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The Journal of Immunology, 2005, 175: 2807-2813.
Copyright © 2005 by The American Association of Immunologists

Follicular Dendritic Cells Catalyze Hepatocyte Growth Factor (HGF) Activation in the Germinal Center Microenvironment by Secreting the Serine Protease HGF Activator

Esther P. M. Tjin*, Richard J. Bende*, Patrick W. B. Derksen*, Anne-Pauline van Huijstee*, Hiroaki Kataoka{dagger}, Marcel Spaargaren* and Steven T. Pals1,*

* Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands; and {dagger} Second Department of Pathology, Miyazaki Medical College, Kiyotake, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ag-specific B cell differentiation, the process that gives rise to plasma cells and memory B cells, involves the formation of germinal centers (GC). Within the GC microenvironment, multiple steps of B cell proliferation, selection, and maturation take place, which are controlled by the BCR in concert with cytokines and contact-dependent signals from follicular dendritic cells (FDCs) and T cells. Signaling by the multifunctional cytokine hepatocyte growth factor (HGF) and its receptor MET has been shown to induce integrin-mediated adhesion of B cells to VCAM-1, which is expressed by FDCs. In the present study we have examined the expression of regulatory components of the HGF/MET pathway, including HGF activator (HGFA), within the secondary lymphoid organ microenvironment. We show that MET is expressed by both centroblasts and plasma cells, and that HGFA is expressed by plasma cells. Because we have shown that HGF is a potent growth and survival factor for malignant plasma cells, HGF may also serve as a survival factor for normal plasma cells. Furthermore, we demonstrate that FDCs are the major source for HGF and its activator within the GC microenvironment. Both HGF and HGFA are expressed by FDCs in the GC dark zone (CD21high/CD23low), but not in the light zone (CD21high/CD23high). These findings suggest that HGF and HGFA provided by dark zone FDCs help to regulate the proliferation, survival, and/or adhesion of MET-positive centroblasts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The germinal center (GC)2 comprises a dynamic microenvironment in which Ag-specific B cell differentiation takes place. Morphological studies have shown that the GC consists of two distinct compartments, the dark and light zones, associated with important separate functions (1, 2, 3, 4). B cells in the dark zone, called centroblasts, undergo rapid clonal expansion and somatic hypermutation of their Ig variable region genes. They subsequently move to the light zone to become centrocytes, which undergo selection based on the affinity of their BCR for Ag presented by follicular dendritic cells (FDCs) (3, 5). Centrocytes with low-affinity receptors and centrocytes that do not receive T cell help undergo apoptosis, whereas centrocytes with a high-affinity BCR receive survival signals from FDCs and T cells and differentiate into plasma cells or memory B cells (1, 2, 3, 5, 6).

Notwithstanding ample insight into the anatomy and function of the GC, the mechanisms orchestrating GC polarity and guiding B cell movement and differentiation within the distinct GC compartments are as yet incompletely understood. Molecules that play a crucial role are chemokines and adhesion receptors. Chemokines, including CXCL13 (B cell-attracting chemokine/B lymphocyte chemoattractant) (7, 8) and CXCL12 (stromal cell-derived factor-1 (SDF-1)), recruit B cells into the GC and regulate their positioning in the dark and light zones (9). The integrin adhesion molecules LFA-1 and VLA-4 mediate attachment of GC B cells to FDCs (10, 11). The integrin activation required for this adhesion presumably is controlled by high-affinity interactions of the BCR with Ag presented by FDCs (12). Apart from establishing physical contact with FDCs, integrin signaling can rescue GC B cells from apoptosis (13, 14) and hence may directly contribute to B cell selection. Once positioned within the GC microenvironment, B cell proliferation, survival, and differentiation are supported by cytokines produced by T cells, FDCs, and other GC accessory cells. A FDC-produced cytokine that may play a role in GC organization as well as in promoting GC B cell proliferation, survival, and differentiation is hepatocyte growth factor/scatter factor (HGF) (15). HGF is a multifunctional cytokine with a domain structure and a proteolytic mechanism of activation similar to those of the blood serine protease plasminogen, but it lacks protease activity (15). Via the tyrosine kinase receptor MET, HGF induces complex biological response in target cells, including growth, survival, and motility. Whereas a functional HGF/MET pathway is indispensable for mammalian development, uncontrolled MET signaling is oncogenic and has been implicated in the development of a variety of human cancers (16, 17). Within the GC microenvironment, MET is expressed by centroblasts, whereas FDCs express and secrete HGF (18). Upon CD40 and BCR stimulation, naive B cells acquire MET expression, and functional studies have shown that HGF induces integrin-dependent adhesion of MET-positive B cells to VCAM-1, expressed by FDCs (18, 19, 20). Together, these data suggest a role for the HGF/MET pathway in regulating B cell-FDC interaction.

Upon secretion, HGF normally retains its 90-kDa single-chain (sc) precursor form, which is probably cell surface or extracellular matrix associated. For biological function, however, proteolytic conversion of scHGF to the heterodimeric active form is essential (21). Plasminogen activators, particularly urokine plasminogen activator and factor XIIa, have been shown to activate scHGF, although at low rates (22, 23). More recently, HGF activator (HGFA), a factor XIIa-related serine protease with efficient HGF-activating activity, was identified (21, 23). HGFA has been implicated in HGF activation at sites of inflammation and tissue repair (24) as well as by tumor cells (25), including multiple myeloma (MM), a malignancy of plasma cells (26). In this study we have examined the mechanism of HGF activation within the GC microenvironment. We show that FDCs derived from the GC dark zone, in contrast to those from the GC light zone, produce HGFA and in this way are able to catalyze HGF activation. In addition, we demonstrate that normal plasma cells express HGFA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

The mAbs used were FITC-conjugated anti-human IgD (DakoCytomation); allophycocyanin-conjugated anti-human CD38 (IgG1; BD Biosciences); PE-conjugated anti-human CD20 (DakoCytomation); anti-human MET DO-24 (IgG2a; Upstate Biotechnology); anti-HGFA (A1 and P1-4, IgG1) (24); anti-factor XIIa, OT-2 (IgG1; Sanquin); anti-human CD21 long isoform (dendritic reticulum cell (DRC)-1); anti-human CD21 (clone 1F8), FITC-conjugated anti-human CD21 (clone 1F8), and anti-CD20 (L26; all from DakoCytomation); anti-CD14 (Leu M3, IgG2b); anti-CD3 (Leu 4, IgG1; BD Biosciences); and isotype controls anti-IgG1, anti-IgG2a, and anti-IgG2b (DakoCytomation). Secondary Abs used were post-Ab for powervision (Immunovision Technologies), HRP-conjugated goat-anti-mouse/rat IgG, and HRP-conjugated anti-FITC (DakoCytomation).

Purification of B cell populations and FDCs

B cells were purified from human tonsils obtained from children undergoing routine tonsillectomy as previously described (13). Briefly, mononuclear cells were isolated by Ficoll-Isopaque density gradient centrifugation. Monocytes and T cells were depleted by plastic adherence and SRBC rosetting, respectively. The total B cells fraction was >97% pure as determined by FACS analysis. To obtain the different B cell populations (naive B cells, memory B cells, GC B cells, and plasma cells), total B cells were stained with FITC-conjugated anti-human IgD, PE-conjugated anti-human CD20, and allophycocyanin-conjugated anti-CD38 and were sorted using a FACS aria (BD Biosciences).

FDC clusters (enriched FDC preparation) were isolated from human tonsils as described previously by Liu et al. (27). Tonsils were cut into pieces and treated with collagenase/DNase mix (200 U/ml collagenase IV and 10 U/ml DNase I; Roche), followed by Ficoll-Isopaque density gradient centrifugation and density sedimentation on a BSA gradient (1.5% BSA).

FDCs (CD21high) were stained with FITC-conjugated anti-CD21 and sorted using FACS aria (BD Biosciences). PE-conjugated anti-CD23 together with anti-CD21 were used to obtain the CD23 FDC populations.

For immunocytochemical staining, FDCs were isolated from an enriched FDC preparation by cell sorting of large cells. Cell purity was ~60–80%, as judged by staining for DRC-1, a FDC-specific marker.

Assay for HGF activation

HGF activation was assayed as described previously (25). In brief, scHGF (R&D Systems) was incubated with FDC-conditioned medium. Conditioned medium was obtained as described previously (28). For HGF activation, 20 µl of conditioned medium were pretreated with 1 U of thrombin and added to 0.1 µg of scHGF. Inhibitor studies were performed in the presence of leupeptin (500 µg/ml), neutralizing Ab against FXII (OT-2; provided by E. Hack, Sanquin, Amsterdam, The Netherlands), or neutralizing Ab against HGFA (P1-4; 40 µg/ml).

FDC sarcoma

The FDC sarcoma, which is a rare neoplasm that arises from lymph nodes as well as extranodal regions (29), was obtained from a 38-year-old male patient and was diagnosed at the Department of Pathology, Academic Medical Center (Amsterdam, The Netherlands). The tumor showed >95% CD21- and DRC-1-positive cells, indicating almost complete replacement of the normal follicle structure by the neoplasm.

Immunocytochemistry

Immunocytochemical stainings were performed on acetone-fixed cytospins. The cytospins were preincubated with 1% BSA in PBS for 15 min. After incubating with the primary Ab (overnight at 4°C), endogenous peroxidase was blocked with 0.1% NaN3 and 0.3% H2O2 in PBS for 10 min. Subsequently, the cytospins were stained with post-Ab of Powervision (Immunovision Technologies) for 15 min, followed by poly-HRP-conjugated goat anti-mouse/rabbit IgG for 30 min. Substrate was developed with 3,3-amino-9-ethylcarbazole (Sigma-Aldrich).

RNA isolation, cDNA synthesis, and RT-PCR

RNA from B cell fractions and cultures was isolated with Nucleospin RNA 11 (Macherey-Nagel). For sorted fractions of plasmablasts and FDCs, Pico Pure RNA isolation kit (Arcturus) was used. After RNA isolation, cDNA was synthesized using 2 nmol Pd(N)6 primer (Pharmacia Biotech) and 160 U of mouse Moloney leukemia virus reverse transcriptase (Invitrogen Life Technologies). The reaction mixture also contained 8 mmol/l DTT, 1 mmol/l of each dNTP, 1x first-strand buffer (50 mmol/l Tris-HCl (pH 8.3), 75 mmol/l KCl, and 3 mmol/l MgCl2), and 24 U of RNase inhibitor (Roche). The reaction was performed for 15 min at 37°C, after which the enzyme was inactivated for 10 min at 95°C.

PCR was performed using Taq DNA polymerase (Invitrogen Life Technologies), 10 µM dNTPs (Pharmacia Biotech), and 1.5 mM MgCl2 in 1x PCR buffer (Invitrogen Life Technologies). The primers used were as follows: HGFA forward, 5'-AGGACACAAGTGCCAGATTG-3; HGFA reverse, 5'-GTTGATCCAGTCCACACATAGT-3'; MET forward, 5'-GAGACTCATAATCCAACTG-3'; MET reverse, 5'-AGCATACAGTTTCTTGCAG-3'; HGF forward, 5'-CAGCATGTCCTCCTGCATCTCC-3'; and HGF reverse, 5'-TCGTGTGGTATCATGGAACTCC-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MET is expressed in human GC B cells and plasma cells

We investigated the expression of MET mRNA (Fig. 1B) and protein (Fig. 1A) in human tonsillar B cell subpopulations using purified B cell subsets isolated by FACS sorting or by FACS analysis of unsorted B cell fractions, respectively. As shown in Fig. 1, we detected MET mRNA (Fig. 1B) and protein expression by GC B cells (Fig. 1, A and C), but not by naive (IgD+,CD38–) and memory B (IgD–,CD38–) cells. In addition, we observed that MET is expressed by plasma cells (Fig. 1, A–C), which represent a small (~1–4%) subset of tonsillar cells characterized by a high expression of CD38 (30). Consistent with the results obtained by studying isolated B cell subsets, immunohistochemical studies of tissue sections of human tonsils and lymph nodes also demonstrated MET expression on GC B cells (Fig. 1D). This expression was most prominent in the GC dark zones, indicating preferential expression of MET on centroblasts. Taken together, these data confirm our previous observation that MET is expressed by centroblasts (18) and, moreover, demonstrate that plasma cells also express MET.



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  		FIGURE 1. A, Expression of MET by B cell subsets. B cell subsets were identified by IgD and CD38 staining as being naive B cells (IgD+,CD38–), GC founder B cells (IgD+,CD38+), GC B cells (IgD–,CD38+), plasma cells (IgD–,CD38++), and memory B cells (IgD–,CD38–; left panel). Unsorted B cells were analyzed for the expression of MET using a CD38/IgD/MET triple-FACS staining. Results are shown as the mean fluorescence intensity (MFI) minus the MFI of the isotype control (right panel). B, MET mRNA is expressed in human GC B cells and plasma cells. Tonsillar B cell subsets were isolated by FACS sorting using CD20/CD38/IgD triple staining. After RNA isolation and cDNA synthesis, RT-PCR for MET was performed. {beta}2-Microglobulin was used as housekeeping gene control. C, Expression of MET protein in human GC B cells and plasma cells. B cell subsets were collected by FACS sorting, and the expression for MET was analyzed using immunoblotting. The MM cell line LME-1 was used as a positive control. D, MET expression in human tonsil. Immunohistochemical double staining of tonsillar sections for MET (blue) and IgD (brown), showing MET expression on GC B cells. DZ, dark zone; LZ, light zone.

 
Plasma cells and FDCs express the serine protease HGFA

We have previously shown that FDCs express and secrete HGF, the ligand of MET (18). However, for biological function, proteolytic conversion of scHGF to the active heterodimeric form is essential (21). In a recent study we demonstrated that MM plasma cells produce HGFA and in this way may activate HGF in the bone marrow microenvironment (26). To assess whether HGFA is expressed and mediates HGF conversion in normal lymphoid tissue, we first assessed the expression of HGFA mRNA and protein by B cells and plasma cells. Interestingly, purified plasma cells strongly expressed HGFA mRNA (Fig. 2A) and protein (Fig. 2B), whereas HGFA expression in naive, memory, and GC B cells was either weak or absent. In contrast to HGFA, HGF mRNA was not detected in purified B cell subsets or plasma cells. The observed expression of HGF mRNA in the unsorted B cell fractions is explained by the presence of FDCs (see below).



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  		FIGURE 2. A, Expression of HGFA in B cell subsets. Tonsillar B cell populations were isolated by FACS sorting using CD20/CD38/IgD triple staining. After RNA isolation and cDNA synthesis, RT-PCR for HGFA and HGF was performed. {beta}2-Microglobulin was used as housekeeping gene control. B, Plasma cells express HGFA. Highly purified B cell populations were collected after FACS sorting, and the expression of HGFA was analyzed using immunoblotting. The MM cell line LME-1 was used as a positive control. C, HGFA expression in total tonsillar B cell preparations. Immunocytochemical staining of unsorted B cell preparations for HGFA, the B cell marker CD20, and the monocyte marker CD14 was performed. The arrowhead indicates a plasmacytoid cell (inset), whereas the arrow indicates a FDC. D, HGFA is expressed by FDCs. FDCs were isolated from an enriched FDC preparation by cell sorting of large cells. The cell purity was determined by staining for the FDC marker DRC-1 and the B cell marker CD20. The mAb A-1 against HGFA was used to detect HGFA expression. The arrow indicates a FDC. E, MET and HGFA expression in sorted FDC preparations. Purified FDC preparations were analyzed by immunoblotting for the expression of MET and HGFA.

 
To confirm and extend these observations, we also performed immunocytochemistry on human tonsillar B cells depleted of T cells and monocytes (Fig. 2C). This population consisted of >97% CD20-positive cells and did not contain monocytes (CD14+) or T cells (CD3+). Of note, scattered cells within this population showed a strong expression of HGFA (Fig. 2C). Apart from a few cells with a plasmacytoid morphology, these HGFA-positive cells showed morphological features of FDCs, i.e., large oval cells with an eccentric or double nucleus (Fig. 2C). Indeed, immunocytochemical staining of FACS-sorted FDCs showed a distinct granular HGFA expression pattern (Fig. 2D), whereas immunoblotting confirmed the expression of HGFA (Fig. 2E). Hence, both plasma cells and FDCs express HGFA.

HGF and HGFA are specifically expressed by GC dark zone FDCs

GCs consist of dark and light zones. The FDCs within these distinct functional compartments show marked phenotypic differences: whereas both dark and light zone FDCs express the long isoform of CD21, FDCs in the light zone express additional molecules, including CD23, Fc{gamma}RII, VCAM-1, and CXCL13 (1, 8, 9, 31, 32). To assess whether HGF and HGFA are also differentially expressed in dark and light zone FDCs, these cells were separated based on differential CD23 expression (Fig. 3A; Ref.33). Although no expression of HGF and HGFA mRNA was detected in the CD23high FDCs, the expression of both transcripts was prominent in the CD23low FDCs (Fig. 3B). Hence, HGF and HGFA expression is a feature of dark zone FDCs. In addition to differential HGF and HGFA expression, we also observed differential expression of CXCL13 mRNA in the CD23-sorted FDCs, confirming the study by Allen et al. (9) showing that this chemokine is predominantly expressed by GC light zone FDCs.



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  		FIGURE 3. A, Dark zone FDCs express HGF and HGFA. Highly purified FDCs were collected from enriched FDC preparation after FACS sorting using the markers CD21 and CD23. The purity of both sorted FDC fractions was determined by restaining the cells with FITC-conjugated anti-human CD21, followed by HRP-conjugated anti-FITC and detection with 3,3-amino-9-ethylcarbazol. Inset, High magnification of the cells. B, CD21+ FDCs, CD23low FDCs (GC dark zone), CD23high (GC light zone), and FDC sarcoma were analyzed for the expression of HGF, HGFA, CD21, and CXCL13 using RT-PCR. {beta}2-Microglobulin was used as a housekeeping gene control.

 
Expression of HGF and HGFA by FDC sarcoma

To corroborate our finding that FDCs express both HGF and HGFA, we also studied the expression of HGF and HGFA mRNA in FDC sarcoma, a rare neoplasm composed of malignant FDCs (29). As in normal dark zone FDCs, both HGF and HGFA were prominently expressed by this tumor (Fig. 3B). We were unable to detect MET expression on the FDC sarcoma cells (data not shown), indicating the absence of an autocrine HGF/MET loop in this tumor.

FDCs convert HGF by producing HGFA

Subsequently, we investigated whether FDCs are able to process scHGF to its active form. Conditioned medium of FDCs effectively converted scHGF (Fig. 4). This required thrombin, whereas the conversion was completely inhibited by the serine protease inhibitor leupeptin (Fig. 4A). Because proteases other than HGFA are, although with low efficiency, capable of activating scHGF in vitro, we explored whether the conversion of scHGF by FDCs could be inhibited by specific interference with HGFA activity. We observed that the anti-HGFA mAb P1-4, which blocks HGFA function, effectively inhibits scHGF conversion by FDCs (Fig. 4A). These findings identify HGFA as the (major) serine protease responsible for the conversion of scHGF by FDCs and identify FDCs as important regulators of HGF activity in the GC microenvironment.



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  		FIGURE 4. FDCs autocatalyze HGF activation by producing HGFA. A, Conditioned medium of an enriched FDC preparation was incubated with scHGF for 6 h in the presence of thrombin combined with either the serine protease inhibitor, leupeptin, or neutralizing Ab against HGFA (P1-4) or against factor XII, as indicated. B, As a positive control, HGF conversion by recombinant HGFA is shown. HGF conversion was determined by immunoblotting with anti-HGF. {alpha}HGF, {alpha}-chain of HGF; HC, H chain of Ig.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Proteolytic activation of HGF in the extracellular milieu is a critical limiting step in HGF/MET signaling (21), In this study we present data indicating an important role of the serine protease HGFA in regulating HGF activation in normal lymphoid tissue. We demonstrate that HGFA is expressed by plasma cells, but not by other B-lineage populations. Furthermore, we show that HGFA is strongly expressed by FDCs and is confined to FDCs in the GC dark zone. These cells coexpress HGF and are in close contact with MET-positive centroblasts. Our findings suggest specific functions of the HGF/MET pathway in Ag-specific B cell differentiation, affecting plasma cell and centroblast functions.

Previous studies from other and our own laboratories have implicated the HGF/MET pathway in the pathogenesis of the plasma cell malignancy MM (34, 35, 36, 37). Our current study represents the first report of HGF/MET pathway components in normal plasma cells. We observed that normal plasma cells express the receptor tyrosine kinase MET, but do not express its ligand, HGF (Figs. 1 and 2A). In addition, we show that HGFA is expressed by plasma cells, but not by other B-lineage populations (Fig. 2, A and B). The coexpression of MET and HGFA in plasma cells is of interest because it indicates that plasma cells are well equipped to receive paracrine HGF signals. Indeed, tonsillar as well as bone marrow stromal cells have been reported to produce HGF, and it is hence conceivable that plasma cells receive paracrine stimulation from the microenvironment (18, 20, 38). Contrary to their malignant counterparts (35), normal plasma cells do not express HGF and hence do not possess an autocrine HGF/MET loop. Although the functional consequences of HGF/MET signaling in the plasma cell have not yet been explored, as in B cells (18, 20, 39), the pathway might regulate integrin-mediated adhesion and promote migration. HGFA expression by plasma cells could thus play a role in their homing to the bone marrow and control their integrin-mediated interaction with bone marrow stromal cells. These stromal cells produce cytokines that support plasma cell survival, including IL-5, IL-6, TNF-{alpha}, and SDF-1. In view of the potent effects of HGF on the survival of MM plasma cells (37), it is conceivable that HGF produced by bone marrow stromal cells also contributes to plasma cell survival.

Differential protein expression within the GC dark and light zones reflects the distinct processes that take place within these compartments. One of the key findings of our study is that FDCs are able to autocatalyze HGF activation by producing both HGF and HGFA (Fig. 4) and that HGFA and HGF expression by FDCs is confined to the CD23high subset, which resides in the GC dark zone (Fig. 3B). Combined with our previous observation that MET is selectively expressed by B cells in the GC dark zone, i.e., by centroblasts, these observations suggest specific functions for the HGF/MET pathway in the GC dark zone. HGF stimulation of B cells has been shown to mediate integrin activation, promoting B cell adhesion to VCAM-1 and ICAM-1 (11, 19), two important integrin ligands on FDCs (10, 11). Hence, active HGF in the GC dark zone could play a role in initiating physical contact of MET-positive centroblasts, which have down-regulated their BCR, with FDCs. Upon transition to the light zone, the B cells, now centrocytes, re-express their BCR, which engages in interactions with Ag presented by FDCs. At this stage, high-affinity interactions of the BCR with Ag could take over the regulation of integrin activity required for B cell-FDC interaction (12). Alternatively, MET-mediated growth and survival signals might directly contribute to the expansion of centroblasts. These signals could be crucial at a differentiation stage at which the cells are largely devoid of BCR expression and may not receive sufficient growth and survival signals.

Cell migration within the lymphoid organs is directed by chemokines and is essential during GC reaction. The chemokine CXCL13 produced by follicular stromal cells is required for recruiting Ag-activated B cells to the GC (8). We observed that CD23high FDCs expressed high levels of CXCL13 mRNA compared with CD23low FDCs (Fig. 3B). This observation confirms that by Allen et al. (9), who, by immunohistochemical staining of the GC and by microdissection of the different GC regions, also demonstrated that CXCL13 is more abundantly expressed in the GC light zone than in the dark zone. Mice lacking CXCL13 (B lymphocyte chemoattractant) or its receptor, CXCR5, show defects in GC localization and size, indicating an important role in GC organization (8, 40). CXCR4 and its ligand, SDF-1, also contribute to GC organization, specifically to sorting of centroblasts into the GC dark zone (9).

Several studies have implicated the HGF/MET pathway in the pathogenesis of B cell neoplasia (18, 19, 34, 35, 36, 37, 39). The expression of MET has been demonstrated in MMs and a subset of non-Hodgkin’s lymphomas. Activation of the pathway in these tumors may involve autocrine stimulation, because coexpression of HGF and MET has been observed (35, 41, 42, 43). In MMs, MET activation promotes proliferation and survival (37). We observed that MMs, compared with normal plasma cells, strongly expressed HGFA as well as MET protein (Fig. 2B). Overexpression of HGFA by tumor cells may enhance HGF/MET signaling, promoting cell growth and survival, and may contribute to disease progression. In the FDC sarcoma tested (Fig. 3B), we observed strong expression of both HGF and HGFA, suggesting that the tumor was related to GC dark zone FDCs. However, the tumor cells did not express MET, excluding a role for autocrine HGF/MET signaling in this tumor.

In summary, our study indicates that FDCs regulate the bioavailability of HGF within the GC microenvironment, a function that may contribute to the control of B cell growth, survival, and adhesion during normal B cell differentiation within lymphoid tissue (Fig. 5).



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  		FIGURE 5. Components of the HGF/MET pathway in the GC and bone marrow microenvironments. During B cell differentiation, HGF and HGFA produced by dark zone (DZ) FDCs (CD23–) may act as proliferative and survival factors for the MET-positive centroblasts (CB). In the light zone (LZ), where the FDC network is more extensive, the centrocytes (CC) are found in tight association with FDCs (CD23+), and via direct physical contact, the CCs receive survival signals. After B cell differentiation, the plasma cells home to the bone marrow (BM), where BM stromal cells secrete HGF. The MET-positive plasma cells autoactivate HGF by producing HGFA, which may contribute to plasma cell survival.

 

   Acknowledgments
 
We thank Berend Hooibrink for sorting the B cells and FDCs and the coworkers of the Department of Otolaryngology of the Saint Lucas/Andreas Hospital (Amsterdam, The Netherlands) for their kind support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Steven T. Pals, Department of Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail address: s.t.pals{at}amc.uva.nl Back

2 Abbreviations used in this paper: GC, germinal center; FDC, follicular dendritic cell; HGF, hepatocyte growth factor; HGFA, HGF activator; MM, multiple myeloma; DRC, dendritic reticulum cell; scHGF, single-chain HGF; SDF-1, stromal cell-derived factor-1. Back

Received for publication December 29, 2004. Accepted for publication June 10, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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