD14+ monocytes, <1% CD2+ CD3+ T cells, <1% CD16+ NK cells, and >90% CD20+ B cells [14 ]. The CD4+ T cells contained <0.1% esterase-positive monocytes, <0.5% CD20+ B cells, <2% CD8+ cells, and >96% CD4+ T cells [14 , 15 ]. In some experiments, B cells were fractionated further into IgD+ B cells and IgD- B cells using Dynal CELLectionTM Pan Mouse IgG kit (Dynal) conjugated with anti-human IgD mAb (murine IgG1; Immunotech). IgD+ B cells thus prepared contained >90% IgD+ B cells. The characteristic features of the IgD+ B cells were comparable with those found in the previous study [16 ], as determined by stimulation with Staphylococcus aureus Cowan I and IL-2. Peripheral blood monocytes were prepared by glass dish adherence, as described previously [17 ].

Induction of in vitro production of IgM and IgG
Routine cultures were carried out in duplicate in a total volume of 200 µl in wells of a 96-well microtiter plate with round-bottomed wells (no. 3799, Costar). B cells (5.0–10.0x104/well) were cultured with or without autologous monocytes (5x104/well), autologous CD4+ T cells (1.0–2.0x105/well), or bone marrow CD34+ cells stimulated with SCF and GM-CSF (2.5–5.0x104/well) in the presence or absence of IL-2 (0.1 U/ml) and IL-10 (10 ng/ml). The cells were incubated routinely for 10 days at 37°C in a humidified atmosphere of 5% CO2 and 95% air. In some experiments, bone marrow CD34+ cells (1x105/well) were stimulated with SCF and GM-CSF in a 24-well microtiter plate for 3–4 weeks, after which culture supernatant was replaced with fresh medium, and fresh B cells (5x105/well) were added with culture inserts so that B cells and bone marrow-derived MLC might be allowed to be in contact or be separated from each other.

Measurement of IgM and IgG
Microtiter plates (Dynex, Chantilly, VA) coated with F(ab')2 fragments of goat anti-human IgM or anti-human IgG (Organon Teknika, Durham, NC) were incubated with cell-free culture supernatants or IgM or IgG standards in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (Miles, Elkhart, IN). Bound IgM or IgG was detected with peroxidase-conjugated F(ab')2 fragments of goat anti-human IgM or IgG (Organon Teknika) as described previously [18 ].

Immunofluorescence staining and analysis
Purified bone marrow CD34+ cells were expanded carefully with SCF (10 ng/ml) and GM-CSF (1 ng/ml) for 3–4 weeks in a 24-well microtiter plate with flat-bottomed wells (no. 3524, Costar; 1x105/well). After the incubation, the cells were stained with fluorescein isothiocyanate (FITC)-anti-CD19 mAb (mouse IgG1; Immunotech), FITC-anti-HLA-DR mAb (mouse IgG2b; Immunotech), phycoerythrin (PE)-conjugated anti-CD14 mAb (mouse IgG2a; Immunotech), PE-conjugated anti-CD3 mAb (mouse IgG1; Immunotech), or PE- or FITC-conjugated, isotype-matched control mAb (Dako, Glostrup, Denmark). Briefly, the cells were washed with 2% normal human serum in PBS, pH 7.2, and 0.1% sodium azide (staining buffer), and then the cells were stained with saturating concentrations of a variety of mAb at 4°C for 30 min. The cells were then washed three times with staining buffer and were fixed with 1% paraformaldehyde in PBS for at least 5 min at room temperature. The cells were analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL) equipped with an argon-ion laser at 488 nm. A combination of low-angle and 90° light-scatter measurements (forward-scatter vs. side-scatter) was used to generate a bit-map gating to identify bone marrow cells using CYTO-TROLTM control cells (Coulter) and Immuno-TrolTM cells (Coulter) as standards, as described previously [11 ]. The percentages of cells stained positively for each mAb were determined by integration of cells above a specified fluorescence channel calculated in relation to the staining with isotype-matched control mAb.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotypic analysis of bone marrow CD34+ cells stimulated with SCF and GM-CSF
Figure 1 shows the representative dual-parameter four-quadrant scattergrams of bone marrow CD34+ cells from an RA patient stimulated with SCF and GM-CSF for 3 weeks. At this time, the stimulated bone marrow cells did not express CD34 (unpublished results). More than 90% of the cells expressed CD14 and HLA-DR, whereas there were substantially no CD19+ cells or CD3+ cells (<0.5%) within these populations. Under microscopy, most of the stimulated bone marrow cells had the appearance of monocytes or dendritic cells with small numbers of fibroblast-like cells [11 ]. Bone marrow CD34+ cells from OA patients or cord blood CD34+ cells showed similar results (unpublished results). These results indicate that bone marrow CD34+ cells differentiate mostly into CD14+ HLA-DR+ MLC without contamination of CD3+ T cells or CD19+ B cells after stimulation with SCF and GM-CSF.



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  		Figure 1. Two-color flow cytometric analysis of the phenotypes of bone marrow CD34+ cells cultured in the presence of SCF and GM-CSF. CD34+ cells from the bone marrow of a RA patient (1x104/well) were cultured in the presence of SCF (10 ng/ml) and GM-CSF (1 ng/ml). After 3 weeks, the cells were harvested and stained with PE-conjugated anti-CD14, FITC-conjugated anti-HLA-DR, PE-conjugated anti-CD3, FITC-conjugated anti-CD19, and PE- or FITC-conjugated, isotype-matched control mAbs. The cells were then analyzed by flow cytometry.

 
The capacity of bone marrow-derived MLC to induce Ig production by peripheral blood B cells
The next experiments examined the capacity of bone marrow-derived MLC to induce Ig production of peripheral B cells from normal, healthy individuals. Highly purified B cells from normal individuals were cultured with or without bone marrow-derived MLC in the presence or absence of IL-2 and IL-10. As shown in Figure 2 , RA bone marrow-derived MLC induced the production of IgG modestly but not IgM in the presence of either IL-2 or IL-10. Of note, IL-2 and IL-10 synergistically enhanced IgG production induced by the bone marrow-derived MLC. Similar results were obtained when the bone marrow from the femoral head of an RA patient or that from the iliac crest of the same patient was used (unpublished results). Autologous peripheral blood monocytes also induced the production of IgG in the presence of IL-2 and IL-10 but less effectively than the bone marrow-derived MLC. The RA bone marrow-derived MLC or autologous monocytes alone stimulated by IL-2 and IL-10 could not produce IgG or IgM (unpublished results). These results indicate that RA bone marrow-derived MLC have the capacity to induce the production of IgG much more effectively than that of IgM by peripheral blood B cells. More importantly, the data indicate that the optimal induction of the IgG production by bone marrow-derived MLC requires the presence of IL-2 and IL-10. Furthermore, the data show that peripheral blood monocytes also retain the capacity to stimulate IgG production of B cells.



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  		Figure 2. MLC derived from RA bone marrow (BM) CD34+ cells induce IgG production of peripheral blood B cells in the presence of IL-2 and IL-10. B cells (1x105/well) were cultured with or without autologous monocytes (M{phi}) (5x104/well) or bone marrow CD34+ cells from an RA patient that had been stimulated with SCF (10 ng/ml) and GM-CSF (1 ng/ml) for 4 weeks (5x104/well). IL-2 (0.1 U/ml) and IL-10 (10 ng/ml) were added as indicated. After 10 days of incubation, the supernatants were harvested and were assayed for IgM and IgG contents by enzyme-linked immunosorbent assay (ELISA). The mean and SD values of the results of six independent experiments are shown.

 
CD34+ cells from cord blood or from bone marrow of patients with OA have the capacity to induce IgG production after stimulation with SCF and GM-CSF
The next experiments explored whether the capacity of bone marrow-derived MLC to induce the production of IgG might be specific for RA. CD34+ cells were similarly purified from cord blood or from the bone marrow of patients with OA and were stimulated with SCF and GM-CSF for 3–4 weeks. As shown in Figure 3 , cord blood-derived MLC or OA bone marrow-derived MLC as well as RA bone marrow-derived MLC induced the production of IgG much more effectively than that of IgM. There were no statistically significant differences in the enhancement of Ig production among the three groups. The MLC derived from the RA or OA bone marrow or from the cord blood by themselves produced <10 ng/ml IgG or IgM upon stimulation with IL-2 and IL-10. Therefore, the results indicate that the capacity to induce IgG production by peripheral blood B cells is not specific for the RA bone marrow-derived MLC.



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  		Figure 3. The comparable capacity of MLC induced from cord blood CD34+ cells or RA or OA bone marrow CD34+ cells by stimulation with SCF and GM-CSF to induce IgG production of peripheral blood B cells. B cells (1x105/well) were cultured in the presence of IL-2 (0.1 U/ml) and IL-10 (10 ng/ml) with or without CD34+ cells obtained from bone marrow of 23 RA patients or 11 OA patients or from 8 cord blood samples that had been stimulated with SCF (10 ng/ml) and GM-CSF (1 ng/ml) for 3–4 weeks (2.5–5.0x104/well). After 10 days of incubation, the supernatants were harvested and were assayed for IgM and IgG contents by ELISA. The increase in the amounts of IgM and IgG as a result of the addition of MLC induced from CD34+ cells was assessed by subtracting the production of IgM and IgG in cultures without the GM-CSF-stimulated CD34+ cells. The significance of the effects of MLC on the production of IgM and IgG was evaluated by Wilcoxon’s signed rank test. The significance of the differences in the enhancement of Ig production among MLC derived from cord blood CD34+ cells and those from RA and OA bone marrow CD34+ cells was evaluated by Student’s t-test.

 
Bone marrow-derived dendritic cells have the capacity to induce IgG production
Previous studies revealed that bone marrow CD34+ cells from RA or OA patients or cord blood CD34+ cells stimulated with SCF and GM-CSF for 4 weeks have the appearance of monocytes, dendritic cells, and fibroblast-like cells [11 ]. We therefore examined whether bone marrow-derived dendritic cells and nurse-like cells with fibroblast-like morphology might induce the production of IgG from peripheral blood B cells. Bone marrow-derived dendritic cells were induced by stimulation of bone marrow CD34+ cells with SCF, GM-CSF, and IL-4 for 4 weeks as described previously [11 ]. Nurse-like cells were cloned from RA bone marrow cells as described previously [4 ]. As can be seen in Table 1 , bone marrow-derived dendritic cells induced the production of IgG as effectively as bone marrow-derived MLC. By contrast, RA nurse-like cells (RA87 and RA91) did not induce the production of IgM or IgG. Therefore, these results indicate that bone narrow-derived dendritic cells, but not nurse-like cells, also have the capacity to induce IgG production from peripheral blood B cells.


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  		Table 1. Comparison of the Effects of Various Bone Marrow-Derived Cells on the Production of IgM and IgG

 
The induction of IgG production by bone marrow-derived MLC requires direct interactions with peripheral blood B cells
The next experiments were carried out to examine whether direct interactions between bone marrow-derived MLC and B cells are required for the induction of the IgG production. Highly purified B cells were cultured with the RA bone marrow-derived MLC in the presence of IL-2 and IL-10 such that they were separated by a filter membrane or were allowed to be in contact with each other. As can be seen in Figure 4 , the production of IgG was decreased markedly when bone marrow-derived monocytes were physically separated from B cells. These results therefore confirm that stimulation of IgG production of peripheral blood B cells requires direct cellular interactions between B cells and bone marrow-derived MLC. It should be noted, however, that there was already increased production of IgG in the presence of IL-2 and IL-10 even when B cells were separated from MLC. It is therefore suggested that there might be an additional soluble factor produced by MLC that enhances IgG production.



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  		Figure 4. Optimal induction of IgG production by bone marrow-derived MLC requires physical contact between bone marrow-derived MLC and B cells (B). B cells (5x105/well) were cultured in the presence of IL-2 (0.1 U/ml) and IL-10 (10 ng/ml) with or without bone marrow CD34+ cells (1x105/well) from RA patients that had been stimulated with SCF (10 ng/ml) and GM-CSF (1 ng/ml) for 3–4 weeks in wells of 24-well microtiter plates equipped with an inner chamber with membrane filter so that B cells (B) and the bone marrow-derived MLC (BM) might be allowed to be in contact or be separated from each other. The cultures were carried out in a total volume of 1 ml. After 10 days of incubation, the supernatants were harvested and were assayed for IgM and IgG contents by ELISA. The mean and SD values of five independent experiments are shown.

 
The collaboration between bone marrow-derived monocytes and B cells does not involve CD154-CD40 interactions
Some studies have shown that CD154 is expressed on activated T cells and plays a critical role in their capacity to provide contact-dependent help to resting B cells [19 , 20 ]. Of note, mRNA for the human CD154 also has been found to be expressed in mast cells and basophils [21 ] as well as in NK cells and monocytes [22 ]. Therefore, it was possible that the interactions between bone marrow-derived MLC and peripheral blood B cells might involve CD154. To address this point, experiments were carried out in which bone marrow-derived MLC and B cells were cocultured with IL-2 and IL-10 in the presence of anti-CD154 mAb or control mAb. As shown in Table 2 , anti-CD154 did not inhibit the production of IgG induced by bone marrow-derived MLC, although anti-CD154 inhibited Ig production of B cells induced by autologous CD4+ T cells. These results indicate that the interactions between bone marrow-derived MLC and B cells, which lead to IgG production, do not involve the CD154 molecule. Of note, it has been shown that CD27-CD70 interactions are also involved in T cell-dependent B cell activation [23 ]. However, anti-CD70 did not inhibit the IgG production induced by bone marrow-derived MLC, although it inhibited Ig production stimulated by T cells. It was therefore unlikely that the bone marrow-derived MLC were contaminated with activated T cells. Of note, previous studies showed that bone marrow-derived nurse-like cells can rescue B cells from spontaneous apoptosis and facilitate Ig production in a mechanism that was blocked by anti-CD106 mAb [4 ]. However, anti-CD106 did not inhibit the IgG production by bone marrow-derived MLC either. In this regard, the interactions between bone marrow-derived MLC and B cells to induce IgG production are different from those between bone marrow-derived nurse-like cells and B cells as described previously [4 ]. In fact, bone marrow-derived nurse-like cells did not induce IgG production in the presence of IL-2 and IL-10 (Table 1) .


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  		Table 2. Differential Effects of Anti-CD154 mAb on the Induction of the Production of IgG by Bone Marrow-Derived MLC and by CD4+ T cells

 
Bone marrow-derived MLC interact preferentially with peripheral blood IgD- B cells
Bone marrow-derived MLC induced the production of IgG much more effectively than that of IgM. It was therefore possible that the bone marrow-derived MLC might induce class switch. To test this hypothesis, we conducted experiments in which peripheral blood B cells were fractionated into IgD+ B cells and IgD- B cells. The capacity of bone marrow-derived MLC to induce the production of IgM and IgG from each population was then examined. As shown in Figure 5 , the RA bone marrow-derived MLC induced the production of IgG, but very modestly that of IgM, from unfractionated B cells as well as from IgD- B cells. Although IgD+ B cells produced substantial amounts of IgM in the presence of IL-2 and IL-10, the bone marrow-derived MLC did not affect it significantly. Moreover, the bone marrow-derived MLC induced the production of IgG from IgD+ B cells very modestly. Rather, the induction of IgG production by the bone marrow-derived MLC turned out to be dependent on the enhancement of IgG production of IgD- B cells. These results obviate the possibility that bone marrow-derived MLC might induce class switch. Moreover, the data demonstrate that bone marrow-derived MLC interact preferentially with IgD- B cells to induce IgG production.



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  		Figure 5. Bone marrow-derived MLC activate IgD- B cells preferentially to produce IgG. Unfractionated, IgD+, or IgD- B cells (1x105/well) were cultured in the presence of IL-2 (0.1 U/ml) and IL-10 (10 ng/ml) with or without bone marrow CD34+ cells from RA patients that had been stimulated with SCF (10 ng/ml) and GM-CSF (1 ng/ml) for 3 weeks. After 10 days of incubation, the supernatants were harvested and were assayed for IgM and IgG contents by ELISA. The mean and SD values of six experiments are shown. The statistical significance was evaluated by Wilcoxon’s signed rank test. *, Significant at P < 0.05 as compared with cultures without RA bone marrow-derived MLC. +, Significant at P < 0.05.

 
Finally, we compared the capacity of the bone marrow-derived MLC and BAFF to induce the production of IgM and IgG. As shown in Figure 6A , soluble BAFF, but not bone marrow-derived MLC, enhanced the production of IgM significantly from IgD+, IgD-, and unfractionated B cells. By contrast, bone marrow-derived MLC enhanced the production of IgG significantly from IgD+, IgD-, and unfractionated B cells, whereas soluble BAFF enhanced IgG production of IgD- and unfractionated B cells. Of note, bone marrow-derived MLC enhanced the production of IgG from IgD+, IgD-, and unfractionated B cells more effectively than soluble BAFF (Fig. 6B) . The results suggest that the interactions between B cells and bone marrow-derived MLC might be different from those involving BAFF in that bone marrow MLC and BAFF display differential capacities to induce the production of IgM and IgG.



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  		Figure 6. Differential effects of bone marrow-derived MLC and BAFF on the production of IgM and IgG. (A) Unfractionated, IgD+, or IgD- B cells (1x105/well) were cultured with RA bone marrow CD34+ cells that had been stimulated with SCF (10 ng/ml) and GM-CSF (1 ng/ml) for 3 weeks (2.5x104/well) or with soluble BAFF (10 ng/ml) in the presence of IL-2 (0.1 U/ml) and IL-10 (10 ng/ml). After 10 days of incubation, the supernatants were harvested and assayed for IgM and IgG contents by ELISA. The statistical significance was evaluated by Wilcoxon’s signed rank test. *, Significant at P < 0.05 as compared with cultures without BAFF or RA bone marrow-derived MLC. (B) Comparison of the enhancement of the production of IgM and IgG from IgD+, IgD-, and unfractionated B cells by BAFF or RA bone marrow-derived MLC. {Delta}IgM/{Delta}IgG were calculated by subtracting the production of IgM/IgG in cultures without BAFF or RA bone marrow-derived MLC from that in cultures with BAFF or RA bone marrow-derived MLC. The statistical significance was evaluated by Wilcoxon’s signed rank test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results in the current studies demonstrate clearly that after stimulation with SCF and GM-CSF for 3–4 weeks, bone marrow CD34+ cells give rise to MLC, which display the capacity to activate resting peripheral blood B cells to produce IgG. Of note, the IgG production was decreased markedly when the bone marrow-derived MLC were physically separated from B cells by a filter membrane. Moreover, the capacity of the bone marrow-derived MLC to stimulate IgG production was preserved after fixation with paraformaldehyde. Therefore, the results indicate that activation of B cells by bone marrow-derived MLC requires direct contact-dependent interactions. In this regard, the activation of B cells by bone marrow-derived MLC is comparable to that by activated T cells [13 ]. Conversely, it should be pointed out that bone marrow CD34+ cells stimulated with SCF, GM-CSF, and IL-4, which have the characteristics of dendritic cells morphologically and phenotypically [11 ], also displayed the comparable capacity to induce the production of IgG. Because several studies showed that dendritic cells are generated from bone marrow-derived monocytes [24 25 26 ], the data indicate that the capacity to induce IgG production is preserved after differentiation of MLC into dendritic cells.

Analysis on flow cytometry revealed that more than 90% of bone marrow CD34+ cells stimulated with SCF and GM-CSF were CD14+ HLA-DR+. There were less than 0.5% of CD3+ cells or CD19+ cells within these populations. Moreover, no CD3+ cells were detected in 5- to 10-day cultures of the bone marrow-derived MLC and B cells in the presence of IL-2 and IL-10 (unpublished results). Therefore, it is most likely that CD14+ HLA-DR+ MLC, but not contaminating T cells, can activate resting B cells. In fact, the activation of B cells induced by bone marrow-derived MLC is different from that induced by activated T cells in several aspects. For example, it has been disclosed that the activation of B cells by activated T cells induces the production of IgM and IgG [27 ], as has also been shown in the present study. By contrast, the activation of B cells by bone marrow-derived MLC induced the production of IgG, but very modestly that of IgM. Furthermore, B cell activation induced by activated T cells was inhibited by anti-CD154 mAb as well as anti-CD70 mAb as is consistent with previous studies [23 , 28 ], whereas B cell activation induced by bone marrow-derived MLC was not blocked by anti-CD154 mAb or anti-CD70 mAb.

Of note, previous studies disclosed that nurse-like cells from bone marrow and synovium of RA patients promote survival and enhance the function of human B cells [4 ]. Although most of bone marrow CD34+ cells stimulated with SCF and GM-CSF were phenotypically CD14+ HLA-DR+, it was possible that this population was contaminated with nurse-like cells. It should be noted that the function of bone marrow-derived nurse-like cells to support B cell responses was blocked by anti-CD106 mAb [4 ]. However, anti-CD106 mAb did not inhibit the production of IgG bone marrow-derived MLC. Moreover, bone marrow-derived nurse-like cell clones could not elicit the production of IgG from peripheral blood B cells in the presence of IL-2 and IL-10. It is therefore unlikely that the capacity of the bone marrow-derived MLC to induce IgG production might be mediated by contaminating nurse-like cells. Taken together, the data suggest that the activation of B cells induced by bone marrow-derived MLC might involve unique cellular interactions that have not been identified yet.

Recent studies have disclosed the presence of a novel ligand of the TNF family designated BAFF [29 ] or BLyS (B-lymphocyte stimulator) [30 ] on T cells [29 ], dendritic cells [29 ], and monocytes [30 ]. BAFF and BLyS have been found to induce proliferation of peripheral blood B cells stimulated with anti-IgM or S. aureus Cowan I, whereas they were unable to activate resting B cells without stimulation of anti-IgM or S. aureus Cowan I [29 , 30 ]. Therefore, it is suggested that BAFF and BLyS might be only a costimulator of B cells, although they play an important role in monocyte-driven B cell activation [29 , 30 ]. Moreover, treatment of BALB/cAnNCR mice by BLyS resulted in the elevation of IgM and IgA, but not IgG in the serum [30 ]. Of note, in the present study, bone marrow-derived MLC induced IgG production more effectively than BAFF, although they were less potent in inducing IgM production than BAFF. Consistently, the results disclosed that soluble BAFF alone induced the production of IgM by IgD+ B cells as well as that by IgD- B cells in the presence of IL-2 and IL-10. Thus, the results demonstrate for the first time that beyond its role as a costimulator of B cells shown in the previous studies [29 , 30 ], BAFF by itself can induce Ig production from resting B cells in the presence of IL-2 and IL-10. However, the data suggest that the interactions between B cells and bone marrow-derived MLC shown in the current studies might be different from those that involve BAFF or BLyS.

Previous studies have shown that dendritic cells provide naive B cells with signals that are essential for class switch [31 ]. In the present study, the bone marrow-derived MLC were found to contain a small proportion of cells of dendritic cell morphology (unpublished results), which are considered to correspond to CD14dull HLA-DRbright cells on flow cytometry [32 ]. Thus, it was possible that such dendritic cells within the bone marrow-derived MLC might induce class switch in naive B cells. In fact, bone marrow-derived cells, which had been generated by stimulation of bone marrow CD34+ cells with SCF, GM-CSF, and IL-4, also induced the production of IgG. However, the bone marrow-derived MLC only modestly stimulated IgD+ B cells to produce IgG in the presence of IL-2 and IL-10. Moreover, IgG production could not be elicited from IgD+ B cells or cord blood B cells even when they had been cocultured with the bone marrow-derived MLC and thereafter restimulated with immobilized anti-CD3-activated CD4+ T cells (unpublished results). Therefore, the results indicate that the bone marrow-derived MLC in the current studies do not provide signals essential for class switch. Rather, the data support the conclusion that the bone marrow-derived MLC stimulate IgD- B cells preferentially through direct cellular interactions. Thus, it is suggested that IgD- B cells might express such molecules that are involved in the interactions with bone marrow-derived MLC more abundantly than IgD+ B cells.

Of note, the optimal IgG production induced by bone marrow-derived MLC required the presence of IL-2 and IL-10. Previous studies showed that IL-2 and IL-10 synergistically enhance Ig production of B cells stimulated with anti-CD40 or S. aureus Cowan I [33 , 34 ]. Moreover, it has been shown that the synergistic effects of IL-2 and IL-10 involve a mechanism that is different from the up-regulation of the expression of IL-2 receptors [34 ]. In fact, IL-2 and IL-10 still displayed additive effects on Ig production in the presence of high doses of cyclosporine that blocked the IL-10-mediated up-regulation of IL-2 receptors, indicating that IL-2 and IL-10 provide signals that are mutually independent [34 ]. Thus, it is possible that the production of IgG induced by bone marrow-derived MLC might require two mutually independently signals delivered by IL-2 and IL-10. Further studies to delineate the nature of signals in B cells delivered by IL-2 and IL-10 would be important for a complete understanding of the mechanism of synergy between IL-2 and IL-10 as well as the mechanism of activation of memory B cells by bone marrow-derived MLC.

It has been shown that the spontaneous generation of CD14+ MLC from bone marrow CD14- precursor cells is accelerated in RA [6 ], possibly resulting in the facilitated entry of such CD14+ cells into the synovium [6 ]. Although the features of bone marrow-derived MLC shown in the current studies were not specific for RA, it is likely that the promoted entry of such cells into the synovium might result in the sustained activation of B cells. It is therefore likely that the capacity of bone marrow-derived MLC to activate B cells might play a role in the local B cell activation and the collection of plasma cells in the synovium, which are characteristic features in RA [35 36 37 ].

In summary, the results in the current studies disclosed that bone marrow-derived MLC generated from CD34+ progenitor cells by stimulation with SCF and GM-CSF displayed the capacity to stimulate IgD- B cells preferentially to produce IgG through as-yet undetermined contact-dependent interactions in the presence of IL-2 and IL-10. Further studies to explore the nature of such interactions would be important for our understanding of the pathogenesis of RA as well as the mechanisms of monocyte-driven B cell activation.


   ACKNOWLEDGEMENTS
 
This work was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion, and Productive Review of Japan. The authors thank Peter E. Lipsky for critical reading of the manuscript and Chise Kawashima for preparing the illustrations and the manuscript.

Received November 27, 2000; revised September 24, 2001; accepted February 2, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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